Article

Cite This: J. Med. Chem. 2019, 62, 7160−7184 pubs.acs.org/jmc

Structure-Based Development of (1-(3′-

Mercaptopropanamido)methyl)boronic Acid Derived Broad-
Spectrum, Dual-Action Inhibitors of Metallo- and Serine-β-
lactamases
Yao-Ling Wang,†,§ Sha Liu,†,§ Zhu-Jun Yu,† Yuan Lei,† Meng-Yi Huang,† Yu-Hang Yan,† Qiang Ma,†
Yang Zheng,† Hui Deng,‡ Ying Sun,‡ Chengyong Wu,‡ Yamei Yu,‡ Qiang Chen,‡ Zhenling Wang,‡
Yong Wu,*,† and Guo-Bo Li*,†
†
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Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan Engineering Laboratory for
Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy,
Sichuan University, Sichuan 610041, China
Downloaded via UNIV DE ALCALA on December 12, 2022 at 08:30:37 (UTC).

‡
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University,
Chengdu 610041, China
*
S Supporting Information

ABSTRACT: The emergence and spread of bacterial pathogens acquired metallo-β-lactamase (MBL) and serine-β-lactamase
(SBL) medicated β-lactam resistance gives rise to an urgent need for the development of new dual-action MBL/SBL inhibitors.
Application of a pharmacophore fusion strategy led to the identification of (2′S)-(1-(3′-mercapto-2′-methylpropanamido)-
methyl)boronic acid (MS01) as a new dual-action inhibitor, which manifests broad-spectrum inhibition to representative MBL/
SBL enzymes, including the widespread VIM-2 and KPC-2. Guided by the VIM-2:MS01 and KPC-2:MS01 complex structures,
further structural optimization yielded new, more potent dual-action inhibitors. Selectivity studies indicated that the inhibitors
had no apparent inhibition to human angiotensin-converting enzyme-2 and showed selectivity across serine hydrolyases in E.
coli and human HEK293T cells labeled by the activity-based probe TAMRA-FP. Moreover, the inhibitors displayed potentiation
of meropenem efficacy against MBL- or SBL-positive clinical isolates without apparent cytotoxicity. This work will aid efforts to
develop new types of clinically useful dual-action inhibitors targeting MBL/SBL enzymes.

■ INTRODUCTION
The β-lactam antibiotics are the most widely used class of
broad-spectrum antibacterial agents; however, they have been
plagued by the ongoing problems of resistance.1−5 Resistance
to β-lactam antibiotics is mainly due to the production of β-
lactamases, which can be classified into two categories
according to their hydrolytic mechanisms.6−8 The first
category is serine-β-lactamases (SBLs; ambler classes A, C,
and D), which employ an active site nucleophilic serine to
destroy the β-lactam ring (Figure 1A). Clinically important
SBL families include KPC, TEM, SHV, CTX-M, GES, SME,
AmpC, OXA, and others.4,9−13 The second category is metallo-
β-lactamases (MBLs; ambler class B), which use one or two
active site zinc ions to activate a nucleophilic water molecule
and thereby cleave the β-lactam ring (Figure 1A).14 MBLs are
further divided into subclasses B1, B2, B3, and somewhere B4,
primarily by difference in active site zinc coordination
features.15,16 The B1 MBLs (e.g., NDM, VIM, and IMP) and
B3 MBLs (e.g., GOB, AIM, SMB, and L1) are catalyticlly
active with two zinc ions and show a broad substrate profile,
while the monozinc B2 MBLs, such as CphA and Sfh-I, have a
Received: May 5, 2019
Published: July 3, 2019

© 2019 American Chemical Society 7160 DOI: 10.1021/acs.jmedchem.9b00735

J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
Figure 1. (A) Mechanisms for metallo- (MBL) and serine-β-lactamase (SBL) catalyzed hydrolysis of carbapenems. (B) Dual-action MBL/SBL
inhibitors.
narrow specificity profile, solely hydrolyzing carbapenems.
Among them, the dizinc B1 MBLs, such as NDM-1, are of
widespread clinical significance.14,17−20
Several SBL inhibitors have been used in combination with a
β-lactam partner to overcome resistance, such as, for example,
clavulanate/amoxicillin, tazobactam/piperacillin, sulbactam/
ampicillin, and avibactam/ceftazidime.2,4 Recently vaborbac-
tam, a boron-heterocyclic SBL inhibitor, was approved for use
with meropenem for treating complicated urinary tract
infections and pyelonephritis.21,22 By contrast, there are no
equivalent inhibitors for MBLs, which can hydrolyze almost all
β-lactam antibiotics including carbapenems (i.e., “last resort”
antibiotics) as well as the β-lactam SBL inhibitors (e.g.,
clavulanate, tazobactam, and sulbactam).18,19,23 More concern-
ing is the continuous emergence and spread of multidrug-
resistant pathogens acquired MBL and SBL genes,24,25 leading
to an urgent need for the development of broad-spectrum,
dual-action MBL/SBL inhibitors to overcome MBL or MBL/
SBL-mediated resistance.
Until recently, there have been a few reports of dual-action
MBL/SBL inhibitors (Figure 1B). Cyclic boronates are the
most desirable dual-action inhibitors, which can potently
inhibit all classes of MBL and SBL enzymes via a mechanism
involving mimicking of their common natural tetrahedral
intermediates as revealed by crystallographic analyses.26,27 The
benzo[b]thiophen-2-ylboronic acids28,29 and cyclobutanones30
(Figure 1B) inhibit multiple MBL and SBL enzymes by an
analogous manner to cyclic boronates (i.e., mimicking the
tetrahedral intermediates). Using combined computational and
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experimental assays, we recently identified aromatic bi-
sphosphonates and rosmarinic acid as dual-action MBL/SBL
inhibitors,31,32 which are only active against some types of
MBL and SBL enzymes. Here, we report structure-based
development of (1-(3′-mercaptopropanamido)methyl)boronic
acid derived new broad-spectrum, dual-action MBL/SBL
inhibitors.
Considering the structural and mechanistic difference
between MBL and SBL enzymes, we proposed a pharmaco-
phore fusion strategy to obtain dual-action MBL/SBL
inhibitors. Application of this strategy led to the identification
of the compound (2′S)-(1-(3′-mercapto-2′-
methylpropanamido)methyl)boronic acid (MS01, Figure
1B), which manifested broad-spectrum inhibition to all classes
of representative MBL and SBL enzymes, including subclass
B1 VIM-2, subclass B1 NDM-1, subclass B2 Sfh-I, subclass B3
GOB-18, class A KPC-2, class A TEM-1, class C AmpC, and
class D OXA-48 enzymes; with the exception of TEM-1 and
AmpC, these enzymes are carbapenemases that are able to
inactivate carbapenems and other β-lactams.11,33 Crystallo-
graphic analyses reveal insights into the dual-action inhibition
modes of MS01 with VIM-2 and KPC-2, providing important
basis for structural optimization. The subsequent multitarget
structure−activity relationship (SAR) studies identified new
(1-(3′-mercaptopropanamido)methyl)boronic acid derivatives
as broad-spectrum, dual-action inhibitors. Some of them were
observed by crystallographic analyses to bind with VIM-2 and
KPC-2 via unexpected binding modes. The inhibitors were
further evaluated for their selectivity to human angiotensin-
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Scheme 1. Synthesis of Compounds MS01−MS04a

a
(a) CH2Br2, n-BuLi, THF, −78 °C, 2 h; CH3SO2OH, 0 °C, 1 h; pinacol, 0 °C, 1 h; (b) LiHMDS, THF, −78 °C to rt, overnight; (c) HCl/1,4-
dioxane, Et2O, −60 °C to rt, 3 h; (d) TBTU, DIPEA, DMF, −10 °C, 2 h; (e) 2-methylpropylboronic acid, HCl (aq), CH3OH/hexane (1:1), rt
overnight.

Table 1. Inhibitory Potency (Ki, μM) of MS01−MS04 to the Representative MBL and SBL Enzymesa

a
The method for measuring IC50/Ki values is described in Experimental Section, and the dose−response curves of compounds inhibiting the
selected enzymes are given in Supporting Information Figure S2.

converting enzyme (ACE)-2 and to the serine hydrolyases in E.
coli and human HEK293T cells by activity-based protein
profiling using TAMRA-FP, as well as their efficacy to
potentiate meropenem activity against MBL- or SBL-positive
clinical isolates.
structures of MBL enzymes in complex with small-molecule
inhibitors,21,34 we observed that most of MBL inhibitors
involve direct active site metal chelation via metal-binding
pharmacophores (MBPs),35 such as (S)-3-mercapto-2-methyl-
propanamido,36−38 5-aminothiazole-4-carboxylic acid,39 (Z)-2-

■ RESULTS AND DISCUSSION

Design of (2′S)-(1-(3′-Mercapto-2′-
methylpropanamido)methyl)boronic Acid (MS01) as a
Dual-Action MBL/SBL Inhibitor. By analyses of crystal
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mercapto-3-phenylacrylic acid,40 2-(mercaptomethyl)benzoic
acid,41 and phthalic acid.42 Comparatively, most of SBL
inhibitors are positioned to covalently bind with the active site
nucleophilic serine, e.g., via the boronic acid group.29,43−47
With the aim to obtain dual-action inhibitors targeting MBL
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Figure 2. Crystallographic analyses reveal compound MS01 binding modes to VIM-2 MBL and KPC-2 SBL. (A) The mode of MS01 binding as
defined by the mFo − DFc electron density maps observed in the VIM-2:MS01 structure (PDB code 6J8R), indicating that MS01 fits well with the
electron density and chelates to and bridges with the two active site zinc ions. (B) Mode of MS01 binding as defined by the mFo − DFc electron
density maps observed in the KPC-2:MS01 structure (PDB code 6J8Q), indicating that MS01 fits well with the electron density and forms a
covalent bond with the residue Ser70. (C) View from the VIM-2:MS01 structure (PDB code 6J8R) reveals that the inhibitor binds to form
hydrophobic and electrostatic interactions with residues on the L3 and L10 loops (e.g., hydrogen-bonding interactions with Asn233 and Arg228 and
hydrophobic interactions with Phe61). (D) View from the KPC-2:MS01 structure (PDB code 6J8Q) reveals that the inhibitor binds to make
hydrogen-bonding interactions with the residues Asn132, Glu166, Asn170, and Thr237 in the active site.

and SBL enzymes, we came up with a strategy of integrating
the key pharmacophores of MBL and SBL inhibitors. As an
initial attempt, we designed and synthesized the compound
(2′S)-(1-(3′-mercapto-2′-methylpropanamido)methyl)boronic
acid (MS01, Figure 1B and Scheme 1). The (2′S)-3′-
mercapto-2′-methylpropanamido moeity of MS01, as a key
motif of captopril (an old drug used in clinical for more than
30 years), may have potential biocompatibility and achieve
broad-spectrum inhibition to multiple MBL enzymes, partic-
ularly B1 MBL enzymes (e.g., NDM-1 and VIM-2), whereas
the α-aminoboronic acid moiety of MS01 is expected to target
the catalytic serine residue of SBL enzymes. Meanwhile, we
synthesized the non-thiol analogues MS02 and MS03 (Table 1
and Scheme 1), to test whether thiol is essential for MBL
inhibition, and the non-boronic acid compound MS04 (Table
1 and Scheme 1), bearing a carboxylic acid group instead of
boronic acid, to examine the necessity of the boronic acid
group for SBL inhibition.
To investigate the MBL/SBL inhibitory activity of MS01−
MS04, we selected a representative panel of clinically relevant
MBLs, including VIM-2 (subclass B1), NDM-1 (subclass B1),
Sfh-I (subclass B2), and GOB-18 (subclass B3), and SBLs,
including KPC-2 (class A), TEM-1 (class A), AmpC (class C),
and OXA-48 (class D). All of these recombinant enzymes were
expressed and purified from E. coli DH5α and BL21 cultures;
the details are described in the Experimental Section. Since
FC5 has been demonstrated as a robust fluorescent substrate
for the dizinc B1 MBLs (e.g., VIM-2 and NDM-1),48 we here
examined whether FC5 could be used as a substrate for activity
test for other classes of β-lactamases. The enzyme kinetic
analyses revealed that all of the tested β-lactamases exhibited
substantial hydrolytic activity toward FC5 resulting in
measurable fluorescent signals, albeit with apparent difference
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in their kinetic parameters (Figure S1 and Table S1),
indicating that FC5 was a suitable substrate for these enzymes.
Consequently, we determined IC50 values of MS01−MS04
with all the representative MBL and SBL enzymes using FC5.
Given the differences in the enzyme kinetics, we calculated the
inhibition constant (Ki) values from the corresponding IC50
values using the Cheng−Prusoff equation49 to compare the
inhibitory potency for the compounds (for details see the
Experimental Section).
As anticipated, MS01 displayed inhibitory activity against all
of the tested MBL and SBL enzymes (Table 1 and Figure S2).
For the subclass B1 MBLs, MS01 showed Ki values of 0.76 and
67.13 μM to the widespread VIM-2 and NDM-1, respectively,
comparable to L-captopril, a well-known MBL inhibitor (Table
1), while the non-thiol compounds MS02 and MS03, bearing a
boronic acid group, had low inhibition to VIM-2 and NDM-1
(Table 1), suggesting the importance of the thiol group (rather
than boronic acid) for B1 MBL inhibition. Another compound
MS04, which contains a thiol group but no boronic acid,
displayed potent inhibition to these two B1 MBLs as MS01
(Table 1), probably owing to the contributions of both the
thiol and carboxylate group, similar to previously reported
inhibitors.36−38 Notably, MS01 can also inhibit subclass B2
Sfh-I and B3 GOB-18 with Ki values of 7.34 and 2.23 μM,
respectively (Table 1). The non-thiol compound MS02
exhibited comparable inhibition as MS01 to Sfh-I and GOB-
18; conversely, the thiol compounds MS04 and L-captopril did
not show marked inhibition against Sfh-I and GOB-18 (Table
1), indicating that the thiol group is likely not essential for
inhibiting these B2/B3 enzymes.
For the tested SBLs, MS01 manifested Ki values of 2.70,
40.97, 6.46, and 70.19 μM, respectively, to class A SBL KPC-2
and TEM-1, class C AmpC, and class D OXA-48 (Table 1 and
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Table 2. Inhibitory Potency (Ki, μM) of MS05−MS12, with Modifications of the (S)-3′-Mercapto-2′-methylpropanamido
Moiety, to the MBL and SBL Enzymesa
a
The dose−response curves of compounds inhibiting the selected enzymes are given in Supporting Information Figure S8.
Figure S2). Similarly, the boronic acid compounds MS02 and
MS03 displayed inhibitory activity against these SBL enzymes,
whereas structurally similar non-boronic acid compound MS04
had no or weak SBL inhibition, suggesting that the boronic
acid group is likely important for SBL inhibition. These SAR
results demonstrated feasibility of integrating the key
pharmacophores of MBL and SBL inhibitors to develop
broad-spectrum, dual-action inhibitors.
X-ray Structure Determination of the VIM-2:MS01
and KPC-2:MS01 Complexes. We then conducted crystallo-
graphic analyses for the VIM-2:MS01 and KPC-2:MS01
complexes to investigate the dual inhibition modes of MS01.
Details of crystallization and structure determinations are
described in Experimental Section and Table S2. Cocrystalliza-
tion experiments yielded high-resolution crystal structures of
the VIM-2:MS01 (PDB code 6J8R) and KPC-2:MS01 (PDB
code 6J8Q) complexes. VIM-2:MS01 crystallized in the
previously described space group P21221,50 with one molecule
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in the asymmetric unit (ASU) (Table S3). In the VIM-2:MS01
structure, the observed clear Fo − Fc electron density adjacent
to the two zinc ions allow unambiguous modeling of MS01
(Figures 2A and S3A). KPC-2:MS01 crystallized in space
group P321 with four molecules in the ASU, different from
those previously reported45,51,52 (Table S4); MS01 could also
be well-modeled into clearly defined electron density in the
active site of KPC-2 (Figures 2B and S3B).
The VIM-2:MS01 structure reveals that MS01 works via a
thiol-participating metal chelating mode of inhibition, similar
to that observed for previously reported thiol-based MBL
inhibitors.36−38,53 In VIM-2:MS01, the thiol binds to displace
the zinc-bridging nucleophilic hydroxide that is observed in the
native VIM-2 active site (PDB code 4BZ3),36 and chelates
with the two active site zinc ions (2.2−2.3 Å) (Figure 2C),
demonstrating the importance of the thiol for VIM-2
inhibition, consistent with the SAR data shown in Table 1.
The amidocarbonyl group of MS01 is positioned to make
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Scheme 2. Synthetic Routes for 1-Substituted (1-(3′-Mercaptopropanamido)methyl)boronic Acid Derivatives MS05 and
MS13−MS31a

a
(a) Nis(pinacolato)diboron, CuBr, NaOtBu, DMF, Ar, 80 °C, 10 h; (b) (+)-pinanediol or (−)-pinanediol, Et2O, rt, overnight; (c) CH2Cl2, n-
BuLi, ZnCl2, THF, −100 °C to rt, overnight; (d) LiHMDS, THF, −78 °C to t., overnight; HCl/1,4-dioxane, Et2O, −60 °C to rt, 3 h; (e) TBTU,
DIPEA, DMF, −10 °C, 2 h; (f) 2-methylpropylboronic acid, HCl (aq), CH3OH/hexane (1:1), rt overnight. (+)-Pinanediol was used as chiral
auxiliary reagent to generate final (1R)-enantiomers, while (−)-pinanediol was used as chiral auxiliary reagent to generate final (1S)-enantiomers.

hydrogen-bonding interactions with the residue Asn233 (3.0 Å)
on the L10 loop of VIM-2 (Figure 2C), a conserved residue
observed in active site of subclass B1 MBLs (e.g., NDM-1
Asn233, IMP-1 Asn233, and BcII Asn233),36 and its boronic acid
form hydrogen-bonds with Arg228 (3.5 Å) (Figure 2C), which
is structurally equivalent to the catalytic important positively
charged residue on L10 loop of B1 MBLs, for example, NDM-
1 Lys211, IMP-1 Lys224, and BcII Lys224.36 This may indicate
that MS01 has a similar inhibition mode to subclass B1 MBLs,
including NDM-1. By contrast, for B2 MBL Shf-I and B3 MBL
GOB-18, MS01 appears to bind via boronic acid (rather than
thiol)-involving zinc ion contacts as predicted by molecular
docking analyses (Figure S4), similar to that observed in
crystal structures of B2 Sfh-I:L-CS319 (a thiol containing
compound) (PDB code 5EW0)53 and B2 CphA:D-captopril
(PDB code 2QDS);54 this distinct inhibition mode of MS01 to
B2/B3 MBLs may explain the differences in the SAR trends
(e.g., MS01 vs MS02 and MS01 vs MS04; see Table 1).
The KPC-2:MS01 structure reveals continuous electron
density connecting the boron atom of MS01 with the terminal
hydroxyl group of the catalytic Ser70 (Figures 2B and S3B),
demonstrating that MS01 binds with KPC-2 via formation of a
covalent adduct between its boronic acid and Ser70 (Figure
2D), consistent with the SAR results in Table 1; this inhibition
mode is similar to that of previously reported boronic acid-
based SBL inhibitors.22,46,47,55,56 The KPC-2:MS01 structure
represents a covalent inhibition mode (i.e., the formed
tetrahedral boron atom) that mimics the tetrahedral transition
state of substrate binding, which is transiently present on the
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hydrolytic pathway.43 Moreover, MS01 binding is further
stabilized by multiple hydrogen-bonding interactions with the
catalytically important residues Ser70 (2.8 Å), Thr237 (2.8 Å),
Asn170 (2.7 Å), Glu166 (2.7 Å), and Asn132 (2.9 Å) (Figure 2D).
Superimposing structures of KPC-2 with TEM-1 (PDB code
1M40),57 AmpC (PDB code 1FSY),58 and OXA-48 (PDB
code 5FAQ)59 revealed that despite differences in their active
sites, MS01 may have a similar binding mode with these SBL
enzymes (Figure S5).
The VIM-2:MS01 and KPC-2:MS01 structures not only
reveal the substrate-competitive, dual-action MBL/SBL
inhibition modes but also provide important basis for further
structural optimization. On superimposing VIM-2:MS01 with
other crystal structures of VIM-2 complexed with 3-
mercaptopropanamido-containing inhibitors,36−38 we observed
that compared to other inhibitors, MS01 only makes weak
hydrophobic interactions with Phe61 and Tyr67 on the flexible
L3 loop that is important for inhibitor capture as previously
described36−38,50 (Figure S6). Further comparison of KPC-
2:MS01 and reported KPC-2 structures45,56,60 revealed that
MS01 did not occupy the subpocket around the residues
Trp105, Ser130, and Thr235 (Figure S7). These results suggested
that introduction of a substituent at the 1-position of MS01
might be able to improve the potency to VIM-2 and KPC-2 as
well as other MBL/SBL enzymes.
Structure-Based Optimization of (1-(3′-
Mercaptopropanamido)methyl)boronic Acid Deriva-
tives. On the basis of the VIM-2:MS01 and KPC-2:MS01
structures, we performed fragment-growing analyses on the 1-
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position using the LEADOPT program, a tool for structure-

based lead optimization.61 The results suggested that the
introduced fragments in (R)-configuration might be better for
binding with VIM-2 and KPC-2. According to the synthetic
accessibility, we synthesized a compound MS05, bearing a
benzyl group at 1-position in (R)-enantiomer (Table 2), via
the route shown in Scheme 2. MS05 displayed potent
inhibition to VIM-2 (Ki = 0.36 μM), comparable to MS01
(Ki = 0.76 μM), while it showed slightly less potent inhibition
to KPC-2 (Ki = 6.85 μM) as compared with MS01 (Ki = 2.70
μM) (Table 2 and Figure S8). MS05 also manifested
inhibition to subclass B1 MBL NDM-1, B2 Sfh-I, B3 GOB-
18, and class A SBL TEM-1, class C AmpC, and class D OXA-
48, with Ki values of 3.88, 3.48, 9.03, 15.41, 7.87, and 25.83
μM, respectively; by comparison with MS01, MS05 had
slightly improved potency to NDM-1, TEM-1, and OXA-48
(Table 2 and Figure S8).
We obtained a crystal structure of KPC-2:MS05 (PDB code
6JN3) to 2.2 Å resolution (Tables S2 and S4), in which MS05
could be confidently fitted with the electron density maps in
the KPC-2 active site (Figure S9). Similar to MS01, MS05 was
observed to form a covalent bond with the catalytic residue
Ser70 and make multiple hydrogen bonds with the residues
Ser70 (3.0 Å), Thr237 (2.7 Å), Asn170 (2.6 Å), Glu166 (2.7 Å),
and Asn132 (3.1 Å) (Figures 2B and 3A). The introduced
benzyl group of MS05 bound to the subpocket located
between the L3 and L5 loops and made edge-to-face aromatic
interactions with Trp105 (Figure 3A). By superimposing the
structures of KPC-2:MS05 and KPC-2:MS01, we observed
that the binding of MS05 resulted in substantial rotation of the
side chain of Trp105 to accommodate the introduced benzyl
group, providing evidence for flexibility in the conformation of
Trp105 and suggesting its important role in substrate/inhibitor
capture (Figure 3B and Figure S7).45,56,60
Exploring the Modifications of (2′S)-3′-Mercapto-2′-
methylpropanamide Motif. We then explored modifications
of the (2′S)-3′-mercapto-2′-methylpropanamido moiety, with
fixing the benzyl group at 1-position (Table 2), to examine
whether it is possible to improve the inhibitory activity. The
replacement of the 2′-methyl group of (2′S)-3′-mercapto-2′- Figure 3. Crystallographic analyses reveal MS05 binding mode to
methylpropanamido with relatively larger chemical groups, KPC-2. (A) View from the KPC-2:MS05 structure (PDB code 6JN3)
including the benzyl (MS06), 1,3-dioxoisoindolin-2-yl reveals that the inhibitor binds to form a covalent bond with the
(MS07), and benzylamino (MS08) groups (Scheme 3), had catalytic residue Ser70, makes hydrogen-bonding interactions with the
residues Ser70, Thr237, Asn170, Glu166, and Asn132, and makes edge-to-
no improvement in the inhibitory potency to the tested MBL
face aromatic interactions with Trp105. (B) Comparison of KPC-
and SBL enzymes as compared with MS05 and even led to a 2:MS05 and KPC-2:MS01 (PDB code 6J8Q) reveals evidence for the
decrease in ligand efficiency (Table 2 and Figure S8). In important role of Trp105 in substrate/inhibitor capture.45,56,60
contrast, the removal of the 2′-methyl group (MS09) appeared
to improve the inhibitory activity for subclass B2 Sfh-I (Ki = optimal moiety to achieve broad-spectrum inhibition to the
0.89 μM), B3 GOB-18 (Ki = 1.64 μM) and class C AmpC (Ki representative MBL and SBL enzymes.
= 1.14 μM) but reduce the potency to subclass B1 VIM-2 (Ki Substitutions at 1-Position Improve the Dual-Action
= 4.04 μM) and NDM-1 (Ki = 190.53 μM) (Table 2 and MBL/SBL Inhibitory Activity. With fixing the optimal (2′S)-
Figure S8), compared with MS05. Similarly, MS10, with 2′- 3′-mercapto-2′-methylpropanamide group, we further explored
mercaptoacetamide instead of (2′S)-3′-mercapto-2′-methyl- the influences of substitutions and enantiomers at 1-position to
propanamide, exhibited decreased activity to VIM-2 (Ki = 3.34 the dual-action MBL/SBL inhibition. Compounds MS13−
μM) and NDM-1 (Ki = 165 μM), and comparable activity to MS31 were synthesized via the routes shown in Scheme 2, and
other tested MBL and SBL enzymes as MS05 and MS09 their SARs with the representative MBLs and SBLs are given in
(Table 2 and Figure S8). The non-thiol analogues MS11 and Table 3. Compared with MS05, the (1S)-enantiomer MS13,
MS12 displayed low inhibitory activity to VIM-2 and NDM-1 with a benzyl group at 1-position, showed comparable
and also had decreased inhibitory activity to the tested SBL inhibition to subclass B1 VIM-2 (Ki = 1.04 μM) and NDM-
enzymes (Table 2), similar to that observed for MS02 and 1 (Ki = 16.86 μM), B2 Sfh-I (Ki = 1.00 μM), and B3 GOB-18
MS03 (Table 1). These results indicated that the (2′S)-3′- (Ki = 4.38 μM) (Table 3). In contrast, for the tested SBLs,
mercapto-2′-methylpropanamide moiety was a relatively MS13 showed decreased potency to class A KPC-2/TEM-1
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Scheme 3. Synthesis of MS06−MS12 and MS32−MS33 Modified at (S)-3′-Mercapto-2′-methylpropanamide Motifa
a
(a) TBTU, DIPEA, DMF, −10 °C, 2 h; (b) 2-methylpropylboronic acid, HCl (aq), CH3OH/hexane (1:1), rt overnight.
and class D OXA-48 (Table 3), probably due to the benzyl
group in (1S)-configuration making certain influences for the
formation of a covalent bond between the boronic acid and
active site catalytic Ser70. The enantiomer pairs MS14 vs
MS15, MS16 vs MS17, and MS18 vs MS19 were observed to
have similar inhibition profiles to the tested SBL enzymes as
MS05 and MS13, e.g., the (1R)-enantiomers MS14, MS16,
and MS18 showing comparable or slightly better (<5-fold)
potency to KPC-2, TEM-1, and AmpC than the corresponding
(1S)-enantiomers MS15, MS17, and MS19 (Table 3 and
Figure S10). Notably, the (1R)-enantiomer MS18, bearing a
(benzofuran-3-yl)methyl substituent at 1-position, displayed
broad-spectrum inhibition to all of the tested MBL/SBL
enzymes with the exception of class D OXA-48; in particular, it
manifested nanomolar potent inhibition to subclass B1 VIM-2
(Ki = 0.44 μM), B2 Sfh-I (Ki = 0.26 μM), B3 GOB-18 (Ki =
0.13 μM), class A KPC-2 (Ki = 0.61 μM), and class C AmpC
(Ki = 0.11 μM) (Table 3 and Figure S10). Compared with
KPC-2, TEM-1, and AmpC, OXA-48 bears a distinct active
site,57−59 e.g., with several hydrophic residues (Val120, Leu158,
Ile102, and Trp105) and the lack of a structurally equivalent
residue to KPC-2/TEM-1 Asn132 and AmpC Asn152 (Figure
S5) to form hydrogen bonds with the amido-carbonyl of MS18
(see subsequent crystallographic analyses for MS22 and
MS23), partly explaining why MS18 does not potently inhibit
OXA-48. More interestingly, the (1S)-enantiomer MS19
inhibited the subclass B1 MBLs VIM-2 (Ki = 0.07 μM) and
NDM-1 (Ki = 6.06 μM) more potently (>6-fold) than the
(1R)-enantiomer MS18 (with Ki values of 0.44 μM and 37.95
μM to VIM-2 and NDM-1, respectively) (Table 3 and Figure
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S10). To investigate the binding mode of MS19 with VIM-2, a
high-quality crystal structure of the VIM-2:MS19 complex to
1.60 Å resolution was obtained (Tables S2 and S3). MS19 was
well-fitted with the electron density maps found in the VIM-2
active site (Figure S11).
The VIM-2:MS19 structure revealed that similar to MS01,
the (2′S)-3′-mercapto-2′-methylpropanamido moiety of MS19
was positioned to chelate with the two zinc ions and form
hydrogen-bonding interactions with Asn233 (3.0 Å) that is a
conserved residue on the L10 loop in subclass B1 MBLs;36,50
the boronic acid made hydrogen bonds with the residue Arg228
(2.8 Å) (Figure 4A) that is structurally equivalent to B1 MBLs,
e.g., NDM-1 Lys211, IMP-1 Lys224, and BcII Lys224, which are
important to recognize β-lactam 2-C carboxylic acid.36,50
Unexpectedly, the structure revealed an unusual binding mode
of the benzofuran moiety of MS19, i.e., which was positioned
to make orthophoric face-to-face π−π stacking interactions
with residue Phe61 on the VIM-2 L3 loop (Figure 4A), partly
explaining why MS19 had very potent inhibition to VIM-2 (Ki
= 0.07 μM). Notably, superimposition of the VIM-2:MS19 and
VIM-2:MS01 structures revealed that the L3 loop of VIM-2
was positioned away from the active zinc ions (2.8 and 0.8 Å
movements of the C-α atoms of Phe61 and Tyr67 respectively,
Figure 4B) for MS19 binding to VIM-2, providing further
evidence for the proposal that the L3 loop contributes to the
highly efficient nature of MBL catalysis by capturing the
potential substrates and delivering them to the zinc ions for
hydrolysis.50
Further comparison of the enantiomeric pairs, with the
phenyl (MS20 vs MS21), para-fluorophenyl (MS22 vs MS23),
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Table 3. Inhibitory Potency (Ki, μM) of 1-Substituted (1-(3′-Mercaptopropanamido)methyl)boronic Acid Derivatives MS13−
MS31 and MS32−MS33 against the Selected MBL and SBL Enzymesa

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Table 3. continued
a
The dose−response curves of compounds inhibiting the selected enzymes are given in Supporting Information Figure S10.
para-methylphenyl (MS24 vs MS25), and naphthyl (MS26 vs
MS27) moiety at 1-position (Table 3), revealed that the (1R)-
enantiomeric compounds MS20, MS22, and MS24 had
comparable inhibition as the corresponding (1S)-enantiomeric
compounds MS21, MS23, and MS25 to the tested B1/B2/B3
MBL enzymes; (1R)-enantiomer MS26, with a rigid substitent
of naphthyl, inhibited subclass B1 VIM-2 and NDM-1 more
potently than (1S)-enantiomer MS27, while MS26 had
comparable potency to B2 Sfh-I and B3 GOB-18 as MS27
(Table 3 and Figure S10). By comparison, for the SBL
enzymes KPC-2, TEM-1, and AmpC, these (1R)-enantiomeric
compounds generally displayed more potent inhibitory
activities compared with their corresponding (1S)-enantio-
meric compounds, similar to that observed for other
enantiomeric pairs in Table 3. Consequently, we conducted
crystallographic analyses for KPC-2 in complex with the (1R)-
enantiomer MS22 and (1S)-enantiomer MS23, with the aim to
investigate the difference in their binding modes. The crystal
structures of KPC-2:MS22 (PDB code 6JN4) and KPC-
2:MS23 (PDB code 6JN5) were obtained and solved to 1.90
and 1.97 Å resolution, respectively (Table S4); both
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compounds were well-modeled into the observed electron
density maps in the active site (Figures S12 and S13).
The structures revealed that both MS22 and MS23 bound to
form a covalent bond with the catalytic residue Ser70 and make
multiple hydrogen bonds with the active site residues Ser70,
Thr237, Asn170, Glu166, and Asn132 (Figure 5A−C), similar to
that observed for MS01 and MS05 (Figures 2D and 3A).
Similar to MS05, MS22 and MS23 were positioned to occupy
the subpocket between the L3 and L5 loops and made
hydrophobic interactions with the flexible residue Trp105
(Figure 5A,B). Notably, in the KPC-2:MS22 structure, the
amido group adopts a trans-conformation and makes hydro-
gen-bonding interactions with Thr237 (2.9 Å) and Asn132 (3.0
Å) (Figure 5A), same as that observed in the structure of (1R)-
enantiomer MS05 complexed with KPC-2 (Figures 3A and
S14). By contrast, the amido group of the (1S)-enantiomer
MS23 was unexpectedly observed to bind in a cis-conformation
and only form hydrogen bonds with Asn132 (2.9 and 3.5 Å)
(Figure 5B). These differences may, in part, explain why (1R)-
enantiomers inhibit the SBL enzymes more potently than (1S)-
enantiomers. Additionally, these structures provided important
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Journal of Medicinal Chemistry
Figure 4. Crystallographic analyses reveal an unexpected mode of
MS19 binding to VIM-2. (A) Close-up view from the VIM-2:MS19
structure (PDB code 6JN6) reveals that the inhibitor binds to form
hydrophobic and electrostatic interactions with residues on the L3
and L10 loops (e.g., hydrogen-bonding interactions with Asn233 and
Arg228 and, unexpectedly, π−π stacking interactions with Phe61). (B)
Superimposition of VIM-2:MS19 and VIM-2:MS01 (PDB code
6J8R) reveals evidence for flexibility in the conformation of Phe61 and
Tyr67 on the L3 loop, suggesting they may play important roles in
capturing substrates and delivering them to the zinc ions for
hydrolysis.50
Figure 5. Crystallographic analyses reveal similar binding modes of (1R)-enantiomer MS22 and (1S)-enantiomer MS23 with KPC-2. (A) Close-up
view from a structure of MS22 complexed with KPC-2 (PDB code 6JN4). (B) Close-up view from a structure of MS23 complexed with KPC-2
(PDB code 6JN5). (C) Superimposition of KPC-2:MS22 and KPC-2:MS23 reveals MS22 and MS23 bind in similar modes involving covalent
bonding and hydrogen-bonding interactions. Note that unlike MS22, MS23 is bound with cis-amide conformation.
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structural basis for developing chiral α-aminoboronic acid-
based SBL inhibitors.
We then compared the MBL/SBL inhibitory activity for
compounds MS28−MS31, which bear small moieties at 1-
position (Table 3). We observed that for the tested MBL
enzymes, the (1R)-enantiomeric compounds MS28 and MS30
have comparable activity as the corresponding (1S)-enantio-
meric compounds MS29 and MS31, with the exception of
MS29; MS29 has relatively weak inhibition (5-fold lower) to
subclass B2 Shf-I and B3 GOB-18. By comparison with MS18,
compounds MS28−MS31 mostly had less potent inhibition to
the SBL enzymes. Interestingly, to class A KPC-2/TEM-1 and
class C AmpC, the (1R)-enantiomer MS28 was more potent
than (1S)-enantiomer MS29, whereas (1R)-enantiomer MS30
had less inhibitory activity than (1S)-enantiomer MS31 (Table
3 and Figure S10). Comparison of the dual-action inhibition
potencies of MS32/MS33 with MS19 further demonstrated
the importance of the (2′S)-3′-mercapto-2′-methylpropanami-
do group for achieving broad-spectrum MBL inhibition,
particularly for the subclass B1 VIM-2 and NDM-1.
The overall SAR studies of various thiol- and/or boronic
acid-containing compounds with the representative MBL/SBL
enzymes (Table 1−3 and Figure S15) revealed the substantial
influences of the thiol moiety and substitutions/enantiomers at
1-position on the dual-action MBL/SBL inhibition. To
subclass B1 MBLs, the (2′S)-3′-mercapto-2′-methylpropana-
mide moiety was demonstrated to be an optimal MBP for
targeting VIM-2 and NDM-1, similar to that previously
reported,36−38 while the inhibitory activities were synthetically
affected by the 1-position substituents and enantiomeric
conformations (Table 3). By contrast, for subclass B2 Shf-I
and B3 GOB-18, no significant differences were observed
between the tested enantiomer pairs (Table 3), implying that
the inhibitors may have different inhibition modes to subclass
B1 and B2/B3 MBLs. To class A KPC-2/TEM-1 and class C
AmpC, the (1R)-enantiomeric compounds, in most cases,
displayed better activities than the corresponding (1S)-
enantiomeric compounds (Table 3 and Figure S15), possibly
owing to the difference in their amino binding conformations
as observed by crystallographic analyses (Figure 5). Yet, almost
all of the tested compounds had no or weak inhibition to the
structurally different class D OXA-48 (Figure S5). Notably, the
crystallographic analyses revealed that the flexible loops in the
active sites of MBL and SBL enzymes played important roles in
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Figure 6. In vitro selectivity profile of the representative inhibitors across serine hydrolases in E. coli cells (A, B) and human 293T cells (C, D) as
determined by competitive activity-based protein profiling using broad-spectrum probe TAMRA-FP. (A) E. coli cells or (C) human 293T cells were
lysed and treated with inhibitors MS01, MS05, MS06, MS18, MS19, MS20, and MS21 at 50 μM for 30 min, followed by the treatment of activity-
based probe TAMRA-FP (750 nM, 20 min). The heat-maps (B) and (D) showing the quantification of data of gels in (A) and (C), respectively;
data represent average values, n = 3 per group (Table S8).

capturing various inhibitors, which may be one of the critical
aspects to understand the relatively complicated multitarget
SAR data. More importantly, the SAR studies identified new
more potent dual-action inhibitors compared with the starting
compound MS01. For example, MS18 manifested potent
nanmolar inhibition to several tested MBL and SBL enzymes,
including the clinically widespread carbapenemases subclass B1
VIM-2 (Ki = 0.44 μM) and class A KPC-2 (Ki = 0.61 μM);
MS18 was observed to be a reversible inhibitor to both VIM-2
and KPC-2, which had a relatively slow dissociation rate for
KPC-2 compared with other tested inhibitors, including
tazobactam (Figure S16).
Selectivity to Metalloenzyme ACE-2 and Serine
Hydrolyases. Since these dual-action inhibitors have the
(2′S)-3′-mercapto-2′-methylpropanamido moiety, which is a
key scaffold of captopril, an angiotensin-converting enzyme
(ACE) inhibitor, we tested the dual-action inhibitors MS01,
MS05, MS06, and MS13−MS33 to examine their selectivity to
the commercially available human ACE-2 (hACE-2) enzyme.
With the exception of MS06, all these compounds have no
apparent inhibitory activity to hACE-2 at the concentration of
100 μM (Figure S17 and Table S7); MS06 displayed
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inhibition of 63.2% and 44.6% to hACE-2 at 100 μM and 50
μM, respectively (Table S7 and Figure S17).
We also examined the selectivity of the representative
inhibitors MS01, MS05, MS06, MS13, MS18, MS19, MS20,
and MS21 across serine hydrolases in E. coli cells and human
HEK293T cells by competitive activity-based protein profiling
using TAMRA-FP, since they bear an α-aminoboronic acid
moiety. TAMRA-FP is a well-characterized broad-spectrum
serine hydrolase activity-based probe, which provides selectiv-
ity profile assays across a broad array of serine hydrolases. As
shown in Figure 6, the tested inhibitors have weak inhibitory
activity against most of the labeled serine hydrolases in E. coli
and HEK293T cells. Particularly, MS01 showed high
selectivity with no significant off-targets (enzyme inhibition
are all below 50%, Table S8) identified in both E. coli and
HEK293T cell lysates (Figure 6 and Table S8). Of note, the
control compound AN2728, an FDA-approved boronate-
containing drug, exhibited less selectivity compared with
most of the tested inhibitors with several off-targets (Figure
6 and Table S8). These results indicate that the dual-action
inhibitors are selective among serine hydrolases in E. coli and
HEK293T cells labeled by TAMRA-FP.
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Journal of Medicinal Chemistry
Table 4. Susceptibility Testing Data of Clinical Isolates Harboring NDM-1, KPC-2, or AmpC to Meropenem alone and in
Combination with the Inhibitors MS01, MS05, MS13, MS18, and MS19 at 50 μM
bacterial strain characterized β-lactamase DMSO MS01
E. coli BAA-2452 blaNDM‑1 32
K. pneumoniae 13249 blaNDM‑1 64
E. coli BAA-2340 blaKPC‑2 4 0.5
E. coli 11119 blaKPC‑2 16
K. pneumoniae BAA-1705 blaKPC‑2 16
K. pneumoniae 5846 blaKPC‑2 16
K. pneumoniae C660 blaAmpC 32
Inhibitors Potentiate Meropenem Activity against
Clinical Isolates Harboring MBL or SBL Enzymes. We
then tested the activity of the inhibitors MS01, MS05, MS13,
MS18, and MS19 (L-captopril and tazobactam as control
compounds) against seven multidrug-resistant Gram-negative
clinical isolates: E. coli BAA-2452 (blaNDM‑1), K. pneumoniae
13249 (blaNDM‑1), E. coli BAA-2340 (blaKPC‑2), E. coli 11119
(blaKPC‑2), K. pneumoniae BAA-1705 (blaKPC‑2), K. pneumoniae
5846 (blaKPC‑2), and K. pneumoniae C660 (blaAmpC). The
minimum inhibitory concentration (MIC) values of merope-
nem against these bacterial strains were determined with or
without the inhibitors. The results showed that MS01, MS05,
MS13, MS18, and MS19 were able to potentiate the efficacy of
meropenem against the clinically relevant E. coli and K.
pneumonia strains, particularly harboring KPC-2 and AmpC, as
evidenced by the substantial reductions of the MIC values (≤8
μg·ml−1), which are better than or comparable to L-captopril
and tazobactam (Table 4); these compounds did not cause
marked inhibition of bacterial growth at 64 μg·mL−1, indicating
that they exhibited synergistic antibacterial effects when used
along with meropenem. Besides, none of these inhibitors
showed cytotoxicity in human HEK293T and MCF-7 cells
when treated at concentrations up to 100 μM (Table S9 and
Figure S18).
■
CHEMISTRY
As shown in Scheme 1A, triisopropyl borate (1) was prestirred
with dibromomethane and n-butyllithium (n-BuLi) in
tetrahydrofuran (THF) at −78 °C for 2 h, and the mixture
was then reacted with methanesulfonic acid and pinacol at 0
°C to afford compound 2.62 A nucleophilic substitution
reaction was then carried out from bromide 2 which was
stirred in THF with lithium bis(trimethylsilyl)amide
(LiHMDS) at −78 °C to obtain bis-silylamine intermediate
3. The amine hydrochloride 4 was synthesized by deprotection
from 3 using 4 N hydrochloride/1,4-dioxane solution and
diethyl ether at −60 °C.63 Compound 4 was then subjected to
a coupling reaction with diverse carboxylic acid using O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate (TBTU), N,N-diisopropylethylamine (DIPEA), and
N,N-dimethylformamide (DMF) at −10 °C to generate 5a−
c, 64 followed by the deprotection reaction using 2-
methylpropylboronic acid and 3 N hydrochloric acid solution
in methanol and hexane to give MS01−MS03. Similarly,
treatment of glycine ethyl ester hydrochloride (6) with (S)-(3-
acetylsulfanyl-2-methyl)propionic acid in DMF gave 7, which
was then subjected to deprotection reaction to yield MS04
(Scheme 1B).
As outlined in Scheme 2, various bromides 8a−d were
carried out with borylation reaction by bis(pinacolato)diboron,
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meropenem MIC (μg·mL−1)
MS05 MS13 MS18 MS19 L-cap Tazo
16 16 32 32 16 8 16
64 64 64 64 32 32 64
2 2 1 2 4 2
4 16 16 8 8 16 8
8 16 16 8 16 16 16
4 8 16 8 8 8 8
8 16 16 8 8 32 16
cupric bromide (CuBr), sodium tert-butoxide (NaOtBu) in
DMF to obtain boronic acid pinacol esters 9a−d,65 which were
then subjected to transesterification reaction with
(1S,2S,3R,5S)-(+)-2,3-pinanediol ((+)-pinanediol) in diethyl
ether (Et2O) to generate boronic acid (+)-pinanediol esters
11a−d. Compounds 11e−j were produced by esterification
reaction from diverse boronic acid 10e−j with (+)-pinanediol
in Et2O. Subsequent Matteson reaction of 11a−j with
dichloromethane (CH2Cl2) and n-butyllithium (n-BuLi) in
THF at −100 °C afforded 12a−j with (S)-configuration,66−68
followed by SN2 reaction with LiHMDS in THF at −78 °C,
generating 13a−j with inverted (R)-configuration (obtained as
hydrochloride salts).69,70 Next, 13a−j were reacted with (S)-
(3-acetylsulfanyl-2-methyl)propionic acid to form 14a−j,
which were then subjected to deprotection reaction to yield
(1R)-1-substituted (1-(3′-mercaptopropanamido)methyl)-
boronic acid derivatives (including MS05, MS14, MS16,
MS18, MS20, MS22, MS24, MS26, MS28, and MS30).
Followed the same synthetic routes as described above, the
corresponding (1S)-enantiomers (including MS13, MS15,
MS17, MS19, MS21, MS23, MS25, MS27, MS29, and
MS31) were obtained using (1R,2R,3S,5R)-(−)-2,3-pinanediol
((−)-pinanediol) instead of (+)-pinanediol.
The intermediate (R)-(1-amino-2-phenylethyl)boronic acid
(1S,2S,3R,5S)-(+)-2,3-pinanediol ester 13a was subjected to a
coupling reaction with various carboxylic acids to provide
15a−g, followed by the deprotection reactions to give MS06−
MS12 (Scheme 3A). Similarly, (S)-(1-amino-2-(3′-
benzofuran)ethyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinane-
diol ester 13d′ reacted with (3-acetylsulfanyl)propionic acid or
(2-acetylsulfanyl)acetic acid to form 16a or 16b, which then
deprotected to give MS32 or MS33 (Scheme 3B).
■
CONCLUSION
Overall, the results revealed the potential of a pharmacophore
fusion strategy targeting the key active site features of MBL
and SBL enzymes for the identification of dual-action
inhibitors. The exquisite fusion of (2′S)-3′-mercapto-2′-
methylpropanamido with acyclic α-aminoboronic acid led to
the identification of new dual-action MBL/SBL inhibitors. The
crystallographic analyses demonstrated the hypothetical MBL/
SBL inhibition modes, i.e., (2′S)-3′-mercapto-2′-methylpropa-
namido positioned to chelate with active site zinc ions of at
least subclass B1 MBL enzymes, and α-aminoboronic acid
covalently bound with the catalytic residue Ser70 of SBL
enzymes. These results may prompt a fresh start for using
pharmacophore fusion to obtain new dual-action inhibitor
chemotypes targeting MBL and SBL enzymes as well as other
structurally and mechanistically distinct enzymes.
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Journal of Medicinal Chemistry
The structure-guided optimization studies yielded inter-
preted multitarget structure−activity relationships and resulted
in new more potent, broad-spectrum, dual-action MBL/SBL
inhibitors, e.g., MS18. The crystal structures revealed insights
into the binding modes of these inhibitors with VIM-2 and
KPC-2, providing important structural basis for developing
dual-action MBL/SBL inhibitors. The structural results also
provided evidence for the flexibility of the loops near active
sites of MBL and SBL enzymes, suggesting their important
roles in substrate/inhibitor capture, which hence need to be
considered in inhibitor design and development.
Selectivity studies indicated that the inhibitors had no
apparent inhibition to human ACE-2 and showed selectivity
across serine hydrolyases in E. coli and HEK293T cells labeled
by TAMRA-FP. The inhibitors could reduce MICs of
meropenem against MBL- or SBL-positive K. pneumoniae
and E. coli clinical isolates without apparent cytotoxicity,
indicating that they are suitable for further structural
optimization, possibly along with membrane permeability
and intracellular accumulation, to obtain clinically useful
dual-action MBL/SBL inhibitors.
■
EXPERIMENTAL SECTION
Chemistry. Unless otherwise noted, all reactions were carried out
under anhydrous conditions, argon atmosphere, and dry solvents.
Reactions were monitored by thin-layer chromatography (TLC) and
were visualized using UV light or phosphomolybdic acid (10% in
ethanol). TLC characterization was performed with precoated silica
gel GF254 (0.2 mm). The purification of products by silica gel
column chromatography was carried out on silica gel (100−200
mesh). All organic extracts were dried over MgSO4, and solvents were
removed with a rotary evaporator, followed by high vacuum oil pump
pressure. NMR spectra were taken on a Varian INOVA 400 or 600
MHz (Varian, Palo Alto, CA, USA). Chemical shifts were expressed in
parts per million (δ, ppm), with tetramethylsilane (TMS) functioning
as the internal standard, and coupling constants (J) were expressed in
Hz. The following abbreviations were used to identify the multi-
plicities: s = singlet, d = doublet, dd = doublet of doublets, dt =
doublet of triplets, t = triplet, q = quartet, m = multiplet, br = broad,
and all combinations thereof can be explained by their integral parts.
High resolution mass spectroscopy (HRMS) data of the products
were collected on a SCIEX X500 QTOF mass spectrometer
instrument. High performance liquid chromatography (HPLC)
analyses were run on a Agilent 1200 HPLC analytical system
(monitoring at a wavelength of 210 or 254 nm) with a Amethyst C18-
H column (4.6 mm × 250 mm, 5 μm) and mixtures of methanol and
H2O containing 0.03% triethylamine and 0.05% trifluoacetic acid as
eluent. All final tested compounds were >95% pure by HPLC
analyses. Commercial reagents were from Best-reagent or Astatech
Chemical Technology Co, Ltd. All reagents were used without further
purification.
Synthesis. Boronic Acid Pinanediol Ester. The key inter-
mediate (R)-(1-amino-2-phenylethyl)boronic acid (1S,2S,3R,5S)-
(+)-2,3-pinanediol ester (13a) was synthesized as described in
Scheme 2 from benzyl bromide (8a) as starting material. 1-
Substituted amine intermediates were generated by similar synthetic
method from corresponding starting material using either
(1S,2S,3R,5S)-(+)-2,3-pinanediol ester or (1R,2R,3S,5R)-(−)-2,3-
pinanediol ester as chiral auxiliary reagent. The products were
obtained as hydrochloride salts.67,68
General Method of Coupling Reaction for Synthesis of
Amide Compounds (5a−c, 7, 14a−j, 14a′−j′, 15a−g, 16a,
16b). TBTU (385 mg, 1.5 mmol) was added into a solution of amine
(1 mmol), carboxylic acid (1 mmol), and DIPEA (387 mg, 3 mmol)
in DMF (10 mL) at −10 °C. The reaction solution was stirred at the
same temperature until the TLC indicates complete disappearance of
amine. Dilute the reaction mass with ethyl acetate (30 mL), and wash
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with water followed by brine. The organic layer was collected, dried
over Na2SO4, and concentrated under reduced pressure. The resulting
residue was purified by silica gel chromatography with dichloro-
methane/methanol combinations as eluent, giving rise to the amide
compounds.
(1-Bromomethyl)boronic Acid Pinacol Ester (2). To a solution of
triisopropyl borate (10 g, 55 mmol) and dibromomethane (4.2 mL,
60 mmol) in dried THF (80 mL) at −78 °C under argon, n-BuLi (20
mL, 50 mmol; 2.5 N in hexane) was added dropwise, and the mixture
was stirred at the same temperature for 2 h. The reaction mixture was
then warmed to 0 °C, methanesulfonic acid (3.2 mL, 50 mmol) was
added, and the mixture was stirred at room temperature for 1 h.
Subsequently, the obtained mixture was cooled to 0 °C, pinacol (5.9
g, 50 mmol) was added, and the mixture was stirred at room
temperature for another 1 h. The solvents were evaporated under
reduced pressure from the reaction mixture and then the obtained
residue was distilled under reduced pressure (62−63 °C, 10 mmHg),
obtaining the product 2 7.5 g in 68% yield as colorless oil. 1H NMR
(400 MHz, CDCl3): δ 1.29 (s, 12H), 2.59 (s, 2H).
(Bis(trimethylsilyl)aminomethyl)boronic Acid Pinacol Ester (3).
To a solution of (1-bromomethyl)boronic acid pinacol ester (2, 4.0 g,
18.1 mmol) in dried THF (36 mL) was added LiHMDS (22.6 mL,
22.6 mmol; 1 N in THF) dropwise under argon atmosphere at −78
°C. The reaction mixture was allowed to warm to room temperature
and stirred overnight. The resulting solution was concentrated under
reduced pressure and reconstituted in hexane (50 mL) and filtered
through Celite. The organic layer was dried with Na2SO4 and
evaporated to give crude N-bis(trimethylsilyl)amine product 3 as
light-brown oil. The product was used for the next step without
purification.
(1-Aminomethyl)boronic Acid Pinacol Ester (4). The crude light-
brown oil (3, 9 mmol) was dissolved in diethyl ether (25 mL) and
cooled down to −60 °C. To the mixture was added 4 N HCl/1,4-
dioxane solution (6.75 mL, 27 mmol), and the reaction was allowed
to stir for 3 h. The hydrochloride salt crashed out of solution and was
filtered. The final product was washed three times with cold diethyl
ether, resulting in a white solid with 1.5 g in 88% yield. 1H NMR (400
MHz, DMSO-d6): δ 1.24 (s, 12H), 2.36 (q, J = 7.6 Hz, 2H), 7.83 (br,
3H).
(S)-(1-(3′-Acetylsulfanyl-2′-methylpropanamido)methyl)boronic
Acid Pinacol Ester (5a). Followed general method using (1-
aminomethyl)boronic acid pinacol ester (4) as amine and (S)-(3-
acetylsulfanyl-2-methyl)propionic acid as carboxylic acid. Colorless
oil, 212 mg in 74% yield. 1H NMR (400 MHz, CDCl3): δ 1.23 (d,
3H, J = 6.8 Hz), 1.27 (s, 12H), 2.10 (s, 3H), 2.22 (s, 2H), 2.62 (m,
1H), 2.78 (m, 1H), 3.00−3.15 (m, 1H), 7.66 (br, 1H).
(1-(n-Butyramido)methyl)boronic Acid Pinacol Ester (5b).
Followed general method using (1-aminomethyl)boronic acid pinacol
ester (4) as amine and n-butyric acid as carboxylic acid. Colorless oil,
96 mg in 42% yield. 1H NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J =
7.2 Hz), 1.23 (s, 12H), 1.53−1.63 (m, 2H), 2.18−2.24 (m, 2H), 2.29
(s, 2H).
(1-(n-Propanamido)methyl)boronic Acid Pinacol Ester (5c).
Followed general method using (1-aminomethyl)boronic acid pinacol
ester (4) as amine and n-propionic acid as carboxylic acid. Colorless
oil, 98 mg in 46% yield. 1H NMR (400 MHz, CDCl3): δ 1.11 (t, 3H, J
= 7.2 Hz), 1.23 (s, 12H), 2.34 (q, 2H, J = 7.2 Hz), 2.64 (s, 2H).
(S)-2-(3′-Acetylsulfanyl-2′-methylpropanamido)acetic Acid Ethyl
Ester (7). Followed general method using glycine ethyl ester
hydrochloride (6) as amine and (S)-(3-acetylsulfanyl-2-methyl)-
propionic acid as carboxylic acid. Colorless oil, 150 mg in 61%
yield. 1H NMR (400 MHz, CDCl3): δ 1.21−1.31 (m, 6H), 2.33 (s,
3H), 2.47−2.56 (m, 1H), 2.95 (dd, 1H, J = 13.6, 6.4 Hz), 3.11 (dd,
1H, J = 13.6, 7.6 Hz), 4.00−4.05 (m, 2H), 4.21 (q, 2H, J = 7.2 Hz),
6.20 (s, 1H).
(1-Phenylmethyl)boronic Acid Pinacol Ester (9a). To a solution of
benzyl bromide (8a, 2.0 g, 11.69 mmol) in degassed DMF (40 mL)
were added bis(pinacolato)diboron (4.45 g, 17.54 mmol), CuBr (340
mg, 2.34 mmol), and NaOtBu (1.69 g, 17.54 mmol). The solution
was stirred under argon atmosphere at 80 °C for 10 h, and the
DOI: 10.1021/acs.jmedchem.9b00735
J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
contents of the flask were cooled to room temperature. The reaction
mixture was filtered out, and the organic layer was extracted with ethyl
acetate (50 mL) and washed with water (20 mL × 2) followed by
brine (20 mL × 2). The organic layer was collected, dried over
Na2SO4, and concentrated under reduced pressure. The resulting
residue was purified by silica gel chromatography with hexane/ethyl
acetate (40:1) combinations as eluent, giving rise to the product 9a
1.78 g in 70% yield as colorless oil. 1H NMR (400 MHz, CDCl3): δ
1.23 (s, 12H), 2.29 (s, 2H), 7.11 (t, 1H, J = 7.2 Hz), 7.16−7.19 (m,
2H), 7.21−7.25 (m, 2H).
(1-Phenylmethyl)boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol
Ester (11a). To a solution of (1-phenylmethyl)boronic acid pinacol
ester (9a, 1.0 g, 4.58 mmol) in diethyl ether (20 mL) was added
(1S,2S,3R,5S)-(+)-2,3-pinanediol ester (859 mg, 5.04 mmol). The
reaction mixture was stirred at room temperature overnight and then
washed with water (20 mL × 2) followed by brine (20 mL × 2). The
organic layer was collected, dried over Na2SO4, and concentrated
under reduced pressure. The resulting residue was purified by silica
gel chromatography with hexane/ethyl acetate (50:1) combinations as
eluent, giving rise to the product 11a 1.13 g in 91% yield as colorless
oil. 1H NMR (400 MHz, CDCl3): δ 0.82 (s, 3H), 1.06 (d, 1H, J =
10.8 Hz), 1.27 (s, 3H), 1.38 (s, 3H), 1.79−1.84 (m, 1H), 1.85−1.90
(m, 1H), 2.04 (t, 1H, J = 5.6 Hz), 2.14−2.21 (m, 1H), 2.26−2.33 (m,
1H), 2.34 (s, 2H), 4.27 (dd, 1H, J = 8.8, 2.0 Hz), 7.09−7.14 (m, 1H),
7.18−7.21 (m, 2H), 7.22−7.26 (m, 2H).
(S)-(1-Chloro-2-phenylethyl)boronic Acid (1S,2S,3R,5S)-(+)-2,3-
Pinanediol Ester (12a). In a three-neck flask equipped with
thermometer, a solution of CH2Cl2 (0.8 mL) in freshly distilled
THF (10 mL) was cooled to −100 °C (in ethanol/liquid nitrogen
bath) under argon atmosphere. n-BuLi (2.5 mL, 6.25 mmol; 2.5 N in
hexane) was added via syringe by running it down the cold wall of the
reaction flask over a period of 20 min. White precipitate of LiCHCl2
was formed, and after 20 min of stirring, cold solution of 1-
(phenylmethyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(11a, 1.13 g, 4.18 mmol) in 5 mL of dry THF was added via syringe
by running it down the cold wall of the reaction flask over a period of
15 min. After 20 min of stirring at −100 °C, ZnCl2 solution (4.2 mL,
4.2 mmol; 1.0 N in THF) was added via syringe by running it down
the cold wall of the reaction flask over a period of 10 min. The
solution was stirred overnight, and the temperature was allowed to
rise to room temperature. The reaction mixture was evaporated and
redissolved in hexane. The organic phase was washed with satd
NH4Cl (20 mL × 2), water (20 mL), and brine (20 mL), then dried
and concentrated to give about 70% crude product as colorless oil.
The product was used for the next step without purification. 1H NMR
(400 MHz, CDCl3): δ 0.83 (s, 3H), 1.06 (d, 1H, J = 11.2 Hz), 1.28
(s, 3H), 1.34 (s, 3H), 1.83−1.91 (m, 2H), 2.06 (t, 1H, J = 5.6 Hz),
2.14−2.22 (m, 1H), 2.30−2.36 (m, 1H), 3.10 (dd, 1H, J = 14.0, 8.8
Hz), 3.21 (dd, 1H, J = 14.0, 7.6 Hz), 3.66 (t, 1H, J = 8.0 Hz), 4.35 (d,
1H, J = 9.2 Hz), 7.20−7.25 (m, 2H), 7.26−7.30 (m, 3H).
(R)-(1-Amino-2-phenylethyl)boronic Acid (1S,2S,3R,5S)-(+)-2,3-
Pinanediol Ester (13a). To a solution of crude (S)-1-chloro-2-
(phenylethyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(12a, 4.0 mmol) in dried THF (10 mL) was added LiHMDS (4.0
mL, 4 mmol; 1 N in THF) dropwise under argon atmosphere at −78
°C. The reaction mixture was stirred overnight and allowed to warm
to room temperature. The resulting solution was concentrated under
reduced pressure and reconstituted in hexane (25 mL) and filtered
through Celite. The organic layer was dried with Na2SO4 and
evaporated to give crude N-bis(trimethylsilyl)amine product as light-
brown oil. The crude light-brown oil was dissolved in diethyl ether
(15 mL) and cooled down to −60 °C. To the mixture was added 4 N
HCl/1,4-dioxane solution (3.0 mL), and the reaction was allowed to
stir for 3 h. The hydrochloride salt crashed out of solution and was
filtered. The final product was washed three times with cold diethyl
ether, resulting in white solid with 811 mg in 68% yield as
hydrochloride salt.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-
phenylethyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol Ester
(14a). Followed general method using (R)-(1-amino-2-phenylethyl)-
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boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester (13a) as amine
and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as carboxylic acid.
White solid, 226 mg in 51% yield. 1H NMR (600 MHz, CDCl3): δ
0.86 (s, 3H), 1.18 (d, 3H, J = 7.2 Hz), 1.28 (s, 3H), 1.36 (d, 1H, J =
6.0 Hz), 1.38 (s, 3H), 1.83−1.88 (m, 2H), 2.01 (t, 1H, J = 5.4 Hz),
2.11−2.15 (m, 1H), 2.30 (s, 3H), 2.34 (m, 1H), 2.47 (q, 1H, J = 6.6
Hz), 2.74−2.78 (m, 1H), 2.95−3.00 (m 2H), 3.03−3.07 (m, 2H),
4.29 (d, 1H, J = 7.8 Hz), 6.35 (s, 1H), 7.19−7.24 (m, 3H), 7.26−7.33
(m, 2H). 13C NMR (100 MHz, CDCl3): δ 16.98, 23.91, 26.29, 27.07,
28.68, 30.45, 31.64, 36.01, 37.03, 37.89, 38.36, 39.65, 51.89, 76.78,
84.09, 126.69, 128.44 (2C), 128.75 (2C), 140.17, 177.31, 195.02.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-
phenylethyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol Ester
(14a′). Followed general method using (S)-(1-amino-2-phenylethyl)-
boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester (13a′) as amine
and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as carboxylic acid.
White solid, 275 mg in 62% yield. 1H NMR (600 MHz, CDCl3): δ
0.83 (s, 3H), 1.16 (d, 3H, J = 6.6 Hz), 1.25 (s, 3H), 1.33 (d, 1H, J =
6.0 Hz), 1.35 (s, 3H), 1.80−1.86 (m, 2H), 1.98 (t, 1H, J = 5.4 Hz),
2.08−2.12 (m, 1H), 2.28 (s, 3H), 2.29−2.32 (m, 1H), 2.44 (q, 1H, J
= 6.6 Hz), 2.74 (dd, 1H, J = 13.8, 10.8 Hz), 2.96 (dd, 2H, J = 13.8, 5.4
Hz), 3.02 (dd, 2H, J = 13.8, 7.8 Hz), 4.26 (d, 1H, J = 7.8 Hz), 6.33 (s,
1H), 7.16−7.20 (m, 3H), 7.25−7.28 (m, 2H). 13C NMR (150 MHz,
CDCl3): δ 16.65, 24.06, 26.38, 27.19, 28.79, 30.47, 32.04, 36.12,
37.17, 38.03, 38.69, 39.80, 51.87, 76.93, 84.22, 126.16, 128.43 (2C),
128.88 (2C), 140.23, 176.96, 195.59.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-(4′-
fluorophenyl)ethyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol
Ester (14b). Followed general method using (R)-(1-amino-2-(4′-
fluorophenyl)ethyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol
ester (13b) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic
acid as carboxylic acid. White solid, 118 mg in 25% yield. 1H NMR
(400 MHz, CDCl3): δ 0.83 (s, 3H), 1.20 (d, 3H, J = 6.8 Hz), 1.24 (m,
4H), 1.35 (s, 3H), 1.78−1.88 (m, 2H), 1.98 (t, 1H, J = 5.2 Hz),
2.06−2.11 (m, 1H), 2.28 (m, 4H), 2.43−2.50 (m, 1H), 2.68−2.75
(m, 1H), 2.89−2.96 (m, 2H), 3.01−3.07 (m, 2H), 4.25 (d, 1H, J =
8.4 Hz), 6.41 (s, 1H), 6.93−6.98 (m, 2H), 7.12−7.17 (m, 2H). 13C
NMR (100 MHz, CDCl3): δ 16.06, 23.12, 25.38, 26.24, 27.87, 29.55,
30.92, 35.08, 35.33, 37.12, 37.87, 38.82, 50.85, 76.11, 83.48, 114.15,
114.36, 129.34, 129.41, 134.78, 159.31, 176.21, 194.87.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-(4′-
fluorophenyl)ethyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol
Ester (14b′). Followed general method using (S)-(1-amino-2-(4′-
fluorophenyl)ethyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol
ester (13b′) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic
acid as carboxylic acid. White solid, 106 mg in 23% yield. 1H NMR
(400 MHz, CDCl3): δ 0.85 (s, 3H), 1.19 (d, 3H, J = 6.8 Hz), 1.27 (m,
4H), 1.37 (s, 3H), 1.80−1.88 (m, 2H), 1.98−2.02 (m, 1H), 2.10−
2.16 (m, 1H), 2.31 (m, 4H), 2.41−2.50 (m, 1H), 2.72−2.78 (m, 1H),
2.93−2.99 (m, 2H), 3.02−3.09 (m, 2H), 4.28 (d, 1H, J = 8.4 Hz),
6.20 (s, 1H), 6.95−7.00 (m, 2H), 7.15−7.19 (m, 2H). 13C NMR (100
MHz, CDCl3): δ 16.79, 24.14, 26.44, 27.26, 28.83, 30.60, 32.20,
36.09, 36.40, 38.15, 39.07, 39.84, 51.87, 77.14, 84.62, 115.19, 115.40,
130.37, 130.45, 135.82, 160.35, 176.99, 195.79.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-(4′-
methoxyphenyl)ethyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinane-
diol Ester (14c). Followed general method using (R)-(1-amino-2-
(4′-methoxyphenyl)ethyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinane-
diol ester (13c) as amine and (S)-(3-acetylsulfanyl-2-methyl)-
propionic acid as carboxylic acid. White solid, 238 mg in 50% yield.
1
H NMR (400 MHz, CDCl3): δ 0.86 (s, 3H), 1.21 (d, 3H, J = 7.2
Hz), 1.28 (s, 3H), 1.33 (d, 1H, J = 10.4 Hz), 1.38 (s, 3H), 1.82−1.90
(m, 2H), 2.01 (t, 1H, J = 5.6 Hz), 2.10−2.16 (m, 1H), 2.29 (s, 3H),
2.31−2.36 (m, 1H), 2.43−2.49 (m, 1H), 2.70 (d, 1H, J = 14.4, 10.4
Hz), 2.89−2.99 (m, 2H), 3.02−3.08 (m, 2H), 3.79 (s, 3H), 4.28 (d,
1H, J = 8.8 Hz), 6.17 (s, 1H), 6,84 (d, 2H, J = 8.8 Hz), 7.12 (d, 2H, J
= 8.8 Hz). 13C NMR (100 MHz, CDCl3): δ 16.14, 23.13, 25.41,
26.26, 27.87, 29.55, 30.96, 35.11, 35.20, 37.13, 38.03, 38.84, 50.87,
54.25, 76.10, 83.49, 112.98 (2C), 128.87 (2C), 131.10, 157.11,
175.82, 194.78.
DOI: 10.1021/acs.jmedchem.9b00735
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Journal of Medicinal Chemistry
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-(4′-
methoxyphenyl)ethyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinane-
diol Ester (14c′). Followed general method using (S)-(1-amino-2-
(4′-methoxyphenyl)ethyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pina-
nediol ester (13c′) as amine and (S)-(3-acetylsulfanyl-2-methyl)-
propionic acid as carboxylic acid. White solid, 171 mg in 36% yield.
1
H NMR (400 MHz, CDCl3): δ 0.86 (s, 3H), 1.19 (d, 3H, J = 6.8
Hz), 1.28 (s, 3H), 1.34 (d, 1H, J = 10.4 Hz), 1.38 (s, 3H), 1.82−1.90
(m, 2H), 2.01 (t, 1H, J = 5.6 Hz), 2.11−2.17 (m, 1H), 2.30−2.37 (m,
4H), 2.43−2.48 (m, 1H), 2.68−2.74 (m, 1H), 2.91−3.06 (m, 4H),
3.79 (s, 3H), 4.29 (d, 1H, J = 8.8 Hz), 6.17 (s, 1H), 6,84 (d, 2H, J =
8.8 Hz), 7.14 (d, 2H, J = 8.8 Hz). 13C NMR (100 MHz, CDCl3): δ
16.79, 24.15, 26.47, 27.28, 28.86, 30.59, 32.17, 36.17, 36.29, 38.15,
38.99, 39.88, 51.93, 55.27, 77.09, 84.45, 113.98 (2C), 129.91 (2C),
132.20, 158.13, 176.83, 195.70.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-(3′-
benzofuran)ethyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol
Ester (14d). Followed general method using (R)-(1-amino-2-(3′-
benzofuran)ethyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(13d) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as
carboxylic acid. White solid, 392 mg in 81% yield. 1H NMR (400
MHz, CDCl3): δ 0.83 (s, 3H), 1.19 (d, 3H, J = 7.2 Hz), 1.25 (m, 4H),
1.33 (s, 3H), 1.78−1.87 (m, 2H), 1.96−2.00 (m, 1H), 2.08−2.15 (m,
1H), 2.22 (s, 3H), 2.29−2.36 (m, 1H), 2.46−2.55 (m, 1H), 2.84−
2.87 (m, 1H), 2.96 (m, 1H), 2.99−3.07 (m, 2H), 3.14−3.17 (m, 1H),
4.25 (d, 1H, J = 7.2 Hz), 6.83 (s, 1H), 7.21 (t, 1H, J = 7.6 Hz), 7.26
(t, 1H, J = 7.6 Hz), 7.40−7.49 (m, 2H), 7.59 (d, 1H, J = 7.6 Hz). 13C
NMR (150 MHz, CDCl3): δ 16.75, 24.10, 25.26, 26.49, 27.26, 28.84,
30.49, 32.06, 36.25, 38.10, 38.80, 38.91, 39.88, 51.97, 77.04, 84.37,
111.47, 118.45, 119.78, 122.43, 124.32, 127.96, 141.97, 155.41,
177.28, 195.73.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-(3′-
benzofuran)ethyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol
Ester (14d′). Followed general method using (S)-(1-amino-2-(3′-
benzofuran)ethyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol
ester (13d′) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic
acid as carboxylic acid. White solid, 365 mg in 75% yield. 1H NMR
(400 MHz, CDCl3): δ 0.85 (s, 3H), 1.18 (d, 3H, J = 6.8 Hz), 1.26 (m,
4H), 1.36 (s, 3H), 1.81−1.90 (m, 2H), 2.00 (t, 1H, J = 5.2 Hz),
2.12−2.18 (m, 1H), 2.25 (s, 3H), 2.28−2.37 (m, 1H), 2.44−2.50 (m,
1H), 2.88−2.92 (m, 1H), 2.96−3.05 (m, 3H), 3.11−3.18 (m, 1H),
4.28 (d, 1H, J = 8.8 Hz), 6.56 (s, 1H), 7.22 (t, 1H, J = 7.6 Hz), 7.28
(t, 1H, J = 7.6 Hz), 7.46 (d, 1H, J = 7.6 Hz), 7.49 (s, 1H), 7.59 (d,
1H, J = 7.6 Hz). 13C NMR (100 MHz, CDCl3): δ 16.81, 24.15, 25.28,
26.52, 27.30, 28.85, 30.51, 32.11, 36.25, 38.16, 38.60, 38.94, 39.93,
52.01, 77.15, 84.49, 111.51, 118.48, 119.83, 122.46, 124.35, 128.03,
142.03, 155.47, 177.19, 195.71.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-
phenylmethyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol Ester
(14e). Followed general method using (R)-(1-amino-1-
phenylmethyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(13e) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as
carboxylic acid. White solid, 144 mg in 33% yield. 1H NMR (600
MHz, CDCl3): δ 0.81 (s, 3H), 1.21 (d, 1H, J = 10.2 Hz), 1.25 (s,
3H), 1.34−1.36 (m, 6H), 1.57 (d, 1H, J = 9.2 Hz), 1.79 (br, 1H),
2.03−2.10 (m, 2H), 2.19−2.23 (m, 1H), 2.34 (s, 3H), 2.68−2.71 (m,
1H), 3.04 (dd, 1H, J = 13.8, 6.0 Hz), 3.18 (dd, 1H, J = 13.8, 7.2 Hz),
4.11 (s, 1H), 4.22 (d, 1H, J = 8.4 Hz), 6.84 (br, 1H), 7.18−7.21 (m,
3H), 7.29−7.32 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 17.00,
24.08, 26.39, 27.21, 28.64, 30.63, 31.78, 36.06, 38.14, 38.50, 39.80,
51.87, 76.88, 77.23, 84.99, 126.21, 126.31 (2C), 128.40 (2C), 140.16,
178.57, 196.16.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-
phenylmethyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol Ester
(14e′). Followed general method using (S)-(1-amino-1-
phenylmethyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester
(13e′) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as
carboxylic acid. White solid, 126 mg in 29% yield. 1H NMR (400
MHz, CDCl3): δ 0.79 (s, 3H), 1.18−1.22 (m, 4H), 1.20 (d, 3H, J =
7.2 Hz), 1.31 (s, 3H), 1.47−1.52 (m, 1H), 1.75 (br, 1H), 1.93−2.04
(m, 2H), 2.13−2.20 (m, 1H), 2.34 (s, 3H), 2.65−2.74 (m, 1H), 3.07
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(dd, 1H, J = 14.0, 6.0 Hz), 3.15 (dd, 1H, J = 14.0, 7.6 Hz), 4.01 (s,
1H), 4.16 (d, 1H, J = 7.6 Hz), 7.15−7.18 (m, 3H), 7.25−7.30 (m,
2H). 13C NMR (150 MHz, CDCl3): δ 16.80, 24.09, 26.43, 27.23,
28.76, 30.61, 31.78, 36.20, 38.07, 38.49, 39.81, 52.02, 76.94, 77.23,
84.31, 126.01, 126.21 (2C), 128.29 (2C), 140.53, 178.17, 195.92.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-(4′-
fluorophenyl)methyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol
Ester (14f). Followed general method using (R)-(1-amino-1-(4′-
fluorophenyl)methyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol
ester (13f) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic
acid as carboxylic acid. White solid, 268 mg in 60% yield. 1H NMR
(400 MHz, CDCl3): δ 0.80 (s, 3H), 1.23 (s, 3H), 1.27 (d, 3H, J = 4.4
Hz), 1.32 (s, 3H), 1.51 (d, 1H, J = 14.0 Hz), 1.77 (m, 1H), 1.89−1.99
(m, 2H), 2.01−2.06 (m, 1H), 2.21 (m, 1H), 2.32 (s, 3H), 2.69 (q,
1H, J = 6.8 Hz), 2.99−3.04 (m, 1H), 3.11−3.17 (m, 1H), 3.99 (s,
1H), 4.16 (d, 1H, J = 7.2 Hz), 6.94−6.98 (m, 2H), 7.10−7.14 (m,
2H), 7.31 (br, 1H). 13C NMR (100 MHz, CDCl3): δ 17.11, 24.10,
26.43, 27.23, 28.82, 30.61, 31.88, 36.22, 38.09, 38.72, 39.80, 52.03,
69.59, 77.27, 84.37, 115.00 (2C), 127.77 (2C), 136.37, 162.58,
178.03, 196.07.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-(4′-
fluorophenyl)methyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol
Ester (14f′). Followed general method using (S)-(1-amino-1-(4′-
fluorophenyl)methyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol
ester (13f′) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic
acid as carboxylic acid. White solid, 333 mg in 74% yield. 1H NMR
(400 MHz, CDCl3): δ 0.79 (s, 3H), 1.23 (s, 3H), 1.28 (d, 3H, J = 8.0
Hz), 1.31 (s, 3H), 1.48 (d, 1H, J = 14.0 Hz), 1.76 (m, 1H), 1.90−2.03
(m, 3H), 2.20 (m, 1H), 2.34 (s, 3H), 2.68 (q, 1H, J = 6.8 Hz), 3.03−
3.09 (m, 1H), 3.11−3.17 (m, 1H), 3.95 (s, 1H), 4.16 (d, 1H, J = 8.0
Hz), 6.97 (t, 2H, J = 8.4 Hz), 7.11−7.15 (m, 2H), 7.49 (br, 1H). 13C
NMR (150 MHz, CDCl3): δ 16.86, 24.04, 26.38, 27.18, 28.77, 30.57,
31.96, 36.20, 38.02, 38.54, 39.75, 52.02, 68.99, 76.96, 84.24, 114.96,
115.10, 127.71, 127.76, 136.32, 160.48, 178.02, 195.83.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-(4′-
methylphenyl)methyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinane-
diol Ester (14g). Followed general method using (R)-(1-amino-1-
(4′-methylphenyl)methyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinane-
diol ester (13g) as amine and (S)-(3-acetylsulfanyl-2-methyl)-
propionic acid as carboxylic acid. White solid, 235 mg in 53% yield.
1
H NMR (400 MHz, CDCl3): δ 0.79 (s, 3H), 1.24 (s, 3H), 1.26 (d,
3H, J = 6.0 Hz), 1.31 (s, 3H), 1.54 (d, 1H, J = 14.0 Hz), 1.74−1.80
(m, 1H), 1.87−1.98 (m, 2H), 1.99−2.08 (m, 1H), 2.17−2.23 (m,
1H), 2.29 (s, 3H), 2.31 (s, 3H), 2.62−2.68 (m, 1H), 2.98−3.05 (m,
1H), 3.08−3.16 (m, 1H), 3.97 (s, 1H), 4.16 (d, 1H, J = 8.4 Hz),
7.04−7.11 (m, 4H), 7.22 (br, 1H). 13C NMR (100 MHz, CDCl3): δ
17.14, 21.05, 24.10, 26.42, 27.25, 28.83, 30.59, 31.86, 36.20, 38.05,
38.70, 39.82, 52.01, 69.57, 77.03, 84.25, 126.27 (2C), 129.03 (2C),
135.46, 137.48, 177.70, 196.04.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-(4′-
methylphenyl)methyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinane-
diol Ester (14g′). Followed general method using (S)-(1-amino-1-
(4′-methylphenyl)methyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pina-
nediol ester (13g′) as amine and (S)-(3-acetylsulfanyl-2-methyl)-
propionic acid as carboxylic acid. White solid, 317 mg in 71% yield.
1
H NMR (400 MHz, CDCl3): δ 0.80 (s, 3H), 1.23 (s, 3H), 1.27 (d,
3H, J = 6.4 Hz), 1.34 (s, 3H), 1.56 (d, 1H, J = 14.0 Hz), 1.75−1.80
(m, 1H), 1.92−2.08 (m, 3H), 2.15−2.20 (m, 4H), 2.30 (s, 3H),
2.62−2.69 (m, 1H), 3.02−3.10 (m, 1H), 3.11−3.18 (m, 1H), 4.00 (s,
1H), 4.19 (d, 1H, J = 8.4 Hz), 6.94 (br, 1H), 7.08−7.15 (m, 4H). 13C
NMR (100 MHz, CDCl3): δ 16.89, 21.06, 24.10, 26.42, 27.24, 28.78,
30.63, 32.14, 36.16, 38.07, 38.92, 39.81, 51.99, 69.55, 77.17, 84.45,
126.39 (2C), 129.08 (2C), 135.58, 137.40, 177.38, 195.97.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-(2′-
naphthyl)methyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol
Ester (14h). Followed general method using (R)-(1-amino-1-(2′-
naphthyl)methyl)boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(13h) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as
carboxylic acid. White solid, 389 mg in 81% yield. 1H NMR (400
MHz, CDCl3): δ 0.77 (s, 3H), 1.21 (s, 3H), 1.27 (d, 3H, J = 10.0
Hz), 1.32 (s, 3H), 1.48 (d, 1H, J = 14.0 Hz), 1.72 (br, 1H), 1.88−1.92
DOI: 10.1021/acs.jmedchem.9b00735
J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
(m, 1H), 1.95−1.98 (m, 1H), 2.00−2.05 (m, 1H), 2.10−2.17 (m,
1H), 2.32 (s, 3H), 2.69−2.78 (m, 1H), 3.02−3.07 (m, 1H), 3.17 (dd,
1H, J = 13.6, 7.6 Hz), 4.17 (d, 1H, J = 8.4 Hz), 4.19 (s, 1H), 7.27−
7.34 (m, 2H), 7.37−7.46 (m, 2H), 7.57 (s, 1H), 7.76−7.79 (m, 3H).
13
C NMR (150 MHz, CDCl3): δ 17.13, 24.05, 26.43, 27.21, 28.82,
30.59, 31.78, 36.15, 38.03, 38.50, 39.80, 52.03, 76.93, 77.12, 84.24,
123.86, 125.12, 125.40, 125.83, 127.57, 127.58, 127.89, 132.16,
133.45, 138.32, 178.32, 196.03.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-1-(2′-
naphthyl)methyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol
Ester (14h′). Followed general method using (S)-(1-amino-1-(2′-
naphthyl)methyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol
ester (13h′) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic
acid as carboxylic acid. White solid, 317 mg in 66% yield. 1H NMR
(400 MHz, CDCl3): δ 0.75 (s, 3H), 1.20 (s, 3H), 1.24 (d, 3H, J =
10.0 Hz), 1.30 (s, 3H), 1.48 (d, 1H, J = 14.0 Hz), 1.71 (br, 1H), 1.95
(t, 1H, J = 5.2 Hz), 1.98−2.05 (m, 1H), 2.08−2.16 (m, 2H), 2.31 (s,
3H), 2.66−2.72 (m, 1H), 3.02 (dd, 1H, J = 13.6, 6.0 Hz), 3.14 (dd,
1H, J = 13.6, 7.6 Hz), 4.12−4.15 (m, 2H), 7.30 (d, 1H, J = 8.4 Hz),
7.36−7.45 (m, 3H), 7.54 (s, 1H), 7.72−7.78 (m, 3H). 13C NMR (150
MHz, CDCl3): δ 16.12, 23.04, 25.42, 26.20, 27.81, 29.58, 30.77,
35.14, 37.02, 37.49, 38.79, 51.02, 75.92, 76.11, 83.23, 122.85, 124.11,
124.39, 124.82, 126.56, 126.57, 126.88, 131.15, 132.44, 137.31,
177.31, 195.02.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-
methylpropyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol Ester
(14i). Followed general method using (R)-(1-amino-2-methylpropyl)-
boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester (13i) as amine
and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as carboxylic acid.
White solid, 238 mg in 60% yield. 1H NMR (400 MHz, CDCl3): δ
0.85 (s, 3H), 0.97 (dd, 6H, J = 6.4, 4.4 Hz), 1.24 (d, 3H, J = 6.8 Hz),
1.27−1.30 (m, 4H), 1.40 (s, 3H), 1.82−1.91 (m, 2H), 1.92−2.00 (m,
1H), 2.02 (t, 1H, J = 5.6 Hz), 2.13−2.21 (m, 1H), 2.29−2.38 (m,
4H), 2.47−2.57 (m, 1H), 2.94−3.03 (m, 2H), 3.09 (dd, 1H, J = 13.6,
7.6 Hz), 4.30 (d, 1H, J = 8.4 Hz), 6.23 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 17.42, 19.92, 20.32, 24.13, 26.42, 27.16, 28.83, 29.90,
30.64, 32.39, 35.83, 38.14, 39.70, 40.14, 51.52, 77.42, 84.95, 175.76,
196.32.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-2-
methylpropyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol Ester
(14i′). Followed general method using (S)-(1-amino-2-
methylpropyl)boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester
(13i′) as amine and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as
carboxylic acid. White solid, 255 mg in 64% yield. 1H NMR (400
MHz, CDCl3): δ 0.85 (s, 3H), 0.97 (d, 6H, J = 6.8 Hz), 1.24 (d, 3H, J
= 6.8 Hz), 1.28 (s, 3H), 1.30 (d, 1H, J = 11.6 Hz), 1.40 (s, 3H),
1.83−1.91 (m, 2H), 1.92−1.97 (m, 1H), 2.02 (t, 1H, J = 5.6 Hz),
2.13−2.20 (m, 1H), 2.29−2.38 (m, 4H), 2.48−2.57 (m, 1H), 2.90 (t,
1H, J = 5.6 Hz), 3.00 (dd, 1H, J = 13.6, 6.4 Hz), 3.09 (dd, 1H, J =
13.6, 7.6 Hz), 4.30 (d, 1H, J = 8.4 Hz), 6.29 (s, 1H). 13C NMR (100
MHz, CDCl3): δ 17.14, 19.92, 20.27, 24.08, 26.40, 27.12, 28.75,
29.78, 30.57, 32.38, 35.81, 38.08, 39.67, 39.88, 51.52, 77.30, 84.77,
175.74, 196.17.
[(1R)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-3-
methylbutyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol Ester
(14j). Followed general method using (R)-(1-amino-3-methylbutyl)-
boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester (13j) as amine
and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as carboxylic acid.
White solid, 292 mg in 71% yield. 1H NMR (400 MHz, CDCl3): δ
0.83 (s, 3H), 0.91 (d, 6H, J = 6.0 Hz), 1.22 (d, 3H, J = 6.8 Hz), 1.26
(s, 3H), 1.31 (d, 1H, J = 10.8 Hz), 1.38 (s, 3H), 1.42 (t, 2H, J = 7.6
Hz), 1.60−1.69 (m, 1H), 1.80−1.87 (m, 2H), 2.00 (t, 1H, J = 5.6
Hz), 2.10−2.17 (m, 1H), 2.30 (m, 4H), 2.47−2.54 (m, 1H), 2.92−
3.02 (m, 2H), 3.04−3.12 (m, 1H), 4.23 (d, 1H, J = 7.6 Hz), 6.39 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 17.16, 22.11, 23.13, 24.13,
25.68, 26.37, 27.23, 28.81, 30.60, 32.18, 36.01, 38.15, 38.72, 39.44,
39.80, 40.33, 51.75, 77.13, 84.64, 176.19, 196.24.
[(1S)-1-((2′S)-(3′-Acetylsulfanyl-2′-methylpropanamido))-3-
methylbutyl]boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol Ester
(14j′). Followed general method using (S)-(1-amino-3-methylbutyl)-
boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester (13j′) as amine
7176
Article
and (S)-(3-acetylsulfanyl-2-methyl)propionic acid as carboxylic acid.
White solid, 279 mg in 68% yield. 1H NMR (600 MHz, CDCl3): δ
0.84 (s, 3H), 0.90−0.94 (m, 6H), 1.22 (d, 3H, J = 6.6 Hz), 1.26 (s,
3H), 1.31 (d, 1H, J = 10.2 Hz), 1.37 (s, 3H), 1.44 (t, 2H, J = 7.6 Hz),
1.62−1.67 (m, 1H), 1.80−1.83 (m, 1H), 1.87 (m, 1H), 1.98−2.02
(m, 1H), 2.12−2.15 (m, 1H), 2.32 (m, 4H), 2.47−2.51 (m, 1H),
2.95−3.00 (m, 2H), 3.05−3.09 (m, 1H), 4.26 (d, 1H, J = 8.4 Hz),
6.25 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 16.99, 22.09, 23.16,
24.15, 25.69, 26.42, 27.25, 28.79, 30.61, 32.35, 36.03, 38.16, 38.78,
39.53, 39.81, 40.34, 51.77, 77.24, 84.68, 175.95, 196.28.
[( 1R )-1- (3′ -Acetylsulfanyl-2 ′-benzylpropanamido)-2-
phenylethyl]boronic Acid (1S,2S,3R,5S)-(+)-2,3-Pinanediol Ester
(15a). Followed general method using (R)-(1-amino-2-phenylethyl)-
boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester (13a) as amine
and (3-acetylsulfanyl-2-benzyl)propionic acid as carboxylic acid.
White solid, 214 mg in 41% yield. 1H NMR (600 MHz, CDCl3): δ
0.88 (s, 3H), 1.27−1.32 (m, 4H), 1.40 (s, 3H), 1.84−1.94 (m, 2H),
2.03 (br, 1H), 2.13−2.16 (m, 1H), 2.31−2.37 (m, 4H), 2.44−2.52
(m, 1H), 2.60−2.65 (m, 1H), 2.84−3.07 (m, 4H), 3.09−3.16 (m,
2H), 4.34 (m, 1H), 6.10 (s, 1H), 6.87 (d, 1H, J = 7.2 Hz), 7.05 (d,
1H, J = 7.2 Hz), 7.17−7.23 (m, 5H), 7.24−7.28 (m, 2H), 7.33 (t, 1H,
J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 24.13, 26.41, 27.24,
28.77, 30.60, 30.82, 35.98, 37.11, 37.78, 38.16, 39.83, 46.78, 51.78,
77.23, 84.86, 126.20, 126.81, 128.38, 128.47, 128.62, 128.77, 128.92,
128.98, 129.06, 129.12, 137.85, 139.88, 175.88, 195.65.
[(R)-1-(2′-Acetylsulfanylacetamido)-2-phenylethyl]boronic Acid
(1S,2S,3R,5S)-(+)-2,3-Pinanediol Ester (15e). Followed general
method using (R)-(1-amino-2-phenylethyl)boronic acid
(1S,2S,3R,5S)-(+)-2,3-pinanediol ester (13a) as amine and (2-
acetylsulfanyl)acetic acid as carboxylic acid. White solid, 154 mg in
37% yield. 1H NMR (400 MHz, CDCl3): δ 0.84 (s, 3H), 1.27 (s, 3H),
1.33 (d, 1H, J = 10.0 Hz), 1.37 (s, 3H), 1.81−1.93 (m, 2H), 1.98−
2.05 (m, 1H), 2.10−2.20 (m, 1H), 2.25−2.46 (m, 4H), 2.75−2.82
(m, 1H), 2.90−3.00 (m, 1H), 3.17−3.22 (m, 1H), 3.44−3.55 (m,
2H), 4.30 (t, 1H, J = 9.2 Hz), 6.38 (s, 1H), 7.12−7.36 (m, 5H). 13C
NMR (100 MHz, CDCl3): δ 23.07, 25.29, 26.14, 27.58, 29.14, 30.34,
34.63, 35.71, 37.16, 38.60, 50.49, 76.67, 84.55, 125.33, 127.46 (2C),
128.05 (2C), 138.58, 168.54, 193.85.
[(S)-1-(3′-Acetylsulfanylpropanamido)-2-(3′-benzofuran)ethyl]-
boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol Ester (16a). Followed
general method using (S)-(1-amino-2-(3′-benzofuran)ethyl)boronic
acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester (13d′) as amine and (3-
acetylsulfanyl)propionic acid as carboxylic acid. White solid, 244 mg
in 52% yield. 1H NMR (400 MHz, DMSO-d6): δ 0.80 (s, 3H), 1.20
(s, 3H), 1.23 (s, 3H), 1.27 (d, 1H, J = 10.4 Hz), 1.59−1.64 (m, 1H),
1.74 (br, 1H), 1.80 (t, 1H, J = 5.6 Hz), 1.90−1.97 (m, 1H), 2.13−
2.21 (m, 1H), 2.31 (s, 3H), 2.54 (t, 2H, J = 6.8 Hz), 2.61−2.69 (m,
1H), 2.78−2.82 (m, 2H), 3.01 (t, 2H, J = 6.8 Hz), 4.05 (d, 1H, J = 8.0
Hz), 7.22−7.31 (m, 2H), 7.55 (dd, 2H, J = 12.4, 8.0 Hz), 7.76 (s,
1H), 9.33 (s, 1H).
[(S)-1-(2′-Acetylsulfanylacetamido)-2-(3′-benzofuran)ethyl]-
boronic Acid (1R,2R,3S,5R)-(−)-2,3-Pinanediol Ester (16b). Followed
general method using (S)-(1-amino-2-(3′-benzofuran)ethyl)boronic
acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester (13d′) as amine and (2-
acetylsulfanyl)acetic acid as carboxylic acid. White solid, 168 mg in
37% yield. 1H NMR (400 MHz, DMSO-d6): δ 0.78 (s, 3H), 1.15 (d,
1H, J = 10.4 Hz), 1.20 (s, 3H), 1.21 (s, 3H), 1.58−1.62 (m, 1H), 1.73
(br, 1H), 1.81 (t, 1H, J = 5.6 Hz), 1.88−1.97 (m, 1H), 2.13−2.22 (m,
1H), 2.36 (s, 3H), 2.68−2.84 (m, 2H), 2.92 (m, 1H), 3.69 (s, 2H),
4.11 (d, 1H, J = 8.0 Hz), 7.22−7.31 (m, 2H), 7.53 (d, 1H, J = 8.0
Hz), 7.59 (d, 1H, J = 7.6 Hz), 7.74 (s, 1H), 9.07 (s, 1H).
General Method of Hydrolysis Reaction for Synthesis of
MS01−MS33. 3 N aqueous HCl solution (0.2 mL) was added into a
solution of protected amide (1 equiv), 2-methylpropylboronic acid (4
equiv) in methanol/hexane (1:1, 10 mL) at 0 °C. The reaction
solution was stirred at the same temperature until the TLC indicates
complete disappearance of amide. The reaction mixture was extracted
with hexane (5 mL) thrice, and the aqueous methanol layer was
concentrated at temperature below 30 °C. The residue was treated
with ice and basified with 2 N aqueous NaOH solution and extracted
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J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
with dichloromethane 5 times. The aqueous layer was then acidified
with 3 N aqueous HCl solution and extracted with dichloromethane
until the TLC indicates complete disappearance of product in
aqueous layer. The dichloromethane layer was collected, dried over
Na2SO4, and concentrated under reduced pressure to give a solid
residue, which was purified by flash chromatography on high
performance silica gel to obtain the final compounds MS01−MS33.
(S)-(1-(3′-Mercapto-2′-methylpropanamido)methyl)boronic
Acid (MS01). Followed general method using (S)-(1-(3′-acetylsul-
fanyl-2′-methylpropanamido)methyl)boronic acid pinacol ester (5a)
as amide. White solid. HPLC purity: 96.2%. 1H NMR (400 MHz,
CDCl3): δ 1.25 (d, 3H, J = 6.8 Hz), 1.64 (br, 1H), 2.31 (s, 2H), 2.60
(m, 1H), 2.76 (m, 1H), 2.98−3.14 (m, 1H), 6.37 (br, 1H). 13C NMR
(100 MHz, CDCl3): δ 17.04, 27.73, 31.95, 41.49, 178.34. MS (ESI+):
148.1 [M + H − H2O]+.
(1-(n-Butyramido)methyl)boronic Acid (MS02). Followed general
method using (1-(n-butyramido)methyl)boronic acid pinacol ester
(5b) as amide. White solid. HPLC purity: 98.0%. 1H NMR (400
MHz, CDCl3): δ 0.84 (t, 3H, J = 7.2 Hz), 1.54 (m, 2H), 2.14 (t, 2H, J
= 7.2 Hz), 2.36 (s, 2H), 7.80 (br, 1H). 13C NMR (100 MHz, CDCl3):
δ 13.65, 18.65, 36.82, 41.28, 176.80. MS (ESI+): 128.1 [M + H −
H2O]+. HRMS (ESI) m/z: calcd for C5H12BNO3[M + H − H2O]+,
128.0877, found, 128.0873; calcd for C5H12BNO3 [M + Na]+,
168.0802, found, 168.0807.
(1-(n-Propanamido)methyl)boronic Acid (MS03). Followed gen-
eral method using (1-(n-propanamido)methyl)boronic acid pinacol
ester (5c) as amide. White solid. HPLC purity: 97.8%. 1H NMR (400
MHz, CDCl3): δ 1.16 (t, 3H, J = 7.2 Hz), 2.41 (q, 2H, J = 7.2 Hz),
2.68 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 11.15, 21.35, 26.41,
179.41. MS (ESI+): 114.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C4H10BNO3 [M + H − H2O]+, 114.0721, found, 114.0718.
(S)-2-(3′-Mercapto-2′-methylpropanamido)acetic Acid (MS04).
Followed general method using (S)-2-(3′-acetylsulfanyl-2′-
methylpropanamido)acetic acid ethyl ester (7) as amide. White
solid. HPLC purity: 95.4%. 1H NMR (400 MHz, DMSO-d6): δ 1.07
(d, 3H, J = 6.8 Hz), 2.22 (t, 1H, J = 8.0 Hz), 2.40−2.49 (m, 2H), 2.66
(dt, 1H, J = 12.4, 6.8 Hz), 3.74 (s, 2H), 8.46 (br, 1H), 8.86 (br, 1H).
13
C NMR (100 MHz, DMSO-d6): δ 17.48, 27.91, 41.03, 43.61,
171.80, 174.83. MS (ESI+): 178.0 [M + H] +. HRMS (ESI) m/z:
calcd for C6H11NO3S [M + H]+, 178.0532, found, 178.0534; calcd for
C6H11NO3S [M + Na]+, 200.0352, found, 200.0354.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-
phenylethyl]boronic Acid (MS05). Followed general method using
[(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
phenylethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(14a) as amide. White solid. HPLC purity: 95.8%. 1H NMR (400
MHz, CDCl3): δ 1.19 (d, 3H, J = 6.8 Hz), 1.46 (t, 1H, J = 8.8 Hz),
2.52−2.59 (m, 2H), 2.67−2.74 (m, 2H), 2.93 (dd, 1H, J = 14.4, 5.2
Hz), 3.01−3.08 (m, 1H), 6.74 (br, 1H), 7.10−7.19 (m, 3H), 7.19−
7.24 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 15.19, 26.40, 35.72,
41.94, 76.22, 125.04, 127.47 (2C), 128.07 (2C), 139.76, 177.90. MS
(ESI+): 250.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for
C12H18BNO3S [M + H − H2O]+, 250.1068, found, 250.1066; calcd
for C12H18BNO3S [M + Na]+, 290.0993, found, 290.0993.
[(1R)-1-(3′-Mercapto-2′-benzylpropanamido)-2-phenylethyl]-
boronic Acid (MS06). Followed general method using [(1R)-1-(3′-
acetylsulfanyl-2′-benzylpropanamido)-2-phenylethyl]boronic acid
(1S,2S,3R,5S)-(+)-2,3-pinanediol ester (15a) as amide. White solid.
HPLC purity: 96.8%. 1H NMR (600 MHz, CDCl3): δ 1.70 (br, 1H),
2.56−2.84 (m, 5H), 2.89−2.98 (m, 2H), 3.08−3.11 (m, 1H), 6.15
(br, 1H), 6.85 (d, 1H, J = 7.2 Hz), 7.03 (d, 1H, J = 7.2 Hz), 7.15−
7.22 (m, 5H), 7.23−7.26 (m, 2H), 7.30−7.33 (m, 1H). 13C NMR
(100 MHz, CDCl3): δ 28.96, 35.77, 37.10, 49.77, 77.22, 126.14,
126.69, 128.38, 128.44, 128.61, 128.88, 128.91, 128.96, 129.05,
129.51, 137.54, 139.86, 175.17. MS (ESI+): 326.1 [M + H − H2O]+.
HRMS (ESI) m/z: calcd for C18H22BNO3S [M + H − H2O]+,
326.1381, found, 326.1377; calcd for C18H22BNO3S [M + Na]+,
366.1306, found, 366.1299.
[(1R)-1-((2′S)-(3′-Mercapto-2′-(1″,3″-dioxoisoindolin-2″-yl)-
propanamido))-2-phenylethyl]boronic Acid (MS07). Followed
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general method using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-(1″,3″-
dioxoisoindolin-2″-yl)propanamido))-2-phenylethyl]boronic acid
(1S,2S,3R,5S)-(+)-2,3-pinanediol ester (15b) as amide. White solid.
HPLC purity: 96.2%. 1H NMR (400 MHz, CDCl3): δ 1.61 (t, 1H, J =
8.8 Hz), 2.75−2.83 (m, 2H), 2.97−3.03 (m, 1H), 3.06−3.13 (m, 1H),
3.22−3.27 (m, 1H), 3.59 (t, 1H, J = 6.8 Hz), 6.59 (br, 1H), 7.21−
7.34 (m, 5H), 7.85−7.97 (m, 4H). 13C NMR (100 MHz, CDCl3): δ
27.81, 35.94, 63.93, 77.44, 123.11, 123.26, 125.95, 128.44 (2C),
129.17 (2C), 132.14 (2C), 132.41 (2C), 141.07 166.46, 166.67,
177.42. MS (ESI+): 381.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C19H19BN2O5S [M + Na]+, 421.1000, found, 421.1011.
[(1R)-1-((2′S)-(3′-Mercapto-2′-benzylaminopropanamido))-2-
phenylethyl]boronic Acid (MS08). Followed general method using
[(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-benzylaminopropanamido))-2-
phenylethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(15c) as amide. White solid. HPLC purity: 95.4%. 1H NMR (400
MHz, CDCl3): δ 1.56 (t, 1H, J = 8.4 Hz), 2.51−2.60 (m, 2H), 2.95−
3.00 (m, 1H), 3.31−3.37 (m, 1H), 3.53−3.57 (m, 1H), 3.91 (t, 1H, J
= 6.8 Hz), 4.68 (dd, 2H, J = 43.6, 16.6 Hz), 6.76 (br, 2H), 7.11−7.22
(m, 5H), 7.26−7.39 (m, 5H). 13C NMR (100 MHz, CDCl3): δ 28.68,
35.56, 52.41, 57.68, 76.21, 125.10, 125.34, 127.33, 127.37, 127.43,
127.51, 127.98, 128.05, 128.09, 128.24, 139.67, 139.78, 171.19. MS
(ESI+): 341.2 [M + H − H2O]+. HRMS (ESI) m/z: calcd for
C18H23BN2O3S [M + H − H2O]+, 341.1490, found, 341.1496; calcd
for C18H23BN2O3S [M + H]+, 359.1595, found, 359.1606.
[(R)-1-(3′-Mercaptopropanamido)-2-phenylethyl]boronic Acid
(MS09). Followed general method using [(R)-1-(3′-acetylsulfanyl-
propanamido)-2-phenylethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pi-
nanediol ester (15d) as amide. White solid. HPLC purity: 96.1%. 1H
NMR (400 MHz, CDCl3): δ 1.66 (t, 1H, J = 8.0 Hz), 2.54 (t, 2H, J =
6.8 Hz), 2.72−2.79 (m, 3H), 2.99 (dd, 1H, J = 14.4, 5.2 Hz), 3.05−
3.14 (m, 1H), 6.80 (br, 1H), 7.18−7.25 (m, 3H), 7.25−7.31 (m, 2H).
13
C NMR (100 MHz, CDCl3): δ 22.06, 29.69, 36.57, 77.22, 126.09,
128.42 (2C), 129.04 (2C), 140.77, 175.13. MS (ESI+): 236.1 [M + H
− H2O]+. HRMS (ESI) m/z: calcd for C11H16BNO3S [M + H −
H2O]+, 236.0911, found, 236.0912; calcd for C11H16BNO3S [M +
Na]+, 276.0836, found, 276.0833.
[(R)-1-(2′-Mercaptoacetamido)-2-phenylethyl]boronic Acid
(MS10). Followed general method using [(R)-1-(2′-acetylsulfanyla-
cetamido)-2-phenylethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinane-
diol ester (15e) as amide. White solid. HPLC purity: 95.4%. 1H NMR
(400 MHz, CDCl3): δ 1.88 (br, 1H), 2.70−2.78 (m, 1H), 2.96 (dd,
1H, J = 14.4, 5.2 Hz), 3.06−3.10 (m, 1H), 3.31 (d, 2H, J = 8.4 Hz),
7.19−7.22 (m, 3H), 7.27−7.32 (m, 2H), 7.37 (br, 1H). 13C NMR
(100 MHz, CDCl3): δ 31.59, 36.56, 77.23, 126.12, 128.53 (2C),
128.97 (2C), 140.64, 174.02. MS (ESI+): 222.1 [M + H − H2O]+.
HRMS (ESI) m/z: calcd for C10H14BNO3S [M + H − H2O]+,
222.0755, found, 222.0749; calcd for C10H14BNO3S [M + Na]+,
262.0680, found, 262.0684.
[(R)-1-(n-Butyramido)-2-phenylethyl]boronic Acid (MS11). Fol-
lowed general method using [(R)-1-(n-butyramido)-2-phenylethyl]-
boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester (15f) as amide.
White solid. HPLC purity: 96.0%. 1H NMR (400 MHz, CDCl3): δ
0.92 (t, 3H, J = 7.2 Hz), 1.62 (q, 2H, J = 7.2 Hz), 2.21 (t, 2H, J = 7.2
Hz), 2.58−2.79 (m, 1H), 2.79−2.99 (m, 1H), 3.00−3.09 (m, 1H),
6.72 (br, 1H), 7.15−7.22 (m, 3H), 7.24−7.28 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ 13.67, 18.67, 34.89, 36.84, 77.29, 125.85,
128.37 (2C), 129.10 (2C), 141.23, 176.82. MS (ESI+): 218.1 [M + H
− H2O]+. HRMS (ESI) m/z: calcd for C12H18BNO3[M + H −
H2O]+, 218.1347, found, 218.1347; calcd for C12H18BNO3[M + Na]+,
258.1272, found, 258.1268.
[(R)-1-(n-Propanamido)-2-phenylethyl]boronic Acid (MS12).
Followed general method using [(R)-1-(n-propanamido)-2-
phenylethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(15g) as amide. White solid. HPLC purity: 96.6%. 1H NMR (400
MHz, CDCl3): δ 1.13 (t, 3H, J = 7.2 Hz), 2.32 (q, 2H, J = 7.2 Hz),
3.08−3.15 (m, 1H), 3.19−3.26 (m, 1H), 3.67 (t, 1H, J = 8.0 Hz),
7.22−7.34 (m, 5H). 13C NMR (100 MHz, CDCl3): δ 10.78, 34.13,
39.86, 77.08, 125.19, 128.97 (2C), 129.90 (2C), 141.12, 176.82. MS
(ESI+): 204.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for
DOI: 10.1021/acs.jmedchem.9b00735
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Journal of Medicinal Chemistry
C11H16BNO3[M + H − H2O]+, 204.1190, found, 204.1148; calcd for
C11H16BNO3[M + Na]+, 244.1115, found, 244.1061.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-
phenylethyl]boronic Acid (MS13). Followed general method using
[(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
phenylethyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester
(14a′) as amide. White solid. HPLC purity: 95.6%. 1H NMR (400
MHz, CDCl3): δ 1.20 (d, 3H, J = 6.8 Hz), 1.59 (t, 1H, J = 8.8 Hz),
2.44−2.60 (m, 2H), 2.72−2.82 (m, 2H), 3.03 (dd, 1H, J = 14.0, 4.8
Hz), 3.07−3.14 (m, 1H), 6.57 (br, 1H), 7.16−7.26 (m, 3H), 7.27−
7.32 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 16.47, 27.91, 36.83,
41.99, 77.24, 125.95, 128.42 (2C), 129.14 (2C), 141.07, 177.52. MS
(ESI+): 250.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for
C12H18BNO3S [M + H − H2O]+, 250.1068, found, 250.1069; calcd
for C12H18BNO3S [M + Na]+, 290.0993, found, 290.0992.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-(4′-
fluorophenyl)ethyl]boronic Acid (MS14). Followed general method
using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
(4′-fluorophenyl)ethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol
ester (14b) as amide. HPLC purity: 97.4%. White solid. 1H NMR
(400 MHz, CDCl3): δ 1.20 (d, 3H, J = 6.8 Hz), 1.61 (t, 1H, J = 8.8
Hz), 2.53−2.58 (m, 1H), 2.62−2.66 (m, 1H), 2.69−2.77 (m, 2H),
2.92 (dd, 1H, J = 14.0, 5.2 Hz), 3.01−3.09 (m, 1H), 6.84 (br, 1H),
6.95 (t, 2H, J = 8.8 Hz), 7.16 (dd, 2H, J = 8.8, 5.6 Hz). 13C NMR
(100 MHz, CDCl3): δ 16.15, 27.67, 35.98, 41.27, 76.21, 115.05,
115.19, 130.33, 130.38, 136.25, 162.16, 178.03. MS (ESI+): 268.1 [M
+ H − H2O]+. HRMS (ESI) m/z: calcd for C12H13BFNO3S [M + H
− H2O]+, 268.0973, found, 268.0974; calcd for C12H13BFNO3S [M +
Na]+, 308.0898, found, 308.0896.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-(4′-
fluorophenyl)ethyl]boronic Acid (MS15). Followed general method
using [(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
(4′-fluorophenyl)ethyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinane-
diol ester (14b′) as amide. White solid. HPLC purity: 97.6%. 1H
NMR (400 MHz, CDCl3): δ 1.15 (d, 3H, J = 6.8 Hz), 1.56 (t, 1H, J =
8.8 Hz), 2.42−2.58 (m, 2H), 2.63−2.72 (m, 2H), 2.88 (dd, 1H, J =
14.0, 4.8 Hz), 2.97−3.06 (m, 1H), 6.64 (br, 1H), 6.90 (t, 2H, J = 8.8
Hz), 7.12 (dd, 2H, J = 8.8, 5.6 Hz). 13C NMR (100 MHz, CDCl3):
δ15.40, 26.79, 34.94, 40.76, 76.21, 114.06, 114.27, 129.38, 129.46,
135.34, 161.62, 176.97. MS (ESI+): 268.1 [M + H − H2O]+. HRMS
(ESI) m/z: calcd for C12H13BFNO3S [M + H − H2O]+, 268.0973,
found, 268.0969; calcd for C12H13BFNO3S [M + Na]+, 308.0898,
found, 308.0893.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-(4′-
methoxyphenyl)ethyl]boronic Acid (MS16). Followed general
method using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanami-
do))-2-(4′-methoxyphenyl)ethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-
pinanediol ester (14c) as amide. White solid. HPLC purity: 96.0%. 1H
NMR (400 MHz, CDCl3): δ 1.21 (d, 3H, J = 6.8 Hz), 1.68 (br, 1H),
2.55−2.79 (m, 4H), 2.93 (dd, 1H, J = 14.0, 5.2 Hz), 3.06−3.09 (m,
1H), 3.76 (s, 3H), 6.82 (d, 2H, J = 8.8 Hz), 7.16 (d, 2H, J = 8.8 Hz).
13
C NMR (150 MHz, CDCl3): δ 16.16, 27.77, 35.83, 41.31, 55.18,
77.20, 113.79 (2C), 129.98 (2C), 132.69, 157.86, 177.81. MS (ESI+):
280.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for C13H20BNO4S
[M + H − H 2O]+ , 280.1173, found, 280.1178; calcd for
C13H20BNO4S [M + Na]+, 320.1098, found, 320.1098.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-(4′-
methoxyphenyl)ethyl]boronic Acid (MS17). Followed general
method using [(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanami-
do))-2-(4′-methoxyphenyl)ethyl]boronic acid (1R,2R,3S,5R)-
(−)-2,3-pinanediol ester (14c′) as amide. White solid. HPLC purity:
96.9%. 1H NMR (400 MHz, CDCl3): δ 1.21 (d, 3H, J = 6.8 Hz), 1.64
(t, 1H, J = 8.8 Hz), 2.50−2.79 (m, 4H), 2.94 (dd, 1H, J = 14.0, 5.2
Hz), 3.05−3.15 (m, 1H), 3.77 (s, 3H), 6.82 (d, 2H, J = 8.8 Hz), 7.15
(d, 2H, J = 8.8 Hz). 13C NMR (100 MHz, CDCl3): δ 16.43, 27.87,
35.81, 41.84, 55.24, 77.26, 113.84 (2C), 130.04 (2C), 132.82, 157.91,
177.74. MS (ESI+): 280.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C13H20BNO4S [M + H − H2O]+, 280.1173, found,
280.1174; calcd for C13H20BNO4S [M + Na]+, 320.1098, found,
320.1094.
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[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-(3′-
benzofuran)ethyl]boronic Acid (MS18). Followed general method
using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
(3′-benzofuran)ethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol
ester (14d) as amide. White solid. HPLC purity: 96.2%. 1H NMR
(400 MHz, CDCl3): δ 1.17 (d, 3H, J = 7.2 Hz), 1.55 (t, 1H, J = 8.4
Hz), 2.45−2.56 (m, 2H), 2.70−2.87 (m, 3H), 3.18−3.21 (m, 1H),
7.18−7.24 (m, 2H), 7.43 (d, 1H, J = 8.0 Hz), 7.51 (s, 1H), 7.59 (d,
1H, J = 8.0 Hz). 13C NMR (150 MHz, CDCl3): δ 15.34, 23.67, 26.77,
40.84, 76.22, 110.32, 117.85, 119.03, 121.30, 123.14, 127.42, 141.00,
154.28, 176.93. MS (ESI+): 290.1 [M + H − H2O]+. HRMS (ESI)
m/z: calcd for C14H18BNO4S [M + H − H2O]+, 290.1017, found,
290.1021; calcd for C14H18BNO4S [M + Na]+, 330.0942, found,
330.0942.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-(3′-
benzofuran)ethyl]boronic Acid (MS19). Followed general method
using [(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
(3′-benzofuran)ethyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol
ester (14d′) as amide. White solid. HPLC purity: 96.1%. 1H NMR
(600 MHz, CDCl3): δ 1.18 (d, 3H, J = 7.2 Hz), 1.56 (t, 1H, J = 8.4
Hz), 2.44−2.49 (m, 1H), 2.53−2.58 (m, 1H), 2.71−2.77 (m, 1H),
2.81−2.87 (m, 1H), 3.01−3.04 (m, 1H), 3.18−3.25 (m, 1H), 6.81 (s,
1H), 7.20 (t, 1H, J = 7.2 Hz), 7.25 (t, 1H, J = 7.2 Hz), 7.43 (d, 1H, J
= 7.8 Hz), 7.52 (s, 1H), 7.59 (d, 1H, J = 7.8 Hz). 13C NMR (100
MHz, CDCl3): δ 16.38, 24.73, 27.83, 41.90, 77.24, 111.36, 118.90,
120.08, 122.34, 124.18, 128.46, 142.04, 155.34, 177.94. MS (ESI+):
290.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for C14H18BNO4S
[M + H − H 2 O]+ , 290.1017, found, 290.1022; calcd for
C14H18BNO4S [M + Na]+, 330.0942, found, 330.0940.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-
phenylmethyl]boronic Acid (MS20). Followed general method using
[(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-1-
phenylmethyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(14e) as amide. White solid. HPLC purity: 95.0%. 1H NMR (400
MHz, CDCl3): δ 1.13 (d, 3H, J = 6.4 Hz), 1.59 (br, 1H), 2.48−2.70
(m, 3H), 3.79 (s, 1H), 7.03−7.18 (m, 5H). 13C NMR (100 MHz,
CDCl3): δ16.23, 27.45, 42.94, 77.24, 126.02, 126.08, 128.53, 128.58,
134.50, 138.07, 175.02. MS (ESI+): 236.1 [M + H − H2O]+. HRMS
(ESI) m/z: calcd for C11H16BNO3S [M + H − H2O]+, 236.0911,
found, 236.0912; calcd for C11H16BNO3S [M + Na]+, 276.0836,
found, 276.0840.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-
phenylmethyl]boronic Acid (MS21). Followed general method using
[(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-1-
phenylmethyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester
(14e′) as amide. White solid. HPLC purity: 95.3%. 1H NMR (400
MHz, CDCl3): δ 1.19 (d, 3H, J = 5.2 Hz), 1.60 (br, 1H), 2.49−2.70
(m, 3H), 3.87 (s, 1H), 7.08−7.17 (m, 5H). 13C NMR (100 MHz,
CDCl3): δ 16.18, 27.41, 42.88, 77.19, 126.07 (2C), 128.54 (2C),
134.47, 138.09, 174.25. MS (ESI+): 236.1 [M + H − H2O]+. HRMS
(ESI) m/z: calcd for C11H16BNO3S [M + H − H2O]+, 236.0911,
found, 236.0908; calcd for C11H16BNO3S [M + Na]+, 276.0836,
found, 276.0833.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-(4′-
fluorophenyl)methyl]boronic Acid (MS22). Followed general
method using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanami-
do))-1-(4′-fluorophenyl)methyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-
pinanediol ester (14f) as amide. White solid. HPLC purity: 95.8%. 1H
NMR (400 MHz, CDCl3): δ 1.24 (d, 3H, J = 6.8 Hz), 1.52 (t, 1H, J =
8.4 Hz), 2.55−2.71 (m, 3H), 3.73 (d, 1H, J = 23.6 Hz), 6.77−6.83
(m, 2H), 6.86−6.91 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 16.23,
27.45, 42.93, 77.23, 114.48, 114.69, 127.11, 127.19, 136.91, 159.69,
178.56. MS (ESI+): 254.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C11H15BFNO3S [M + H − H2O]+, 254.0817, found,
254.0819; calcd for C11H15BFNO3S [M + Na]+, 294.0742, found,
294.0740.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-(4′-
fluorophenyl)methyl]boronic Acid (MS23). Followed general
method using [(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanami-
do))-1-(4′-fluorophenyl)methyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-
DOI: 10.1021/acs.jmedchem.9b00735
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Journal of Medicinal Chemistry
pinanediol ester (14f′) as amide. White solid. HPLC purity: 95.3%.
1
H NMR (400 MHz, DMSO-d6): δ 1.14 (d, 3H, J = 6.0 Hz), 2.28 (m,
1H), 2.60−2.68 (m, 3H), 3.52 (s, 1H), 6.90−6.97 (m, 4H), 9.34 (br,
1H). 13C NMR (100 MHz, DMSO-d6): δ 16.90, 27.41, 43.13, 77.29,
114.36, 114.57, 127.99, 128.07, 138.93, 159.38, 178.45. MS (ESI+):
254.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for C11H15BFNO3S
[M + H − H 2O]+ , 254.0817, found, 254.0819; calcd for
C11H15BFNO3S [M + Na]+, 294.0742, found, 294.0740.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-(4′-
methylphenyl)methyl]boronic Acid (MS24). Followed general
method using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanami-
do))-1-(4′-methylphenyl)methyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-
pinanediol ester (14g) as amide. White solid. HPLC purity: 95.7%.
1
H NMR (400 MHz, CDCl3): δ 1.15 (br, 3H), 1.53 (m, 1H), 2.28 (s,
3H), 2.52−2.65 (m, 3H), 3.77 (d, 1H, J = 22.8 Hz), 6.75−6.85 (m,
2H), 6.93−6.96 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 16.24,
21.07, 27.64, 42.95, 77.23, 126.01, 126.08, 128.52, 128.58, 134.49,
138.06, 175.02. MS (ESI+): 250.1 [M + H − H2O]+. HRMS (ESI)
m/z: calcd for C12H18BNO3S [M + H − H2O]+, 250.1068, found,
250.1069; calcd for C12H18BNO3S [M + Na]+, 290.0993, found,
290.0989.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-(4′-
methylphenyl)methyl]boronic Acid (MS25). Followed general
method using [(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanami-
do))-1-(4′-methylphenyl)methyl]boronic acid (1R,2R,3S,5R)-
(−)-2,3-pinanediol ester (14g′) as amide. White solid. HPLC purity:
96.1%. 1H NMR (400 MHz, CDCl3): δ 1.15 (d, 3H, J = 4.4 Hz), 1.53
(m, 1H), 2.28 (s, 3H), 2.43−2.64 (m, 3H), 3.75 (d, 1H, J = 22.4 Hz),
6.78−6.85 (m, 2H), 6.92−6.95 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 16.54, 21.07, 27.52, 41.42, 77.23, 126.06 (2C), 128.54
(2C), 134.46, 138.09, 174.24. MS (ESI+): 250.1 [M + H − H2O]+.
HRMS (ESI) m/z: calcd for C12H18BNO3S [M + H − H2O]+,
250.1068, found, 250.1064; calcd for C12H18BNO3S [M + Na]+,
290.0993, found, 290.0991.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-(2′-
naphthyl)methyl]boronic Acid (MS26). Followed general method
using [(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-1-
(2′-naphthyl)methyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol
ester (14h) as amide. White solid. HPLC purity: 95.7%. 1H NMR
(600 MHz, DMSO-d6): δ 1.06 (d, 3H, J = 6.6 Hz), 1.50 (t, 1H, J = 8.4
Hz), 2.57−2.60 (m, 1H), 2.64−2.72 (m, 2H), 3.75 (d, 1H, J = 8.4
Hz), 7.00 (d, 1H, J = 7.8 Hz), 7.39−7.43 (m, 4H), 7.66 (d, 1H, J =
7.8 Hz), 7.75 (d, 1H, J = 7.8 Hz), 9.08 (br, 1H). 13C NMR (100
MHz, DMSO-d6): δ17.03, 27.50, 43.16, 77.38, 122.98, 125.03,
126.09, 127.11, 127.59, 127.83, 130.13, 131.69, 133.41, 141.02,
178.39. MS (ESI+): 286.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C15H18BNO3S [M + H − H2O]+, 286.1068, found,
286.1043; calcd for C15H18BNO3S [M + Na]+, 326.0993, found,
326.0971.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-1-(2′-
naphthyl)methyl]boronic Acid (MS27). Followed general method
using [(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-1-
(2′-naphthyl)methyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol
ester (14h′) as amide. White solid. HPLC purity: 96.6%. 1H NMR
(400 MHz, CDCl3): δ 1.10 (d, 3H, J = 6.8 Hz), 1.50 (t, 1H, J = 8.8
Hz), 2.62−2.69 (m, 1H), 2.70−2.77 (m, 2H), 3.72 (d, 1H, J = 8.8
Hz), 7.02 (d, 1H, J = 6.8 Hz), 7.37−7.44 (m, 4H), 7.58 (d, 1H, J =
6.8 Hz), 7.67 (d, 1H, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3):
δ17.10, 27.33, 42.81, 77.11, 122.94, 125.03, 125.61, 126.11, 127.12,
127.57, 127.85, 131.70, 133.45, 141.03, 178.32. MS (ESI+): 286.1 [M
+ H − H2O]+. HRMS (ESI) m/z: calcd for C15H18BNO3S [M + H −
H2O]+, 286.1068, found, 286.1071; calcd for C15H18BNO3S [M +
Na]+, 326.0993, found, 326.0990.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-
methylpropyl]boronic Acid (MS28). Followed general method using
[(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
methylpropyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(14i) as amide. White solid. HPLC purity: 95.4%. 1H NMR (400
MHz, CDCl3): δ 0.91 (t, 6H, J = 7.2 Hz), 1.23 (d, 3H, J = 6.8 Hz),
1.60−1.71 (m, 1H), 1.83−1.93 (m, 1H), 2.57−2.66 (m, 3H), 2.75−
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2.81 (m, 1H). 13C NMR (150 MHz, CDCl3): δ 16.44, 20.14, 20.67,
27.72, 29.36, 41.70, 77.24, 177.91. MS (ESI+): 202.1 [M + H −
H2O]+. HRMS (ESI) m/z: calcd for C8H18BNO3S [M + H − H2O]+,
202.1068, found, 202.1066; calcd for C8H18BNO3S [M + Na]+,
242.0993, found, 242.0987.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-2-
methylpropyl]boronic Acid (MS29). Followed general method using
[(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-2-
methylpropyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester
(14i′) as amide. White solid. HPLC purity: 95.1%. 1H NMR (400
MHz, CDCl3): δ 0.91 (d, 3H, J = 6.4 Hz), 0.94 (d, 3H, J = 6.4 Hz),
1.24 (d, 3H, J = 6.4 Hz), 1.66 (t, 1H, J = 8.4 Hz), 1.90−1.96 (m, 1H),
2.57−2.65 (m, 3H), 2.76−2.82 (m, 1H), 7.05 (br, 1H). 13C NMR
(150 MHz, CDCl3): δ 16.48, 20.24, 20.58, 27.80, 29.30, 41.99, 77.16,
177.37. MS (ESI+): 202.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C8H18BNO3S [M + H − H2O]+, 202.1068, found, 202.1065;
calcd for C8H18BNO3S [M + Na]+, 242.0993, found, 242.0984.
[(1R)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-3-
methylbutyl]boronic Acid (MS30). Followed general method using
[(1R)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-3-
methylbutyl]boronic acid (1S,2S,3R,5S)-(+)-2,3-pinanediol ester
(14j) as amide. White solid. HPLC purity: 97.2%. 1H NMR (400
MHz, CDCl3): δ 0.89 (d, 3H, J = 1.6 Hz), 0.91 (d, 3H, J = 1.6 Hz),
1.26 (d, 3H, J = 6.8 Hz), 1.32−1.39 (m, 1H), 1.47−1.54 (m, 1H),
1.60−1.70 (m, 2H), 2.58−2.66 (m, 2H), 2.78−2.84 (m, 1H), 2.92−
2.97 (m, 1H), 7.23 (br, 1H). 13C NMR (150 MHz, CDCl3): δ 16.32,
22.78, 23.08, 25.99, 27.87, 40.33, 41.69, 77.20, 177.25. MS (ESI+):
216.1 [M + H − H2O]+. HRMS (ESI) m/z: calcd for C9H20BNO3S
[M + H − H2O]+, 216.1224, found, 216.1220; calcd for C9H20BNO3S
[M + Na]+, 256.1149, found, 256.1142.
[(1S)-1-((2′S)-(3′-Mercapto-2′-methylpropanamido))-3-
methylbutyl]boronic Acid (MS31). Followed general method using
[(1S)-1-((2′S)-(3′-acetylsulfanyl-2′-methylpropanamido))-3-
methylbutyl]boronic acid (1R,2R,3S,5R)-(−)-2,3-pinanediol ester
(14j′) as amide. White solid. HPLC purity: 95.6%. 1H NMR (400
MHz, CDCl3): δ 0.88 (t, 6H, J = 6.8 Hz), 1.22 (d, 3H, J = 6.8 Hz),
1.31−1.41 (m, 1H), 1.44−1.53 (m, 1H), 1.56−1.72 (m, 2H), 2.54−
2.69 (m, 2H), 2.76−2.83 (m, 1H), 2.92 (m, 1H), 7.27 (br, 1H). 13C
NMR (150 MHz, CDCl3): δ 16.31, 22.67, 23.11, 26.01, 27.83, 40.20,
41.72, 77.21, 177.48. MS (ESI+): 216.1 [M + H − H2O]+. HRMS
(ESI) m/z: calcd for C9H20BNO3S [M + H − H2O]+, 216.1224,
found, 216.1219; calcd for C9H20BNO3S [M + Na]+, 256.1149, found,
256.1140.
[(S)-1-(3′-Mercaptopropanamido))-2-(3′-benzofuran)ethyl]-
boronic Acid (MS32). Followed general method using [(S)-1-(3′-
acetylsulfanylpropanamido)-2-(3′-benzofuran)ethyl]boronic acid
(1R,2R,3S,5R)-(−)-2,3-pinanediol ester (16a) as amide. White
solid. HPLC purity: 95.9%. 1H NMR (400 MHz, DMSO-d6): δ
1.56 (t, 1H, J = 8.4 Hz), 2.49 (t, 2H, J = 6.8 Hz), 2.71−2.79 (m, 3H),
2.98−3.03 (m, 1H), 3.06−3.11 (m, 1H), 6.77 (br, 1H), 7.17−7.26
(m, 2H), 7.48−7.53 (m, 2H), 7.71 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 20.70, 29.79, 42.47, 77.30, 111.34, 118.88, 120.05,
122.33, 124.16, 128.43, 142.04, 155.30, 177.94. MS (ESI+): 276.1 [M
+ H − H2O]+. HRMS (ESI) m/z: calcd for C13H16BNO4S [M + H −
H2O]+, 276.0860, found, 276.0869; calcd for C13H16BNO4S [M +
Na]+, 316.0785, found, 316.0787.
[(S)-1-(2′-Mercaptoacetamido))-2-(3′-benzofuran)ethyl]boronic
Acid (MS33). Followed general method using [(S)-1-(2′-
acetylsulfanylacetamido)-2-(3′-benzofuran)ethyl]boronic acid
(1R,2R,3S,5R)-(−)-2,3-pinanediol ester (16b) as amide. White
solid. HPLC purity: 95.2%. 1H NMR (400 MHz, CDCl3): δ 1.60
(t, 1H, J = 8.8 Hz), 2.84−2.92 (m, 1H), 2.96−3.09 (m, 2H), 3.47 (d,
2H, J = 6.8 Hz), 6.74 (br, 1H), 7.30−7.39 (m, 2H), 7.60−7.67 (m,
2H), 7.83 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 25.26, 36.24,
77.13, 111.41, 118.42, 119.81, 122.45, 124.43, 127.01, 142.01, 157.45,
177.17. MS (ESI+): 262.1 [M + H − H2O]+. HRMS (ESI) m/z:
calcd for C12H14BNO4S [M + H − H2O]+, 262.0704, found,
262.0710; calcd for C12H14BNO4S [M + Na]+, 302.0629, found,
302.0633.
DOI: 10.1021/acs.jmedchem.9b00735
J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
The NMR and HRMS spectra of MS01−MS33 are given in
Supporting Information.
Constructs, Protein Expression, and Purification. The NDM-
1 (aa 1−270), Sfh-I (aa 3−234), GOB-18 (aa 1−290), KPC-2 (aa
29−289), TEM-1 (aa 24−286), AmpC (aa 20−377), and OXA-48
(aa 1−265) (protein sequences given in Table S5) were cloned into
pET28a vectors for expression of N-terminally His6-Tagged proteins
that can be cleaved by TEV protease; the cloned VIM-2 (aa 27−266)
plasmids were obtained from C. J. Schofield group in University of
Oxford. All the proteins were overexpressed in E. coli Transetta (DE3)
cells (Novagen) at 37 °C using LB medium supplemented with 50 μg·
mL−1 ampicillin and 50 μg·mL−1 chloramphenicol (for KPC-2,
additionally complemented with 200 mM sorbitol and 5 mM
betaine). When cells grew to an OD600 of 0.6, the temperature was
lowered to 20 °C (for VIM-2 and NDM-1), 16 °C (for Sfh-1, GOB-
18, KPC-2, AmpC, and OXA-48), or 27 °C (for TEM-1), and 0.5 mM
IPTG was then added to induce the protein expression for 18−20 h.
Cells were harvested by centrifugation (15 min, 4000 rpm),
resuspended in lysis buffer A (20 mM Tris-HCl, pH 8.0, 250 mM
NaCl) supplemented with EDTA-free protease inhibitor, and lysed by
using an ultrahigh-pressure homogenizer (JNBIO). Cellular debris
were removed by centrifugation of 15 000 rpm for 30 min, and the
supernatant were loaded onto an Ni-NTA column (Roche), followed
by extensive washing with buffer B (20 mM Tris-HCl, pH 8.0, 250
mM NaCl, 5 mM imidazole) to remove nonspecifically binding
proteins. The target proteins were eluted with buffer C (20 mM Tris-
HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole). Fractions
containing the purified enzymes were concentrated using Amicon
Ultra 10K (Millipore) and then desalted using a HiTrap desalting
column (GE Healthcare) into the assay buffer (see Table S6) for
enzyme kinetic analyses.
The His-Tags of VIM-2 and KPC-2 proteins were further removed
by TEV protease cleavage (1:100 w/w) at 4 °C overnight and
captured on Ni-NTA resin (Roche). Proteins were then loaded onto a
Superdex S75 column equilibrated in buffer D (20 mM Tris-HCl, pH
7.5, 200 mM NaCl, 0.5 mM TCEP) for VIM-2 or buffer E (20 mM
Tris-HCl, pH 8.0, 300 mM NaCl) for KPC-2, and concentrated for
crystallization. The protein concentrations were determined using a
NanoDrop 2000 spectrophotometer (Thermo Scientic). All the
proteins were stored at −80 °C.
Enzyme Kinetic Assays. All determinations were performed at
room temperature (∼25 °C) by using a Thermo microplate reader
and 96-well flat-bottom black plates. All the enzymes and substrates
were dissolved in the assay buffers (Table S6). The activity for all the
purified enzymes was determined using the fluorescent substrate
FC548 and monitored by following the variation in fluorescence at λex
of 380 nm and λem of 460 nm. Ten different concentrations of FC5
were used to determine the kinetic parameters (KM and kcat), which
were obtained by fitting the initial velocity data to the Michaelis−
Menten equation using GraphPad Prism software (La Jolla, CA).
Compound IC50/Ki Determinations. All the compounds were
freshly prepared in 100 mM DMSO stock solutions. The IC50 values
were determined by preincubation of the appropriate amount of
enzymes with the desired compound (with 10 different concen-
trations in 3-fold dilution) in the assay buffer for 10 min, followed by
adding the substrate FC5 to initiate the reactions and monitoring the
fluorescence at λex of 380 nm and λem of 460 nm. All determinations
were tested in triplicate. The IC50/pIC50/se pIC50 values were
obtained from the plot of activity versus inhibitor concentration using
the GraphPad Prism software. All the IC50 curves are given in
Supporting Information Figures S2, S8, and S10. The Ki values were
calculated from the IC50 values using the Cheng−Prusoff equation:49
IC50
Ki =
[S]
1+ KM
where [S] is the FC5 concentration for activity test (Table S6) and
KM is the Michaelis constant of the enzymes (Table S1).
Jump Dilution Recovery Assays. The reversibility of the VIM-2
inhibition by MS01, MS05, MS18, MS19, or L-captopril was
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examined by the jump-dilution recovery method.71 Incubation of 20
nM VIM-2 enzymes (the enzyme concentration equal to 100-fold of
that used in the activity assays) was with 9.6 μM MS01, 4.5 μM
MS05, 5.6 μM MS18, 0.89 μM MS19, or 6.5 μM L-captopril (i.e., the
concentration of inhibitor equal to 10-fold of their respective IC50
value to VIM-2) for 10 min at room temperature. Then, the samples
were rapidly diluted 100-fold by adding the substrate FC-5 containing
buffer, followed by measuring the enzyme activity. The reversibility of
the KPC-2 inhibition by MS01, MS05, MS18, MS19, or tazobactam
was determined similarly as described above.
X-ray Crystallography. The crystal structures of VIM-2:MS01
(PDB code 6J8R) and VIM-2:MS19 (PDB code 6JN6), KPC-2:MS01
(PDB code 6J8Q), KPC-2:MS05 (PDB code 6JN3), KPC-2:MS22
(PDB code 6JN4), and KPC-2:MS23 (PDB code 6JN5) were
obtained by cocrystallization experiments. The purified VIM-2 and
KPC-2 proteins were freshly prepared to a concentration of 10 mg·
mL−1 in the crystallization buffer (for VIM-2, 20 mM Tris-HCl, pH
7.5, 200 mM NaCl, and 0.5 mM TCEP; for KPC-2, 20 mM Tris-HCl,
pH 8.0, 300 mM NaCl) and were then added with 5 mM inhibitor.
The protein/inhibitor mixtures were incubated for 1 h at 4 °C prior to
crystallization. The crystallization drops constituted a 1:1 ratio of
protein/inhibitor mixtures/reservoir solutions. The crystallization
conditions for VIM-2 are 23−30% PEG 3350, 0.2 M magnesium
formate, and for KPC-2, conditions are 32−35% PEG 8000, 0.1 M
lithium sulfate, 0.05 M sodium acetate, pH 4.5. The VIM-2 and KPC-
2 protein crystals were cryoprotected using a solution of 19% (v/v)
glycerol and perfluoropolyether oil (from Aladdin), respectively, and
flash-cooled in liquid nitrogen. X-ray diffraction data were obtained at
the Shanghai Synchrotron Radiation Facility (SSRF) BL19U1
beamline and processed using HKL2000. The molecular replacement
and refinement were carried out as previously described.37,38,50
Crystallization conditions are in Table S2, and data collection and
refinement statistics are in Tables S3 and S4.
ACE-2 Inhibition Assays. The ACE-2 proteins were purchased
from Sigma-Aldrich (catalog no. SAE0064), and the substrate Mca-
Ala-Pro-Lys(Dnp)-OH (CAS no. 305336-82-7) was purchased from
Leon Biological Technology (catalog no. P180529-3). The ACE-2
enzymes (4 nM) were incubated with each compound (100 μM or 50
μM with a final DMSO concentration ≤0.5%) for 10 min at 37 °C.
Then, the substrate Mca-Ala-Pro-Lys(Dnp)-OH (2 μM) was added to
start the reactions, and the fluorescence was recorded at λex of 328 nm
and λem of 393 nm at 37 °C using a Thermo microplate reader. All
determinations were performed in triplicate.
Serine Hydrolyase Inhibition Assays. Cell Culture and Protein
Preparation. Human embryonic kidney 293T cells (HEK293T) were
cultured in Dulbecco’s modified Eagle medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and
100 U/mL streptomycin at 37 °C in a humidified 5% CO2
atmosphere. Cells were passaged every 2−3 days and harvested by
suspending them in 10 mL of medium. The suspension was
centrifuged for 5 min at 1000 rpm, and the supernatant was removed.
The cell pellet was flash-frozen in liquid nitrogen and stored at −80
°C. Cell pellets were thawed on ice and resuspended in lysis buffer
(20 mM HEPES, pH 7.2, 1 mM MgCl2, 0.25 M sucrose, 2 mM DTT,
25 U/mL Benzonase). The suspension was homogenized by pipet.
Protein concentration was determined by NanoDrop 2000
spectrophotometer (Thermo Scientic). The protein fractions were
diluted to a total protein concentration of 2 mg/mL and stored in
small aliquots at −80 °C.
E. coli Transetta (DE3) cells (Novagen) were growing at 37 °C
using LB medium as described above. Cells were harvested by
centrifugation (15 min, 4000 rpm), followed by resuspending in the
lysis buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl) supplemented
with EDTA-free protease inhibitor, and then lysed using an ultrahigh-
pressure homogenizer (JNBIO). Cellular debris was removed by
centrifugation of the lysate at 15 000 rpm for 30 min, and the
supernatant was resuspended in the lysis buffer (20 mM HEPES, pH
7.2, 1 mM MgCl2, 0.25 M sucrose, 2 mM DTT, 25 U/mL
Benzonase). The protein fractions were diluted to a total protein
DOI: 10.1021/acs.jmedchem.9b00735
J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
concentration of 5 mg/mL and stored in small aliquots at −80 °C for
later use.
Selectivity Profile by Activity-Based Protein Profiling (ABPP). For
gel-based ABPP experiments, the extracted proteome from HEK293T
and E. coli cells was preincubated for 30 min with vehicle (DMSO) or
inhibitor (50 μM) at rt, followed by the treatment with activity-based
probe TAMRA-FP (750 nM, final concentration) (from Thermo
Fischer Scientific) for 20 min at rt. The reactions were quenched with
5 μL of standard 5× SDS−PAGE loading buffer. The samples were
directly loaded and resolved on SDS−PAGE gel (12% acrylamide).
The gels were then scanned with a ChemiDoc MP system (Cy 3
settings, 605/50 filter) and analyzed using Image Lab 4.1.
Microbiological Susceptibility Testing. The inhibitors MS01,
MS18, and MS19 (L-captopril and tazobactam as control com-
pounds) were tested against seven clinically isolated bacterial strains
producing NDM-1, KPC-2 or AmpC, including E. coli BAA-2452
(blaNDM‑1), K. pneumoniae 13249 (blaNDM‑1), E. coli BAA-2340
(blaKPC‑2), E. coli 11119 (blaKPC‑2), K. pneumoniae BAA-1705
(blaKPC‑2), K. pneumoniae 5846 (blaKPC‑2), and K. pneumoniae C660
(blaAmpC). The MIC values of meropenem against the bacterial strains
were determined with or without the inhibitors by broth micro-
dilution according to the Clinical Laboratory Standards Institute
(CLSI) guidelines.72 Meropenem and inhibitors were first dissolved in
DMSO and then diluted in cation-adjusted Mueller−Hinton II broth
(CAMHB). The 0.5 McFarland suspension of the bacterial strains in
0.85% NaCl was diluted in CAMHB broth. The inoculum were
seeded in 96-well microtiter plates to make the final concentration of
5 × 105 CFU/mL and treated with meropenem (128−0.25 μg/mL in
2-fold dilution) and/or the inhibitors (50 μM). The microtiter plates
were incubated at 37 °C for 16−20 h, followed by visual evaluation of
the bacterial growth.
Cytotoxicity Assays. HEK293 cells were plated at ∼5000 cells/
well in 96-well plate 24 h prior to treatment with the inhibitors at a
concentration of 100 μM and incubated for another 72 h. 20 μL of
MTT solution (5.0 mg/mL) was added to the medium and incubated
for another 4 h. Then, 150 μL of DMSO was added to each well after
removing MTT solution. The absorbance was read at 570 nm
wavelength on an automatic microplate spectrophotometer.
Molecular Docking and LEADOPT Optimization. The
molecular docking simulations were carried out by using AutoDock
Vina program.73 The crystal structures of Sfh-I (PDB code 3SD9)53
and GOB-18 (PDB code 5K0W)74 were used as the protein
templates. The protein and ligand structures were prepared as
previously described.32,75 The docking poses were viewed using the
PyMOL program. The LEADOPT program61 was used to perform
fragment-growing analyses, which involves a fragment library
consisting of 6877 unique fragments. The binding conformations of
MS01 with VIM-2 (PDB code 6J8R) and KPC-2 (PDB code 6J8Q)
were kept unchanged, and the two hydrogens at the 1-position of
MS01 were set as the fragment growing points. According the binding
site information, LEADOPT generated a number of potential
derivatives with predicted binding affinities. The top-ranked
compounds were visually inspected and further considered with
respect to the synthetic accessibility.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.9b00735.
Tables showing kinetic data for FC5 with MBL and SBL
enzymes, enzymatic assay conditions, crystallization
conditions, crystallographic data collection and refine-
ment statistics; figures showing Michaelis−Menten plots
of enzymes catalyzed hydrolysis of FC5, IC50/Ki curves
of dual-action inhibitors, modes of inhibitor binding as
defined by electron density maps, and structure
comparison; synthetic methods and characterization
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data for the intermediates that are not included in the
main text; and NMR and HRMS spectra for the target
compounds (PDF)
Molecular formula strings and the MBL/SBL Ki data
(CSV)
Molecular docking model of GOB-18:MS01 (PDB)
Molecular docking model of Sfh-I:MS01 (PDB)
Accession Codes
The coordinates and structure factors of VIM-2:MS01 (PDB
code 6J8R), VIM-2:MS19 (PDB code 6JN6), KPC-2:MS01
(PDB code 6J8Q), KPC-2:MS05 (PDB code 6JN3), KPC-
2:MS22 (PDB code 6JN4), and KPC-2:MS23 (PDB code
6JN5) complex structures have been deposited with the
Protein Data Bank. Authors will release the atomic coordinates
and experimental data upon article publication.
■
AUTHOR INFORMATION
Corresponding Authors
*Y.W.: phone, +86-028-85503235; e-mail, wyong@scu.edu.cn.
*G.-B.L.: phone, +86-135-5016-1826; e-mail, liguobo@scu.
edu.cn.
ORCID
Yong Wu: 0000-0003-0719-4963
Guo-Bo Li: 0000-0002-4915-6677
Author Contributions
§
Y.-L.W. and S.L. are co-first-authors. G.B.L. and Y.W.
designed the project. Y.-L.W, M.-Y.H., Y.L., Q.M., and Y.Z.
performed molecule design and chemical synthesies and
analyzed the NMR and mass spectra, guided by G.-B.L. and
Y.W.; S.L., Z.-J.Y., and Y.-H.Y. performed protein production
and kinetic experiments, guided by G.-B.L.; S.L., G.-B.L., C.W.,
Y.Y., and Q.C. performed crystal experiments and structural
refinements; S.L. and H.D. performed the selectivity assays;
Y.S. and Z.W. performed microbiological susceptibility testing;
G.-B.L., Y.-L.W., and S.L. wrote the manuscript. All authors
read and approved the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the funds from the National
Natural Science Foundation of China (Grants 81874291,
8177357, 81502989, and 21807076), the Sichuan Science and
Technology Program (Grant 2018HH0100), the Scientic
Research Foundation of Sichuan University (Grants
2082604401098 and 20822041A4193), and the Fundamental
Research Funds for the Central Universities. The authors
thank the staff of BL19U1 beamline of the National Center for
Protein Science Shanghai at Shanghai Synchrotron Radiation
Facility for assistance during data collection.
■
ABBREVIATIONS USED
MBL, metallo-β-lactamase; SBL, serine-β-lactamase; SAR,
structure−activity relationship; MBP, metal-binding pharma-
cophore; ACE, angiotensin-converting enzyme; VIM-2, Verona
integron-encoded MBL-2; NDM-1, New Delhi MBL-1; KPC-
2, Klebsiella pneumoniae carbapenemase-2; OXA-48, oxacilli-
nase-48; TAMRA-FP, carboxytetramethylrhodamine fluoro-
phosphonate; IPTG, isopropyl β-D-1-thiogalactopyranoside;
EDTA, ethylenediaminetetraacetic acid; TCEP, tris(2-
carboxyethyl)phosphine; DMSO, dimethyl sulfoxide; DTT,
DOI: 10.1021/acs.jmedchem.9b00735
J. Med. Chem. 2019, 62, 7160−7184
Journal of Medicinal Chemistry
dithiothreitol; SDS−PAGE, sodium dodecyl sulfate−polyacry-
lamide gel electrophoresis; MTT, 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide; CFU, colony-forming
unit; aa, amino acids; PDB, Protein Data Bank; n-BuLi, n-
butyllithium; THF, tetrahydrofuran; LiHMDS, lithium bis-
(trimethylsilyl)amide; TBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate; DIPEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
NaOtBu, sodium tert-butoxide; Et2O, diethyl ether; CH2Cl2,
dichloromethane; HCl, hydrogen chloride; CuBr, cupric
bromide; Na2SO4, sodium sulfate; TLC, thin-layer chromatog-
raphy; NaOH, sodium hydroxide
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