RhII-Catalyzed De-symmetrization of Ethane-1,2-dithiol and Propane-1,3-dithiol Yields Metallo-β-lactamase Inhibitors

Article abstract of DOI:10.1002/cmdc.202100344

Diversity-oriented synthesis (DOS) is a rich source for novel lead structures in Medicinal Chemistry. In this study, we present a DOS-compatible method for synthesis of compounds bearing a free thiol moiety. The procedure relies on Rh(II)-catalyzed coupling of dithiols to diazo building blocks. The synthetized library was probed against metallo-β-lactamases (MBLs) NDM-1 and VIM-1. Biochemical and biological evaluation led to identification of novel potent MBL inhibitors with antibiotic adjuvant activity.

Full text of DOI:10.1002/cmdc.202100344

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Very Important Paper
RhII-Catalyzed De-symmetrization of Ethane-1,2-dithiol and
Propane-1,3-dithiol Yields Metallo-β-lactamase Inhibitors
Mikhail Krasavin,*[a] Daniil Zhukovsky,[a] Igor Solovyev,[a] Darina Barkhatova,[a] Dmitry Dar’in,[a]
Denia Frank,[b] Giada Martinelli,[b] Lilia Weizel,[c] Anna Proschak,[c] Marco Rotter,[c]
Jan S. Kramer,[c] Steffen Brunst,[c] Thomas A. Wichelhaus,[b] and Ewgenij Proschak*[c]
Diversity-oriented synthesis (DOS) is a rich source for novel lead
structures in Medicinal Chemistry. In this study, we present a
DOS-compatible method for synthesis of compounds bearing a
free thiol moiety. The procedure relies on Rh(II)-catalyzed
coupling of dithiols to diazo building blocks. The synthetized
library was probed against metallo-β-lactamases (MBLs) NDM-1
and VIM-1. Biochemical and biological evaluation led to
identification of novel potent MBL inhibitors with antibiotic
adjuvant activity.
Introduction
been developed which reached very significant binding
potency in vitro,[5] none of them reached clinical evaluation yet.
Previously we showed that approved drugs exhibiting free thiol
moieties potently inhibit different MBLs in vitro.[6] Our efforts to
follow the SOSA (selective optimization of side activities)
approach[7] to optimize the approved drugs thiorphan and
captopril towards efficient MBL inhibitors revealed numerous
challenges in the development of thiol-based MBL inhibitors.[8]
Recently, we discovered an efficient Rh(II)-catalyzed SÀ H
insertion reaction of α-diazo-γ-butyrolactams with a variety of
aromatic and aliphatic thiols.[9] Interestingly, the same reaction
with ethane-1,2-dithiol led to the formation of the mono-
insertion product in good chemical yield. To the best of our
knowledge, the latter reaction represents the first example to a
general approach to de-symmetrization of symmetrical aliphatic
dithiols 2 using chemistry of diazo compounds 1 (Figure 1). In
this article, we describe an application of this novel synthetic
approach towards the linking of a thiol moiety to a range of
chemically diverse aliphatic scaffolds which led to identification
of potent MBL inhibitors among resulting alkylthio-substituted
thiols 3 (Figure 1).
Multiresistant ESKAPE (Enterococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudo-
monas aeruginosa, and Enterobacter species) pathogens are a
major global threat for human health. Among other resistance
factors, β-lactamases are most prevalent and effectively protect
the bacteria against the different kind of β-lactam antibiotics,
including last resort penems and cephalosporins.[1] The β-
lactamase-mediated mechanism of β-lactam hydrolysis relies
either on nucleophilic serine residue (in serine β-lactamases,
SBLs) or metal ions (in metallo- β-lactamases, MBLs) in the
active site of the enzyme. Although in general a β-lactamase
inhibitor does not exhibit antimicrobial activity itself, it prevents
the rapid degradation of β-lactam antibiotics and thereby acts
as antibiotic adjuvant.[2] While SBL inhibitors are widely
established, agents inhibiting MBLs or both type of β-
lactamases are still under clinical evaluation.[3] Fast evolution of
β-lactamases, caused by high selection pressure make the
search for new inhibitors highly urgent. One of the possible
MBL inhibition mechanism involves inhibitors possessing a thiol
moiety. Thiols bind tightly to the Zn2+ ions in the MBL active
site.[4] Although a great variety of thiol-based inhibitors have
[a] Prof. M. Krasavin, D. Zhukovsky, I. Solovyev, D. Barkhatova, Prof. D. Dar’in
Institute of Chemistry, Saint Petersburg State University
26 Universitetskii prospect, Peterhof 198905 (Russia)
E-mail: m.krasavin@spbu.ru
[b] D. Frank, G. Martinelli, Prof. T. A. Wichelhaus
Institute of Medical Microbiology and Infection Control
University Hospital Frankfurt
Paul-Ehrlich-Straße 40, 60596 Frankfurt (Germany)
[c] L. Weizel, Dr. A. Proschak, M. Rotter, Dr. J. S. Kramer, S. Brunst,
Prof. E. Proschak
Institute of Pharmaceutical Chemistry
Goethe-University Frankfurt
Max-von-Laue Str. 9, 60438 Frankfurt a.M. (Germany)
E-mail: proschak@pharmchem.uni-frankfurt.de
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
Figure 1. De-symmetrization of symmetrical aliphatic dithiols 2 via Rh(II)-
catalyzed SÀ H insertion of diazo compounds 1 exploited in this work.
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Results and Discussion
With the arsenal of 18 structurally diverse α-diazocarbonyl
compounds 1a–r in hand, we proceeded with coupling the
respective Rh(II) carbenes to either ethane-1,2-dithiol or
propane-1,3-dithiol, or both, which led to the expected de-
symmetrization of the latter and the formation of alkylthio-
substituted thiols 3a–x in modest to good yields (Scheme 2).
Synthesis of alkylthio-substituted aliphatic thiols 3
The arsenal of diazo compounds 1a–i selected for this study
has been reported previously as prepared via the recently
developed ‘sulfonyl-azide-free’ (SAFE) protocol (vide infra).[10–12]
α-Diazo-γ-butyrolactams 1j–n were prepared via the Danheiser
diazo transfer protocol using 4-nitrobenzene sulfonyl azide as
diazo transfer reagent.[13,14] It should be noted that all afore-
mentioned diazo compounds are stable to storage except for
1j which undergoes a rapid dimerization[14] and had, therefore,
been used immediately[13] in the subsequent Rh(II)-catalyzed
SÀ H insertion reaction without isolation (Figure 2).
Biochemical evaluation
In order to investigate the structure-activity relationships of the
dithiol library, we measured the inhibitory activity of 3a–x in a
fluorescence-based enzyme activity assay (Table 1). Two rele-
vant β-lactamase isoforms, NDM-1 and VIM-1 were selected for
in vitro assays. The fluorogenic substrate fluorocillin[15] was
prepared according to literature and its conversion was
monitored as described previously.[6] In general, a linear
relationship between the potency of 3a–x towards both
enzymes could be observed, however, inhibitory potency
towards VIM-1 was almost tenfold higher (Figure 3). A very clear
preference for the propane-1,3-dithiol over ethane-1,2-dithiol
compounds could be observed from matched molecular pairs.
The most potent compound with a balanced inhibitory activity
towards both enzymes was the N-phenyl-γ-lactam derivative
In addition to known α-diazocarbonyl compounds 1a–n, we
synthesized a small set of α-diazo acetamides 1o–r. Amines 4
reacted with 2,2,6-trimethyl-4H-1,3-dioxin-4-one (5) in refluxing
xylene which led to ring opening towards α-acetyl acetamides
6. The latter, after change of the solvent to acetonitrile, were
subjected to the SAFE diazo transfer protocol.[12] After the diazo
transfer was complete, brief reaction workup and removal of
the acetyl group by treatment with KOH solution in aqueous
acetonitrile led to the formation of α-diazo acetamides 1o–r.
The latter were isolated by chromatography in modest yields as
calculated over 3 chemical steps (Scheme 1).
Figure 2. Previously reported diazo compounds 1a–n employed in this
study.
Scheme 2. Preparation of alkylthio-substituted thiols 3a–x (aRh2(OAc)4
(1 mol%) for the preparation of compounds 3m–t; Rh2(esp)2 (0.5 mol%)–in
all other cases).
Scheme 1. Preparation of α-diazo acetamides 1o–r.
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of Asn220. The carbonyl oxygen of the cyclopentanone moiety
forms a hydrogen bond towards the side chain of Lys211. Both
interactions towards Asn220 and Lys211 are described to be
important for recognition of the carboxylate moiety of β-
lactam.[17] The binding mode explains the preference of the
propane-1,3-dithiol over ethane-1,2-dithiol derivatives, due to
the optimal distance of three carbons between the thiol and
the thioether groups. (Figure 4)
The docking of the most potent derivative 3o reveals the
same preferred interactions as the simplified analogue 3a.
(Figure 3B) The N-phenyl ring reaches out to a flat subpocket
which is only partially lipophilic and open to solvent. Due to its
open nature, a certain variability can be assumed in this area.
This hypothesis fits to the observation that various moieties fit
this subpocket without significant loss in activity e.g. N-phenyl
derivative 3o (IC50(NDM-1)=0.3 μM), N-(3-CF3)-phenyl derivative
3q (IC50(NDM-1)=0.48 μM), or N-(2-thiazolyl) derivative 3s
(IC50(NDM-1)=0.39 μM). Notably, only (S)-enantiomers of 3a
and 3o were able to display these favourable binding modes
while (R)-enantiomers were unable to form all directed inter-
actions in a low-energy conformation. This observation paves
the way for future investigations of the enantioselective
synthesis route and subsequent biochemical evaluation of the
enantiomers.
Table 1. In vitro inhibition of recombinantly expressed and purified MBLs
NDM-1 and VIM-1 by compounds 3a–3x.
Compound
IC50 (NDM-1) [μM]
IC50 (VIM-1) [μM]
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
0.48�0.07
3.91�0.42
0.25�0.06
5.63�1.19
0.60�0.03
1.34�0.32
0.43�0.02
3.63�0.47
0.88�0.16
1.55�0.42
7.79�1.30
0.44�0.11
1.58�0.22
1.41�0.07
0.30�0.04
2.08�0.11
0.48�0.01
1.54�0.03
0.39�0.05
1.19�0.12
3.31�0.12
5.50�0.30
0.58�0.03
2.40�0.13
0.17�0.04
0.22�0.03
0.07�0.01
0.23�0.01
0.23�0.05
0.18�0.01
0.14�0.00
0.46�0.03
0.19�0.01
0.90�0.06
0.73�0.05
0.04�0.01
0.18�0.00
0.08�0.00
0.02�0.00
0.22�0.01
0.07�0.01
0.04�0.02
0.04�0.01
0.10�0.02
0.30�0.02
0.33�0.02
0.10�0.01
0.40�0.03
3s
3t
3u
3v
3w
3x
Figure 3. Plot of pIC50 values against NDM-1 and VIM-1 by compounds 3a–
3x.
3o, which inhibited NDM-1 with an IC50 of 0.3 μM and VIM-1
with an IC50 of 0.02 μM.
In order to rationalize the structure-activity relationships of
the prepared library, molecular docking experiments with the
most potent derivative 3o and the most simple analogue 3a
were conducted. Therefore, structures of both possible enan-
tiomers of 3o and 3a were docked into the X-ray structure of
NDM-1 (PDB code 4EXS[16]) in complex with a thiol-containing
inhibitor L-captoptril. The obtained docking mode of 3a
revealed that the free thiol group, which was assumed to be
negatively charged, is located between the Zn2+ ions in the
catalytic center. It thereby displaces the polarized water
responsible for β-lactam hydrolysis. Furthermore, the thioether
moiety forms a directed hydrogen bond towards backbone NH
Figure 4. Proposed binding modes of compounds 3a (A) and 3o (B) bound
to NDM-1. Binding modes are generated by molecular docking, the
compounds are shown as golden sticks, the protein side chains are displayed
as green lines, Zn2+ ions are represented as cyan balls. The surface around
the phenyl substituent of 3o is coloured by lipophilicity (green: lipophilic,
purple: hydrophilic).
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For further biological evaluation we concentrated on the
double enthalpy ΔH=À 49 kJ/mol compared to 3a and 3s
(ΔH=À 22 kJ/mol and À 27 kJ/mol).
compounds with the balanced potency towards both, NDM-1
and VIM-1, 3o and 3s. Furthermore, the minimalistic derivative
3a was used for comparison to ensure that the aromatic
derivative does not change the mode of action of the
compound. Some classes of MBL inhibitors do not directly bind
to the active site but act as zinc chelators and withdraw
catalytically essential zinc ions.[18] We verified the direct
inhibitory mode of action by adding 100 μM ZnCl2 to the
recombinant MBL in vitro assay. As Figure 5 shows, addition of
zinc ions does not impair the inhibitory activity of 3a, 3o, and
3s, suggesting direct inhibition and binding to the active site.
The next step was the investigation of the binding
thermodynamics of 3a, 3o, and 3s. For these experiments we
used a closely related enzyme VIM-2 which is an isoform of VIM-
The considerable inhibition of purified NDM-1 and VIM-1
in vitro suggested that compound 3a, 3o, or 3s, which
themselves exhibit no intrinsic antimicrobial activity (Table 2),
can potentially restore the activity of imipenem against
bacterial isolates producing MBL.
To investigate this, the MIC of imipenem or combined with
various concentrations of compound 3a, 3o, or 3 s was
determined against E. coli transformants producing NDM-1 or
VIM-1 (Table 3 and 4). Especially compound 3o and 3 s
substantially reduced the MIC of imipenem against NDM-1 or
VIM-1 producing bacteria up to 8-fold and 32-fold, respectively.
1 and can be recombinantly expressed in very high concen- Conclusion
trations, suitable tor isothermal titration calorimetry (ITC)
experiments (Figure 6). ITC titration of 250 μM of 3a, 3o, or 3s,
respectively, into 50 μM of VIM-2 revealed potent entropy-
driven binding of all three compounds, with 3a being the
weakest (Kd =0.88 μM). Notably, binding of 3o displays almost
In this study we could show that Rh(II)-catalyzed introduction of
dithiols is a highly useful method for diversity-oriented syn-
thesis of chemical libraries which are intended to contain free
sulfhydryl groups. We prepared a diverse library of thiol-based
inhibitors of MBLs and evaluated them in vitro. Biochemical and
biological evaluation of the prepared library showed that
potent MBL inhibitors with antibiotic adjuvant activity could be
generated.
Figure 5. In vitro inhibition of recombinantly expressed and purified MBLs
NDM-1 and VIM-1 by compounds 3a, 3o, and 3s in presence and in absence
of 100 μM ZnCl2.
Table 3. Synergistic antimicrobial activity of imipenem with 3a, 3o, and
3s. against E. coli NDM-1 (T2359).
Imipenem MIC in mg/L
+ μg/ml
3a
3o
3 s
0
128
32
64
64
64
128
128
128
128
16
32
128
16
32
64
64
64
128
128
128
64
32
16
8
32
128
128
128
128
4
2
Table 4. Synergistic antimicrobial activity of imipenem with 3a, 3o, and
3s. against E. coli VIM-1 (T2544).
Figure 6. Corrected heat rate of ITC measurements. A) 250 μM 3a into 50 μM
VIM-2; B) 250 μM of 3o into 50 μM VIM-2; C) 250 μM 3s into 50 μM VIM-2.
Imipenem MIC in mg/L
+ μg/ml
3a
3o
3 s
0
32
8
32
1
2
32
2
4
128
64
32
16
8
4
2
Table 2. Intrinsic antimicrobial activity of 3a, 3o, and 3s.
16
16
16
16
32
32
8
8
MIC in mg/L
3a
Transformants
16
16
16
16
16
16
16
32
3o
3 s
E. coli NDM-1 (T2359)
E. coli VIM-1 (T2544)
>512
>512
>512
>512
>512
>512
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(br s), 46.5, 34.2. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for
C10H10ClN3NaO 246.0405; Found 246.0408.
Experimental Section
Chemical synthesis
2-Diazo-1-(4-tosylpiperazin-1-yl)ethanone (1r)
General methods
Yield 72 mg (24%). Yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.70–7.57
(m, 2H), 7.41–7.34 (m, 2H), 4.94 (s, 1H), 3.52 (s, 4H), 3.01 (t, J=
5.1 Hz, 4H), 2.46 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.8, 144.3,
132.0, 129.9, 127.7, 46.7, 45.9, 43.2 (br s), 21.5. HRMS (ESI/Q-TOF)
m/z: [M+Na]+ Calcd for C13H16N4NaO3S 331.0835; Found 331.0829.
Known diazocarbonyl compounds 1a–n were prepared according
to literature procedures,[10–14] other reagents were obtained from
commercial sources and used without any additional purification.
Solvents were distilled over suitable drying agents. Mass spectra
were recorded with a Bruker Maxis HRMS-ESI-qTOF spectrometer
(electrospray ionization mode). NMR spectroscopic data were
1
recorded with Bruker Avance 400 spectrometer (400.13 MHz for H
General procedure for the preparation of compounds 3a-x
and 100.61 MHz for 13C) in CDCl3 and were referenced to residual
solvent proton peaks (δH =7.28) and solvent carbon peaks (δC =
77.0). Melting points were determined with a Stuart SMP50 instru-
ment in open capillary tubes.
To a vigorously stirred solution of diazo compound 1 (0.5 mmol)
and symmetrical dithiol (5 mmol) in dichloromethane (10 mL) an
appropriate rhodium(II) catalyst (0.005 mmol of Rh2(OAc)4 for
lactams 3m–t and 0.0025 mmol of Rh2(esp)2 in all other cases) was
added. The reaction mixture was stirred at room temperature over
18 h. The volatiles were removed using a rotary evaporator and the
desired product was isolated by a column chromatography on silica
gel using ethyl acetate–n-hexane 1:4 as eluent.
Preparation of α-diazo acetamides 1o-r
A mixture of appropriate amine 4 (1 mmol) and 2,2,6-trimethyl-4H-
1,3-dioxin-4-one (5, 1 mmol) in o-xylene (6 mL) was heated at reflux
for 1 h and the solvent was removed under reduced pressure. The
residue was dissolved in acetonitrile (8 mL) and a mixture of 3-
(chlorosulfonyl)benzoic acid (292 mg, 1.34 mmol), sodium azide
(98 mg, 1.5 mmol) and potassium carbonate (276 mg, 2 mmol) in
water (8 mL), pre-stirred over 30 min, was added. The resulting
emulsion was stirred for 2 h at room temperature whereupon the
diazo transfer was complete. The reaction mixture was extracted
with chloroform (2×10 mL). The chloroform solution was separated,
dried over anhydrous Na2SO4, filtered and concentrated to dryness.
The residue was dissolved in acetonitrile (20 mL) and was treated
with a solution of KOH (140 mg, 5 mmol) in water (4 mL). The
resulting mixture was stirred for 3 h at room temperature and
extracted with chloroform (2×10 mL). The organic phase was dried
over anhydrous Na2SO4, filtered and the solvent was removed
under reduced pressure. The residue was purified by column
chromatography using ethyl acetate-n-hexane 1:4 as eluent.
2-((3-Mercaptopropyl)thio)cyclopentanone (3a)
Yield 48%, 46 mg. Purple amorphous solid. 1H NMR (400 MHz,
CDCl3) δ 3.13–3.04 (m, 1H), 2.82–2.73 (m, 1H), 2.70–2.57 (m, 3H),
2.49–2.39 (m, 1H), 2.31–2.03 (m, 3H), 1.96–1.79 (m, 4H), 1.37 (t, J=
8.1 Hz, 1H).13C NMR (101 MHz, CDCl3) δ 213.7, 47.0, 35.9, 32.9, 30.2,
29.2, 23.4, 20.4. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for
C8H14NaOS2 213.0378; Found 213.0377.
2-((2-Mercaptoethyl)thio)-1-(p–tolyl)ethanone (3b)
Yield 27%, 31 mg. Yellow amorphous solid. 1H NMR (400 MHz,
CDCl3, in equilibrium with cyclic semi-thioacetal) δ 7.88 (d, J=
8.3 Hz, 2H), 7.29 (d, J=8.0 Hz, 2H), 3.81 (s, 2H), 2.85–2.80 (m, 2H),
2.80–2.71 (m, 2H), 2.43 (s, 3H), 1.73–1.66 (m, 1H). 13C NMR (101 MHz,
CDCl3, in equilibrium with cyclic semi-thioacetal) δ 194.2, 144.5,
132.6, 129.5, 128.9, 36.8, 36.1, 24.1, 21.7. HRMS (ESI/Q-TOF) m/z: [M
+Na]+ Calcd for C11H14NaOS2 249.0378; Found 249.0388.
1-(4-(4-Chlorobenzyl)piperazin-1-yl)-2-diazoethan-1-one (1o)
1
Yield 139 mg (50%). Orange semi-solid. H NMR (400 MHz, CDCl3) δ
7.41–7.30 (m, 2H), 7.30–7.25 (m, 2H), 4.98 (s, 1H), 3.87–2.93 (m, 6H),
2.60–2.11 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 164.7, 136.2, 133.0,
130.3, 128.5, 62.1, 52.7, 46.4, 43.8. HRMS (ESI/Q-TOF) m/z: [M+H]+
Calcd for C13H16ClN4O 279.1007; Found 279.0999.
2-((3-Mercaptopropyl)thio)-1-(p–tolyl)ethanone (3c)
Yield 62%, 74 mg. Yellow amorphous solid. 1H NMR (400 MHz,
CDCl3) δ 7.88 (d, J=8.3 Hz, 2H), 7.28 (d, J=8.0 Hz, 2H), 3.78 (s, 2H),
2.70 (t, J=7.1 Hz, 2H), 2.61 (q, J=7.1 Hz, 2H), 2.43 (s, 3H), 1.91 (p,
J=7.0 Hz, 2H), 1.37 (t, J=8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
194.2, 144.3, 132.7, 129.4, 128.9, 37.0, 32.7, 30.6, 23.3, 21.7. HRMS
(ESI/Q-TOF) m/z: [M+Na]+ Calcd for C12H16NaOS2 263.0535; Found
263.0540.
2-Diazo-N-((1S*,2S*)-2-hydroxycyclohexyl)-N-methylacetamide
(1p)
Yield 75 mg (38%). Yellow amorphous solid. 1H NMR (400 MHz,
CDCl3) δ 5.06 (s, 1H), 3.67–3.40 (m, 1H), 2.81 (s, 3H), 2.62 (s, 1H),
2.25–2.10 (m, 1H), 1.84–1.68 (m, 3H), 1.59–1.13 (m, 5H). 13C NMR
(101 MHz, CDCl3) δ 167.8, 167.4 (br s), 78.4, 70.0 (br s), 68.4, 60.9,
59.5 (br s), 47.0, 34.8 (br s), 30.6, 29.0 (br s), 28.6, 28.5, 25.0, 24.4,
23.9, 23.8. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for C9H15N3NaO2
220.1056; Found 220.1060.
1-(4-Chlorophenyl)-2-((2-mercaptoethyl)thio)ethanone (3d)
Yield 36%, 44 mg. Yellow oil. Thiol: 1H NMR (400 MHz, CDCl3) δ
7.97–7.83 (m, 2H), 7.49–7.44 (m, 2H), 3.79 (s, 2H), 2.94–2.65 (m, 4H),
1.69 (t, J=7.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 193.2, 140.0,
1
133.3, 130.2, 129.1, 36.7, 36.1, 24.1. Cyclic semi-thioacetal: H NMR
(400 MHz, CDCl3) δ 7.61 (d, J=8.7 Hz, 2H), 7.36 (d, J=8.7 Hz, 2H),
4.78 (s, 1H), 3.51–3.41 (m, 1H), 3.12–2.98 (m, 1H), 2.94–2.64 (m, 2H).
13C NMR (101 MHz, CDCl3) δ 142.1, 134.2, 128.6, 126.5, 75.9, 43.9,
29.0, 27.8. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for C10H11ClNaOS2
268.9832; Found 268.9838.
N-(4-Chlorobenzyl)-2-diazo-N-methylacetamide (1q)
1
Yield 64 mg (29%). Yellow oil. H NMR (400 MHz, CDCl3) δ 7.33 (d,
J=8.4 Hz, 2H), 7.20 (d, J=8.1 Hz, 2H), 4.99 (s, 1H), 4.51 (s, 2H), 2.86
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 166.2, 135.7, 133.1, 128.8, 50.9
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TOF) m/z: [M+Na]+ Calcd for C13H17NNaO4S2 338.0491; Found
338.0477.
1-(4-Fluorophenyl)-2-((3-mercaptopropyl)thio)ethanone (3e)
1
Yield 56%, 68 mg. Yellow oil. H NMR (400 MHz, CDCl3) δ 8.09–7.99
(m, 2H), 7.20–7.13 (m, 2H), 3.78 (s, 2H), 2.72 (t, J=7.1 Hz, 2H), 2.63
(q, J=7.1 Hz, 2H), 1.92 (p, J=7.0 Hz, 2H), 1.38 (t, J=8.1 Hz, 1H). 13C
NMR (101 MHz, CDCl3) δ 192.9, 165.9 (d, J=255.5 Hz), 131.5 (d, J=
9.3 Hz), 131.5, 115.9 (d, J=22.0), 37.0, 32.6, 30.6, 23.3. HRMS (ESI/Q-
TOF) m/z: [M+Na]+ Calcd for C11H13FNaOS2 267.0284; Found
267.0280.
Dimethyl 2-((3-mercaptopropyl)thio)succinate (3l)
1
Yield 62 mg, 49%. Yellow oil. H NMR (400 MHz, CDCl3) δ 3.79–3.74
(m, 3H), 3.73–3.63 (m, 4H), 3.01 (ddt, J=16.9, 9.5, 1.2 Hz, 1H), 2.86–
2.73 (m, 2H), 2.73–2.58 (m, 3H), 1.96–1.85 (m, 2H), 1.37 (t, J=8.1 Hz,
1H). 13C NMR (101 MHz, CDCl3) δ 172.1, 171.0, 52.6, 52.0, 41.4, 36.3,
32.8, 29.8, 23.2. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for
C9H16NaO4S2 275.0382; Found 275.0390.
2-((2-Mercaptoethyl)thio)-1-(4-methoxyphenyl)ethanone (3f)
1
Yield 27%, 33 mg. Yellow oil. H NMR (400 MHz, CDCl3) δ 8.00–7.92
(m, 2H), 7.05–6.91 (m, 2H), 3.88 (s, 3H), 3.78 (s, 2H), 2.85–2.79 (m,
2H), 2.78–2.71 (m, 2H), 1.69 (t, J=8.0 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 193.2, 163.8, 131.1, 128.0, 113.9, 55.5, 36.6, 36.1, 24.2.
HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for C11H14NaO2S2 265.0327;
Found 265.0326.
3-((3-Mercaptopropyl)thio)-1-methylpyrrolidin-2-one (3m)
1
Yield 54 mg, 53%. Yellow oil. H NMR (400 MHz, CDCl3) δ 3.50–3.39
(m, 2H), 3.35–3.25 (m, 1H), 3.02–2.93 (m, 1H), 2.86 (s, 3H), 2.85–2.77
(m, 1H), 2.68–2.61 (m, 2H), 2.46 (dtd, J=15.1, 8.5, 6.5 Hz, 1H), 1.98–
1.85 (m, 3H), 1.41 (t, J=8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
173.1, 47.4, 42.7, 33.2, 30.0, 29.4, 26.3, 23.4. HRMS (ESI/Q-TOF) m/z:
[M+Na]+ Calcd for C8H15NNaOS2 228.0487; Found 228.0477.
2-((3-Mercaptopropyl)thio)-1-(4-methoxyphenyl)ethanone (3g)
1
Yield 17%, 22 mg. Yellow oil. H NMR (400 MHz, CDCl3) δ 8.00–7.95
(m, 2H), 6.99–6.93 (m, 2H), 3.89 (s, 3H), 3.76 (s, 2H), 2.71 (t, J=
7.1 Hz, 2H), 2.62 (q, J=7.1 Hz, 2H), 1.92 (p, J=7.0 Hz, 2H), 1.37 (t,
J=8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 193.3, 163.8, 131.1,
128.1, 113.9, 55.5, 36.8, 32.7, 30.6, 23.3. HRMS (ESI/Q-TOF) m/z: [M+
Na]+ Calcd for C12H16NaO2S2 279.0484; Found 279.0491.
3-((2-Mercaptoethyl)thio)-1-phenylpyrrolidin-2-one (3n)
1
Yield 86 mg, 67%. Yellow oil. H NMR (400 MHz, CDCl3) δ 7.66–7.60
(m, 2H), 7.44–7.37 (m, 2H), 7.24–7.13 (m, 1H), 3.99 (dt, J=9.6, 7.4 Hz,
1H), 3.82 (ddd, J=9.6, 8.2, 4.0 Hz, 1H), 3.70 (dd, J=8.5, 4.6 Hz, 1H),
3.31–3.21 (m, 1H), 3.00–2.91 (m, 1H), 2.91–2.81 (m, 2H), 2.61 (dtd,
J=13.4, 8.3, 7.2 Hz, 1H), 2.05 (ddt, J=13.5, 7.5, 4.3 Hz, 1H), 1.74 (t,
J=8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 172.1, 139.2, 128.9,
124.9, 120.0, 46.7, 44.1, 35.3, 26.1, 24.7. HRMS (ESI/Q-TOF) m/z: [M+
Na]+ Calcd for C12H15NNaOS2 276.0487; Found 276.0495.
3-((2-Mercaptoethyl)thio)dihydrofuran-2(3H)-one (3h)
1
Yield 55%, 49 mg. Purple oil. H NMR (400 MHz, CDCl3) δ 4.50–4.42
(m, 1H), 4.36 (td, J=8.6, 4.1 Hz, 1H), 3.58 (dd, J=8.4, 4.3 Hz, 1H),
3.23–3.13 (m, 1H), 2.97–2.88 (m, 1H), 2.87–2.79 (m, 2H), 2.75–2.63
(m, 1H), 2.14 (ddt, J=13.6, 7.0, 4.1 Hz, 1H), 1.72 (t, J=8.1 Hz, 1H).
13C NMR (101 MHz, CDCl3) δ 175.2, 66.8, 38.9, 35.2, 29.9, 24.4. HRMS
(ESI/Q-TOF) m/z: [M+Na]+ Calcd for C6H10NaO2S2 201.0014; Found
201.0008.
3-((3-Mercaptopropyl)thio)-1-phenylpyrrolidin-2-one (3o)
Yield 69 mg, 52%. Yellow amorphous solid. 1H NMR (400 MHz,
CDCl3) δ 7.68–7.56 (m, 2H), 7.45–7.33 (m, 2H), 7.23–7.11 (m, 1H),
3.97 (dt, J=9.5, 7.3 Hz, 1H), 3.80 (ddd, J=9.6, 8.2, 4.1 Hz, 1H), 3.65
(dd, J=8.5, 4.6 Hz, 1H), 3.12–2.98 (m, 1H), 2.88 (dt, J=12.9, 7.3 Hz,
1H), 2.67 (dt, J=8.2, 7.0 Hz, 2H), 2.63–2.54 (m, 1H), 2.11–1.92 (m,
3H), 1.43 (t, J=8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 172.2, 139.3,
128.9, 124.8, 120.0, 46.7, 44.2, 33.2, 29.6, 26.1, 23.5. HRMS (ESI/Q-
TOF) m/z: [M+Na]+ Calcd for C13H17NNaOS2 268.0824; Found
268.0830.
Methyl 2-(4-chlorophenyl)-2-((2-mercaptoethyl)thio)acetate (3i)
1
Yield 56%, 78 mg. Yellow oil. H NMR (400 MHz, CDCl3) δ 7.45–7.40
(m, 2H), 7.37–7.32 (m, 2H), 4.62 (s, 1H), 3.76 (s, 3H), 2.82–2.74 (m,
2H), 2.72–2.66 (m, 2H), 1.67 (t, J=8.1 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 170.8, 134.4, 131.5, 129.9, 129.0, 52.9, 51.3, 35.8, 24.3.
HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for C11H13ClNaO2S2
298.9938; Found 298.9933.
3-((2-Mercaptoethyl)thio)-1-(3-(trifluoromethyl)phenyl)
pyrrolidin-2-one (3p)
Ethyl 2-((2-mercaptoethyl)thio)-2-(4-nitrophenyl)acetate (3j)
Yield 100 mg, 62%. Yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.98–7.85
(m, 2H), 7.56–7.48 (m, 1H), 7.46–7.41 (m, 1H), 4.02 (dt, J=9.5, 7.4 Hz,
1H), 3.84 (ddd, J=9.5, 8.2, 3.9 Hz, 1H), 3.72 (dd, J=8.5, 4.5 Hz, 1H),
3.32–3.19 (m, 1H), 3.01–2.91 (m, 1H), 2.90–2.79 (m, 2H), 2.64 (dtd,
J=13.5, 8.4, 7.3 Hz, 1H), 2.08 (ddt, J=13.6, 7.6, 4.2 Hz, 1H), 1.75 (t,
J=8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 172.4, 139.7, 131.3 (q,
J=32.4 Hz), 129.5, 123.9 (q, J=272.5 Hz), 122.8, 121.3 (q, J=3.8 Hz),
116.3 (q, J=3.8 Hz), 46.5, 43.9, 35.3, 25.9, 24.6. HRMS (ESI/Q-TOF)
m/z: [M+Na]+ Calcd for C13H14F3NNaOS2 322.0542; Found 322.0532.
1
Yield 87 mg, 58%. Yellow oil. H NMR (400 MHz, CDCl3) δ 8.22 (d,
J=8.8 Hz, 2H), 7.68 (d, J=8.8 Hz, 2H), 4.70 (s, 1H), 4.32–4.15 (m, 2H),
2.89–2.76 (m, 2H), 2.74–2.68 (m, 2H), 1.67 (t, J=8.1 Hz, 1H), 1.28 (t,
J=7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.6, 147.7, 143.3,
129.6, 123.9, 62.4, 51.4, 36.0, 24.2, 14.1. HRMS (ESI/Q-TOF) m/z: [M+
Na]+ Calcd for C12H15NNaO4S2 324.0335; Found 324.0346.
Ethyl 2-((3-mercaptopropyl)thio)-2-(4-nitrophenyl)acetate (3k)
1
3-((3-Mercaptopropyl)thio)-1-(3-(trifluoromethyl)phenyl)
pyrrolidin-2-one (3q)
Yield 122 mg, 73%. Yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.99–7.81
(m, 2H), 7.54–7.48 (m, 1H), 7.45–7.41 (m, 1H), 4.00 (dt, J=9.5, 7.4 Hz,
1H), 3.83 (ddd, J=9.4, 8.2, 4.1 Hz, 1H), 3.66 (dd, J=8.5, 4.6 Hz, 1H),
Yield 90 mg, 57%. Yellow oil. H NMR (400 MHz, CDCl3) δ 8.28–8.17
(m, 2H), 7.73–7.65 (m, 2H), 4.64 (s, 1H), 4.30–4.16 (m, 2H), 2.76–2.65
(m, 2H), 2.65–2.56 (m, 2H), 1.93–1.82 (m, 2H), 1.34 (t, J=8.1 Hz, 1H),
1.29 (t, J=7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.7, 147.7,
143.5, 129.6, 123.8, 62.3, 51.5, 32.6, 30.3, 23.2, 14.1. HRMS (ESI/Q-
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3.10–2.98 (m, 1H), 2.92–2.83 (m, 1H), 2.71–2.55 (m, 3H), 2.08 (ddt,
J=13.5, 7.5, 4.3 Hz, 1H), 2.03–1.90 (m, 2H), 1.42 (t, J=8.1 Hz, 1H).
13C NMR (101 MHz, CDCl3) δ 172.5, 139.8, 131.3 (q, J=32.4 Hz),
129.5, 123.9 (q, J=272.5 Hz), 122.8, 121.2 (q, J=3.8 Hz), 116.3 (q,
J=3.9 Hz), 46.5, 44.1, 33.1, 29.6, 25.9, 23.4. HRMS (ESI/Q-TOF) m/z:
[M+Na]+ Calcd for C14H16F3NNaOS2 358.0518; Found 358.0529.
N-(4-Chlorobenzyl)-2-((2-mercaptoethyl)
thio)-N-methylacetamide (3w)
Yield 71%, 103 mg. Yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.38–7.27
(m, 2H), 7.24–7.12 (m, 2H), 4.62–4.51 (m, 2H), 3.39–3.30 (m, 2H), 3.00
(s, 3H), 2.96–2.89 (m, 2H), 2.85–2.75 (m, 2H), 1.74–1.68 (m, 1H). 13C
NMR (101 MHz, CDCl3) δ 169.5, 169.2, 135.5, 134.8, 133.7, 133.3,
129.3, 129.2, 128.8, 127.9, 53.3, 50.5, 36.2, 36.0, 35.6, 34.1, 33.0, 32.9,
24.3. HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C12H17ClNOS2
290.0435; Found 290.0443.
3-((2-Mercaptoethyl)thio)-1-(thiazol-2-yl)pyrrolidin-2-one (3r)
1
Yield 77 mg, 59%. Yellow oil. H NMR (400 MHz, CDCl3) δ 7.51 (d,
J=3.6 Hz, 1H), 7.06 (d, J=3.6 Hz, 1H), 4.29–4.12 (m, 2H), 3.98–3.69
(m, 1H), 3.30–3.14 (m, 1H), 3.05–2.91 (m, 1H), 2.91–2.79 (m, 2H),
2.73–2.65 (m, 1H), 2.31–2.00 (m, 1H), 1.74 (t, J=8.1 Hz, 1H). 13C NMR
(101 MHz, CDCl3) δ 171.5, 157.6, 137.8, 114.2, 46.0, 43.1, 35.4, 26.2,
24.6. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for C9H12N2NaOS3
261.0185; Found 261.0184.
2-((2-Mercaptoethyl)thio)-1-(4-tosylpiperazin-1-yl)ethanone (3x)
Yield 50%, 94 mg. White solid, mp 100.5-101.4 C. 1H NMR
°
(400 MHz, CDCl3) δ 7.69–7.58 (m, 2H), 7.38–7.33 (m, 2H), 3.70 (t, J=
5.1 Hz, 2H), 3.58 (t, J=5.0 Hz, 2H), 3.26 (s, 2H), 3.09 (t, J=5.0 Hz,
2H), 3.00 (t, J=5.1 Hz, 2H), 2.84–2.75 (m, 2H), 2.72–2.63 (m, 2H),
2.45 (s, 3H), 1.56 (t, J=8.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
167.6, 144.2, 132.3, 129.9, 127.8, 46.0, 45.9, 45.8, 41.1, 36.1, 32.8,
24.2, 21.6. HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C15H23N2O3S3
375.0865; Found 375.0882.
3-((3-Mercaptopropyl)thio)-1-(thiazol-2-yl)pyrrolidin-2-one (3s)
1
Yield 64 mg, 47%. Yellow oil. H NMR (400 MHz, CDCl3) δ 7.50 (d,
J=3.5 Hz, 1H), 7.05 (d, J=3.5 Hz, 1H), 4.25–4.12 (m, 2H), 3.73 (dd,
J=8.6, 4.6 Hz, 1H), 3.04 (ddd, J=12.9, 7.5, 6.3 Hz, 1H), 2.88 (dt, J=
12.8, 7.3 Hz, 1H), 2.73–2.57 (m, 3H), 2.12 (ddt, J=13.7, 7.2, 4.7 Hz,
1H), 2.06–1.89 (m, 2H), 1.41 (t, J=8.1 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 171.6, 157.7, 137.7, 114.2, 46.1, 43.2, 33.0, 29.8, 26.3, 23.4.
HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd for C10H14N2NaOS3
297.0160; Found 297.0171.
Biological evaluation
Inhibition of VIM-1and NDM-1metallo-β -lactamases
Activity assays for MBLs were performed at room temperature in
black polystyrol 96well plates (Corning, Corning, NY, USA) using
dicefalotinodifluorofluorescein (Fluorocillin) as a substrate. Proteins
were diluted in assay buffer (HEPES 50 mM, pH 7.5 containing
0.01%Triton X-100), with final protein concentrations of NDM-1:
3 nM, or VIM-1: 4 nM. Samples were supplemented with an
equimolar amount of ZnCl2. An amount of 1 μL of compounds 3a–
3x at different concentrations was incubated with 89 μL of enzyme
in assay buffer. After an incubation period of 30 min at room
temperature, 10 μL of Fluorocillin substrate was added to yield the
final assay volume of 100 μL. Final concentration of DMSO was 1%.
The fluorescence emitted by the fluorescent product difluorofluor-
escein was monitored every 45 s for 30 cycles using a Tecan
fluorescent plate reader (Infinite 200; excitation at 495 nm and
emission at 525 nm) and was compared with a standard curve. The
rate of the enzymatic reaction was obtained by dividing the
quantity of the fluorescent product (RFU) by time (min). Negative
controls were measured in the absence of enzyme, whereas the
positive controls were measured in the presence of enzyme and in
the absence of inhibitors. The inhibitory effect of each substance
was measured in triplicate in three independent experiments. IC50
values were calculated using data obtained from measurements
with at least eight different inhibitor concentrations, applying a
sigmoidal dose–response (variable slope with four parameters)
equation using GraphPad Prism 5 (GraphPad Software, La Jolla, CA,
USA) software. In order to investigate the inhibitory activity of 3a,
3o, or 3s in the presence of a high Zn2+ concentration, assay buffer
containing an additional 100 μM ZnCl2 was used. The assay was
performed as described above with NDM-1 or VIM-1 and 10 μM of
3a, 3o, or 3s.
4-(3-((2-Mercaptoethyl)thio)-2-oxopyrrolidin-1-yl)benzonitrile
(3t)
1
Yield 50 mg, 36%. Yellow oil. H NMR (400 MHz, CDCl3) δ 7.84–7.77
(m, 2H), 7.72–7.65 (m, 2H), 4.00 (dt, J=9.6, 7.5 Hz, 1H), 3.84 (ddd,
J=9.5, 8.3, 3.8 Hz, 1H), 3.72 (dd, J=8.4, 4.3 Hz, 1H), 3.28–3.17 (m,
1H), 2.97–2.90 (m, 1H), 2.90–2.82 (m, 2H), 2.64 (dq, J=13.6, 8.2 Hz,
1H), 2.09 (ddt, J=13.6, 7.7, 4.0 Hz, 1H), 1.74 (t, J=8.1 Hz, 1H). 13C
NMR (101 MHz, CDCl3) δ 172.8, 143.0, 133.1, 119.4, 118.7, 107.6,
46.3, 44.0, 35.4, 25.7, 24.6. HRMS (ESI/Q-TOF) m/z: [M+Na]+ Calcd
for C13H14N2NaOS2 301.0440; Found 301.0431.
1-(4-(4-Chlorobenzyl)piperazin-1-yl)-2-((3-mercaptopropyl)thio)
ethan-1-one (3u)
1
Yield 122 mg, 68%. Yellow oil. H NMR (400 MHz, CDCl3) δ 7.31 (d,
J=8.3 Hz, 2H), 7.27 (d, J=8.5 Hz, 2H), 3.63 (t, J=5.1 Hz, 2H), 3.54–
3.45 (m, 4H), 3.30 (s, 2H), 2.77 (t, J=7.1 Hz, 2H), 2.68–2.60 (m, 2H),
2.47 (t, J=5.0 Hz, 2H), 2.43 (t, J=5.2 Hz, 2H), 1.94 (p, J=7.0 Hz, 2H),
1.39 (t, J=8.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 167.6, 136.3,
133.0, 130.3, 128.5, 62.0, 53.0, 52.7, 46.4, 41.8, 33.2, 32.9, 30.5, 23.3.
HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C16H24ClN2OS2 359.1013;
Found 359.1008.
N-((1S*,2S*)-2-Hydroxycyclohexyl)-2-((2-mercaptoethyl)
thio)-N-methylacetamide (3v)
1
Yield 28%, 37 mg. Yellow oil. H NMR (400 MHz, CDCl3) δ 4.36–4.22
(m, 0.5H), 3.71–3.41 (m, 2H), 3.41–3.29 (m, 1.5H), 2.99 (s, 1.5H), 2.95–
2.83 (m, 3H), 2.83–2.74 (m, 2H), 2.19–2.06 (m, 1H), 1.92–1.67 (m, 4H),
1.63–1.08 (m, 5H), 1.02–0.78 (m, 0.5H). 13C NMR (101 MHz, CDCl3) δ
170.8, 170.2, 70.5, 69.6, 63.8, 59.3, 36.1, 35.9, 35.2, 34.6, 33.8, 33.5,
30.3, 29.4, 28.5, 27.3, 25.1, 25.0, 24.5, 24.4, 24.3, 24.3, 24.3. HRMS
(ESI/Q-TOF) m/z: [M+H]+ Calcd for C11H22NO2S2 264.1086; Found
264.1095.
Kd determination using isothermal titration calorimetry
VIM-2 expression and purification was published elsewhere.[19]
Before the measurement the VIM-2 protein was dialyzed against
500 times the volume of buffer (50 mM Tris, 500 mM NaCl, 5% (v/v)
glycerol with a pH=8) using a dialysis membrane with a 3.5 kDa
MWCO. For the measurements the compounds 3a, 3o, or 3s,
respectively, were diluted using the dialyses buffer to a final
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concentration of 250 μM containing a final DMSO concentration of
1%. The VIM-2 protein was diluted to a final concentration of
50 μM, supplemented with 1% DMSO using the dialysis buffer and
pure DMSO. For the control measurements dialysis buffer was
supplemented with pure DMSO to a final concentration of 1%. The
measurements were performed using an “Affinity ITC” (TA-Instru-
ments) in reversed mode, with a stir rate of 75 rpm and a
Conflict of Interest
The authors declare no conflict of interest.
Keywords: multiresistant bacteria · metallo β lactamases · thiol
inhibitors · Rh(II) catalysis · diazo compounds
°
temperature of 25 C. The VIM-2 was placed in the cell and the
respective compound in the syringe. For blank measurements
either the protein or compound dilution were exchanged with
buffer containing 1% DMSO. For the measurements of 3a and 3s
one time 0.5 μl was injected followed by 50 injections of 2 μl with a
spacing of 240 s. 3o was measured with one injection of 0.5 μl
followed by 35 injections of 2 μl with a spacing of 240 s. The data
were analyzed using the “NanoAnalyze Data Analysis” software
(version 3.10.0; TA-Instruments).
[1] D. M. P. De Oliveira, B. M. Forde, T. J. Kidd, P. N. A. Harris, M. A. Schembri,
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2019, 94, 1427–1440.
[6] F.-M. Klingler, T. A. Wichelhaus, D. Frank, J. Cuesta-Bernal, J. El-Delik,
H. F. Müller, H. Sjuts, S. Göttig, A. Koenigs, K. M. Pos, D. Pogoryelov, E.
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[8] D. Büttner, J. S. Kramer, F.-M. Klingler, S. K. Wittmann, M. R. Hartmann,
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Minimal inhibitory concentrations (MICs) of imipenem monohydrate
(Sigma-Aldrich)�compound 3a, 3o, or 3s against transformed E.
coli strains producing NDM-1 or VIM-1 were determined according
to microdilution method established by Clinical and Laboratory
Standards Institute (CLSI).20 The checkerboard assay was performed
to test for synergy in vitro. The microtiter-plates were set up with
serial doubling dilutions of compound 3a, 3o, or 3s (2-128 mg/L)
and imipenem (0.125-128 mg/L).
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Acknowledgements
This research was supported by the German Research Foundation
and Russian Basic Research Foundation (DFG-RBRF joint research
grant PR/1405/8-1; 21-53-12001). E.P. thanks DFG for Heisenberg-
Professorship PR1405/7-1. Open access funding enabled and
organized by Projekt DEAL.
Manuscript received: April 27, 2021
Accepted manuscript online: June 29, 2021
Version of record online: ■■■, ■■■■
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
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FULL PAPERS
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