Structural Basis of Metallo-β-lactamase Inhibition by N-Sulfamoylpyrrole-2-carboxylates

Article abstract of DOI:10.1021/acsinfecdis.1c00104

Metallo-β-lactamases (MBLs) can efficiently catalyze the hydrolysis of all classes of β-lactam antibiotics except monobactams. While serine-β-lactamase (SBL) inhibitors (e.g., clavulanic acid, avibactam) are established for clinical use, no such MBL inhibitors are available. We report on the synthesis and mechanism of inhibition of N-sulfamoylpyrrole-2-carboxylates (NSPCs) which are potent inhibitors of clinically relevant B1 subclass MBLs, including NDM-1. Crystallography reveals that the N-sulfamoyl NH2 group displaces the dizinc bridging hydroxide/water of the B1 MBLs. Comparison of crystal structures of an NSPC and taniborbactam (VRNX-5133), presently in Phase III clinical trials, shows similar binding modes for the NSPC and the cyclic boronate ring systems. The presence of an NSPC restores meropenem efficacy in clinically derived E. coli and K. pneumoniae blaNDM-1. The results support the potential of NSPCs and related compounds as efficient MBL inhibitors, though further optimization is required for their clinical development.

Full text of DOI:10.1021/acsinfecdis.1c00104

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Article
Structural Basis of Metallo-β-lactamase Inhibition by
N‑Sulfamoylpyrrole-2-carboxylates
§
§
̃
Alistair J. M. Farley, Yuri Ermolovich, Karina Calvopina, Patrick Rabe, Tharindi Panduwawala,
̈
Jurgen Brem, Fredrik Björkling,* and Christopher J. Schofield*
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ABSTRACT: Metallo-β-lactamases (MBLs) can efficiently catalyze the
hydrolysis of all classes of β-lactam antibiotics except monobactams.
While serine-β-lactamase (SBL) inhibitors (e.g., clavulanic acid,
avibactam) are established for clinical use, no such MBL inhibitors are
available. We report on the synthesis and mechanism of inhibition of N-
sulfamoylpyrrole-2-carboxylates (NSPCs) which are potent inhibitors of
clinically relevant B1 subclass MBLs, including NDM-1. Crystallography
reveals that the N-sulfamoyl NH2 group displaces the dizinc bridging
hydroxide/water of the B1 MBLs. Comparison of crystal structures of an
NSPC and taniborbactam (VRNX-5133), presently in Phase III clinical
trials, shows similar binding modes for the NSPC and the cyclic boronate
ring systems. The presence of an NSPC restores meropenem efficacy in
clinically derived E. coli and K. pneumoniae blaNDM-1. The results
support the potential of NSPCs and related compounds as efficient MBL inhibitors, though further optimization is required for their
clinical development.
KEYWORDS: antimicrobial resistance, sulfonamide, metallo-β-lactamase, taniborbactam, NDM-1
Reported MBL inhibitors include bicyclic boronates, thiols,
and succinate derivatives (Figure 1).15,16 Recently, substituted
pyrroles and related compounds have been described as MBL
inhibitors in the patent and scientific literature.17−20 Wachino et
al. have reported that substituted pyrroles and furans bearing α-
carboxylic acid and N-sulfamoyl functional groups are effective
MBL inhibitors, in particular of the B1 subclass.21 N-1
Sulfamoylpyrrole-2-carboxylates have also been reported as B1
MBL inhibitors,22 though no structures of them in complex with
MBLs are reported. Structurally related sulfonamide-based
inhibitors of metalloenzymes are used therapeutically, e.g. as
carbonic anhydrase inhibitors with broad clinical utility.23−25
The N-sulfamoylpyrrole compounds are of mechanistic interest
because the (initial) binding mode of some classes of potent β-
lactamase inhibitors can mimic that of substrates (e.g., clavulanic
acid) or tetrahedral intermediates (boronates).26 We envisaged
that the approximately tetrahedral geometry about the sulfamoyl
sulfur27,28 may mimic the tetrahedral intermediate formed
during β-lactam hydrolysis, and the Lewis basicity of the oxygen
he β-lactams are one of the most important antibacterial
1
Tclasses;
however, their efficacy is increasingly being
eroded by resistance, most importantly by β-lactamases.2 Even
carbapenems, often used as “last resort” antibiotics, are often no
longer effective due to production of carbapenemases by
Enterobacteriaceae strains.3 Ambler class A, C, and D β-
lactamases are nucleophilic serine enzymes (serine-β-lacta-
mases, SBLs), whereas class B are metallo-β-lactamases
(MBLs).4 Combinations of a β-lactam antibiotic and a β-lactam
containing SBL inhibitor have long been used as a treatment
option for bacterial infections; however, no MBL combinations
are clinically approved.
Presently, only a few subclasses of SBLs,5,6 e.g. Class A KPCs
and Class D OXAs, are reported to efficiently hydrolyze
carbapenems, and these can be countered by clinically available
SBL inhibitors, e.g. avibactam.7,8 Class B MBLs, however, can
hydrolyze all carbapenems and β-lactam containing SBL
inhibitors.9−11 MBL inhibition is challenging in part because
of the need to obtain activity against a range of relevant enzymes,
which vary in their active site details.12,13 Subclass B1 and B3
MBLs are dizinc ion enzymes, whereas B2 MBLs employ one
zinc ion. The B1 MBLs are the most important from a clinical
perspective and include the IMP (imipenemase), NDM (New
Delhi MBL), and VIM (Verona integron-encoded MBL) MBL
subfamilies.14
Received: February 26, 2021
Published: May 18, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acsinfecdis.1c00104
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Figure 1. (a) Classes of β-lactam antibiotics. (b) Outline of the MBL hydrolysis mechanism. Ligands around the zinc ions are not shown for clarity. (c)
Representative SBL inhibitors. (d) Representative MBL inhibitors29−31 with sulfonamide ANT268119 and related sulfonamide and sulfamoyl
inhibitors.21,22,32 Zinc-chelating functional groups are highlighted in blue.
and nitrogen atoms may enable effective coordination to the zinc
ion. Here, we report on the mechanism of action and B1 MBL
potencies of N-sulfamoyl-substituted pyrrole-2-carboxylic acids
(NSPCs).
gave C2-substituted benzyl ester 3. The N-Cbz protected
sulfamoyl group was installed in good yield by tetrabutylammo-
nium fluoride (TBAF)-mediated N-sulfonyl deprotection,
followed by deprotonation of the pyrrole NH with sodium
hydride and then electrophilic trapping with zwitterionic
sulfamoylating reagent 7.38 Subsequent Pd-catalyzed Suzuki−
Miyaura cross-coupling with 4-fluorophenylboronic acid
afforded the C3-aryl derivative 5 as a sodium sulfonylazanide
salt which, upon hydrogenation, gave sodium carboxylate 6b
(93%). The free acid 6a was obtained from 5 by acidification
with aqueous HCl, followed by global deprotection with Pd/C/
H2 and purification by reverse phase HPLC in 69% yield.
With a robust synthesis of 6 in hand, we synthesized seven
other NSPC derivatives varying the C3-pyrrole substituent, with
bromopyrrole 4 providing a convenient vector for late-stage
diversification with aryl and heteroaryl groups via Suzuki−
Miyaura coupling, followed by hydrogenation (8−14, see
Supporting Information). Unexpectedly, under the hydro-
genation conditions used for the preparation of amino-
RESULTS AND DISCUSSION
■
We targeted the synthesis of NSPC 6a, which has a para-
fluorophenyl substitution at its C3 position, because this
substituent has been identified as being preferred in a related
series of published pyrrole inhibitors (Scheme 1).33,34 We aimed
to employ mild hydrogenolytic deprotection to prepare the
NSPCs because of potential competitive decarboxylation of
pyrrole-2-carboxylic acids35,36 and N1-sulfonyl group cleavage
under acidic or basic conditions.35−37 The efficient synthesis of
6a was readily achieved in seven steps from pyrrole (1) (12%
overall yield, Scheme 1). Initial N-sulfonylation of 1 with
PhSO2Cl was followed by regioselective electrophilic C3-
bromination using Br2. Subsequent directed ortho-metalation
and electrophilic trapping with benzylchloroformate (CbzCl)
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a
Scheme 1. Synthesis of 6a and NSPCs 8-14
a
(a) NaH, then PhSO2Cl, DMF, 0 °C, 2 h; (b) Br2, AcOH, reflux, 1 h, 54% yield over two steps; (c) i-Pr2NLi, then CbzCl, THF, −78 to 0 °C; (d)
1 M TBAF, THF, rt, 2 h, 63% over two steps; (e) NaH, then 7, THF, 0 °C to reflux, 4 h, 69%; (f) 4-FC6H4B(OH)2, 5% Pd(dppf)Cl2, Na2CO3,
dioxane: H2O 2:1, MW, 100 °C, 3 h, 70%; (g) 1 M aq HCl, then 10% Pd/C, H2 atmosphere, MeOH, rt, overnight, 69%; (h) 10% Pd/C, H2
atmosphere, MeOH, rt, overnight, 93%; (i) structure previously disclosed.
pyrimidine 11, near equimolar quantities of zwitterionic cyclic
guanidine 12 were also formed due to over-reduction;39 both
products were separated by preparative HPLC. For the
preparation of 14, it was necessary to first install the pinacol
boronate ester at the C3 position of pyrrole 4 by Pd-catalyzed
Miyaura borylation; the intermediate boronate ester then
underwent coupling with the commercial heteroaryl bromide.
The NSPCs were screened against four of the currently most
clinically relevant B1 MBLs, i.e. VIM-1, VIM-2, NDM-1, and
IMP-1 (Table 1), using an assay employing the “fluorogenic”
cephalosporin FC5.40 The NSPCs inhibit all 4 MBLs, with
potencies in the submicromolar range (pIC50 s 6.5−8.5), though
manifesting different inhibition profiles. Notably, the cyclic
guanidine 12 is a highly potent VIM-1 inhibitor (pIC50 8.5),
with a similar potency to the bicyclic boronate taniborbactam
(formerly VNRX-5133), which is in Phase III clinical trials.41
Interestingly, the cyclic guanidine 12 showed higher activity
toward VIM-1 than the unsaturated aminopyrimidine 11;
however, 12 is a less potent inhibitor of NDM-1. Most of the
NSPCs showed submicromolar activity (IC50) for inhibition of
NDM-1 and VIM-2, and 6a, 6b, 10, and 13 showed nanomolar
potency against NDM-1. Furthermore, some NSPCs are ∼150-
to >1500-fold more potent (pIC50 values 7.3−9.2) than the
bicyclic boronates CB2 or taniborbactam (pIC50 6.0 and 5.6,
respectively) against IMP-1. Within the compound set tested by
us, no stand-out compound potently inhibiting all four MBLs
was identified, revealing the scope for further optimization of the
NSPC scaffold.
Table 1. Activity of N-Sulfamoyl Pyrrole Carboxylate
Derivatives against Clinically Relevant MBLs
pIC50
VIM-1
NDM-1
VIM-2
IMP-1
CB2
taniborbactam
6a
6b
8
7.142
8.141
6.9
6.9
7.1
7.543
8.041
8.1
8.2
7.9
8.543
8.341
7.7
7.5
6.8
6.043
5.641
9.2
8.9
8.6
Our pIC50 values for 9 obtained from the fluorescence-based
assay are higher than the previously reported pIC50 values for
AMRC272 when using a nitrocefin-based assay.22 The
discrepancy likely reflects different enzymatic assay conditions,
protein constructs, and enzyme purification procedures;
however, it should be noted that both assays show a clear
preference for inhibition of IMP-1 over VIM-2.22
9
6.5
7.9
6.7
9
10
11
12
13
14
7.1
7.4
8.5
6.6
8.1
7.9
6.5
8.8
7.8
7.3
7.9
6.8
8.9
8.2
7.3
>9.2
8.3
6.422
7.422
5.722
6.322
6.621
Crystallography. We investigated the NSPC ligand−
enzyme interaction by crystallography and obtained a structure
for VIM-1 complexed with 6 (space group: P121 1, 1.21 Å
resolution, Figure 2). The structure was solved by molecular
replacement (PDB: 5N5G),44 with iterative fitting of 6 at the
active site. The two zinc ions (coordinated by H114, H116,
D118 (Zn1), H179, C198, and H240(Zn2)) were refined with
occupancies of 0.75 for both Zn1 and Zn2. The reduced
occupancy for Zn1 is due to partial oxidation of Cys198 to a 3-
sulfino alanine residue (Csd198), which was modeled and
refined in a ratio of Cys (75%) to Csd (25%), as shown in
previous work on VIM-1 (PDB 5FQA).45
7.4
7.9
8.2
a c
,
AMRC272
AMRC276
AMRC364
AMRC439
6.122
6.822
5.622
6.422
6.021
4.522
4.222
4.422
4.322
7.721
a c
,
a c
,
a c
,
b
SPC
a
b
Determined using 100 μM nitrocefin.22 Determined by measuring
hydrolysis of imipenem.21 Structure in Figure 1d. Assay details are
given in the Supporting Information. Enzyme concentration: 100 pM
(VIM-1), 20 pM (NDM-1), 20 pM (IMP-1), and 500 pM (VIM-2);
the concentration of FC5 was 5 μM. Note: inhibition data are
reported as pIC50 values (pIC50 = −log10IC50) and repeated in
quadruplicate.
c
Uncomplexed VIM-1:Zn2 (PDB: 5N5G)44 has a dizinc
bridging hydroxide/water, as do other B1MBls.26 The NH2- of
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Figure 2. VIM-1 active site binding mode of sulfamoyl inhibitor 6 and comparison with that of a bicyclic boronate and SPC. (a) Polder omit map48 of
VIM-1:Zn2:6 (PDB: 7AYJ, 1.21 Å resolution, 3.0 σ contour level) showing the NSPC sulfamoyl NH2 group replaces the dizinc bridging water. (b)
Superimposition of VIM-1:Zn2:CB2 (PDB: 7AYJ, yellow) and VIM-2:Zn2:CB2 (PDB: 5FQC, teal)43 structures reveals related binding modes. Note,
whereas binding of CB2 to VIM-2 has an impact on the Zn−Zn distance compared to unligated VIM-2, the effects of binding of 6 on this distance are
negligible (see Supporting Information Figure S1e−g); note also that the aryl side chains of CB2 and 6 project in different directions. (c)
Superimposition of VIM-1:Zn2:SPC (PDB: 7AYJ, yellow) and Vim-2:Zn2:NSPC (PDB: 6KZN, salmon)21 structures reveal the same binding mode.
Table 2. MIC Values of Meropenem (MEM)-SPC Combination against NDM-1 Producing Enterobacteriaceae
MIC (μg mL−1
)
species,
genotype
[I]
MEM
MEM
6a
MEM
6b
MEM
10
MEM
11
MEM
12
MEM
13
MEM
14
a
MEM (μg mL−1
)
VNRX
MEM 8 MEM 9
strain
IR57
E. coli
64
0.5
2
8
0.5
2
8
0.5
2
8
0.5
2
8
1
1
0.5
1
1
1.5
4
4
1.5
blaNDM-1
≤0.25
≤0.25
0.5
≤0.25
≤0.25
4
≤0.25
≤0.25
≤0.25
≤0.25
≤0.25
≤0.25
≤0.25
0.5
≤0.25
≤0.25
2
≤0.25
≤0.25
≤0.25
≤0.25
≤0.25
≤0.25
≤0.25
0.5
≤0.25
≤0.25
1
≤0.25
≤0.25
0.375
≤0.25
≤0.25
≤0.25
≤0.25
0.5
≤0.25
≤0.25
1
≤0.25
≤0.25
0.5
≤0.25
≤0.25
≤0.25
≤0.25
1
≤0.25
≤0.25
1.5
≤0.25
≤0.25
0.5
≤0.25
≤0.25
0.375
≤0.25
1
≤0.25
0.5
1.5
≤0.25
0.5
≤0.25
0.375
16
≤0.25
0.5
32
≤0.25
0.5
0.5
0.5
1
≤0.25
0.5
8
0.5
0.5
0.5
≤0.25
0.5
≤0.25
≤0.25
3
≤0.25
≤0.25
4
≤0.25
≤0.25
1.5
≤0.25
≤0.25
0.5
0.5
1
B68-1
S117
IR43
K. pneumoniae
blaNDM-1
64
E. coli
blaNDM-1
256
128
K. pneumoniae
blaNDM-1
0.5
≤0.25
≤0.25
16
≤0.25
0.375
0.25
a
MIC values of meropenem (MEM) and VRNX were at 10 μg mL−1.41 MIC experiments were repeated in triplicate.
sulfamoyl group of 6 replaces this “hydrolytic” water, probably in
its deprotonated form, though this cannot be discerned from the
crystal structure. The C2-carboxylate of 6 ligates to Zn2, as
observed in substrate derived complexes and those of inhibitors
with analogously placed carboxylates, including CB2/VNRX-
5133 (Figure 1).41−43
was performed in a minimum inhibition concentration (MIC)
antimicrobial assay format with 4 clinically relevant NDM-1
producing strains of Escherichia coli and Klebsiella pneumoniae
(Table 2). At a fixed concentration of 8 μg mL−1, the N-
sulfamoyl pyrroles reduce the meropenem MIC from 64 to
∼0.375 μg mL−1. In all cases, the MICs for the NSPCs were
better than those for VNRX-5133. At a concentration of 0.5 μg
mL−1 of the NSPC, the analogue potencies can be compared; 6
and 8−10 exhibit greater potency compared to the higher mass
compounds 13 and 14, bearing 4-morpholinophenyl or bicyclic
heteroaromatic groups at C3, respectively. These differences
may reflect differences in uptake as the pIC50s of 13/14 against
NDM-1 are consistent with the other analogues (Table 1).
Furthermore, the comparative data for aminopyrimidine 11 and
its saturated analogue 12 show the latter is significantly less
active at 0.5 μg mL−1, in particular with the B68-1, S117, and
IR47 strains, likely reflecting their relative NDM-1 pIC50 values
(Table 1). These data correlate with the submicromolar/
nanomolar enzymatic inhibition for NDM-1 and show the
ability of the series to penetrate the cell membrane, at least in the
studied Enterobacteriaceae clinical isolates.
The binding mode of 6 is related to that of bicyclic boronate
MBL inhibitors, (e.g., VIM-2:Zn2:CB2 (PDB: 5FQC, super-
imposition Figure 2b)),43 in which the binding of the two boron-
bound oxygens mimics the binding modes proposed for the two
oxygens in the oxyanion intermediate in MBL catalysis.
However, in the NSPC complex, one of these oxygens is
“replaced” by the tetrahedral sulfamoyl amino group (NR2-S-
NH2 108°; NR2SO(1) 105°; NR2−S-O(2) 114°, O(1)-S-NH2
111°, see Figure S1a). Comparison of the Zn(1)−Zn(2)
distances in the VIM-1:Zn2:6 and the VIM-2:Zn2:CB2 complex
reveals differences. The Zn(1)−Zn(2) distance is increased in
the VIM-2:Zn2:CB2 complex to 4.34 Å compared to 3.47 and
3.62 Å44 in the unligated VIM-2:Zn2 (PDB: 5N5G) and VIM-
1:Zn2 (PDB: 4NQ2) complexes, respectively46 (see Supporting
Information Figure S1e). Binding of 6, however, does not
substantially increase the Zn(1)−Zn(2) distance, i.e. it is 3.60 Å
compared to the unligated VIM-1/VIM-2. This distance is
similar to those reported for a hydrolyzed VIM-
1:Zn2:meropenem complex (PDB: 5N5I, Zn(1)−Zn(2): 3.50
Å, Figure S1d)44 and other unligated VIM family members.47
Antimicrobial susceptibility testing of the NSPCs in
combination with Meropenem, following CLSI guidelines,49,50
CONCLUSIONS
■
Our biochemical and microbiological results combined with
recently published data34,22 reveal the NSPCs as promising MBL
inhibitors with particularly potent (low nM) activity against
NDM-1 and IMP-1 and submicromolar activity against VIM-1
and VIM-2 enzymes. The structural studies presented here
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define an NSPC binding mode very similar to that of the α-
carboxylate- and N-sulfamoyl-substituted furans and pyrroles as
MBL inhibitors described by Wachino et al.21 Thus, the likely
deprotonated amino group of the tetrahedral sulfamoyl group
bridges the two zinc ions replacing the hydrolytic water in a
manner reflecting a tetrahedral intermediate in catalysis. The
structural analyses suggest that the relatively low activity against
VIM-1/VIM-2 may in part reflect differences in inhibitor (and
substrate) carboxylate binding mode. Dynamics at the dizinc
center of the protein, which are not observable with cryo-
temperature crystal structures, may also account for the
observed differences in inhibition data between the tested
MBL enzymes. Indeed, the overall highly conserved active site
architecture of the MBL superfamily enzymes supports a wide
range of reactions, including nucleic acid hydrolysis and redox
reactions, and in some cases, human MBLs are being pursued as
drug targets.51,52 The compact and polar nature of the NSPCs
and related scaffolds suggests that they may have wide utility as
inhibitors of MBL superfamily enzymes. However, further
derivatization of the NSPCs is warranted to increase potency
and spectrum of activity toward the most abundant resistance
causing MBLs to restore utility of important β-lactamase
antibacterials.
to buffer B (MeCN:formic acid, 100:0.1 v/v%) from 0 to 100%
B in 3.5 min, then 1 min at 100% B, maintaining a flow rate of 0.8
mL/min. High resolution mass spectra were recorded using a
Bruker μTOF (ESI) spectrometer. The m/z values are reported
in Daltons.
Analytical HPLC was carried out using an Ultimate HPLC
system (Thermo Scientific) consisting of a LPG-3400A pump (1
mL/min), a WPS-3000SL autosampler, and a DAD-3000D
diode array detector (220 and 254 nm) using a Gemini-NX C18
column (4.6 × 250 mm, 3 μm, 110 Å, Phenomenex); gradient
elution 0 to 100% B (MeCN-H2O-TFA 90:10:0.1 v/v/v%) in
solvent A (H2O-TFA 100:0.1 v/v%) over 15 min.
Preparative HPLC (prepHPLC) was carried out using an
Ultimate HPLC system (Thermo Scientific) consisting of a
LPG-3200BX pump (20 mL/min), a Rheodyne 9725i injector, a
10 mL loop, a MWD-300SD detector (220 and 254 nm), and an
AFC-3000SD automated fraction collector using a Gemini-NX
C18 column (21.2 × 250 mm, 5 μm, 110 Å, Phenomenex);
gradient elution 0 to 100% B (MeCN-H2O-formic acid
90:10:0.1 v/v/v%) in solvent A (H2O-formic acid 100:0.1 v/v
%) over 15 min (unless noted otherwise).
Data for both analytical and preparative HPLC were acquired
and processed using Chromeleon software v. 6.80.
3-Bromo-1-(phenylsulfonyl)-1H-pyrrole (2). N1-Sulfo-
nylation.53 Sodium hydride (2.20 g, 55.0 mmol, 60 wt %, 1.1
equiv) was added portionwise to the solution of pyrrole (3.47
mL, 50.0 mmol, 1.0 equiv) in dry DMF (150 mL) at 0 °C. The
obtained mixture was stirred for 1 h at the same temperature
(note for this step: a constant flow of nitrogen gas was used to
reduce foaming). Benzenesulfonyl chloride (7.66 mL, 66.0
mmol, 1.2 equiv) was added slowly over 5 min at 0 °C; the
cooling bath was removed, and the reaction was further stirred
for 0.5 h at room temperature (starting pyrrole was consumed,
TLC). The reaction mixture was carefully quenched with half-
saturated NH4Cl (200 mL) at 0 °C and diluted with 200 mL of
EtOAc. The organic phase was washed with water (4 × 150 mL),
brine (150 mL), dried over Na2SO4, and concentrated under
reduced pressure, providing 10.64 g of crude 1-(phenyl-
sulfonyl)-1H-pyrrole as a beige solid which was taken through
to the next step without further purification.
METHODS
■
The experimental procedures describing the synthesis and
characterization of the compounds, the evaluation of their
biological activity (enzyme assays, in vitro antibacterial
susceptibility testing), and X-ray crystallography studies are
fully described in the Supporting Information.
General Information. Commercially available reagents and
solvents were from Merck or Fluorochem and were used as
received. All manipulations with air- and moisture-sensitive
compounds were carried out under a positive pressure of argon
in flame-dried glassware. Reactions under microwave conditions
were carried out in Biotage Initiator EXP microwave reactor
with Robot Sixty sample processor.
Chromatographic separations/purifications were performed
either using manually packed columns with Silica gel 60 (Merck,
15−40 μm) for dry column vacuum chromatography (DCVC))
or using Reveleris X2 Flash Chromatography Purification
Bromination.54 A solution of bromine (2.57 mL, 50 mmol, 1
equiv) in acetic acid (40 mL) was added dropwise to the
solution of 1-(phenylsulfonyl)-1H-pyrrole (10.64 g, ∼50 mmol,
1 equiv) in AcOH (90 mL). The mixture was refluxed for 1 h,
then cooled to rt, concentrated and coevaporated with toluene
(2 × 150 mL). Purification by DCVC (5% EtOAc - heptane)
afforded 11.53 g of the desired product as a purple oil which
solidified upon standing. Further purification by crystallization
from MeOH (20 mL) gave 7.69 g (54% from pyrrole) of 2 as a
white crystalline solid.
̈
System (BUCHI) with FlashPure Silica prepacked columns.
Reactions were monitored by TLC on silica gel 60 F254 plates
(Merck).
NMR spectra were acquired using a 600 MHz Bruker Avance
III HD machine equipped with a 5 mm DCH cryoprobe and a
400 MHz Bruker Avance II equipped with a 5 mm BBFO probe.
Chemical shifts were referenced to residual protio- and
perdeuterio-solvent resonances (δH 7.26 and δC 77.16 for
CDCl3; δH 2.50 and δC 39.52 for DMSO-d6) as internal
1H NMR (400 MHz, CDCl3) δ 7.90−7.83 (m, 2H), 7.67−
7.59 (m, 1H), 7.58−7.46 (m, 2H), 7.19−7.15 (m, 1H), 7.09 (t, J
= 2.9 Hz, 1H), 6.29 (dd, J = 3.4, 1.6 Hz, 1H); 13C NMR (101
MHz, CDCl3) δ 138.6, 134.4, 129.7, 127.1, 121.4, 119.9, 116.5,
102.4. The analytical data are consistent with those reported in
the literature.54
Benzyl 3-Bromo-1-(phenylsulfonyl)-1H-pyrrole-2-car-
boxylate (3). According to a modified version of the reported
procedure,34 a 1.6 M solution of n-BuLi in cyclohexane (25.0
mL, 40.0 mmol, 1.25 equiv) was slowly added to a precooled
−78 °C stirred solution of i-Pr2NH (5.87 mL, 41.6 mmol, 1.3
equiv) in anhydrous THF (24 mL) under argon atmosphere.
After addition was complete, the reaction mixture was stirred for
standards for 1H NMR and 13C NMR spectra, respectively. 19
F
NMR spectra were referenced indirectly via the 2H signal of the
lock substance (CDCl3 or DMSO-d6) and the Ξ(19F) value. All
NMR spectra were processed with MestReNova software v.
14.1.
Low resolution mass spectrometry (LRMS) data were
obtained using a Waters Acquity H-class UPLC with a Sample
Manager FTN and a TUV dual wavelength detector coupled to a
QDa single quadrupole analyzer using electrospray ionization
(ESI). UPLC separation was achieved with a C18 reversed-
phase column (Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 μm)
operated at 40 °C, using a linear gradient of the binary solvent
system of buffer A (H2O:MeCN:formic acid, 95:5:0.1 v/v/v%)
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10 min at −10 °C, then recooled back to −78 °C. A solution of
pyrrole derivative 2 (9.15 g, 32.0 mmol, 1 equiv) in THF (30
mL) was added over 20 min at −78 °C. The reaction flask was
stirred at the same temperature for 1 h, followed by dropwise
addition (∼15 min) of benzyl chloroformate (CbzCl) (8.22 mL,
57.6 mmol, 1.8 equiv) in 10 mL THF. (Note: the traces of CO2
from the CbzCl solution in THF were removed with the stream
of argon before use.) The reaction mixture was stirred for 30 min
at −78 °C, then slowly warmed to 0 °C (∼2 h), quenched with
50 mL of NH4Clsat, and diluted with EtOAc (100 mL) and H2O
(100 mL). The aqueous layer was extracted with EtOAc (2 ×
100 mL), dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by DCVC (2%, then 10%
EtOAc/Hept) to give 12.16 g of crude product as an orange oil;
impure fractions containing the desired compound were
repurified by DCVC (2%, then 10% EtOAc/Hept) and afforded
ester 3 (10.94 g, purity >80% by 1H NMR) as a pale orange oil
which solidified upon storage in the refrigerator.
reduced pressure to give the desired product as a white solid
(18.0 g, 90%).
1H NMR (400 MHz, DMSO-d6) δ 8.51−8.43 (m, 2H), 7.37−
7.27 (m, 3H), 7.27−7.22 (m, 2H), 6.98−6.90 (m, 2H), 4.87 (s,
2H), 3.22 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 157.5,
156.6, 138.4, 136.9, 128.2, 127.6, 127.6, 106.3, 65.9, 40.0. The
analytical data are consistent with those reported in the
literature.55
Benzyl 1-(N-((Benzyloxy)carbonyl)sulfamoyl)-3-
bromo-1H-pyrrole-2-carboxylate (4). Sodium hydride
(60% in mineral oil, 822 mg, 20.6 mmol, 1.5 equiv) was added
portionwise to a precooled 0 °C solution of benzyl 3-bromo-1-
pyrrole-2-carboxylate (S1) (3.84 g, 13.7 mmol, 1 equiv) in dry
THF (41 mL). After stirring at 0 °C for 30 min, sulfamoylating
reagent 7 (5.06 g, 15.1 mmol, 1.1 equiv) was added, and the
reaction mixture was heated to reflux for 4 h. After cooling to 0
°C, the reaction was quenched by the dropwise addition of water
(40 mL), concentrated under reduced pressure to remove
organic solvents, and extracted into ethyl acetate (3 × 100 mL).
The combined organic phases were washed with brine (40 mL),
dried over Na2SO4, and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
on SiO2 (Reveleris, 20→100% EtOAc/Hept, then 0→20%
MeOH/EtOAc gradients) to give 7.03 g of the product 4 as the
sodium salt as a beige foam. The residue was taken up in EtOAc
(40 mL), sequentially washed with 0.5 M HClaq (2 × 40 mL),
brine (40 mL), dried over Na2SO4, and evaporated to dryness in
vacuo and purified by column chromatography (Reveleris, 10→
50% EtOAc/heptane gradient) to afford 4.68 g (69%) of the
desired compound 4 as a yellowish oil.
1H NMR (400 MHz, CDCl3) δ 7.93−7.88 (m, 2H), 7.64−
7.57 (m, 1H), 7.55 (d, J = 3.4 Hz, 1H), 7.51−7.45 (m, 2H),
7.41−7.31 (m, 5H), 6.40 (d, J = 3.5 Hz, 1H), 5.28 (s, 2H). The
analytical data are consistent with those reported in the
literature.34
Benzyl 3-Bromo-1H-pyrrole-2-carboxylate (S1). A 1 M
solution of TBAF in THF (28.6 mL, 28.6 mmol, 1.1 equiv) was
added dropwise to a solution of N-sulfonylpyrrole 3 (10.94 g,
∼26 mmol, 1 equiv) in dry THF (75 mL) at room temperature.
The obtained reaction mixture was stirred for 2 h, and then H2O
(100 mL) was added. The aqueous layer was extracted with
EtOAc (3 × 75 mL); the organic extracts were combined,
washed with water (2 × 100 mL), brine (100 mL), dried over
Na2SO4, and concentrated to dryness. Purification by DCVC
(5%, then 20% EtOAc/Hept) afforded two fractions containing
the product:
1H NMR (400 MHz, CDCl3) δ 7.50−7.44 (m, 3H), 7.41−
7.24 (m, 9H), 6.32 (d, J = 3.4 Hz, 1H), 5.35 (s, 2H), 5.14 (s,
2H); 13C NMR (101 MHz, CDCl3) δ 159.5, 149.6, 134.7, 134.1,
129.7, 129.1, 128.9, 128.8, 128.8, 128.7, 128.6, 121.6, 114.9,
112.4, 69.5, 68.0. The analytical data are consistent with those
reported in the literature.34
General Procedure A: Suzuki−Miyaura Cross-Cou-
pling Reaction. A microwave vial charged with bromide 4
(197 mg, 0.40 mmol, 1 equiv), the corresponding boronate
(0.52 mmol, 1.3 equiv), Na2CO3 (127 mg, 1.20 mmol, 3 equiv),
Pd(dppf)Cl2·DCM (16.3 mg, 0.02 mmol, 0.05 equiv) and a
degassed dioxane/H2O mixture (2:1, 2 mL) was purged with
argon, sealed, and stirred under microwave irradiation at 100 °C
for 3 h. After cooling to rt, the reaction mixture was diluted with
2 mL of EtOAc and 2 mL of H2O; the organic phase was
separated, and the aqueous layer was extracted with EtOAc (2 ×
2 mL). The combined organic phases were filtered through a pad
of Na2SO4 with Celite on top, and the filtrate was concentrated
under reduced pressure and further purified by column
chromatography on SiO2 (Reveleris purification system) to
afford the desired C3 substituted pyrrole.
1. The less polar fraction (1.32 g) was repurified by column
chromatography on SiO2 (Reveleris, 0→15% EtOAc/
Hept gradient), providing 942 mg of S1 as a white solid.
2. The more polar fraction (5.87 g) was crystallized from 30
mL of a 20% EtOAc/Hept mixture. The precipitate (1.08
g) was discarded, and the mother liquor was concentrated
to give 4.69 g of S1 as a colorless oil, quickly solidifying
upon standing.
The repurified product obtained from both fractions was of
analogous purity (1H NMR) and equally acceptable for the next
chemical step. Total yield −5.64 g (63% over 2 steps from 2).
1H NMR (400 MHz, CDCl3) δ 9.35 (br s, 1H), 7.50−7.44
(m, 2H), 7.43−7.31 (m, 3H), 6.85 (t, J = 3.1 Hz, 1H), 6.35 (t, J =
2.9 Hz, 1H), 5.36 (s, 2H); 13C NMR (101 MHz, CDCl3) δ
160.1, 135.9, 128.7, 128.3, 122.9, 120.1, 115.1, 104.2, 66.5. The
analytical data are consistent with those reported in the
literature.34
((Benzyloxy)carbonyl)((4-(dimethyliminio)pyridin-
1(4H)-yl)sulfonyl)azanide (7). A solution of benzyl alcohol
(6.28 mL, 60.6 mmol, 1.0 equiv) in CH2Cl2 (100 mL) was
cooled to 0 °C followed by the dropwise addition of
chlorosulfonyl isocyanate (5.21 mL, 60 mmol, 1.0 equiv).
After stirring at 0 °C for 10 min, 4-(dimethylamino)pyridine
(14.7 g, 120 mmol, 2 equiv) was added portionwise, and the
reaction mixture allowed to warm to room temperature and
stirred overnight. The resulting mixture was diluted with
CH2Cl2 (100 mL), washed with water (3 × 100 mL), dried
over MgSO4, filtered, and concentrated to dryness under
Sodium ((Benzyloxy)carbonyl)((2-((benzyloxy)-
carbonyl)-3-(4-fluorophenyl)-1H-pyrrol-1-yl)sulfonyl)-
azanide (5). Use of General Procedure A with 4-fluorophenyl)-
boronic acid as the coupling partner gave, after purification by
column chromatography (Reveleris, 20→90% EtOAc/Hept
gradient), the desired compound 5 as a yellow amorphous solid
in 70% yield (149 mg).
1H NMR (600 MHz, CDCl3) δ 7.55 (d, J = 3.1 Hz, 1H), 7.13
(t, J = 7.4 Hz, 1H), 7.10−7.03 (m, 5H), 7.00 (t, J = 7.6 Hz, 2H),
6.86 (dd, J = 8.4, 5.3 Hz, 2H), 6.69−6.61 (m, 4H), 5.84 (d, J =
3.1 Hz, 1H), 4.89 (s, 2H), 4.81 (s, 2H); 13C NMR (151 MHz,
CDCl3) δ 162.2 (d, J = 246.5 Hz), 161.8, 158.4 (br), 136.5,
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Authors
136.1, 134.5, 131.2 (d, J = 3.3 Hz), 130.9 (d, J = 8.1 Hz), 129.7,
128.44, 128.40, 128.3, 128.1, 127.9, 127.8, 118.9, 114.6 (d, J =
21.5 Hz), 110.9, 67.9, 67.2; 19F NMR (376 MHz, CDCl3) δ
−115.4; LRMS (ESI) m/z: [M-Na]− calcd for
C26H20FN2NaO6S 507.1, found 507.2.
Alistair J. M. Farley − Department of Chemistry, Chemistry
Research Laboratory and the Ineos Institute for Antimicrobial
Research, University of Oxford, Oxford OX1 3TA, United
Kingdom; orcid.org/0000-0001-5578-6790
Yuri Ermolovich − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DK-2100 Copenhagen, Denmark
̃
Karina Calvopina − Department of Chemistry, Chemistry
Research Laboratory and the Ineos Institute for Antimicrobial
Research, University of Oxford, Oxford OX1 3TA, United
Kingdom
Patrick Rabe − Department of Chemistry, Chemistry Research
Laboratory and the Ineos Institute for Antimicrobial Research,
University of Oxford, Oxford OX1 3TA, United Kingdom
Tharindi Panduwawala − Department of Chemistry, Chemistry
Research Laboratory and the Ineos Institute for Antimicrobial
Research, University of Oxford, Oxford OX1 3TA, United
Kingdom
General Procedure B: Hydrogenolysis of N-Cbz and O-
Bn Protected Pyrrole-2-carboxylates. A one-necked round-
bottom flask containing a 0.025 M methanol solution of the
corresponding O-Bn- and N-Cbz-protected pyrrole (1 equiv)
was charged with Pd on carbon (10% w/w, 0.2 equiv), sealed,
and evacuated/backfilled with dihydrogen gas (3 times). The
reaction mixture was hydrogenated at atmospheric pressure (H2
balloon) overnight under vigorous stirring, then filtered through
a pad of Celite, and the filter cake was washed with methanol.
The combined filtrates were concentrated to dryness under
reduced pressure.
3-(4-Fluorophenyl)-1-sulfamoyl-1H-pyrrole-2-carbox-
ylic acid (6a). The N-Cbz sodium salt 5 (72.7 mg, 0.137 mmol)
was dissolved in 10 mL of EtOAc, transferred to a separatory
funnel, and successively washed with 1 M HClaq (2 × 5 mL),
brine (5 mL), and dried over Na2SO4. The solvent was removed
in vacuo to give S8 as a yellow oil in 96% yield (67.2 mg).
1H NMR (600 MHz, CDCl3) δ 8.69 (br s, 1H), 7.55 (d, J = 3.3
Hz, 1H), 7.38−7.34 (m, 3H), 7.34−7.28 (m, 3H), 7.27−7.22
(m, 4H, overlapped with solvent peak), 7.04−7.00 (m, 2H),
6.94−6.87 (m, 2H), 6.23 (d, J = 3.3 Hz, 1H), 5.18 (s, 2H), 5.12
(s, 2H); 19F NMR (376 MHz, CDCl3) δ −114.1.
Ju
̈
rgen Brem − Department of Chemistry, Chemistry Research
Laboratory and the Ineos Institute for Antimicrobial Research,
University of Oxford, Oxford OX1 3TA, United Kingdom;
orcid.org/0000-0002-0137-3226
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.1c00104
Author Contributions
§A.J.M.F. and Y.E. contributed equally.
The oil obtained above (67.2 mg) was hydrogenated
according to General Procedure B; then, the crude residue
was further purified by prepHPLC to give the desired compound
6a in 72% yield (27.0 mg) as a white fluffy solid.
Funding
We thank our coworkers and collaborators and the Innovative
Medicines Initiative (European Lead factory and ENABLE
components), the Medical Research Council, the Wellcome
Trust, Cancer Research UK, and the Ineos Institute for
Antimicrobial Research for funding our work on antibiotics,
MBL fold/metalloenzymes, and β-lactamase inhibitors. This
research was funded in whole or in part by the Wellcome Trust
(Grant 106244/Z/14/Z).
1H NMR (600 MHz, DMSO-d6) δ 13.17 (br s, 1H), 8.17 (br
s, 2H), 7.46−7.42 (m, 2H), 7.42 (d, J = 3.2 Hz, 1H), 7.24−7.18
(m, 2H), 6.37 (d, J = 3.2 Hz, 1H); 13C NMR (151 MHz, DMSO-
d6) δ 162.5, 161.5 (d, J = 244.1 Hz), 131.7, 130.75 (d, J = 3.4
Hz), 130.69 (d, J = 8.2 Hz), 125.2, 120.8, 114.8 (d, J = 21.4 Hz),
110.7; 19F NMR (376 MHz, DMSO-d6) δ −115.8; HPLC
analysis tR 11.3 min, purity >99.6%; HRMS (ESI) m/z: [M-H]−
calcd for C11H8FN2O4S 283.0194, found 283.0189.
Notes
The authors declare no competing financial interest.
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AUTHOR INFORMATION
■
Corresponding Authors
Fredrik Björkling − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DK-2100 Copenhagen, Denmark;
Email: fb@sund.ku.dk
Christopher J. Schofield − Department of Chemistry,
Chemistry Research Laboratory and the Ineos Institute for
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