Bicyclic Boronate VNRX-5133 Inhibits Metallo- And Serine-β-Lactamases

Article abstract of DOI:10.1021/acs.jmedchem.9b00911

The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of β-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-β-lactamases (SBLs) and some clinically important metallo-β-lactamases (MBLs), including NDM-1 and VIM

Full text of DOI:10.1021/acs.jmedchem.9b00911

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Article
Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-#-Lactamases
Alen Krajnc, Jürgen Brem, Philip Hinchliffe, Karina Calvopina, Tharindi Panduwawala,
Pauline A. Lang, Jos J.A.G. Kamps, Jonathan M. Tyrell, Emma Widlake, Benjamin
G. Saward, Timothy R. Walsh, James Spencer, and Christopher J. Schofield
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00911 • Publication Date (Web): 27 Aug 2019
Downloaded from pubs.acs.org on August 27, 2019
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Bicyclic Boronate VNRX-5133 Inhibits Metallo- and
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Serine-β-Lactamases
Alen Krajnca, Jürgen Brema, Philip Hinchliffeb, Karina Calvopiñaa, Tharindi D. Panduwawalaa,
Pauline A. Langa, Jos J. A. G. Kampsa, Jonathan M. Tyrrellc, Emma Widlakec, Benjamin G.
Sawarda, Timothy R. Walshc, James Spencerb and Christopher J. Schofielda,*.
aChemistry Research Laboratory, Department of Chemistry, 12 Mansfield Road, University of
Oxford, Oxford, OX1 3TA, United Kingdom.
bSchool of Cellular and Molecular Medicine, Biomedical Sciences Building, University Walk,
University of Bristol, Bristol, BS8 1TD, United Kingdom.
cDepartment of Medical Microbiology & Infectious Disease, Institute of Infection & Immunity,
UHW Main Building, Heath Park, Cardiff, CF14 4XN, United Kingdom.
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KEYWORDS: Boron / boronic acid / boronate drugs; VNRX-5133; Vaborbactam; β-lactam
antibiotics / antibacterials; serine- and metallo-β-lactamase inhibitors; antibiotic resistance;
carbapenems; cephalosporins.
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ABSTRACT
The bicyclic boronate VNRX-5133 is a new type of β-lactamase inhibitor in clinical development.
We report that VNRX-5133 inhibits serine-β-lactamases (SBLs) and some clinically important
metallo-β-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1
and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds
to the class D SBL OXA-10 and NDM-1. The crystallographic results highlight the ability of bicyclic
boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures
imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography we
observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen
onto the boron of the bicyclic core. Different side-chain-dependent binding modes for bicyclic
boronates imply scope for optimisation. The results further support the ‘high energy
intermediate’ analogue approach for broad-spectrum β-lactamase inhibitor development and
highlight the ability of boron-inhibitors to interchange between different hybridization states /
binding modes.
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INTRODUCTION
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β-Lactamase catalysed hydrolysis is the most important resistance mechanism for β-lactams
which are the most important class of antibacterials (Figure 1A, top).1 Inhibitors of one of the two
mechanistic classes of β-lactamases, the nucleophilic serine-β-lactamases (SBLs)2, are established
drugs for use in combination with an appropriate β-lactam antibiotic partner (Figure 1B, bottom).
The well-established SBL inhibitors (clavulanic acid3, 4, sulbactam5, tazobactam6) only inhibit a
subset of SBLs (Ambler class A and some class C enzymes, but not typically class D enzymes) and
are increasingly susceptible to evolved resistance, including via bacterial production of extended-
spectrum serine-β-lactamases (ESBLs). The same issues are compromising the use of
carbapenems, which manifest both antibacterial and β-lactamase inhibition properties.7, 8
The clinical importance of the second mechanistic class of β-lactamases, i.e. the zinc ion
dependent metallo-β-lactamases (MBLs), which have a different fold/evolutionary origin to the
SBLs, is growing.9 This is of particular concern because MBLs catalyse the efficient hydrolysis of
near all β-lactam classes, with the exception of the monobactams which are not currently
hydrolyzed by MBLs at a clinically relevant rate.10 The vulnerability of the β-lactams to β-
lactamases has long stimulated interest in developing non-β-lactam inhibitors of penicillin-
binding proteins (PBPs) and β-lactamases. These studies resulted in the development of
avibactam11, 12, which unlike (at least most) ‘traditional’ β-lactam inhibitors, that act irreversibly
to form acyl-enzyme complexes, inhibits class A, C and some class D SBLs by reversible formation
of an acyl-enzyme type complex via reaction of its diazabicyclooctane core. However, avibactam
does not inhibit MBLs13; moreover, there is evidence that SBLs and MBLs have potential to evolve
to hydrolyse it.14
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Figure 1. Classes of β-lactam antibacterials, β-lactamase inhibitors in clinical use, outline
mechanisms for serine- and metallo-β-lactamase (SBL and MBL) catalysis, and bicyclic
boronates in research and development. (A) Major classes of β-lactam antibiotics and SBL
inhibitors currently in clinical use, including the clinically approved ‘monocyclic’ boron-containing
inhibitor Vaborbactam (which has little MBL activity15); Mode of action of (B) SBLs and (C) MBLs,
exemplified by hydrolysis of a carbapenem. Note that the hydrolysed carbapenem products can
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be produced in different tautomeric forms. Enz-Nu = Enz-(ZnII)n-OH (MBL) or Enz-Ser-OH (SBL);
(D) Note that the ability of boronate inhibitors (e.g. VNRX-5133) to interchange between sp2 and
sp3 forms, can enable them to mimic both substrates (sp2 carbonyl) and the first tetrahedral
intermediate (sp3). Selected examples of bicyclic boronates from research and development are
shown.
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There is thus interest in the development of non-acylating inhibitors of β-lactamases and PBPs.
With this objective in mind, multiple approaches and compounds have been explored, most with
relatively little success. In pioneering work, acyclic boronic acids have been developed as multiple
myeloma drugs, targeting human proteasomes, which employ nucleophilic threonine catalysis.16
After a long gestation period, boronic acids/boronate esters have emerged as β-lactamase
inhibitors with considerable clinical potential.17-20 Boronate-based inhibitors are of mechanistic
interest as in their tetrahedral (sp3 hybridised) forms, they are proposed to be analogues of the
high energy ‘tetrahedral’ intermediates present in the catalytic cycle of the nucleophilic serine
enzymes, such as SBLs (Figure 1B) and PBPs, as well as MBLs (Figure 1C).21, 22 It has also been
proposed that boron in its sp2 hybridised form can mimic the carbonyl group of the β-lactam
substrates, which thus far at least SBLs have evolved to bind highly efficiently.23
Recent work has led to the first clinical introduction of a boronic acid-based SBL inhibitor,
vaborbactam (formerly RPX7009) for use in combination with the carbapenem meropenem.24, 25
Whilst the early boronic acid SBL inhibitors are apparently predominantly acyclic in solution,
vaborbactam, adopts a monocyclic boronate structure, as observed at the active site of an SBL
(CTX-M-15).24 Vaborbactam, however, has limited SBL coverage and only moderately inhibits
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MBLs.15, 26 By contrast, recent studies have indicated that bicyclic boronates can inhibit a broader
range of SBLs and, importantly, some B1 subfamily MBLs.27, 28
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The potential of bicyclic boronates to act as dual action inhibitors of SBLs and MBLs is reported
in the academic27-29 and patent literature.30 VNRX-5133 is in Phase 3 clinical trials as a relatively
broad spectrum β-lactamase inhibitor. However, whilst its activity against SBLs has been
reported31-33, its MBL inhibition activity has been unclear. There are no reported structures of
VNRX-5133 complexed with SBLs or MBLs in the PDB database. To address these issues we
synthesized VNRX-5133 and tested it for inhibition against a panel of SBLs and MBLs. The results
support the potential of bicyclic boronates for broad-spectrum β-lactamase inhibition. Together
with previous studies, they also illustrate, how the ability of boron to readily interchange
between different hydrizidation states and binding modes in water can help to enable potent
inhibition.
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RESULTS AND DISCUSSION
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Synthesis. VNRX-5133 was synthesised via a modified version of the reported stereocontrolled
route in 11 steps from 2-methoxy-3-methylbenzoic acid 1 via Matteson homologation34 (Figure
2A).
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Figure 2. Synthesis and NMR analysis of VNRX-5133. (A) Reagents and conditions: (a) oxalyl
chloride, cat. DMF, CH2Cl2, rt, 90 min then 2-methylpropan-2-ol, 40 °C, 18 h; (b) N-
bromosuccinimide , benzoyl peroxide, CCl4, reflux, UV, 5 h; (c) bis[(+)-pinanediolato]diboron,
Pd(dppf)Cl2, KOAc, 1,4-dioxane, 95 °C, 16 h; (d) CH2Cl2, THF, n-BuLi, -100 °C, 45 min, then 37 in
THF, ZnCl2, -95 °C to rt, overnight; (e) lithium bis(trimethylsilyl)amide, THF, -100 °C to -78 °C , 2 h;
(f) MeOH, THF, -10 °C to rt, 1 h; (g) K2CO3, 1:1 CH2Cl2/H2O, rt, 1 h, then 2-(Boc-amino)ethyl
bromide, benzyltriethylammonium chloride, reflux, 18 h; (h) di-tert-butyl dicarbonate, N,N-
diisopropylethylamine, reflux, 16 h; (i) LiOH, 1:2:1 THF/EtOH/H2O, rt, 5 h; (j) 11, triethylamine,
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1
PyBOP, then crude 7, rt, 75 min; (k) BCl3, CH2Cl2, -78 °C, 1 h. (B) H NMR (600 MHz) of HPLC
purified VNRX-5133 in D2O.
The requisite (+)-pinanediol boronate precursor 4 (87%) was prepared according to reported
procedures.27 Enantioselective one-carbon homologation of 4 using in situ generated
dichloromethyllithium35 gave (S)-chloride 5. Initial yields after chromatographic purification were
low. Following optimisation, 5 was routinely obtained in improved yield (55-61%) in high
diastereomeric purity (d.r., <98.5%, 1H NMR).36 Reaction of 5 with lithium bis(trimethylsilyl)amine
at -90 °C gave bis (trimethylsilyl)-protected amine 6 with inversion of the configuration. To avoid
decomposition, crude N-6 was not separated but immediately treated with stoichiometric
anhydrous methanol (-10 °C to room temperature, THF) to give amine 7.
The desired side chain carboxylic acid with the trans stereochemistry 11 was prepared from
commercial ethyl 2-(trans-4-aminocyclohexyl) acetate hydrochloride 8 in three steps. Initial
attempts to install the desired ethane-1,2-diamine moiety via N-alkylation were unproductive,
possibly due to the low solubility of 8 in the tested organic solvents. Biphasic conditions (1:1,
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CH2Cl2:H2O) employing 5 mol% benzyl triethylammonium chloride as a phase-transfer catalyst
gave 9 (49%) following chromatography, which was Boc-protected to give ethyl ester 10 (82%).
Saponification, followed by ion-exchange chromatography (Amberlite® H-120) gave 11 (67%).
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The formation of the amide linking 7 and 11 was achieved using (benzotriazole-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)37 to give 12 in moderate yield
(34%). One-pot cleavage of the Boc, tert-butyl, methyl ether protecting groups and the chiral
auxiliary using BCl3 (-78 °C, CH2Cl2) followed by acidic workup (likely aiding the spontaneous
bicyclisation) gave VNRX-5133 (13) (42%) following HPLC purification. VNRX-5133 (13) was thus
prepared in 3% overall yield over 6 linear steps (excluding steps e and f, Figure 2A, where
intermediates were not isolated) with 96.7% purity by qHNMR. The route described here
represents an improvement for the asymmetric synthesis of VNRX-5133 compared to that
reported 30, with the number of steps being cut from 16 to 11. There is, however, clear scope for
further optimization.
Biochemical Evaluation. We screened VNRX-5133 for activity against a panel of clinically
relevant SBLs and MBLs (Table 1) using an established method involving hydrolysis of the
‘fluorogenic’ cephalosporin probe FC538 or meropenem for CphA27 (Supplementary Table S1). In
conference reports, VNRX-5133 has been reported to inhibit both SBLs and MBLs.31, 33, 39
Consistent with this and our previously reported results on the inhibition of all classes of β-
lactamases by structurally related (bi)cyclic boronates27-29, VNRX-5133 manifests submicromolar
half-maximal inhibitory concentration (IC50) values (0.53 – 0.008 μM) against all major classes of
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clinically relevant β-lactamases tested, with particularly potent activity, i.e. in the subnanomolar
range (IC50 0.5 nM), against the Verona-Integron-Encoded MBL-2, VIM-2. Notably, VNRX-5133
was less active against the clinically important Imipenemase (IMP)-1 B1 MBL (IC50 2.51 μM) and
the B2 MBL Aeromonas hydrophila CphA (CphA, IC50 2.51 μM), nor did it inhibit subclass B3 MBL
L1 from Stenotrophomonas maltophilia. The comparison of IC50 values reveals that VNRX-5133 is
50 to >50 000-fold more potent against the clinically relevant MBLs compared to vaborbactam15
(VNRX-5133 is, in general, more potent than the structurally related bicyclic boronate CB226,27
against the same enzymes). Variation in the pre-incubation times of VNRX-5133 with sublass B1
MBL NDM-1 did not result in different IC50 values (Supplementary Table S2 and Supplementary
Figure S33), thus supporting the case for the reversible inhibition by VNRX-5133, as observed for
related bicyclic boronate inhibitors.40
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Notably, improved inhibition with respect to vaborbactam is also observed for VNRX-5133
against the class A narrow-spectrum β-lactamase TEM-116 (500-fold increase) and for the tested
class D SBLs (a 10 to 2000-fold increase, Table 1). In particular, VNRX-5133 manifests moderate
inhibition of the narrow spectrum oxacillinase OXA-10 (IC50 0.234 μM), which is not inhibited by
vaborbactam (Table 1). Only moderate inhibition of the OXA-48 carbapenemase was observed
(IC50 0.537 μM). Overall, these results support the proposal that VNRX-5133 possesses an
unusually broad-spectrum inhibitory activity against Ambler Class A (ESBLs), B (NDM and VIM), C
(AmpC from P. aueroginosa) and, to a somewhat lesser extent, D (OXA) β-lactamases.
Nonetheless, the lower activity of VNRX-5133, in particular, against the subclass B1 MBL IMP-1,
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as well as the tested B2/B3 MBLs implies there is scope for further optimization of this new
inhibitor class.
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Table 1. Activities of VNRX-5133 versus representative serine- and metallo-β-lactamases.1
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Class
Enzyme
VNRX-5133
IC50 [μM]
0.12 μM
Vaborbactam15
IC50 [μM]
6 μM
CB227, 28
IC50 [μM]
SBL
A
TEM-116
IMP-1
NDM-1
VIM-1
VIM-2
CphA
0.003 μM27
1 μM27
B1
B1
B1
B1
B2
B3
C
2.51 μM
126 μM
631 μM
398 μM
316 μM
631 μM
336 μM
5 μM
0.01 μM
0.029 μM27
0.085 μM28
0.003 μM27
>100 μM27
Not inhibited41
0.12 μM28
MBLs
0.0079 μM
0.0005 μM
2.51 μM
L1
>10 μM
SBL
AmpC
(P. aureginosa)
0.301 μM
D
D
D
D
OXA-10
OXA-10≠
OXA-48
OXA-48≠
0.234 μM
0.645 μM
0.537 μM
2.39 μM
>400 μM
>400 μM
25 μM
Not available
5.1 μM28
SBLs
Not available
2.6 μM28
32 μM
Crystallography. To investigate the binding mode of VNRX-5133 to β-lactamases, we initiated
crystallographic analyses and obtained structures of it in complex with the class D SBL OXA-10
1 IC50 values of VNRX-5133 against a panel of SBLs and MBLs (see Supplementary Table S1 for
error analysis). SBL, serine-β-lactamase; MBL, metallo-β-lactamase. ≠These assays were run in the
presence of 100 mM aqueous sodium. bicarbonate.
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and the B1 subclass MBL NDM-1, at resolutions of 2.17 Å (space group: P212121) and 1.51 Å (space
group: P212121), respectively. In each strcture, there are two chains (A/B) in the asymmetric unit
(ASU).
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A)
Chain A:
B)
C)
Overlay of bicyclic (60%) and
tricyclic (40 %) VNRX-5133 forms
Chain B:
in chain A.
W93
W93
N233
N233
H250
K224
H122
H122
H120
Zn2
Electron density map for tricyclic
Zn2
Zn1
Zn1
form in Chain B:
K224
H120
C208
H189
H189
NDM-1:VNRX-5133 (bicyclic)
NDM-1:VNRX-5133 (tricyclic)
D)
E)
Chain A:
F)
Overlay of OXA-10:VNRX-5133
in chains A and B:
W102
Electron density map for form in
Chain A of OXA-10:
KCX70
R250
F208
S67
G207
OXA-10:VNRX-5133 (bicyclic)
Figure 3. Structural basis of serine- and metallo-β-lactamase inhibition by VNRX-5133. (A) View
from a crystal structure of VNRX-5133 complexed with NDM-1 (PDB ID: 6RMF) in chain A showing
the major observed bicyclic form (yellow); (B) View from crystal structure of VNRX-5133
complexed with NDM-1 (PDB ID: 6RMF) in chain B showing the tricyclic form (magenta); (C) An
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overlay of bicyclic (60%, yellow) and tricyclic (40%, magenta) forms of VNRX-5133 in chain A or B
of NDM-1 and 2mFo-DFc electron density for the tricyclic inhibitor form in Chain B (contoured to
3σ, grey mesh); (D) Proposed mechanism for formation of the unexpected tricyclic VNRX-5133
complex; (E) Binding mode of VNRX-5133 to the OXA-10 SBL (PDB ID: 6RTN); (F) Comparison of
the binding modes of VNRX-5133 in complex with OXA-10 (PDB ID: 6RTN) in chain A (pale blue)
and chain B (pale yellow); omit electron density for bicyclic form in chain A (contoured at 3σ, grey
mesh).
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After soaking of an NDM-1 crystal with VNRX-5133, there was clear Fo-Fc density corresponding
to bound VNRX-5133 in the active site in both chains A and B. Unexpectedly, the electron density
(Figure 3) indicated the presence of a tricyclic form of VNRX-5133 (Figure 3B, and Supplementary
Figure S2A) which was refined at full occupancy in chain B (with an average B-factor of 18.02 Å2).
In chain A, a mixture of both the bicyclic (as solely observed with OXA-10) (Figure 3A) and tricyclic
forms was modelled (at occupancies of 0.6 and 0.4, respectively). The ethylamino atoms of the
VNRX-5133 side chain in both chains A and B lacked observable electron density and were
removed in the final model. The tricyclic structure is formed by cyclization of the side chain amide
oxygen onto the boron. Although there is no obvious basic amino acid residue close enough to
the side chain amide to catalyse this reaction manifest crystal structure, this type of reaction has
precedent in synthetic chemistry.42
For both bicyclic and tricyclic forms with NDM-1, the bicyclic ‘boronate core’, including the aryl
carboxylate, adopts near identical binding modes (Figure 3C). Notably, binding of VNRX-5133 to
NDM-1 increases the Zn1-Zn2 distance from 3.6 Å (e.g. PDB ID: 5ZGZ43) to 4.3 Å (in both chains
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A and B); a similar increase has been observed on binding / reaction of the antibiotic ampicillin
with NDM-1 (PDB ID: 5ZGE43) and on binding of CB2 to VIM-2.43 Such changes in the positioning
of metal ions induced by inhibitor-substrate interactions in β-lactamase catalysis have also been
observed with extended X-ray absorption fine structure (EXFAS) spectroscopy studies, as shown
in the case of the subclass B3 MBL L1 from Stenotrophomonas maltophilia44 and with other
metallo-enzymes (see e.g.45-47); it may also be that the extent of such metal ion translocations
are not fully reflected in crystallographic compared to solution studies. It should also be noted
that the precipitants used in crystallography experiments for NDM-1:VNRX-5133 and OXA-
10:VNRX-5133 structures were at pH 5.8 and pH 8.0, respectively.
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One boron-bound oxygen effectively bridges the two active site zinc ions in NDM-1 but is closer
to Zn1 (1.9 Å) than Zn2 (3 Å). The boron-bound oxygen that becomes part of the 5-membered
ring of the tricyclic VNRX-5133 (Figure 3B) is positioned to make a significantly weaker interaction
with Zn2 (2.8 Å) than Zn1 and is positioned almost identically to the same boron-bound oxygen
in the bicyclic VNRX-5133 (Figure 3C). For comparison, CB2 binding to VIM-2 results in
interactions of the boron-bound oxygens of 2.6 Å and 1.9 Å to Zn2.27 A number of other
interactions of VNRX-5133 are conserved with respect to other bicyclic boronates (i.e. CB2 and
others27-29), including that of the aryl carboxylate with Zn2 and Lys224 and the ‘endocyclic’
boronate ester oxygen with Zn2. The L3 loop, which is proposed to be involved in binding
inhibitors/substrates,48 is partly disordered in the case of the VNRX-5133 complex (residues 67-
70 and 68-70 could not be modelled in chains A and B, respectively) and has high B-factors
compared to the rest of the main chain, indicating flexibility. These observations suggest that the
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L3 loop may not have an important role in stabilising VNRX-5133 NDM-1 binding (Supplementary
Figure S4).
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The tricyclic form observed in NDM-1 is probably generated by the reaction of the acylamino
sidechain carbonyl group with the VNRX-5133 boron, together with associated loss of
water/hydroxide (Figure 3D). Given that we observed evidence for both the bicyclic and tricyclic
forms in the crystal structure (Figure 3 A,B), it seems likely that tricyclisation occurs at the active
site, though we cannot rule out the presence of the tricyclic form at low concentrations in the
solution phase.42 Further studies will be necessary to exclude the possibility that the observed
tricyclic inhibitor form is not a crystallization artifact; it has been proposed that at least one MBL-
inhibitor complex crystal structure, ie. the mono-zinc carbapenemase MBL CphA in complex with
biapenem, does not necessarily reflect the catalytic pathway in solution.49, 50 Nevertheless, the
observation of a tricycle is a striking example of the ability of boron-based inhibitors to
interchange between different forms, potentially giving a tightly bound enzyme-inhibitor
complex.
In the case of the OXA-10:VNRX-5133 complex structure (Figure 3E), the inhibitor is observed
to bind similarly in chains A and B (Figure 3F), with the boron atom covalently linked to the
nucleophilic serine (Ser67), likely mimicking the tetrahedral intermediate in SBL/MBL catalysis
(Figure 1C, blue box).27, 28 The essential conserved lysine (Lys70)51, which acts as a general
acid/base is, at least predominantly, in its carbamylated (KCX70) form in both chains A and B
(Supplementary Figure S3). Comparison of this structure with that of OXA-10 complexed with the
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structurally related bicyclic boronate inhibitor CB1 (PDB ID: 5FQ927, Figure 3B) reveals that, whilst
the bicyclic cores of both inhibitors manifest similar binding modes, there are substantial
variations in the conformations adopted by their acylamino side chains (see below) (Figure 4).
The boron-containing bicyclic cores and the acylamino sidechains of both VNRX-5133 and CB1
are positioned to make hydrophobic/aromatic interactions with tryptophan (Tyr102) and
methionine (Met99). Although there is some variation in their precise conformations, the VNRX-
5133 and CB1 aryl carboxylates are both positioned to make polar interactions with Gly207 and
Arg250, that bind the analogous carboxylates in β-lactams (Supplementary Figure S1). Note, some
variations of the aryl carboxylate binding modes are anticipated, given the differences in the
precise modes of carboxylate binding employed by different classes of SBL/MBL/PBP.
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The VNRX-5133 acylamino side chain adopts clearly different conformations in the bicyclic
inhibitor forms in both, OXA-10 and NDM-1 structures (Figure 4). Although care should be taken
in assuming that the crystallographically observed binding modes accurately reflect the solution
behaviour, the structural observations imply the optimised VNRX-5133 side chain can enable
potent inhibition of different β-lactamase classes, by adopting different binding modes. Further
structural analyses of the cyclic boronates bound to MBLs and SBLs reveal that the acylamino
side chain adopts two types of orientations (at least in the crystalline state), reflecting binding to
SBLs or MBLs. These observations also imply that there is likely further scope for side chain
optimisation, including with respect to extending the scope and potency of MBL inhibition and
obtaining more potent (bi/tri)cyclic boronate-based PBP inhibitors.
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Importantly, comparison of the OXA-10 and NDM-1 structures with those observed for other
structurally related compounds, i.e. CB1/CB2 (which differ from VNRX-5133 only in their C-3
acylamino side chains) when complexed with SBLs (OXA-1027, CTX-M-1528, AmpC29), MBLs (NDM-
127, VIM-227, BcII27), and a PBP (PBP-527), revealed conservation in the binding mode of the
bicyclic boronate core (Figure 4). Even allowing for the observed tricycle formation with NDM-1,
similar conformations are observed across all Ambler classes of β-lactamases as well as with a
PBP (Figure 4). Superimposition of structures of OXA-10:VNRX-5133 and NDM-1:VNRX-5133 with
analogous structures of the respective enzymes with ‘intermediate’ complexes derived from
substrates (e.g. hydrolysed benzylpenicillin) reveals the binding modes adopted by VNRX-
5133/related inhibitors overlap with those adopted by hydrolysed β-lactams (Supplementary
Figures S1 and S2B).
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F61
F61
E149
N220
9
N233
N233
D263
A119
Zn1
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W93
K224
Zn1
W87
L231
K224
D120
Zn1
D120
D124 Zn2
Zn2
Zn2
VIM-2:CB2
(PDB ID: 5FQC)
BcII:CB2
(PDB ID: 5FQB)
NDM-1:VNRX-5133
(PDB ID: 6RFM)
Overlay of reported bicyclic
boronates complexed with
SBLs / MBLs and a PBP:
L153
Q146
S345
H216
N112
N179
S110
T214
Y177
K342
MBLs
(Z-shaped)
SBLs
(U-shaped)
N373
R248
T343
AmpC:CB1
(PDB ID: 6I30)
PBP5:CB2
(PDB ID: 5J8X)
N107
S240
N173
F208
N135
F208
W102
W102
S67
S67
Fi
S133
T238
R250
G207
G207
R250
KCX70
KCX70
OXA-10:VNRX-5133
OXA-10:CB1
(PDB ID: 5FQ9)
CTX-M-15:CB1
(PDB ID: 5T66)
(PDB ID: 6RTN)
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Figure 4. Overlays of reported bicyclic boronate structures and VNRX-5133 in MBLs, SBLs, and
a PBP reveal different side chain orientations. Views from: NDM-1:VNRX-5133 (yellow, bicyclic
form, PDB ID: 6RFM), VIM-2:CB2 (teal, PDB ID: 5FQC27), BcII:CB2 (pale green, PDB ID: 5FQB27),
AmpC:CB1 (orange, PDB ID: 6I3029), PBP-2:CB2 (grey, PDB ID: 5J8X27), OXA-10:VNRX-5133 (pale
blue, PDB ID: 6RTN), OXA-10:CB1 (brown, PDB ID: 5FQ927) and CTX-M-15:CB1 (wheat, PDB ID:
5T6628). Note that although the orientations of the acylamino sidechains vary, binding modes of
cyclic ring systems and their carboxylate are conserved, including for VNRX-5133. The orientation
of the acylamino side chains adopt two broad orientations, reflecting binding to SBLs (U-shaped)
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Microbiology. Antimicrobial susceptibility testing of VNRX-5133 in combination with
meropenem (carbapenem) or cefepime (cephalosporin) was performed in minimal inhibitory
concentration (MIC) antimicrobial assay format utilising six clinical isolates of NDM-1 producing
clinically relevant strains of Escherichia coli and K. pneumoniae (Table 2). In all cases, the MIC
values of cefepime/meropenem were significantly reduced in the presence of VNRX-5133
compared to its absence (MIC >64 μg/mL). Both cefepime/VNRX-5133 and meropenem/VNRX-
5133 combinations were highly active against all six of the NDM-1 producing clinical isolates
tested, with MIC ranges of 16 – 0.25 μg/mL and 1 – 0.125 μg/mL, respectively. These results
reveal the potential of VNRX-5133 to act against clinically relevant MBLs (i.e. NDM-1) in bacteria,
consistent with the results from clinical trials with VNRX-5133.44.45
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Table 2. Effect of VNRX-5133 on cefepime/meropenem MICs for selected NDM-1 MBL
expressing Enterobacteriaceae.2
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Cef / MEM
MIC
Cef / MEM
+ VNRX-5133
[10 μg/mL]
16 / 1
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Strain Species
Genotype
[μg/mL]
> 64
S117
IR57
B64
Ec
Ec
Kp
Kp
Kp
Ec
NDM-1
NDM-1
NDM-1
NDM-1
NDM-1
NDM-1
> 64
8 / 0.5
> 64
0.25 / 0.25
0.5 / 0.5
B68-1
IR43
91N
> 64
> 64
0.5 / 0.25
4 / 0.125
> 64
CONCLUSIONS
The overall results clearly support the clinical potential39, 52-54 of VNRX-5133 for inhibition of
MBLs as well as SBLs, so potentially extending the current utility of current β-lactam antibiotics.
In this regard, VNRX-5133 is different to the clinically approved boron-containing β-lactamase
inhibitor vaborbactam, which has little activity versus clinically relevant MBLs.15, 26 The coverage
of clinically relevant MBLs (and SBLs) by VNRX-5133, however, is imperfect, with significantly
lower or no inhibition being observed for clinically relevant IMP-1, L1 or OXA-48 (Table 1). There
is thus scope for further optimisation of this promising new class of β-lactamase inhibitor.
2 Ec, Escherichia coli; Kp, Klebsiella pneumoniae; MEM, meropenem; Cef, cefepime.
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The crystallographic results also further imply the potentially unique properties of boron-
containing small molecules to interchange between different binding modes/hybridisation
states, thus potentially enabling (more) potent inhibition. It is possible that bicyclic boronates
can bind to SBLs and MBLs in their sp2 hybridisation state which mimics that of the β-lactam.23
Once bound at the active site they can then react with the SBL nucleophilic serine or MBL-Zn(II)
bridged water/hydroxide, to give a tightly bound sp3 complex, mimicking the tetrahedral
intermediate in catalysis. Note that with the MBLs, in principle the sp3 boronate form could bind
to the active site, with a displacement of the ‘hydrolytic’ water/hydroxide from the active site Zn
(II) ions.27-29
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The potential of boron to enable further reaction when bound to a protein is strikingly
evidenced by the crystallographic observation of the tricyclic inhibitor form in the case of NDM-
1 (Figure 3B). Further biophysical analyses are required to demonstrate relevance of the tricyclic
inhibitor form in solution and thus rule out the possibility that the tricyclic inhibitor form is an
artifact arising from the crystallization conditions.
This ability has been exploited in the case of boron compounds in dynamic combinatorial
chemistry55 and is manifested in the reaction of amidomethylboronic acids inhibitors with two
active site serines in the case of penicillin-binding protein from Actinomadura sp. R39.56 We
propose that the ability of boron compounds to ‘morph’ between states can be further exploited
in inhibitor/modulator design, especially where conformational changes during ligand binding
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are desirable. Such applications will likely require a combination of precise activity/binding assays
coupled with detailed biophysical studies.
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EXPERIMENTAL SECTION
General Procedures. Unless otherwise stated, reactions were performed under argon using
dried glassware and solvents. All commercially available chemicals, reagents and solvents were
used as commercially supplied or purified using appropriate standard procedures. Petroleum
ether (PetEt) refers to distilled light petroleum of fraction 30-40 °C. A cold bath at -100 °C was
prepared by addition of liquid nitrogen to a mixture of 1:1 ethanol/methanol. Reactions were
monitored by thin layer chromatography (TLC) using Merck silica gel 60 F254 aluminium sheets 5
x 7.5 cm and using an LC-MS system (Agilent Technologies 1260 Infinity Series) fitted with a 6120
Quadrupole mass spectrometer and a Merck Chromolith® Performance C18 (100 × 4.2 mm) HPLC
column. TLC analyses were visualized by exposure to UV irradiation (λmax = 254 or 365 nm) and
by dipping the plates in phosphomolybdic acid, potassium permanganate, or ninhydrin followed
by heating with a heat gun. Chromatographic purifications were performed using a Biotage®
Isolera flash purification system with Biotage® pre-packed SNAP KP-Sil or SNAP-ULTRA columns
and analytical grade solvents. 1H, 13C and 11B NMR spectra were recorded using Bruker AVIII HD
400, AVIII HD 500 or AVIII 600 instruments in the solvents indicated. Deuterated solvents were
used as supplied. Chemical shifts (δ), referenced using residual solvent peaks, are reported in
parts per million downfield from tetramethylsilane or residual solvent peak as internal standard.
Multiplicity is given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), app
(apparent) or a combination of these. Coupling constants, J, are reported in hertz (Hz) to the
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nearest 0.5 Hz. COSY, HSQC and/or HMBC spectra were utilized to aid the chemical shift
assignments where appropriate. Infrared (IR) spectra were recorded using a Bruker Tensor 27 FT-
IR spectrometer; wavenumbers (νmax) are quoted in cm-1. Analytical grade solvents and a Perkin
Elmer 341 Polarimeter were used for measurement of optical rotations; [α]T퐷 values are reported
in 10-1deg·cm2g-1 and concentrations (c) are quoted in g per 100 mL; D refers to the D-line of
sodium (589 nm) and temperatures (T) are given in degrees Celsius (°C). Preparative HPLC was
run using a Shimadzu prep-LC system equipped with ACE® 5 µm C18 column (100 mm x 21.2 mm
id). HPLC Preparatory Method A refers to: [binary gradient: 10 mM HCl in water (solvent A),
acetonitrile + 25% MeOH (solvent B), 15 mL/min] 0-5 min gradient 1% B; 5-30 min gradient 10%
B; 31-36 min gradient 90% B, 37-45 min gradient 1% B; UV detection at 254 nm. Low-resolution
mass spectra were recorded using an Agilent 6120 Quadrupole MS instrument. High-resolution
mass spectra (HRMS) were recorded using a Bruker MicroTOF instrument with an ESI source and
Time of Flight (TOF) analyser. LC-MS system (Agilent Technologies 1260 Infinity Series) was fitted
with a 6120 Quadrupole mass spectrometer and a Merck Chromolith® Performance C18 (100 ×
4.2 mm) HPLC column. All compounds synthesized were 95% pure as judged by 1H and 13C NMR,
LC-MS or qHNMR analyses.
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tert-Butyl 2-methoxy-3-methylbenzoate (2) To a solution of 2-methoxy-3-methylbenzoic
acid 1 (2 g, 12.0 mmol, 1 eq) and anhydrous N,N-dimethylformamide (5 drops) in anhydrous
CH2Cl2 (20 mL) was added oxalyl chloride (1.54 mL, 17.9 mmol, 1.5 eq) dropwise under Shlenk
conditions. The resultant mixture was stirred at room temperature (rt) for 90 min, before
volatiles were removed in vacuo. The resulting oil was dissolved in anhydrous 2-methylpropan-
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2-ol (30 mL) and stirred at 40 °C for 18 hours before the volatiles were removed in vacuo. H2O
(30 mL) and CH2Cl2 (60 mL) were added and the layers were separated. The aqueous layer was
extracted with CH2Cl2 (2 x 60 mL). The combined organic layers were washed with brine (30 mL),
dried (MgSO4), then concentrated in vacuo. The crude material was purified by flash
chromatography (0-30% EtOAc in cyclohexane) to afford the desired product 2 as a colourless oil
(1.70 g, 63%). Rf 0.50 (19:1 CH2Cl2–MeOH); 1H NMR (600 MHz, CDCl3) δ 7.52 – 7.42 (m, 1H, H-4),
7.25 – 7.20 (m, 1H, H-2), 6.95 (app t, J = 7.5 Hz, 1H), 3.75 (s, 3H, H-16), 2.24 (s, 3H, H-9), 1.53 (s,
9H, C(CH3)3); 13C NMR (101 MHz, CDCl3) δ 166.1 (C-8), 158.0 (C-6), 134.5 (C-2), 132.6 (C-1), 129.0
(C-4), 126.9 (C-5), 123.5 (C-3), 81.4 (C-12), 61.5 (C-16), 28.4 (C(CH3)3), 16.2 (C-9); νmax / cm-1 (neat):
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2978, 2360, 1720, 1593, 1468, 1416, 1367, 1169; HRMS (ESI-TOF) calcd for C13H18O3 Na [M+Na]+
: 245.11482, found: 245.11487.
tert-Butyl 3-(bromomethyl)-2-methoxybenzoate (3) A mixture of tert-butyl 2-methoxy-
3-methyl benzoate 2 (1.69 g, 7.60 mmol, 1 eq), N-bromosuccinimide (NBS; 1555 mg, 8.74 mmol,
1.15 eq) and benzoyl peroxide (BPO; 368 mg, 1.52 mmol, 0.2 eq) in CCl4 (20 mL) was refluxed in
the presence of a Philips HB175 (75 W, UV type 3) lamp for 5 hours. The reaction mixture was
then cooled to rt, the precipitate removed by filtration, and the filtrate concentrated in vacuo.
The crude material was purified by flash chromatography (0-25% petroleum ether (PetEt) in
cyclohexane) to afford the desired product 3 as a colourless oil (1.33 g, 58%). Rf 0.50 (19:1 CH2Cl2–
MeOH); 1H NMR (400 MHz, CDCl3) δ 7.66 – 7.60 (m, 1H, H-4), 7.48 – 7.41 (m, 1H, H-2), 7.04 (app
t, J = 8.0 Hz, 1H, H-3), 4.52 (s, 2H, H-9), 3.90 (s, 3H, H-16), 1.54 (s, 9H, C(CH3)3);13C NMR (151 MHz,
CDCl3) δ 165.3 (C=O), 158.2 (C-6), 134.6 (C-2), 132.7 (C-1), 132.3 (C-4), 127.0 (C-5), 123.9 (C-3),
81.9 (C(CH3)3), 62.9 (C-16), 28.3 (C(CH3)3), 27.8 (C-9); νmax / cm-1 (neat): 2979, 2360, 1718, 1591,
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1469, 1423, 1080; HRMS (ESI-TOF) calcd for C13H17O3 Br23Na [M+Na]+ : 323.02533, found:
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tert-Butyl
2-methoxy-3-(((4S,6S,7aR)-5,5,7a-trimethylhexahydro-4,6-
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methanobenzo[d][1,3,2] dioxaborol-2-yl)methyl)benzoate (4) tert-Butyl 3-(bromomethyl)-2-
methoxybenzoate 3 (1.7 g, 5.64 mmol, 1 eq), bis[(+)-pinanediolato] diboron (2.43 g, 6.80 mmol,
1.2 eq) and potassium acetate (1108 mg, 98.1 mmol, 2 eq) were suspended in anhydrous 1,4-
dioxane (22.5 mL). The vessel was degassed for 20 min, then Pd(dppf)Cl2 (228 mg, 0.28 mmol, 5
mol%) was added. The reaction mixture was refluxed at 95 °C for 16 hours, then concentrated in
vacuo. The residue was redissolved in EtOAc (20 mL) and filtered through a Celite® pad. The
filtrate was concentrated to dryness under reduced pressure, and the crude material was purified
by flash chromatography (0-20% EtOAc in cyclohexane) to afford the desired product as a
colorless oil (1.79 g, 87%). Rf 0.70 (15:1 PetEt–EtOAc); 1H NMR (400 MHz, CDCl3) δ 7.48-7.42 (m,
1H, H-4), 7.28-7.22 (m, 1H, H-6), 6.94 (app t, J = 7.5 Hz, 1H, H-5), 4.20 (dd, J = 9.0, 2.0 Hz, 1H, H-
13), 3.74 (s, 3H, H-29), 2.25 (s, 2H, H-24), 2.24 – 2.17 (m, 1H, H-15''), 2.13 – 2.10 (m, 1H, H-12),
1.96 (t, J = 5.5 Hz, 1H, H-16), 1.84 – 1.80 (m, 1H, H-15'), 1.77 – 1.74 (m, 1H, H-11''), 1.52 (s, 9H,
C(CH3)3), 1.32 (s, 3H, H-23), 1.21 (s, 3H, H-18), 1.12 (d, J = 10.0 Hz, 1H, H-11'), 0.76 (s, 3H, H-19);
13C NMR (101 MHz, CDCl3) δ 166.2 (C-8), 157.5 (C-2), 134.4 (C-6), 134.1 (C-3), 128.6 (C-4), 126.5
(C-1), 123.5 (C-5), 86.0 (C-13), 81.2 (C(CH3)3), 78.0 (C-14), 61.6 (C-29), 51.4 (C-15), 39.7 (C-12),
39.6 (C-16), 38.3 (C-17), 35.6 (C-24), 28.7 (C-19), 28.4 (C(CH3)3), 27.2 (C-23), 26.5 (C-11), 24.2 (C-
18); 11B NMR (128 MHz, CDCl3) δ 32.8; νmax / cm-1 (neat): 2977, 2918, 2360, 2341, 1721, 1466,
1425. 1367, 1338, 1305, 1279, 1230, 1077, 1028; HRMS (ESI-TOF) calcd for C23H33O5 10B 23Na
[M+Na]+ : 422.23496, found: 422.23471; [휶]푫ퟐퟓ = + 15.0 ° (c 0.8, CHCl3).
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3-((2S)-2-chloro-2-((4S,6S,7aR)-5,5,7a-trimethylhexahydro-4,6-
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methanobenzo[d] [1,3,2]dioxaborol-2-yl)ethyl)-2-methoxybenzoate (5) In an oven-dried three-
necked round bottom flask under an argon flow, a solution of anhydrous CH2Cl2 (0.29 mL, 4.56
mmol, 2.5 eq) in anhydrous THF (5.5 mL) was cooled to -100 °C. Whilst maintaining a low
temperature, n-butyllithium (2.5 M in hexanes, 1.16 mL, 1.6 eq) was added slowly dropwise down
the inside wall of the flask (maintaining the temperature below -90 °C at all times). The resulting
turbid white suspension formed by a formation of microcrystalline dichloromethyllithium was
stirred at -100 °C for 45 min. Batches that turned black in the above process (signalling the
decomposition of dichloromethyllithium) were immediately quenched with isopropanol and
water and discarded. A pre-cooled (-90 °C) solution of boronic ester 4 (730 mg, 1.82 mmol, 1 eq)
in anhydrous THF (1.2 mL) was then added dropwise at -95 °C. The resulting bright yellow solution
was stirred for 20 min at -95 °C, before the freshly prepared anhydrous ZnCl2 solution (0.7 M in
THF, 1.48 mL, 0.8 eq) was added in one-portion. The reaction mixture was then allowed to warm
up slowly to rt overnight without removal of the cooling bath. The resultant solution was cooled
using an ice-bath, quenched with sat. aq. NH4Cl solution (20 mL), extracted with EtOAc (3 x 20
mL), washed with brine (15 mL), dried (Na2SO4), filtered, then concentrated in vacuo. The crude
material was purified by flash chromatography (5-50% PetEt in pentane) to afford the desired
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product 5 as a pale yellow oil (500 mg, 61%). Rf 0.45 (20:1 PetEt–EtOAc); H NMR (500 MHz,
CDCl3) δ 7.62 (dd, J = 7.5, 2.0 Hz, 1H, H-4), 7.39 (dd, J = 7.5, 2.0 Hz, 1H, H-6), 7.05 (app t, J = 7.5
Hz, 1H, H-5), 4.35 (dd, J = 9.0, 2.0 Hz, 1H, H-13), 3.85 (s, 3H, H-29), 3.75 – 3.70 (m, 1H, H-22), 2.26
(dd, J = 14.0, 7.5 Hz, 2H, H-24), 2.25 – 2.17 (m, 1H, H-15’’), 2.10 (m, 1H, H-12), 1.94 – 1.90 (m, 1H,
H-16), 1.84-1.80 (m, 1H, H-15’), 1.78 – 1.74 (m, 1H, H-11’’), 1.45 (s, 9H, C(CH3)3), 1.27 (s, 3H, H-
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23), 1.22 (s, 3H, H-18) 1.19 (d, J = 10.0 Hz, 1H, H-11’), 0.83 (s, 3H, H-19); 13C NMR (126 MHz, CDCl3)
δ 166.2 (C-8), 157.8 (C-2), 137.0 (C-6) , 134.4 (C-3), 128.6 (C-4), 125.5 (C-1), 123.5 (C-5), 86.0 (C-
13), 81.2 (C-25), 78.0 (C-14), 61.6 (C-29), 51.3 (C-15), 39.6 (C-16), 39.2 (C-17), 35.6 (C-24), 28.7
(C-19), 28.3 (C(CH3)3), 27.2 (C-23), 26.5 (C-11), 24.1 (C-18), C-22 not observed due to peak
broadening; νmax / cm-1 (neat): 2979, 2929, 2359, 2341, 1719, 1466, 1421, 1369, 1303, 1254, 1172,
1135, 767; HRMS (ESI-TOF) calcd for C24H34O5 10B35Cl 23Na [M+Na]+ : 470.21164, found:
470.21167; [휶]ퟐ푫ퟓ = - 10.0 ° (c 7.6, CHCl3).
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tert-Butyl
methanobenzo[d] [1,3,2]dioxaborol-2-yl)ethyl)-2-methoxybenzoate (7) To a stirred solution of
tert-butyl 3-((2S)-2-chloro-2-((4S,6S,7aR)-5,5,7a-trimethylhexahydro-4,6-
3-((2R)-2-amino-2-((4S,6S,7aR)-5,5,7a-trimethylhexahydro-4,6-
methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-methoxybenzoate 5 (400 mg, 0.89 mmol, 1 eq)
in anhydrous THF (4 mL) under an argon flow was added a solution of lithium
bis(trimethylsilyl)amide (LiHDMS, 1 M in THF, 0.94 mL, 0.94 mmol, 1.05 eq) dropwise over 30 min
at -100 °C. The resultant mixture was stirred at -78 °C for 120 min before the volatiles were
removed in vacuo. The resultant thick brown oil was immediately used in the next step without
further purification. To a solution of crude bis-TMS-protected amine 6 (500 mg, 0.87 mmol, 1 eq)
in THF (4 mL) was added anhydrous MeOH (2 mL) dropwise at -10 °C. The resultant cloudy
solution was stirred at rt for 60 min before the volatiles were removed in vacuo. The crude amine
7 thus obtained as pale yellow oil was immediately used in the next step without further
purification.
Ethyl 2-((1R,4R)-4-((2-((tert-butoxycarbonyl)amino)ethyl)amino) cyclohexyl)acetate (9)
Ethyl-2-(trans-4-aminocyclohexyl)acetate hydrochloride 8 (2 g, 9.04 mmol, 1 eq) was dissolved in
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1:1 CH2Cl2-H2O (70 mL) and K2CO3 (2740 mg, 20.7 mmoL, 2.3 eq) was added. The resultant
biphasic suspension was vigorously stirred at rt for 60 min, before benzyltriethylammonium
chloride (103 mg, 0.45 mmol, 5 mol%) and 2-(Boc-amino)ethyl bromide (2100 mg, 9.37 mmol,
1.04 eq) were added in one portion. Reaction mixture was refluxed for 18 hours, before it was
poured into sat. aq. NH4Cl (70 mL). The layers were separated, and the aqueous layer was
extracted with CH2Cl2 (3 x 70 mL). The combined organic layers were washed with brine (70 mL),
dried (MgSO4), then concentrated in vacuo. The crude material was purified by flash
chromatography (0-20% MeOH in CH2Cl2) to afford the desired product 9 as a white solid (1.45 g,
49%). Rf 0.80 (1:1 EtOAc–pentane); m.p. 118-120 °C; 1H NMR (600 MHz, CDCl3) δ 5.00 (br s, 1H,
Boc-NH), 4.05 (q, J = 7.0 Hz, 2H, H-14), 3.18 – 3.04 (m, 2H, CH2 9/ CH2 6), 2.74 – 2.61 (m, 2H, , CH2
9/ CH2 6), 2.39 – 2.24 (m, 1H, H-2), 2.11 (d, J = 7.0 Hz, 2H, H-10), 1.90 – 1.82 (m, 2H, CH2), 1.80 –
1.59 (m, 3H, CH2, H-5), 1.37 (s, 9H, C(CH3)3), 1.18 (t, J = 7.0 Hz, 3H, H-15), 1.11 – 1.03 (m, 2H, CH2),
0.99 – 0.92 (m, 2H, CH2); 13C NMR (151 MHz, CDCl3) δ 173.1 (C-11), 156.3 (C-22), 79.3 (C(CH3)3),
60.3 (-CO2CH2CH3), 56.6 (C-2), 46.4 (CH2), 41.8 (C-10), 34.7 (C-5), 33.3 (CH2), 31.7 (CH2), 28.6
(C(CH3)3), 14.4 (-CO2CH2CH3); νmax / cm-1 (neat): 3348, 2953, 2851, 1731, 1711, 1520, 1450, 1391,
1282, 1249, 1172, 1032; HRMS (ESI-TOF) calcd for C17H33O4N2 [M+H]+ : 329.24348, found:
329.24289.
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Ethyl
2-((1R,4R)-4-((tert-butoxycarbonyl)(2-((tert-butoxycarbonyl)
acetate (10) Ethyl 2-((1R,4R)-4-((2-((tert-
amino)ethyl)amino)cyclohexyl)
butoxycarbonyl)amino)ethyl)amino)cyclohexyl)acetate 9 (1.4 g, 4.26 mmol, 1 eq) was dissolved
in anhydrous CH2Cl2 (37 mL) before di-tert-butyl dicarbonate (2326 mg, 10.7 mmol, 2.5 eq) and
N, N-diisopropylethylamine (1.11 mL, 6.39 mmol, 1.5 eq) were added. The reaction mixture was
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refluxed for 16 hours, before the volatiles were removed in vacuo. The residue thus obtained was
resuspended in CH2Cl2 (30 mL) and sat. aq. NH4Cl (50 mL) was added. The layers were separated
and the aqueous layer was extracted with CH2Cl2 (3 x 70 mL). The combined organic layers were
dried (MgSO4), then concentrated in vacuo. The crude material was purified by flash
chromatography (0-20% MeOH in CH2Cl2) to afford the desired product 10 as a yellow oil (1.50 g,
82%). Rf 0.90 (1:1 EtOAc–pentane); 1H NMR (400 MHz, CDCl3) δ 4.11 (q, J = 7.0 Hz, 2H, H-22), 3.31
– 3.10 (m, 4H, H-10, H-11), 2.17 (d, J = 7.0 Hz, 2H, H-7), 1.86 – 1.58 (m, 5H, CH2, H-2 ), 1.65 – 1.32
(m, 19H, C(CH3)3), H-5), 1.25 (t, J = 7.0, 3.0 Hz, 3H, H-21 ), 1.17 – 0.99 (m, 2H, CH2); 13C NMR (101
MHz, CDCl3) δ 172.8 (C-8), 156.4 (C-29), 156.1 (C-18), 79.9 (C(CH3)3), 60.2 (-CO2CH2CH3), 42.5
(CH2), 41.6 (CH), 41.0 (CH2) , 34.0 (CH), 32.2 (CH2), 30.3 (CH2), 28.5 (C(CH3)3), 28.4 (C(CH3)3), 14.3
(-CO2CH2CH3); νmax / cm-1 (neat): 3367, 2978, 2931, 1715, 1689, 1516, 1453, 1408, 1391, 1248,
1170; HRMS (ESI-TOF) calcd for C22H41O6N2 [M+H]+ : 429.29591, found: 429.29582.
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ethyl)amino)
cyclohexyl)acetic acid (11) Ethyl 2-((1R,4R)-4-((tert-butoxycarbonyl)(2-((tert-
butoxycarbonyl)amino)ethyl)amino) cyclohexyl)acetate 10 (1604 mg, 3.74 mmol, 1 eq) was
dissolved in 1:2:1 mixture of THF/EtOH/H2O (10 mL) and LiOH·H2O (224 mg, 9.36 mmol, 2.5 eq)
was added. The resultant cloudy solution was stirred at rt for 5 hours before the volatiles were
removed in vacuo. The residue thus obtained was dissolved in minimum amount of water and
filtered through a short column of Amberlite® IR-120 (H+ form). To the obtained aqueous filtrate
was added EtOAc (50 mL), and the layers were separated. The aqueous layer was extracted with
EtOAc (3x50 mL), washed with brine (20 mL), dried (Na2SO4), then concentrated in vacuo to afford
the desired product 11 as a shiny white solid (1 g, 67%). Rf 0.60 (1:1 EtOAc–pentane); m.p. 97-98
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