Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Article abstract of DOI:10.1016/j.bmc.2018.02.043

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.

Full text of DOI:10.1016/j.bmc.2018.02.043

Accepted Manuscript
Structure activity relationship studies on rhodanines and derived enethiol in-
hibitors of metallo-β-lactamases
Dong Zhang, Marios S. Markoulides, Dmitrijs Stepanovs, Anna M. Rydzik,
Ahmed El-Hussein, Corentin A.M. Bon, Jos J.A.G. Kamps, Klaus-Daniel
Umland, Patrick M. Collins, Samuel T. Cahill, David Y. Wang, Timothy D.W.
Claridge, Jürgen Brem, Michael A. McDonough, Christopher J. Schofield
PII:
S0968-0896(18)30185-8
https://doi.org/10.1016/j.bmc.2018.02.043
BMC 14229
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 January 2018
20 February 2018
22 February 2018
Please cite this article as: Zhang, D., Markoulides, M.S., Stepanovs, D., Rydzik, A.M., El-Hussein, A., Bon, C.A.M.,
Kamps, J.J.A., Umland, K-D., Collins, P.M., Cahill, S.T., Wang, D.Y., Claridge, T.D.W., Brem, J., McDonough,
M.A., Schofield, C.J., Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-
β-lactamases, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.043
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Bioorganic & Medicinal Chemistry
Structure activity relationship studies on rhodanines and derived enethiol inhibitors of
metallo-β-lactamases
Dong Zhanga, Marios S. Markoulidesa, Dmitrijs Stepanovsa,1, Anna M. Rydzika,2 , Ahmed El-Husseina,c3
Corentin A. M. Bona,4, Jos J. A. G. Kampsa, Klaus-Daniel Umlanda,5, Patrick M. Collinsb, Samuel T.
Cahilla, David Y. Wanga, Timothy D. W. Claridgea, Jürgen Brema, Michael A. McDonougha, and
Christopher J. Schofielda,
a Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom.
b Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom.
c The National Institute of Laser Enhanced Science, Cairo University, Egypt.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam
antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by
rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic
analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water.
In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol
inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results
demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a
range of clinically relevant MBLs and provide additional evidence for the potential of
rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-
enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply
that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors
should as a matter of course include testing of their hydrolysis products.
Available online
Keywords:
Metallo -lactamase
Antibiotic resistance
Carbapenemase
Inhibitors
Structure activity relationships
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +44 (0)1865 285 000; fax: +44 (0)1865 285 002; e-mail: christopher.schofield@chem.ox.ac.uk.
1 Present address: Latvian Institute of Organic Synthesis, Aizkraukles Str. 21 Riga, LV-1006, Latvia.
2 Present address: Boehringer Ingelheim Pharma GmbH & Co KG Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany.
3 Present address (on leave from): The National Institute of Laser Enhanced Science, Cairo University, Egypt.
4 Present address: École polytechnique université, Paris saclay Route de Saclay, 91128 Palaiseau.
5 Present address: Bleichestrasse 10, 8400 Winterthur, Switzerland.

1. Introduction
clinical perspective.19 Developing MBL inhibitors with the
breadth of activity required for clinical application is challenging,
because of variations in the mobile regions surrounding the
active sites of B1 MBLs.20 Various types of MBL inhibitors have
been developed,21 most of which chelate to the active site Zn(II)
ion(s); however, few if any, of the reported inhibitors have the
required breadth of potency against the three major B1 MBL
families that are clinically widespread (i.e. the New Delhi MBL
(NDM), Verona integron-encoded MBL (VIM), and
Imipenemase (IMP) MBLs).
Following the clinical introduction of the penicillins in the
1940s, β-lactam antibiotics came to be, and remain, amongst the
most important medicines in use today.1 The remarkable
longevity and the widespread ability of β-lactams to act as
antibiotics has been achieved in the face of multiple resistance
mechanisms,2,3,4 the most prevalent of which is mediated by β-
lactamases which catalyse the hydrolysis of β-lactams.2,5,6,7,8,9,10
There are two mechanistic types of β-lactamase – the serine β-
lactamases (SBLs), which employ a nucleophilic serine (Ambler
classes A, C, D) and the metallo-β-lactamases (MBLs), which
utilise a Zn(II) bound hydroxide in β-lactam hydrolysis (class
B).6,9 Inhibitors of the class A and C SBLs have been used
successfully in combination with β-lactams11. More recently, a
broad spectrum inhibitor of class A, C, and some D SBLs,12
Avibactam, has been introduced for clinical use in combination
with a cephalosporin.13,14 However, no clinically useful MBL
inhibitors are currently available,15,16 and most SBL inhibitors are
susceptible to MBL catalysed hydrolysis (Figure 1).17
We have developed an assay platform for MBLs employing a
fluorogenic cephalosporin substrate,22 which we used to screen
potential β-lactamase inhibitors. As part of this work we tested
the potency of the rhodanine ML302 (Scheme 1), which was
identified following a high-throughput screen, as inhibitor of
VIM-2 and IMP-1.23 Unexpectedly, we found that ML302
undergoes hydrolysis to give the enethiol fragment, ML302F
(Scheme 1), which inhibits MBLs via active site Zn(II) ion
chelation; in the case of VIM-2 we observed the unusual
formation of a ternary complex between the enzyme and two
different ligands, ML302 and ML302F.24 We now describe
structure activity relationship and biophysical studies on the
The class B MBLs all utilise one (subclass B2 and some B3)
or two (subclasses B1 and some B3) Zn(II) ions at their active
site.18 The B1 MBLs are the most important MBLs from a
25
rhodanine derived enethiol MBL inhibitors.
Figure 1 Outlined mechanism for B1 MBL catalysed -lactam hydrolysis as exemplified by hydrolysis of a carbapenem. The anionic intermediate, but not
the tetrahedral intermediate‡, has been observed spectroscopically25.
et al.29 (Scheme 5). All compounds were subject to analysis
(Supplemental information sections 2 and 3)
2. Materials and methods
2.1. Protein production and purification
2.3. Inhibition analyses
Recombinant forms of NDM-1, VIM-2, SPM-1, IMP-1, BcII
Inhibition analyses against bacterial MBLs and SBLs were
and CphA MBLs and TEM-1, CTX-M-15, AmpC and OXA-10
performed as described previously.22,24,26 Residual enzyme
activity was determined for a range of inhibitor concentrations.
Non-linear regression fitting of IC50 curves was carried out using
a three-parameter dose-response curve in GraphPad Prism. Errors
in IC50 values are expressed as:
SBLs were produced as described previously. 22,23,26
2.2. Experimental procedures for synthesis
The syntheses of 3a-s, ML302 analogues 5a-s, and ML302F
analogues 6a-s were performed as previously described.24
Following the procedure of Brem et al.,24 10 was prepared in
two steps from the corresponding aldehyde via Knoevenagel
condensation with rhodanine followed by amide coupling
(Scheme 2).
Additional data are presented in Supplemental Information
(Table S1 and Figures S1-S6).
Following the procedure of Shaffer et al.,27 α-
mercaptocarboxylic acids 13a and 13b were prepared in two
steps from the corresponding α-bromocarboxylic acids via
nucleophilic substitution with potassium thioacetate followed by
basic hydrolysis (Scheme 3).
Following the procedure of Braña et al.,28 α-hydroxycinnamic
acid 7 was prepared in two steps from the corresponding
aldehyde via Erlenmeyer azlactone synthesis of 6 followed by
acid hydrolysis (Scheme 4).
2.4. NMR time course experiments
NMR experiments were carried out using a Bruker Avance III
700 MHz equipped with a TCI inverse cryoprobe or a Bruker
Avance III HD 600 MHz spectrometer equipped with a Prodigy
cryoprobe at 298 K. Data were analysed using Bruker Topspin
3.5. Processing of spectra was done with a Lorentzian line
broadening of 0.3 Hz. Chemical shifts (δ) are given as parts per
1
million (ppm) relative to residual HDO (δH 4.70 ppm for H
NMR).
α-Hydroxy phosphonic acid 22 and α-sulfanyl phosphonic
acid 26 were synthesised according to the procedure of Bebrone

For rhodanine stability studies solutions were buffered in
either freshly prepared NH4HCO3 (50 mM, pH 7.50) or Tris-d11
(50 mM, pH 7.50) both with NaCl (100 mM) in D2O. ML302
stock solution (50 mM in DMSO-d6) was added to a sample to
give a final concentration of 200 µM. When specified, NDM-1
was added to give a final concentration of 1 µM. The reaction
was followed at 1 hour intervals over 18 hours (Figures S7 and
S8).
to that used for the synthesis of the ML302 5a-q and ML302F
6a-q analogues (Scheme 2). The 2,4-dioxo thiazolidineacetic
acid 8 was found to be substantially less reactive towards
2.5. Crystallography
BcII crystals were prepared using the sitting drop vapour
diffusion method (293 K, 200 mM ammonium sulfate, 100 mM
w
bis-Tris buffer pH 5.5, 25% /v polyethylene glycol 3350, and 5
mM inhibitor). The crystals were cryoprotected using well
v
solution diluted to 25% /v glycerol before being flash cooled in
liquid nitrogen. All data sets were collected at 100 K. All data
were autoprocessed at the beamline using xia2.30 The structures
were solved using molecular replacement (using PDB ID 4TYT
as a search model24) within PHASER. The structures were then
fit to electron density and refined using COOT31 and PHENIX32
until Rwork and Rfree no longer converged.
For VIM-2, crystal soaking was performed by directly adding
32.5 nL of a 100 mM stock of ML302F in DMSO to 300 nL
crystal drops using a Labcyte Echo 550 acoustic drop dispenser,
which is part of the XChem pipeline at Diamond Light Source.33
Crystals were soaked with the ligand for 135 minutes before the
v
addition of 300 nL of 50% /v glycerol and flash cooling. X-ray
diffraction data were collected at Diamond Light Source
beamline I04-1, and processed with Diamond’s automated
processing pipelines, using xia228 and XDS,34 with
XChemExplorer35 and Dimple used for electron density
generation. Initial ligand bound electron density was identified
using PanDDA36. Grade37 was used for ligand restraint
generation. Final model preparation was performed by iterative
cycles of refinement using REFMAC38 and model building in
Coot31. Data collection, PDB codes, and refinement statistics for
all structures are given in Table S2.
Scheme 1. Synthesis of enethiol based β-lactamase inhibitors. (a) Route for
preparation of ML302 5a-q analogues and ML302F 6a-q analogues.24 (b) R
groups for 5a-q and 6a-q. MW: microwave irradiation.
Knoevenagel condensation than the analogous 4-oxo-2-thioxo 2
ring system. This reduced reactivity coupled with the broad
spectrum biological importance of these types of ring systems
has led to the development of catalytic protocols for their
3. Results and Discussion
3.1. Synthetic routes to the enethiols and related compounds
synthesis
CH3COONa/AcOH,
(e.g.
pyridine/EtOH,
piperidine/EtOH/AcOH,
NH4OAc,
The synthetic route used for the preparation of ML302 and
ML302F analogues (5a-q and 6a-q, respectively) is shown in
Scheme 1.24 ML302 analogues 5a-q were prepared in two steps,
by Knoevenagel-type condensation of rhodanine-3-acetic acid 2
and the appropriate aldehyde 1a–q to provide, predominantly, the
Z-isomer of the benzylidene-4-oxo-2-thioxo-thiazolidin-3-yl)-
acetic acid derivatives (3a-3q),39,40 which were coupled with
amino-4-methyl-piperazine 4 to give the desired ML302
analogues (5a-q). Base mediated hydrolysis of the ML302
derivatives 5a-q then provided ML302F and its analogues 6a-q
(Scheme 1).
NH4OAc/AcOH,
NH4OAc/toluene etc.).42,43,44,45,46,47,48,49,50,51 However, these
conditions proved to be low yielding in our hands. Applying
longer reaction times (12 hours under microwave conditions),
using the EtOH/piperidine system provided the best result (80%)
Consistent with
a
literature report,41 we observed
decomposition of the ML302F analogues 6a-q in DMSO. Thus,
biochemical assays were performed by using the corresponding
sodium salts (conversion with 100 mM sodium bicarbonate
immediately prior to assay), and characterizations were
performed in MeOD (see section 3. in Supplementary
Information). The (ML302F) 6 analogues showed good stability
as crystalline solids in their acid form, after purification by re-
crystallisation from toluene.
Scheme 2 Synthesis of the 2,4-dione derivative 10 of ML302.
for preparation of 2,4-dioxo thiazolidineacetic acid 9.
For comparison with the enethiols, α-mercaptocarboxylic
acids 13a and 13b were prepared in two steps from the
corresponding α-bromocarboxylic acids (11a and 11b,
respectively), following the procedure of Shaffer et al.27 via
To investigate the effects of changing the electronic properties
of the thiazolidine ring on MBL inhibition, the 2,4-dione
derivative 10 of ML302 was prepared from 8 in a similar manner

nucleophilic substitution with potassium thioacetate followed by
basic hydrolysis (Scheme 3). The α-hydroxycinnamic acid enol
analogue 17 of ML302F was prepared from the corresponding
aldehyde via Erlenmeyer azlactone synthesis of 16 followed by
acid mediated hydrolysis (Scheme 4).28
integron–encoded MBL-2; BcII, Bacillus cereus II MBL; SPM-1,
São Paulo MBL-1; IMP-1, imipenemase MBL-1) and the Class
B2 MBL CphA (carbapenem hydrolysing MBL from Aeromonas
hydrophila).22 The latter MBL (CphA) only utilises one active
site Zn(II) ion in catalysis, whilst the others normally use two
Zn(II) ions. We also screened the inhibitors against
a
representative panel of SBLs from different classes (TEM-1,
Temoneira β-lactamase-1, class A; CTX-M-15, cefotaxime
hydrolysing β-lactamase from Munich 15, class A, extended
spectrum β-lactamase (ESBL); AmpC E. coli, class C, and OXA-
10, oxacillinase-10, class D).
Scheme 3 Synthesis of racemic α-mercaptocarboxylic acids 13a and 13b.26
A number of trends for the rhodanine derived inhibitors
(compound series, 3a-q, 5a-q, and 6a-q) are apparent from the
results (Table 1). In all cases the enethiols (6a-q) were the most
potent inhibitors within a given set of rhodanine/enethiol
derivatives, implying that the enethiols are the prime source of
inhibition. With a few exceptions (mostly in the case of VIM-2
and the atypical subclass B1 MBL, SPM-1), the rhodanine-3-
acetic acids (3a-q) were either inactive (at 50µM) or only weakly
active compared to the enethiols (6a-q). This is also the case for
the amides (ML302, 5a-5q) against certain MBLs, though there
were more exceptions (e.g. 5i-q). It is likely that, at least to some
extent, the activities for the rhodanine-3-acetic acids (3a-q) and
amides (ML302, 5a-5q) result from (partial) hydrolysis of the
compounds to give their corresponding enethiols (6a-q). The
differences in activities between rhodanine-3-acetic acids (3a-q)
or amides (ML302, 5a-5q) may in part reflect the extent of
hydrolysis. Whether or not such hydrolysis is enzyme catalysed
is difficult to judge given the potency of enethiol (6a-q)
inhibition. The proposal of enzyme mediated hydrolysis is
supported by the different results observed for analogous amides
and enethiols. Thus, amide ML302 manifests similar inhibition
compared to the enethiol ML302F for two of the tested B1
subclass MBLs (IMP-1 and VIM-2), whereas for SPM-1, BcII
and NDM-1, ML302 was ~5, ~4 and ~15 fold less active than
ML302F. For the subclass B2 MBL CphA, ML302 (IC50 value
>50 µM) was also significantly less active than ML302F (IC50
value 200 nM). Thus, ML302 may be hydrolysed at different
rates by different MBLs.
Scheme 4 Synthesis of α-hydroxycinnamic acid 17.27
Aside from the previously reported formation of ternary
complexes24 (Figure 2), some of the results do, however, suggest
the intact rhodanines may have inhibitory activity as precedented
by work from Spicer et al.53 Interestingly, although the 2,4-
dioxo-1,3-thiazolidin analogue (10) of ML302 is less active than
ML302 against all tested MBLs, it did manifest activity against
SPM-1, BcII, and VIM-2, being much less active against IMP-1
and particularly, NDM-1. We did not observe hydrolysis of 10 by
Scheme 5 Synthesis of α-hydroxy phosphonic acid 22 and α-sulfanyl
phosphonic acid 26.41-50
To investigate the importance of the carboxylate52 in MBL
inhibition, α-sulfanyl phosphonic acid 26 was synthesised in five
steps in an overall yield of 40% from diisopropyl phosphite
(Scheme 5). Thus, 1,2-addition of diisopropyl phosphite 18 to 4-
bromo-2-fluoro-benzaldehyde 19 afforded the α-hydroxy
phosphonate 20, which was converted to the ^-
nitrobenzenesulfonate derivative 23. Nucleophilic substitution
using potassium thiocyanate gave the thiocyanate derivative 24
which was reduced with sodium borohydride to afford α-sulfanyl
phosphonate 25. Treatment of α-sulfanyl phosphonate 25 with
trimethylsilyliodide^, followed by methanolysis afforded the α-
sulfanyl phosphonic acid 26. α-Hydroxy phosphonic acid 22 was
prepared in a similar way, by hydrolysis of the α-hydroxy
phosphonate 20.
1
NMR on the timescale of the inhibition studies, H NMR (700
MHz) in the presence or absence of NDM-1 MBL (Fig. S1).
Although we cannot rule out the hydrolysis of 10 to form
ML302F at low levels, these results suggest that further SAR
studies on the intact rhodanine scaffold, or preferably more stable
analogues of it, are of interest. One possibility is that the intact
rhodanines can bind to the active site in a manner not involving
metal chelation, as recently reported for another series of MBL
inhibitors.54
INSERT TABLE 1 HERE
The results (Table 1) reveal some SAR trends – some of the
di-/tri-substituted amides and enethiols were clearly more active
than the mono-substituted compounds. However, because the
amides may be under-going hydrolysis/inhibiting via more than
one mode of action, care must be taken in comparing the SAR for
the two series. Thus, in most cases, the amides 5a-5e, showed
almost no inhibition against all the subclass B1 MBLs (IC50
3.2. Inhibition assays
We first screened the inhibitors against a representative set of
presently clinically important and other MBLs22, comprising both
Class B1 enzymes (NDM-1, New Delhi MBL-1; VIM-2, Verona

values ~50 or >50 µM), when the para-position of the phenyl
ring was mono-functionalised with halogen or alkyl groups. By
contrast, e.g., the mono-ortho-substituted 5f-5i (IC50 values 2.0-
52.1 µM), and di-ortho-substituted 5j (IC50 values < 2.0 µM), and
2,3,6-trifluoro 5k (IC50 values < 3.5 µM) analogues did manifest
significant inhibition for all the tested B1 subclass MBLs, with
the exception of NDM-1.
We then screened selected enethiols (6a, 6b, 6g, 6j, 6k, 6l, 6m
and ML302F) in the presence of their amide analogues (5a, 5b,
5g, 5j, 5k, 5l, 5m and ML302) for potential inhibition via the
formation of a ternary complex (Table 3). The use of a 1:1
mixture of ML302 and ML302F (ML302M) manifested a >20-
fold increase in potency (IC50 = 1.8 nM) compared to the use of
either ML302 (IC50 = 60 nM) or ML302F (IC50 = 40 nM)
individually against VIM-2, as observed previously.12 The
mixture ML302M was also moderately more active against IMP-
1 (IC50 = 3 nM), and more potent against CphA (IC50 = 20 nM)
than ML302 (IC50 = 90 nM, >50 µM, respectively) or ML302F
alone (IC50 = 20 nM and 200 nM, respectively). However, the
increased activity with the mixture was not present in the
inhibition of the other subclass B1 MBLs (BcII, SPM-1, NDM-1)
tested, arguing against the formation of a ternary complex in
these cases (Table 3). This observation is consistent with the
results of our previous study,24 i.e. that a ternary complex can be
accommodated by VIM-2, but not BcII, suggesting different
binding modes for these two enzymes. 24 Further, no evidence for
enhanced inhibition relative to the enethiols alone was observed
for any of the other tested mixtures, (5a, 5b, 5g, 5j, 5k, 5l, 5m
and 6a, 6b, 6g, 6j, 6k, 6l, 6m). Thus, the available evidence is
that formation of ternary complex is limited to specific enethiol
inhibitor-MBL combinations.
Similar trends were observed for the enethiols 6a-6l, although
IC50 values are mostly in the nanomolar range across all the
MBLs tested. 6j (IC50 values for SPM-1, IMP-1, BcII, VIM-2 and
NDM-1 of 4.3 nM, 30 nM, 60 nM, 50 nM and 4.4 µM,
respectively), inhibited with a similar potency compared to
ML302F across the panel (IC50 values for SPM-1, IMP-1, BcII,
VIM-2 and NDM-1 of 20 nM, 20 nM, 80 nM, 40 nM and 2.4
µM, respectively). The results with enethiols 6j and 6l led to
further investigations on aromatic substitutions. The di-ortho-
substituted 6m (IC50 values from 10 nM to 2.1 µM), 6p (IC50
values from 60 nM to 41.5 µM) and 6q (IC50 values from 20 nM
to 4.9 µM) showed much better inhibition compared to the 2,3-
substituted (6n) (IC50 values from 200 nM to >50 µM) or 2,5-
substituted (6o) (IC50 values from 800 nM to 46.9 µM)
analogues. Compounds with increased steric bulk, e.g.
thianaphthene 6l or 6r, still manifested potent or moderately
potent inhibition against most of the MBLs, with one clear
exception in each case (NDM-1 with 6l and BcII with 6r).
INSERT TABLE 3 HERE
We then synthesised and tested a set of analogues to
investigate the importance of the different functional groups in
the enethiols (6a-q, ML302F). The hydroxyl analogue of 6c, i.e.
17 (Table S1), was near inactive (at 100 µM), as was the 2-
methyloxazol-5(4H)-one (16), precursor of 17, supporting the
importance of the sulphur atom for binding and inhibition. The
phosphoric acids 22 and 26 were also inactive, implying the
importance of the carboxylate in inhibition (Table S1). The
saturated analogues of 6a, i.e. 13b (IC50 values for SPM-1, IMP-
1, BcII, VIM-2 and NDM-1; 70 nM, 50 nM, 1.4 µM, 70 nM and
38.7 µM, respectively) and its saturated truncated form, 13a (IC50
values for SPM-1, IMP-1, BcII, VIM-2 and NDM-1 of 50 nM, 70
nM, 100 nM, 40 nM and 12.9 µM, respectively) manifested
comparable potency against subclass B1 MBLs compared to 6a
(IC50 values for SPM-1, IMP-1, BcII, VIM-2 and NDM-1; 3 nM,
30 nM, 700 nM, 400 nM and 7.9 µM, respectively). However,
13a/13b were much less active against CphA (IC50 values for 6a,
13a and 13b; 2.1 µM, 71.0 µM and 130.0 µM, respectively). 13a
was more active than 13b against BcII and NDM-1. The
hydroxyl analogues, i.e. mandelic acid and 3-phenylactic acid,
were inactive (at 100 µM), supporting the importance of the
sulphur atom for MBL inhibition (Table 2). The observations
support previous findings for the use of the α-
mercaptocarboxylic acid motif for MBL binding/inhibition.55
Although relative acidity of the functional groups may be a
factor, the increased inhibition observed for the (racemic)
saturated α-mercaptocarboxylic acids (13a, 13b) compared to the
analogous enethiol (6a) could be a result of the different spatial
relationship of the thiol and the carboxylate, enabling the
saturated α-mercaptocarboxylic acids to bind better.
3.3. Structural Studies
Previously, we have reported crystallographic studies of VIM-
2 and the BcII MBLs in complex with ML302F and in the case
of VIM-2, ML302.24 In the case of BcII, a single ML302F
24
molecule was apparent at the active site. Unexpectedly, when
ML302 was co-crystallised with VIM-2, each of the two
molecules in the asymmetric unit had ML302F chelating Zn(II)
at the active site. However, an additional molecule of ML302
that was apparent only near the active site of chain A (and not
chain B), was positioned to interact with ML302F, via staggered
π-stacking between the rhodanine ring of ML302 and the 2,3,6-
trichlorophenyl ring of ML302F (Figure 2).24 In order to further
investigate the mode of enethiol binding to MBLs in relation to
our SAR results, we obtained four additional high resolution
crystal structures of BcII co-crystallised with enethiols 6c
INSERT TABLE 2 HERE
Figure 2. Prior crystallographic analysis revealed that ML302 undergoes
fragmentation to form the enethiol inhibitor ML302F (PDB ID: 4PVO),24
which coordinates to the di-Zn(II) containing active site. All figures are labelled
using the BBL numbering scheme.
All of the enethiols, except 6r (as well as 5d, 5r, which are
intact rhodanines), were near inactive (at 200 µM) against a
panel of SBLs (Table S2). The observed weak SBL inhibition by
5r and 6r (residual activities at 200 µM for TEM-1, CTX-M-15,
AmpC and OXA-10; ~6%, ~40%, ~18% and ~47% and ~1%,
~65%, ~41% and ~47%, respectively) may be due to active site
hydrophobic interactions involving the tri-aromatic ring system.
(Figure S9, PDB ID: 5JMX), 6k (Figure S10, PDB ID: 6EUM),
6l (Figure S11, PDB ID: 6EWE), and 6s (Figure 3, PDB ID:
6F2N). We also obtained a new structure of VIM-2 in complex
with ML302F (Figure S12, PDB ID: 6EW3) using a low volume

soaking method.56 (Note: The geometric restraints generated by
GRADE for the C=C double bond length of the enethiols
reported here are 1.4 Å, whereas that in our previously reported
VIM-2:ML302F structure (PDB: 4PVO24) was slightly longer
(1.5 Å) due to the geometric restraints output by ELBOW.24)
the enethiol inhibitors is rotated about the C3–C4 bond such that
it is not co-planar with the plane of the enethiol alkene, likely
hindering conjugation. For ML302F the skewed arrangement
was thought to be, at least partially, caused by steric hindrance
due to the ortho di-chloro substituents on the phenyl ring as
proposed previously,24 Because enethiols without ortho-
substituents are also observed to retain similar conformations (as
evidenced by a crystal structure of BcII in complex with 6c,
Figure S9), the ortho- substitution may not be an essential factor
in obtaining potent inhibition by the enethiols.
Superimposing the structure of BcII in complex with 6s and
VIM-2 in complex with ML302F, implies that there may be a
steric clash between the naphthalene side chain of 6s and Tyr67
on the L3 loop of VIM-2, suggesting unfavourable binding.
However, the BcII and VIM-2 IC50 values for 6s are comparable
(IC50 = 0.2 µM and 0.1 µM, respectively), indicating 6s might
adopt a different conformation when binding to VIM-2 and/or
that it induces a conformational change of the VIM-2 L3 loop.
4. Conclusions
The overall results reveal that rhodanine derived species have
potential as broad spectrum MBL inhibitors, which might be in
part due to the proposal that enethiol carboxylate binding mimics
that of -lactam hydrolysis product (Figure S13). Their capacity
to inhibit SBLs and penicillin binding proteins appears more
limited, at least among those tested in this study.24,26 Although
the enethiols (6a-6q), which are derived by rhodanine hydrolysis,
are the most potent of the series identified, the SAR on
compounds with intact rhodanine ring structures suggests that
rhodanine related heterocycles that do not chelate via a
thiol/sulphur may also have potential as MBL inhibitors. Recent
work on another series suggests that such compounds have
potential to inhibit without active site metal chelation.57 The
proposal of different binding modes for the enethiols (6a-61) and
rhodanine amides (5a-51) is supported by the observation of only
partially overlapping SAR trends for the two series.
Figure 3. Superimposition of structures of BcII (turquoise) (PDB ID: 5JMX,
6EUM, 6EWE, 6F2N) with VIM-2 (pink) (PDB ID: 6EW3) showing the
similarity in binding modes for 6c (yellow), 6k (salmon), 6l (purple), 6s
(green), and ML302F (wheat). In each MBL the thiolate interacts with both
Zn(II) ions and the inhibitor carboxylate interacts with Zn2 and (Lys-224 of
BcII/Arg233 of VIM-2).
Comparison of the BcII structures in complex with the different
enethiols reveal that the core enethiols have a remarkably similar
binding mode to ML302F, with the thiol(ate) displacing the
bridging water molecule normally located between the two Zn(II)
ions (Zn1 and Zn2) and the inhibitor carboxylate ligating to Zn2.
The enethiol linked phenyl rings of the inhibitors all occupy the
same region of the active site (Figure 3). As observed for
binding of the products of MBL-catalysed β-lactam hydrolysis, in
the structure of NDM-1 (PDB ID: 4EYF)56 (Figure S13), one of
the enethiol carboxylate oxygens is positioned to interact with
We have previously reported structural evidence that
ML302/ML302F can form a ternary complex with VIM-2.24 The
SAR results presented here suggest that formation of such a
ternary complex is not a general feature of rhodanine derived
MBL inhibition and hence, although interesting, is unlikely to be
a productive path for the development of broad spectrum
clinically useful MBL inhibitors.
Rhodanines are characterised as ‘difficult to progress’ and
‘promiscuous’ compounds.57,58,59 Our work reveals further
complexities involved in interpreting assay results involving
rhodanines. Despite their complex nature, one rhodanine is
clinically approved for use in nerve damage due to diabetes
mellitus (Epalrestat®, an aldose reductase inhibitor)60 and other
rhodanine-related heterocycles are in development.61 The results
presented here support the proposal that rhodanines (at least)
have potential as promiscuous enzyme inhibitors/protein binders,
in part owing to their tendency to undergo hydrolysis to products,
including enethiols, which have potential to inhibit the multiple
metallo-enzymes present in cells, including related MBL fold
enzymes, which have important biological roles beyond
antibiotic resistance including in nucleic acid repair and
metabolism.62 Our results also imply that any medicinal
chemistry studies employing rhodanine inhibitors should as a
matter of course include testing of their hydrolysis products.
Figure 4. View from a crystal structure of BcII (turquoise) in complex with 6s
6s (green). Active site residues shown as ball-and-stick with atoms coloured C
(white), O (red), N (blue), Zn (grey spheres), water (red spheres). Ligand
interactions are indicated with black dashed lines. Ligand mFo-DFc OMIT
maps contoured to 3.0 σ are shown as light grey mesh.
Zn2. The other oxygen is positioned to interact with the Nε amino
group of Lys-224 (Figure 4). As observed in the VIM-
2:ML302F complex, the plane of the phenyl side chain on all of

32. Adams P.D., Afonine P.V., Bunkoczi G., Chen V.B., Davis I.W., Echols
N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., McCoy
A.J., Moriarty N.W., Oeffner R., Read R.J., Richardson D.C., Richardson
J.S., Terwilliger T.C., Zwart P.H., Acta Crystallogr. D Biol. Crystallogr.,
2010;66:213-221.
33. Collins P.M., Ng J.T., Talon R., Nekrosiute K., Krojer T., Douangamath
A., Brandao-Neto J., Wright N., Pearce N.M., von Delft F., Acta
Crystallogr. D Struct. Biol., 2017;73:246-255.
Acknowledgments
We thank the following for funding: the Wellcome Trust, the
Medical Research Council, EU H2020 Marie Sklodowska-Curie
No 657314, the Diamond Light Source (proposals mx9306,
mx12346, lb16949). We thank the beamline staff for assistance.
34. Kabsch W., Acta Crystallogr. D Biol. Crystallogr., 2010;66:125-132.
35. Krojer T., Talon R., Pearce N., Collins P., Douangamath A., Brandao-
Neto J., Dias A., Marsden B., von Delft F., Acta Crystallogr. D Struct.
Biol., 2017;73:267-278.
36. Pearce N.M., Krojer T., Bradley A.R., Collins P., Nowak R.P., Talon R.,
Marsden B.D., Kelm S., Shi J.Y., Deane C.M., von Delft F., Nat. Comm.,
2017;8.
Supplementary Material
Supplementary material is available online.
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Table1. Screening results for the inhibition of MBLs by rhodanine derived inhibitors.
IC50 (µM) versus
R
Compound
SPM-1
IMP-1
BcII
VIM-2
NDM-1
3a
5a
6a
>50
>50
0.3 ± 7×10-3
>50
22.7 ± 0.3
0.3 ± 0.03
>50
>50
0.7 ± 0.2
36.8 ± 0.3
16.4 ± 0.1
0.4 ± 0.02
>50
26.7 ± 1.0
7.9 ± 0.1
3b
5b
6b
>50
>50
0.3 ± 7×10-3
>50
>50
0.2 ± 5×10-3
>50
>50
0.7 ± 0.05
32.0 ± 0.7
>50
0.4 ± 0.01
>50
28.0 ± 1.0
3.7 ± 0.1
3c
5c
6c
>50
>50
4.1 ± 0.4
>50
>50
2.7 ± 0.1
>50
>50
8.2 ± 0.1
34.5 ± 0.6
>50
9.7 ± 0.3
>50
>50
5.0 ± 0.4
3d
5d
6d
39.7 ± 0.8
>50
2.3 ± 0.3
>50
28.7 ± 1.2
3.3 ± 0.4
>50
>50
9.9 ± 0.2
>50
44.0 ± 0.4
9.4 ± 0.2
>50
>50
6.3 ± 0.2
3e
5e
6e
>50
>50
0.6 ± 0.1
>50
31.5 ± 3.2
0.8 ± 5×10-3
>50
>50
2.4 ± 0.1
>50
>50
0.4 ± 0.02
>50
48.9 ± 0.7
8.2 ± 0.3
3f
5f
6f
>50
42.7 ± 0.8
0.3 ± 7×10-3
>50
22.6 ± 0.3
1.4 ± 1.0
>50
26.3 ± 1.5
4.0 ± 0.1
17.6 ± 0.7
6.2 ± 0.2
3.3 ± 0.2
>50
>50
12.9 ± 0.4
3g
5g
6g
25.1 ± 1.5
9.8 ± 0.4
0.1 ± 3×10-3
43.2 ± 2.1
7.6 ± 0.1
0.4 ± 0.01
>50
>50
0.9 ± 0.1
6.7 ± 0.2
2.0 ± 0.2
0.4 ± 0.02
>50
>50
5.1 ± 0.5
3h
5h
6h
36.6 ± 0.8
8.1 ± 0.3
3.5 ± 0.3
32.5 ± 1.3
8.3 ± 0.2
0.8 ± 0.06
>50
14.6 ± 0.8
5.8 ± 0.1
>50
11.4 ± 1.0
2.7 ± 0.1
>50
>50
16.2 ± 0.3
3i
5i
6i
2.0 ± 0.5
8.8 ± 0.3
1.4 ± 0.4
22.4 ± 0.4
37.1 ± 2.1
0.7 ± 0.02
31.7 ± 1.0
>50
6.3 ± 0.1
5.9 ± 0.1
37.6 ± 1.0
3.5 ± 0.1
>50
>50
5.0 ± 0.2
3j
5j
6j
1.2 ± 0.3
43.7 ± 0.5
0.3 ± 0.01
>50
2.0 ± 0.2
9.8 ± 2.8
0.8 ± 0.1
>50
>50
0.1 ± 4×10-3
4.3×10-3 ± 3×10-4 0.03 ± 3×10-4 0.06 ± 2×10-3 0.05 ± 1×10-3 4.4 ± 0.2

3k
5k
6k
8.8 ± 0.3
1.5 ± 0.2
0.1 ± 2×10-3
46.5 ± 0.7
1.6 ± 0.4
0.4 ± 0.02
27.0 ± 2.2
3.5 ± 0.2
0.3 ± 0.01
47.6 ± 0.4
2.6 ± 0.1
0.3 ± 0.01
>50
40.1 ± 2.1
3.3 ± 0.1
3l
5l
6l
13.9 ± 0.5
>50
0.4 ± 0.02
>50
>50
0.7 ± 0.05
21.7 ± 2.4
7.2 ± 3.3
3.1 ± 0.3
36.9 ± 0.2
41.8 ± 3.6
0.5 ± 0.03
>50
>50
11.1 ± 0.3
3m
5m
6m
3.5 ± 0.4
0.2 ± 2×10-3
0.01 ± 1×10-4
18.9 ± 0.8
41.8 ± 2.3
0.2 ± 0.3
0.6 ± 0.1
0.3 ± 0.01
>50
28.4 ± 0.4
0.2 ± 4×10-5
0.01 ± 6×10-5 0.07 ± 3×10-3 0.03 ± 2×10-4 0.6 ± 0.1
3n
5n
6n
1.0 ± 0.1
0.3 ± 8×10-3
0.2 ± 7×10-3
46.2 ± 1.4
0.6 ± 0.2
1.0 ± 2
22.1 ± 1.0
1.1 ± 0.3
1.0 ± 0.2
7.6 ± 0.3
39.5 ± 0.5
1.3 ± 0.2
>50
32.8 ± 0.8
46.5 ± 0.7
3o
5o
6o
16.6 ± 1.0
2.8 ± 0.2
0.8 ± 0.06
>50
7.0 ± 0.9
4.7 ± 0.2
>50
15.4 ± 1.0
1.6 ± 0.5
>50
>50
1.5 ± 0.2
>50
>50
45.3 ± 0.6
3p
5p
6p
32.1 ± 2.3
1.3 ± 0.2
0.06 ± 2×10-3
>50
1.3 ± 0.2
0.2 ± 9×10-3
>50
3.0 ± 0.1
0.1 ± 2×10-3
6.8 ± 0.2
41.7 ± 1.9
0.2 ± 0.07
>50
>50
21.5 ± 0.8
3q
5q
6q
>50
0.2 ± 7×10-3
0.02 ± 2×10-4
>50
2.7 ± 0.2
>50
27.0 ± 0.6
18.7 ± 1.0
29.4 ± 1.0
>50
>50
0.02 ± 2×10-4 0.2 ± 2×10-3 0.07 ± 2×10-3 4.9 ± 0.3
ML302A
ML302
ML302F
4.8 ± 0.3
0.1 ± 9×10-3
0.02 ± 2×10-4
9.6 ± 0.4
21.3 ± 0.5
0.3 ± 0.01
3.6 ± 0.7
>50
0.09 ± 2×10-3
0.06 ± 5×10-3 15.6 ± 0.7
0.02 ± 3×10-4 0.08 ± 2×10-3 0.04 ± 2×10-3 2.4 ± 0.1
10
6r
0.5 ± 0.04
3.2 ± 0.2
0.6 ± 0.05
0.2 ± 0.02
>50
3.2 ± 0.2
1.8 ± 3×10-3
>50
0.05 ± 1×10-3 1.7 ± 0.6
6s
0.06 ± 3×10-3
0.08 ± 2×10-3
0.2 ± 0.03
0.1 ± 3×10-3
1.1 ± 1.7
S9

Table 2 — IC50 values for the inhibition of MBLs by α-mercapto, and α-hydroxy, carboxylic acids and enethiols.
IC50 versus (µM)
R
Compound
6a
SPM-1
IMP-1
BcII
VIM-2
NDM-1
CphA
0.3 ± 7×10-3
0.3 ± 0.03
0.7 ± 0.2
0.4 ± 0.02
7.9 ± 0.1
2.1 ± 0.02
0.04 ± 1×10-
13a
13b
0.05 ± 1×10-3
0.07 ± 1×10-3
0.07 ± 3×10-3 0.1 ± 4×10-3
12.9 ± 1.0 71.0 ± 1.0
38.7 ± 4.7 130.0 ± 10
3
0.07 ± 2×10-
0.05 ± 2×10-3
1.4 ± 0.3
3
Mandelic acid
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
3-Phenyllactic acid
NI: No observed inhibition at 100 µM.
Table 3. Observed inhibition of MBLs by a 1:1 mixture of rhodanine amides (5) and enethiols (6) compared to their inhibition by the
separate molecules.
IC50 when R = (µM)
Compound/Mixture Enzyme
a
b
g
J
k
l
m
ML302/F
Amide, 5
Enethiol, 6
5+6 (1:1)
>50
0.3 ±
>50
0.3 ±
9.8 ± 0.4
0.1 ± 4×10-3
1.5 ± 0.2
0.1 ±
2×10-3
>50
0.4 ±
0.02
0.2 ± 2×10-3
0.1 ± 9×10-3
0.1 ±
4.3×10-3
±
SPM-1
IMP-1
BcII
0.01 ± 1×10-4
0.02 ± 2×10-4
0.02 ± 2×10-4
0.02 ± 2×10-3
7×10-3
7×10-3
3×10-3
2.9×10-5
2.3 ± 0.09
42.4 ± 0.7
1.3 ± 0.2
0.1 ± 0.02
0. 4 ± 0.2
0.7 ± 0.3
Amide, 5
22.7 ± 0.3
0.3 ± 0.03
>50
0.2 ±
7.6 ± 0.1
0.3 ± 0.01
0.03 ±
1.6 ± 0.4
>50
0.7 ±
0.05
0.5 ±
0.03
0.2 ± 4×10-3
0.09 ± 2×10-3
0.02 ± 3×10-4
Enethiol, 6
0.4 ± 0.01
0.4 ± 0.02
0.01 ± 6×10-5
5×10-3
0.1 ±
3×10-4
0.02 ±
0.2 ±
5×10-3
7.0×10-3
8×10-5
±
3.0×10-3
4×10-5
±
5+6 (1:1)
0.2 ± 0.01
0.4 ± 0.01
3×10-3
3×10-4
Amide, 5
Enethiol, 6
5+6 (1:1)
>50
>50
>50
2.0 ± 0.2
0.06 ±
3.5 ± 0.2
0.3 ± 0.01
0.6 ± 0.4
7.2 ± 3.3
3.1 ± 0.3
6.3 ± 0.4
1.5 ± 0.2
0.3 ± 0.01
0.7 ± 0.2
1.8 ± 0.3
0.7 ± 0.2
1 ± 0.1
0.9 ± 0.1
3.2 ± 0.06
0.07 ± 3×10-3
0.03 ± 1×10-3
0.08 ± 2×10-3
0.03 ± 1×10-3
2×10-3
0.1 ± 0.01
41.8 ±
3.6
0.5 ±
0.03
Amide, 5
Enethiol, 6
5+6 (1:1)
16.4 ± 0.1
0.4 ± 0.02
0.2 ± 0.01
>50
2.0 ± 0.2
0.4 ± 0.02
0.4 ± 0.02
0.8 ± 0.1
2.6 ± 0.1
0.3 ± 0.01
0.2 ± 0.01
0.3 ± 0.01
0.03 ± 2×10-4
0.02 ± 1×10-4
0.06 ± 5×10-3
0.04 ± 2×10-3
0.05 ±
1×10-3
0.05 ±
1×10-3
VIM-2
0.4 ± 0.01
0.3 ± 0.01
1.8×10-3
3×10-4
±
2.3 ± 0.2
Amide, 5
Enethiol, 6
5+6 (1:1)
26.7 ± 1.0
7.9 ± 0.1
18.5 ± 0.1
28.0 ± 1.0
3.7 ± 0.1
16.2 ± 0.5
>50
>50
40.1 ± 2.1
3.3 ± 0.1
28.6 ± 0.7
>50
11.1 ±
0.3
28.4 ± 0.4
0.6 ± 0.1
1.7 ± 0.3
15.6 ± 0.7
2.4 ± 0.1
1.3 ± 0.4
NDM-1
CphA
5.1 ± 0.5
33.8 ± 1.0
4.4 ± 0.2
2.8 ± 0.4
>50
Amide, 5
Enethiol, 6
5+6 (1:1)
>50
2.1 ± 0.02
2.7 ± 0.04
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
>50
0.8 ± 0.02
0.4 ± 0.01
>50
0.2 ± 4×10-3
0.02 ± 1×10-3
Note that in most cases the mixture is of similar potency to the enethiol alone, but that in a few cases (notably ML302/ML302F) the mixture is more potent.
S10

Graphical Abstract
Structure activity relationship studies on
rhodanines and derived enethiol inhibitors of
metallo-β-lactamases
Leave this area blank for abstract info.
Dong Zhanga, Marios S. Markoulidesa, Dmitrijs Stepanovsa, Anna M. Rydzika, Ahmed El-Husseina,c Corentin A.
M. Bona, Jos J. A. G. Kampsa, Klaus-Daniel Umlanda, Patrick M. Collinsb, Samuel T. Cahilla, David Y. Wanga
Timothy D. W. Claridgea, Jürgen Brema, Michael A. McDonougha, and Christopher J. Schofield.a
a Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford,
OX1 3TA, United Kingdom.
b Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom.
c The National Institute of Laser Enhanced Science, Cairo University, Egypt.
Enethiols inhibit clinically relevant metallo -
lactamases in isolated form and in
antibiotic resistant bacteria.
S11