Discovery of ANT3310, a Novel Broad-Spectrum Serine β-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and Acinetobacter baumannii

Article abstract of DOI:10.1021/acs.jmedchem.0c01535

The diazabicyclooctanes (DBOs) are a class of serine β-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of β-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of β-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen"producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.

Full text of DOI:10.1021/acs.jmedchem.0c01535

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Article
Discovery of ANT3310, a Novel Broad-Spectrum Serine β‑Lactamase
Inhibitor of the Diazabicyclooctane Class, Which Strongly
Potentiates Meropenem Activity against Carbapenem-Resistant
Enterobacterales and Acinetobacter baumannii
́
̂
David T. Davies,* Simon Leiris, Magdalena Zalacain, Nicolas Sprynski, Jerome Castandet,
Justine Bousquet, Clarisse Lozano, Agustina Llanos, Laethitia Alibaud, Srinivas Vasa, Ramesh Pattipati,
Ravindar Valige, Bhaskar Kummari, Srinivasu Pothukanuri, Cyntia De Piano, Ian Morrissey,
Kirsty Holden, Peter Warn, Francesca Marcoccia, Manuela Benvenuti, Cecilia Pozzi, Giusy Tassone,
Stefano Mangani, Jean-Denis Docquier, David Pallin, Richard Elliot, Marc Lemonnier,
and Martin Everett
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ABSTRACT: The diazabicyclooctanes (DBOs) are a class of
serine β-lactamase (SBL) inhibitors that use a strained urea moiety
as the warhead to react with the active serine residue in the active
site of SBLs. The first in-class drug, avibactam, as well as several
other recently approved DBOs (e.g., relebactam) or those in
clinical development (e.g., nacubactam and zidebactam) potentiate
activity of β-lactam antibiotics, to various extents, against
carbapenem-resistant Enterobacterales (CRE) carrying class A,
C, and D SBLs; however, none of these are able to rescue the
activity of β-lactam antibiotics against carbapenem-resistant
Acinetobacter baumannii (CRAB), a WHO “critical priority
pathogen” producing class D OXA-type SBLs. Herein, we describe
the chemical optimization and resulting structure−activity relationship, leading to the discovery of a novel DBO, ANT3310, which
uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity
against OXA-CRAB as well as SBL-carrying CRE pathogens.
are in clinical development (Figure 1). To date, all BLIs in
clinical use are specific to serine β-lactamases (SBLs), which
include class A, C, and D enzymes. Resistance is also increasing
because of the occurrence of the class B metallo β-lactamase
(MBL) enzymes, such as NDM-1,6 and currently, there are no
MBL inhibitors in clinical use.7 Significant advances have been
achieved with the development of boronate SBLIs, such as
vaborbactam (VAB),8 mainly a Klebsiella pneumoniae carbape-
nemase (KPC) inhibitor, and taniborbactam,9 which possesses
broader spectrum β-lactamase inhibitory properties, including
VIM-2 from the MBL family. Recently, a series of publications
have described a new boronate QPX7728,10 representing an
INTRODUCTION
■
Multidrug-resistant Gram-negative bacteria have become an
increasingly serious healthcare threat, exacerbated by major
structural and economic issues underlying the development of
new antibacterial drugs.1 The effectiveness of the most
important class of antibacterial agents, the β-lactam antibiotics,
has been compromised by a main resistance mechanism, the
production of β-lactamase enzymes.2 Co-dosing of β-lactams
with a β-lactamase inhibitor (BLI) has been used as a
successful strategy to combat this type of resistance as far back
as 1981, when the combination of clavulanic acid and
amoxycillin was launched.3 Other established BLIs, such as
tazobactam and sulbactam (Figure 1), have become less
clinically useful because of the increasing resistance attributable
to bacterial production of extended spectrum β-lactamases.4 In
2015, the novel diazabicyclooctane (DBO) BLI avibactam
entered the clinic5 in fixed combination with ceftazidime and
several “fast followers”, including relebactam (approved June
2020), plus nacubactam, zidebactam, and durlobactam, which
Received: September 2, 2020
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Figure 1. Various BLIs.
a
Scheme 1. Synthesis of the Key Acid, Alcohol, and Aldehyde Intermediates
a
(a) KHCO3 and THF; (b) triphosgene, DIPEA, and DCM, 0 °C to RT, 16 h; (c) LiOH·H2O and THF/H2O, RT; (d) LiBH4 and THF/EtOH;
and (e) trichloroisocyanuric acid, TEMPO, and DCM.
advance over taniborbactam as it has improved inhibition of
the NDM-type MBLs.10−12 The whole area has been recently
reviewed.13
In the DBO series, the reactive β-lactam ring of earlier BLIs
has been replaced by a similarly reactive strained bicyclic urea.
Avibactam has proved to be the inspiration for other additions
to the DBO series, such as relebactam, nacubactam,
zidebactam, and durlobactam (Figure 1),14−19 all of which
contain a basic substituent appended to the carboxamide.
However, none of these, with the exception of durlobactam,
penetrate/accumulate into Acinetobacter strains, and because
durlobactam is not active against Enterobacterales, a combina-
tion to treat both carbapenem-resistant Enterobacterales
(CRE) and carbapenem-resistant Acinetobacter baumannii
(CRAB) infections remains elusive. Consequently, CRAB
remains a “critical priority pathogen” as designated by the
WHO.20 Carbapenem resistance in CRAB is mediated by class
D OXA-type enzymes, so a successful inhibitor should be able
to penetrate into Acinetobacter and have activity against OXA-
type enzymes. At the outset of this research, we set ourselves
B
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a
Scheme 2. Synthesis of the CF3 Analogue
a
(a) (Boc)2O, TEA, and DCM, 0 °C to RT, 5 h; (b) Me3SOI, KOtBu, and DMSO/THF; (c) BnONH2-HCl and EtOAc; (d) MsOH and EtOAc
then KHCO3 aq; (e) NaBH3CN, H2SO4, and EtOAc; (f) triphosgene, DIPEA, and DCM, 0 °C to RT, 16 h; (g) H2, Pd/C then Me3NSO3, and
TEA then NaHCO3 aq.
a
Scheme 3. Synthesis of CF2-Thiazolyl Analogue 10
a
(a) TMS-thiazole, Ac2O, and DMSO; (b) DAST and DCM; (c) TiCl4 and DCM; (d) SO3-pyridine, TEA, and DCM; (e) nBu4NOAc and DCM;
and (f) Dowex-Na and H2O.
a
Scheme 4. Synthesis of the CHF2 and CHCl2 Analogues 11 and 12
a
(a) DAST and DCM; (b) PCl5 and DCM, 0 °C to RT, 8 h; (c) H2 and Pd/C; (c) SO3-pyridine, TEA, and DCM; (d) nBu4NOAc and DCM; and
(e) Dowex-Na, H2O.
the objective of achieving a broad-spectrum DBO that restored
carbapenem activity against CRAB and which ideally possessed
little or no intrinsic antibacterial activity in order to minimize
the potential for resistance.
RESULTS AND DISCUSSION
■
Chemistry. Many of the analogues were accessed from
either key acid 1, obtained after the basic hydrolysis of ethyl
ester 2, the primary alcohol intermediate 3, itself prepared by
C
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a
Scheme 5. Synthesis of the CH2F, CH2Cl, and CH2OCF3 Analogues 15, 16, and 17
a
(a) Mesyl chloride, TEA, DMAP, DCM then TBAF, and MeCN, 70 °C; (b) mesyl chloride, TEA, DMAP, DCM then TBACl, and MeCN, 70 °C
(c) TMSCF3, AgOTf, 2-fluoropyridine, KF, Selectfluor, and EtOAc, RT; (d) H2 and Pd/C; (e) SO3-pyridine, TEA, and DCM; (f) nBu4NOAc and
DCM; and (g) Dowex-Na and H2O.
a
Scheme 6. Synthesis of the F, Cl, SCF3, H, and SO2CF3 Analogues 21 to 25
a
(a) AgNO3, selectfluor, and acetone/H2O 4:1, 50 °C, 3 h; (b) Pb(OAc)4, NCS, AcOH, and DMF, 60 °C, 4 h; (c) AgSCF3, selectfluor, 2,6-
lutidine, and acetone; (d) Bu3SnH, AIBN, and Toluene, reflux; (e) mCPBA and DCM, RT; (f) H2 and Pd/C; (g) SO3-pyridine, TEA, and DCM;
(h) nBu4NOAc and DCM; (i) Dowex-Na and H2O.
the LiBH4 reduction of ester 2, or its corresponding aldehyde 4
obtained by the oxidation of alcohol 3 using trichloroisocya-
nuric acid and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl
(TEMPO). Ethyl ester 2 was prepared from commercially
available diamine oxalate salt 5 by conversion to free base 6
and cyclization with triphosgene (Scheme 1).
Certain analogues required bespoke syntheses. For example,
CF3 analogue 7 was synthesized from commercially available
(5S)-5-(trifluoromethyl)pyrrolidine-2-one 8, which was Boc
protected and then converted to 7, following the sequence
reported for avibactam in the literature,21 with the minor
modification that the sulfur trioxide trimethylamine complex
was used to introduce the sulfonic acid functionality and the
D
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a
Scheme 7. Synthesis of CF2CH3 Analogue 31
a
(a) MeNHOMe; (b) MeMgBr and THF; (c) DAST and DCM; (d) H2 and Pd/C; (e) SO3-pyridine, TEA, and DCM; (f) nBu4NOAc and DCM;
and (g) Dowex-Na and H2O.
sodium salt was obtained by treatment with sodium
bicarbonate (Scheme 2).
DBOs with various OXA enzymes (e.g., PDB entries 4WMC,
4WM9, 42SJ, and 5FAQ) shows several interesting features
(Figure 2). First, the orientation of the primary carboxamide
Analogues 10 to 12 were prepared from aldehyde 4. The
reaction of 4 with trimethylsilyl thiazole in the presence of
acetic anhydride afforded the thiazolyl alcohol which was
oxidized in situ to give ketone 13, based on the chemistry of
Dondoni.22 This was converted to the bis-fluoro methylene
derivative 14 by treatment with diethylaminosulfur trifluoride
(DAST). Difluoromethylthaizolyl analogue 10 was then
obtained following the usual synthetic route,21 (Scheme 3)
involving debenzylation, sulfonation/tetrabulylammonium salt
formation, and then ion exchange to give the desired sodium
salt. Difluoromethyl 11 and dichloromethyl 12 analogues were
obtained after treatment of the aldehyde with DAST or PCl5,
respectively, followed again by the usual sequence (Scheme 4).
Compounds 15 to 17 were prepared from alcohol 3. The
activation of the alcohol as a mesylate, then treatment with
TBAF or TBACl in acetonitrile at 70 °C afforded the
fluoromethyl and chloromethyl derivatives 18 and 19,
respectively, which were converted to 15 and 16 by the
usual synthetic pathway. Trifluoromethoxy compound 17 was
obtained following the conditions reported in the literature,
leading to trifluoromethoxy intermediate 2023 (Scheme 5).
Compounds 21 to 25 were prepared from acid 1. Fluoro and
trifluoromethylthio derivatives 26 and 27 were prepared by
decarboxylation and radical treatment with Selecfluor in the
presence of AgNO3 or AgSCF3. Chloro derivative 28 was
produced by the reaction with Pb(OAc)4 in the presence of N-
chlorosuccinimide. This intermediate was reduced to H
analogue 29 with Bu3SnH, and SCF3 intermediate 27 oxidized
using mCPBA to give the corresponding sulfone 30. All these
different intermediates were submitted to the usual chemistry
sequence described previously, affording analogues 21 to 25
(Scheme 6).
Figure 2. (A) Covalently bound avibactam in OXA-48 showing water
molecules in the pocket (PDB entry 4WMC) and (B) covalently
bound FPI1465 (close analogue of nacubactam) in OXA-48 (PDB
entry 5FAQ) showing the inhibitor side chain exiting the pocket.
substituent in the avibactam adduct is not crucial as it does not
seem to make any obvious H-bond with class D β-lactamase
enzymes, and alternative conformations can be seen in the
secondary carboxamides derived from other DBOs (Figure 2).
Second, in OXA-48 there are two water molecules in the active
site pocket around the carboxamide. The original plan was to
make a range of substituents other than amide, which retained
the electron-withdrawing properties of the carboxamide and
which with increasing size might also fill the small pocket
around the carboxamide by the extrusion of the water
moleculesan established strategy in lead optimization.24−26
Our primary screening cascade comprised a panel of SBL
enzymes (Enterobacter cloacae AmpC, CTX-M-15, TEM-1,
OXA-48, and KPC-2) for enzyme inhibition studies plus the
potentiation of the carbapenem antibiotic meropenem (MEM)
against a series of complementary clinical strains of bacteria
(see Table 1). The enzyme inhibition is reported as IC50
valueshowever, this data must be interpreted with caution
because of the irreversible nature of the reaction forming a
covalent product, that is, it is not an equilibrium situation. The
results from this type of assay are dependent on the assay
conditions (in particular, the pre-incubation time of the
enzyme-inhibitor mixture used in the experiment) but
nevertheless provided useful data to rank the order of the
CF2CH3 analogue 31 was prepared also from carboxylic acid
intermediate 1. This was activated through Weinreb amide 32
and treated with Grignard reagent MeMgBr to afford methyl
ketone 33. The reaction with DAST afforded−CF2CH3
intermediate 34, which was converted to 31 following the
usual synthetic route (Scheme 7).
Evaluation of Available Structural Data, Initial
Chemistry Strategy, and Screening Cascade. The
inspection of the X-ray structures of covalent adducts of
E
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Table 1. Enzyme Inhibition Data and Potentiation of MEM Tested on Various Carbapenemase-Producing Clinical Isolates
a
(Indicated in Bold) in the Presence of the 4 μg/mL Inhibitor
IC50 values (μM)
MEM MIC (μg/mL)
K.
E.
cloacae
K. pneumoniae pneumoniae
K.
(TEM-OSBL,
CTX-M-14,
OXA-48)
(TEM-1,
SHV-11,
KPC-3)
pneumoniae
A.
volume
(Å3)
CTX-
M-15 TEM-1
OXA-
48
OXA- (TEM-1,
23
(SHV-11,
OXA-181) (OXA-23)
baumannii
compound
R
AmpC
KPC-2
KPC-2)
none
8
16
0.50
4
128
0.25
64
2
16
1
32
16
32
32
32
16
32
16
32
32
4
avibactam
CONH2
H
35.2
5.1
0.008
>3.0
0.001
1.200
0.119
0.084
0.017
0.178
0.010
0.010
0.002
0.004
0.200
0.019
0.011
0.008
0.002
0.005
0.511
0.492
0.135
0.208
0.403
0.810
0.026
0.001
0.067
0.267
0.216
0.117
0.001
0.001
0.252
1.337
0.154
0.443
0.064
0.178
0.019
0.099
0.015
0.002
0.169
1.107
0.015
0.269
0.175
0.008
>3.0
>3.0
>3.0
>3.0
>3.0
1.604
1.684
0.810
1.320
N.D.
0.059
>3.0
>3.0
>3.0
N.D
0.03
2
24
8
15
CH2F
CH2Cl
CHF2
CHCl2
CF3
26.9
36.0
31.9
51.2
36.9
55.7
72.8
23.5
50.0
90.9
64.3
19.1
10.0
0.304
0.026
0.035
0.052
0.004
0.002
<0.001
0.003
0.313
0.001
0.023
0.004
0.010
0.051
0.028
0.007
0.004
0.006
0.002
0.015
0.008
0.043
0.003
0.013
0.031
0.019
0.125
0.06
0.06
0.06
0.06
0.03
0.06
0.06
0.06
0.125
0.25
0.06
0.03
1
1
16
8
2
16
1
11
2
1
12
16
2
2
16
2
7
2
22
SCF3
8
4
8
25
SO2CF3
CN
8
8
16
WCK4234
0.06
8
0.5
2
0.25
31
10
17
23
21
CF2CH3
CF2-thiaz.
CH2OCF3
Cl
16
16
16
4
32
32
32
32
2
16
8
8
32
64
1
4
F
0.050
0.25
1
a
(N.D = not done).
potency of compounds. The objective was to achieve a broad-
spectrum SBLI but with particular emphasis on the class D
carbapenemase OXA-48 in the panel because of the prevalence
of OXA-type variants as resistant elements in A. baumannii.27,28
The whole cell potentiation assay involved measuring the
MEM MIC against a panel of resistant clinical strains in the
presence of 4 μg/mL of the inhibitor, with the rationale that
functionally active inhibitors would significantly lower the
MEM MIC against these resistant strains (Table 1). A note of
caution regarding this assay is that clinical strains often harbor
more than one resistance mechanism, for example, multiple
enzymes or porin/efflux changes, making interpretation
difficult.
Structure−Activity Relationship of Non-amidic Sub-
stituents. The H analogue 24 was used as a baseline. As
anticipated, this compound was a significantly worse enzyme
inhibitor than avibactam, across this panel (Table 1), but
remarkably it did show very modest MEM potentiation against
some of the clinical strains. The poor enzyme inhibition may
be due to a lower interaction with the target because the amide
substituent is absent, or to a reduced rate of the reaction with
the catalytic serine in the active site because of the absence of
the electron-withdrawing substituent. We decided to explore
novel “non-amidic” substituents at this position because the
−CONH− amido functionality itself does not seem to engage
in significant direct interactions with OXA enzymes, whereas,
in contrast, the various larger side chains on the amide
derivatives (as in nacubactam and zidebactam) do make
significant enzyme interactions. An early first compound,
trifluoromethyl (CF3) analogue 7, showed good enzyme
inhibition and potentiation of MEM against the panel of
clinical strains. In particular, 7 is apparently a better inhibitor
of OXA-48 than avibactam, with an IC50 of 19 nM. This CF3
analogue, however, was not as active in the potentiation assays
as avibactam or the cyano analogue WCK4234.29,30 Never-
theless, encouraged by the activity of 7, we made the mono-
and difluoromethyl analogues 15 and 11, which exhibited a
broadly similar profile, particularly in the potentiation assays.
However, as the monochloro- and dichloromethyl analogues
16 and 12 did not perform well in the potentiation assays, even
though the enzyme data were encouraging, the corresponding
trichloromethyl analogue was not synthesized.
We investigated the introduction of the electron-with-
drawing trifluoromethylthio (SCF3) group. Both the simple
thioether 22 and the corresponding sulphone 25 exhibited very
good enzyme inhibition. However, apart from the E. cloacae
strain, the performance of these analogues in the potentiation
assays was mediocre. Similarly, poor MEM potentiation was
seen with the modestly potent inhibitor 17 bearing the
trifluoromethoxymethyl (CH2OCF3) substituent. In an
attempt to completely fill the small pocket around the
avibactam carboxamide by displacing the water molecules,
we made (1,3-thiazol-2-yl)-difluoromethyl analogue 10, which
modeled extremely well into OXA-48, once the two water
molecules were removed (Figure 3). However, despite being a
good inhibitor of other enzymes in the panel, this compound
was an extremely poor inhibitor of OXA-48. Once again, the
potentiation of this analogue was generally poor. Similarly, the
Figure 3. (A) (1,3-Thiazol-2-yl)-difluoromethyl analogue 10 modeled
into OXA-48 (PDB entry 4S2K), waters removed prior to
minimization and (B) fluoro analogue 21 modeled into OXA-48,
waters left in place before minimization.
F
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replacement of a fluorine atom in trifluoromethyl with a
methyl group gave methyldifluoromethyl (CF2CH3) analogue
31, which did show good inhibition of OXA-48; however, once
more, there was disappointing potentiation of MEM.
Re-evaluation of the Chemistry StrategySmall is
Beautiful and Stereoelectronic Control. Because of the
disappointing potentiation results, the chemistry strategy was
re-evaluated. The accumulation of antibiotics in the periplasm
of Gram-negative bacteria is affected by two opposing
processesentry though water-filled porins31 and efflux via
transporter systems such as the AcrAB-TolC system present in
Escherichia coli.32 In a recent seminal study,33 experimentally
determined guidelines for bacterial accumulation were
proposed, which invoke low globularity, low numbers of
rotatable bonds, and the presence of a primary amine as being
key factors, facilitating accumulation in the periplasm. In our
series, it is apparent that the larger the size of the substituent,
the worse is the potentiation of MEM, for similar levels of
enzyme inhibition. This is unlikely to be because of reduced
access by slower diffusion through the porins because other
analogues with quite large substituents are able to penetrate
effectively. More likely, this phenomenon is related to the
enhanced recognition of the drug molecules by the efflux
pumps. Even the weak enzyme inhibitor 24, with a simple
hydrogen atom substituent, achieves marginally improved
potentiation against the K. pneumoniae strain expressing TEM,
CTX, and OXA variants than does the sulfone analogue 25,
which is a very potent enzyme inhibitor. This structure−
activity relationship (SAR) encouraged us to look at even
smaller electron-withdrawing substituents than trifluoromethyl;
consequently, we considered placing simple halogens at this
position, even though, a priori, this did not seem to be a
chemically attractive option because the RCON-C-halogen
motif is notoriously reactive because of the facile elimination of
the halide, generating an iminium species. Although there are
examples in the literature of such functional group
juxtapositioning, such as in chloroazetidinone34 and fluori-
nated glycosyl donor35 (Figure 4), in all cases these molecules
are functioning as reactive reagents and not as pharmacological
agents.
Figure 5. Stereoelectronic control prevents the elimination of the
halide.
Encouraged by these considerations, we synthesized and
evaluated both chloro analogue 23 and fluoro analogue 21.
These have substituent volumes of 19.1 and 10.0 Å3,
respectively, both smaller than the primary carboxamide of
avibactam (35.2 Å3) and obviously much smaller than the (1,3-
thiazol-2-yl)-difluoromethyl substituent (90.9 Å3). Not only
were both analogues chemically stable but they were modestly
potent enzyme inhibitors. However, there were huge differ-
ences in the potentiation between the two; whereas 23 did not
perform well, 21 showed an excellent profile, even potentiating
the effect of MEM against the difficult-to-kill A. baumannii
strain (Table 1). Subsequent studies on OXA-23 showed that
21 was a very good inhibitor of OXA-23, as well as OXA-48
(Table 1). In contrast, avibactam had no activity against OXA-
23 even though it was a reasonable inhibitor of OXA-48,
offering an explanation for the lack of effectiveness of the
MEM−avibactam combination against OXA-23 producing A.
baumannii (Table 1). The overall profile of 21 in our primary
screens resembled that of WCK423430 but on further analysis
of the potentiation of MEM against a larger panel of clinical
isolates 21 proved to be equipotent against KPC- and OXA-
CRE while superior against OXA-CRAB (Table 2). In fact, the
Table 2. Potentiation of MEM MIC against KPC- and OXA-
Producing CRE and OXA-Producing CRAB at the 4 μg/mL
a
Concentration of SBLI
MIC (μg/mL)
antibiotic (combination)
MIC50
MIC90
minimum
maximum
KPC-Positive CRE (n = 50)
MEM
MEM/21
MEM/WCK4234
CAZ−Avi
>32
0.06
0.06
1
>32
1
1
4
>32
16
16
≤0.03
≤0.03
0.25
2
16
OXA-Positive CRE (n = 48)
MEM
MEM/21
MEM/WCK4234
CAZ−Avi
16
>32
1
0.25
2
2
>32
2
0.5
4
0.12
0.12
0.12
≤0.03
≤0.03
0.25
OXA-Positive CRAB (n = 50)
MEM
MEM/21
MEM/WCK4234
CAZ−Avi
>32
4
16
>32
8
32
8
>32
>32
>32
>32
0.25
0.25
4
Figure 4. Examples of RCON-C-halogen.
>32
>32
a
However, the importance of the stereoelectronic control is
sometimes underestimated in the organic synthesis.36−38 If we
consider the 3D molecular structure of the proposed halo
analogues (Figure 5), it can be seen that the lone pair (shown
in red) and the C-Hal bond are at approximately 60° to each
other, not a suitable orientation for the orbital overlap required
to eliminate halide and form the iminium species. In classical
terms, the resulting iminium species contravenes Bredt’s law,
which states you cannot have a double bond at the bridgehead
position of a small bicyclic system.39
Ceftazidime−avibactam (CAZ−Avi) was used as a comparator.
combination of MEM/21 compares favorably to that of
ceftazidime/avibactam, a drug marketed for the treatment of
KPC-and OXA-CRE, and has the additional coverage of OXA-
CRAB (Table 2).
As anticipated, in contrast to the intrinsic antibacterial
activities of avibactam and zidebactam, the antibacterial activity
of 21 was negligible (Table 3).
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Table 3. Intrinsic Antibacterial Activity (μg/mL) of Avibactam, Zidebactam, and 21 on Various Carbapenemase-Producing
Clinical Isolates (Indicated in Bold)
MIC (μg/mL)
E. cloacae
(TEM-1, KPC-2)
K. pneumoniae
(TEM-OSBL, CTX-M-14, OXA-48)
K. pneumoniae
(TEM-1, SHV-11, KPC-3)
K. pneumoniae
(SHV-11, OXA-181)
A. baumannii
(OXA-23)
compound
avibactam
zidebactam
21
16
0.5
128
16
0.25
64
16
2
128
32
2
128
>128
>128
>128
ADMET Properties of 21. The ADMET properties of 21
were investigated (Table 4). Compound 21 was stable in
Table 5. Mouse IV PK of 21 and Avibactam
parameter
avibactam
21
dose (mg/kg)
T1/2 (h)
AUC (ng·h/mL)
Cl (mL/min/kg)
Vd (L/kg)
1.0
0.37
279
59
1.0
0.64
412
40
Table 4. ADMET Properties of Compound 21
ADME
plasma stability, % remaining after
1 h (mouse/rat/dog/human)
90, 55, 85, 79
hepatocyte Clint
(μL/min/million cells)
(mouse/rat/dog/human)
<3.5/<3.4/<3.0/<3.4
1.9
2.2
compatible to that of MEM, which is the crucial requirement
for an IV drug to be co-dosed with MEM and supported the
investigation of in vivo efficacy of 21 in combination with
MEM.
PPB, % unbound
>93% for all species
>50
(mouse/rat/dog/human)
in vitro
toxicity
CYP inhibition (TDI) IC50 (μM)
1A2, 2C9, 2C19, 2D6, 3A4
HepG2, CC50 (μM)
>100
Efficacy of 21 in a Murine Thigh Infection Model. The
ability of 21 to restore MEM efficacy was investigated in a 9 h
neutropenic murine thigh infection model, with two K.
pneumoniae clinical strains expressing either KPC-2 or OXA-
48 SBLs. This model was specifically developed to demonstrate
the efficacy of carbapenems, which have a very short half-life in
rodents and need to be dosed frequently in order to maintain
plasma levels above the MIC for the correct percent time of
the dosing interval (40% T > MIC). A MEM dose of 300 mg/
kg administered every 2 h (q2 h) was used in these studies.
This dose results in a % T > MIC in mice similar to that of a 2
g dose administered every 8 h by a 3 h infusion in humans,40
and this is the clinical MEM dose/dosing regimen expected to
be used with this combination. The addition of 25, 50, and 100
mg/kg 21 to 300 mg/kg MEM-reduced bacterial burdens
(colony forming units, CFU) in a dose-dependent manner to
levels below that of the initial starting inoculum at the highest
dose (Figure 7a,b). Compared to treatment with MEM alone,
which was not efficacious, a statistically significant reduction in
bacterial counts could be observed in all the groups treated
with the combination, even those that received the lower dose.
In fact, the combination demonstrated similar efficacy to
hERG, IC50 (μM) patch clamp
ames genotoxicity test
single dose doses tested: 30, 100, 300, and 1000 tolerated up to 1000
rat MTD mg/kg mg/kg
>100
negative
mouse, dog, and human plasma (79−90% remaining after 1 h),
although less so in the rat plasma (55% remaining after 1 h)
and was mostly present as a free drug (>93% unbound).
Hepatocyte clearance and cytochrome P450 inhibition (time
dependent) were low for all species, indicating high metabolic
stability and low potential for drug−drug interactions.
Compound 21 showed a low in vitro toxicity profile, with no
observed cytotoxicity (HepG2 cell line), cardiotoxicity
(inhibition of the hERG potassium ion channel), or
genotoxicity (Ames test). Furthermore, 21 was well tolerated
(up to 1000 mg/kg) following a single iv dose in rats.
Pharmacokinetics. The IV PK of 21 in mice compared
favorably to that of avibactam (Figure 6 and Table 5), showing
a longer half-life, greater exposure, slower clearance, and higher
volume of distribution. Again, this is in accord with the
physicochemical properties of the series as a whole. The PK is
Figure 6. Mouse IV PK of 21 and avibactam.
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Figure 7. Efficacy of 21 in a murine thigh infection model. Neutropenic mice (five/group) were inoculated by IM administration into both thighs
with suspensions of either (a) KPC-2 K. pneumoniae NR-48977 or (b) OXA-48 K. pneumoniae AC00783. MEM was dosed IV at 300 mg/kg alone
or in combination with 21 at 25, 50, or 100 mg/kg at 1, 3, 5, and 7 h postinfection. MEM/VAB (dosed IV at 300/50 mg/kg at 1, 3, 5, and 7 h
postinfection) was used as a positive control in (a) tigecycline (dosed IV at 40 mg/kg at 1 h postinfection) was used as a positive control in (b).
Animals were euthanized at 9 h postinfection and thigh CFUs determined. Three-dimensional structure of the OXA-48 class D carbapenemase
covalently bound to 21.
MEM/VAB (Figure 7a) and tigecycline (Figure 7b), which
were used as positive controls in these studies. These data
provided the in vivo proof-of-concept needed to continue the
progression of the compound through preclinical development.
A crystal structure of the OXA-48:21 covalent complex was
obtained at a maximum resolution of 1.85 Å (see Supporting
Information for methods and data collection and refinement
statistics). The asymmetric unit included a single OXA-48
dimer, in which 21 was found to be covalently bound to the
catalytic serine in both subunits (Figure 8A). In addition, the
Lys73 residue was found to be decarboxylated in both
subunits, and a linear CO2 molecule could be fitted in the
extra electron density found in the active site of subunit A
(Figure 8B), as previously observed in the OXA-48:avibactam
complex.41 The carboxylated Lys73 bears functional relevance,
as it is important in both the acylation and deacylation steps of
the catalytic mechanism of class D β-lactamases.42−44
Furthermore, the substituent of the piperidine ring, consisting
of a single F atom and despite being shorter than that of
avibactam (where a carboxamide is found), could be clearly
observed in the electron density map, supporting that the
compound would not undergo significant chemical modifica-
tions after the opening of the five-membered carbamide-
containing ring.
in the avibactam complex (Figure 8C).41 The F substituent
was found in a hydrophobic pocket made of residues Val120,
Leu158, and Trp105 but did not create any significant
interaction with such residues (closest distance, 3.9 Å), only
interacting with a water molecule (observed in subunit B only)
(Figure 8B,C).
When compared to the native enzyme,44 the binding of the
inhibitor was associated with several modifications in the active
site that were similar to that observed in the avibactam
complex, that is, the decarboxylation of the Lys73, the rotation
of the Ser118 side chain, the displacement of the Leu158 side
chain, and minor shifts of the region between helices α3 and
α5.
Overall, these data are in agreement with the predicted
binding mode from earlier modeling, reflecting the conserved
binding mode of strained DBO compounds, mostly differing
(expect durlobactam45) by the nature of the piperidine side
chain. As anticipated, the region of the active site around the
fluorine atom of 21 may still contain two water molecules
(observed in subunit B).
CONCLUSIONS
■
A rapid and focused medicinal chemistry campaign successfully
delivered a new drug in the DBO space, which is clearly
differentiated from previous analogues by its expanded
microbiological spectrum to include the restoration of MEM
antibacterial activity against OXA-producing Acinetobacter
strains (CRAB). A reductionist approach led to the
identification of a single fluorine atom as being the optimum
substituent to achieve the desired properties, and while,
superficially, the fluoro analogue might be expected to have
stability issues, a consideration of the stereoelectronic aspects
of the system mitigated against this possibility. The X-ray
Stabilizing interactions were found between compound 21
and OXA-48 residues, essentially (1) between the oxygen
atoms of the sulfate moiety with the side chains of Ser118
(SxV-conserved motif), Lys208 and Thr209 (KTG-conserved
motif), and Arg250, the latter involved in a strong electrostatic
interaction, and (2) the carbonyl oxygen atom of the covalent
adduct interacting with the N backbone atom of residues Ser70
and Tyr211, forming the so-called oxyanion hole (Figure 8B).
These interactions were very similar to those already observed
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through preclinical development as a broad-spectrum DBO,
with coverage of KPC- and OXA-CRE but with the added
benefit of activity against OXA-CRAB. From a medicinal
chemistry perspective, it is noteworthy that all the synthesized
compounds from this project are described in this paper. From
this very short campaign, we discovered ANT3310, a novel
DBO, with clear differentiating advantages over other drugs in
this chemical class and addressing the increasingly important
global threat of antibiotic resistance.
EXPERIMENTAL SECTION
■
General Procedures. Reactions were performed under argon or
nitrogen using dried glassware and solvents. Commercially available
reagents and solvents were used as supplied or purified using standard
protocols. Reactions were conducted at room temperature unless
otherwise stated and monitored by standard thin-layer chromatog-
raphy or liquid chromatography−mass spectrometry (LC−MS)
techniques. Chromatography was performed with standard silica
columns packed with silica gel (Merck silica gel 40−63 μm) and
eluting with solvent combinations as described. The Dowex Na resin
1
was obtained from Sigma-Aldrich. H spectra were recorded using
500 MHz (Bruker), 400 MHz (Bruker), 400 MHz (Varian), and 300
MHz (Varian) instruments in the deuterated solvents as indicated.
Chemical shifts (δ) are reported in parts per million downfield from
tetramethylsilane or alternatively using the residual solvent peak as an
internal standard. Multiplicity is given as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br (broad). Coupling
constants (J) are reported in Hertz (Hz). Preparative high-
performance liquid chromatography (HPLC) conditions are individ-
ually reported as appropriate. All final compounds tested were >95%
pure as determined by NMR and LC−MS. Modeling of potential
targets into OXA-48 were performed with Flare from the Cresset suite
of software and Discovery Studio 2019 from Biovia. Substituent
volume calculations were undertaken with Marvin from ChemAxon.
Sodium (2R,5R)-2-Fluoro-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-
yl Sulfate (21), (ANT3310). Ethyl (2S,5R)-6-(Benzyloxy)-7-oxo-1,6-
diazabicyclo[3.2.1]octane-2-carboxylate (2). A suspension of
commercially available ethyl (2S,5R)-5-[(benzyloxy)amino]-
piperidine-2-carboxylate oxalate salt (15.0 g, 40.7 mmol) in
tetrahydrofuran (THF) (150 mL) at 0 °C was treated with a solution
of potassium hydrogen carbonate (16.3 g, 163 mmol) in water (150
mL). After 1 h, the mixture was extracted with ethyl acetate and the
combined extracts washed with water then brine, dried (Na2SO4), and
evaporated affording ethyl (2S,5R)-5-[(benzyloxy)amino]piperidine-
2-carboxylate as brown oil (10.0 g, 88%).
Figure 8. Crystal structure of OXA-48 inhibited by 21. (A) Content
of the asymmetric unit, including an OXA-48 dimer, a chloride anion
(green sphere) at the interface between the subunits (shown in
different colors) in which the catalytic Ser70 was covalently bound to
compound 21 (cyan sticks), as well as water molecules (red spheres)
and ethylene glycol (green sticks). (B) View of the active site of the
enzyme (subunit A) showing the network of interactions between the
OXA-48 residues (green sticks) and the inhibitor 21 (see text for
details), the decarboxylated Lys73, and the presence of a linear CO2
A solution of ethyl (2S,5R)-5-[(benzyloxy)amino]piperidine-2-
carboxylate (5.0 g, 18 mmol) in DCM (100 mL) at 0 °C was treated
with N,N-diisopropylethylamine (DIPEA) (12.5 mL, 72 mmol)
followed by the addition of a solution of triphosgene (2.6 g, 9 mmol)
in DCM (10 mL). The mixture was stirred at ambient temperatures
for 16 h, then saturated aqueous potassium bicarbonate solution (100
mL) was added. After 1 h, the phases were separated and the DCM
phase washed with water then brine, dried (Na2SO4), and evaporated
affording brown oil (5.0 g, 92%). LC−MS Anal. Calcd for
molecule (the conserved motifs Ser70-x-x-Lys, Ser118-x-Val and Lys208
-
Thr/Ser−Gly are colored in red, magenta, and blue, respectively).
(C) View of the active site of the OXA-48:21 complex (subunit A,
salmon; water molecules, red spheres) and its superimposition with
the native OXA-48 (white, subunit B of PDB entry 3HBR) and the
OXA-48:avibactam complex (blue, subunit C of PDB entry 4WMC),
showing the impact of inhibitor binding on the position of active
residues.
1
C16H20N2O4, 304.1. Found [M + H] = 305.1. H NMR (500 MHz,
CDCl3): δ ppm 7.43−7.35 (m, 5H), 5.06 (d, J = 11.5 Hz, 1H), 4.90
((d, J = 11.5 Hz, 1H), 4.25−4.22 (m, 2H), 4.09 (d, J = 6.5 Hz, 1H),
3.31 (d, J = 1.5 Hz, 1H), 3.05−3.00 (m, 1H), 2.93 (d, J = 12 Hz, 1H),
2.11−2.04 (m, 3H), 1.71−1.60 (m, 1H), 1.29 (t, J = 7.0 Hz, 3H).
(2S,5R)-6-(Benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-
carboxylic Acid (1). To a solution of ethyl (2S,5R)-6-(benzyloxy)-7-
oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (7.0 g, 23.0 mmol) in
acetone/water (1:1, 120 mL) was added LiOH·H2O (0.97 g, 23.0
mmol) at 0 °C. The resulting reaction mixture was stirred for 2 h. The
mixture was diluted with water (50 mL) and washed with EtOAc (2 ×
100 mL). The aqueous layer was acidified with 1N HCl to
approximately pH3 and then extracted with EtOAc (2 × 100 mL).
The combined organic phases were washed with water, brine, dried
over anhydrous Na2SO4, filtered, and concentrated to obtain (2S,5R)-
crystal structure of OXA-48 bound to 21, revealing a similar
binding and network of interaction with that of other DBO
compounds, also supports the concept that the inhibitor does
not undergo major chemical modifications upon ring opening.
Compound 21, designated as ANT3310, is effective against
CREs and A. baumannii strains and is efficacious in mouse
infection models. ANT3310 is currently being progressed
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6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylic acid
(5 g, 79%) as a white solid, which was used in the next step without
purification.
Sodium (2R,5R)-2-Fluoro-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-
yl Sulfate (21) Procedure (c). A stirred solution tetrabutylammonium
(2R,5R)-2-fluoro-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (220
mg, 0.45 mmol) in water (10 mL) was treated with a Dowex Na resin
(1 g). After 1 h, the mixture was filtered through a pad of the Dowex
Na resin, washing with H2O (5 mL). The combined filtrate was again
treated with the Dowex Na resin (1 g) for 1 h, filtered through a bed
of the Dowex Na resin, and washing with H2O (5 mL). This process
was repeated for another 3 times. The combined filtrates were
lyophilized to obtain the title compound as a white solid (90 mg,
75%). HRMS Anal. Calcd for C6H8O5N2FS [M], 239.0132. Found
239.0143. 1H NMR (500 MHz, DMSO-d6): δ ppm 5.35 (ddd, 1H, J =
46 Hz, 5 Hz, 2.5 Hz, CHF), 4.08−4.06 (m, 1H), 3.26−3.24 (m, 1H),
3.06−3.04 (m, 1H), 1.99−1.68 (m, 4H). Anal. Calcd for
C6H8O5N2FSNa, C, 27.48; H, 3.08; N, 10.68; Na, 8.77. Found: C,
28.32; H, 3.22; N, 10.52; Na, 8.49.
LC−MS Anal. Calcd for C14H16N2O4, 276.1. Found [M + H] =
1
277.2. H NMR (500 MHz, DMSO-d6): δ ppm 13.01 (br s, 1H),
7.45−7.32 (m, 5H), 4.92 (AB q, J = 11 Hz, 2H), 3.86 (d, J = 7 Hz,
1H), 3.62 (s, 1H), 2.95−2.89 (m, 2H), 1.99−1.95 (m, 1H), 1.88−
1.79 (m, 2H), 1.70−1.66 (m, 1H).
(2R,5R)-6-(Benzyloxy)-2-fluoro-1,6-diazabicyclo[3.2.1]octan-7-
one (26) (A) and (2S,5R)-6-(Benzyloxy)-2-fluoro-1,6-diazabicyclo-
[3.2.1]octan-7-one (B). To a stirred solution of (2S,5R)-6-
(benzyloxy)-7-oxo-1, 6-diazabicyclo [3.2.1] octane-2-carboxylic acid
(2.5 g, 9.04 mmol) in acetone: water (4:1, 100 mL) was added
Selectfluor (6.4 g, 18.0 mmol) and AgNO3 (153 mg, 0.90 mmol) at
room temperature. The reaction mixture was heated at 50 °C for 3 h
then evaporated. The resulting residue was extracted with EtOAc
(100 mL), filtered through a Celite pad, and washing the pad with
further EtOAc (10 mL). The filtrate was washed with the NaHCO3
solution (50 mL), water (50 mL), and brine (50 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, and evaporated to
obtain oil. This was chromatographed on silica gel eluting with 10%
EtOAc in hexane as the eluent affording the desired isomer (2R,5R)-
6-(benzyloxy)-2-fluoro-1,6-diazabicyclo[3.2.1]octan-7-one (A) (550
mg, 24%) as pale yellow viscous oil. Further elution with 50−60%
EtOAc in hexane afforded the undesired isomer (2S,5R)-6-
(benzyloxy)-2-fluoro-1,6-diazabicyclo[3.2.1]octan-7-one (B) (300
mg, 13%) as a pale yellow solid.
Sodium {7-Oxo-1,6-diazabicyclo[3.2.1]octan-6-yl}-
oxidanesulfonate (24). (5R)-6-(Benzyloxy)-1,6-diazabicyclo[3.2.1]-
octan-7-one. To a solution of (2R,5R) charcol-6-(benzyloxy)-2-
chloro-1,6-diazabicyclo[3.2.1]octan-7-one 28 (500 mg, 1.87 mmol) in
toluene (15 mL) was added AIBN (30.8 mg, 0.18 mmol) and Bu3SnH
(1.09 g, 3.75 mmol). The reaction mixture was heated at 120 °C for 2
h. The reaction mixture was evaporated. The resulting oil was
chromatographed on silica eluting with 50% EtOAc in hexane as the
eluent, affording (5R)-6-(benzyloxy)-1,6-diazabicyclo[3.2.1]octan-7-
one (350 mg, 81%) as an off-white solid. LC−MS Anal. Calcd for
1
C13H16N2O2, 232.1. Found [M + H] = 233.1. H NMR (500 MHz,
CDCl3): δ ppm 7.43 (d, J = 6.5 Hz, 2H), 7.39−7.34 (m, 3H), 5.06 (d,
J = 11.5 Hz, 1H), 4.89 (d, J = 11.5 Hz, 1H), 3.42−3.38 (m, 1H), 3.29
(s, 1H), 3.18−3.16 (m, 1H), 2.97−2.91 (m, 1H), 2.77−2.73 (m, 1H),
2.12−2.00 (m, 2H), 1.62−1.56 (m, 2H).
(5R)-6-Hydroxy-1,6-diazabicyclo[3.2.1]octan-7-one. According to
Procedure (a): (5R)-6-(benzyloxy)-1,6-diazabicyclo[3.2.1]octan-7-
one (350 mg, 1.50 mmol) was converted the desired compound
(190 mg, 90%) as an off-white solid, which was used immediately next
step without purification. LC−MS Anal. Calcd for C6H10N2O2, 142.1.
Found [M + H] = 143.2.
Tetrabutylammonium (5R)-7-Oxo-1,6-diazabicyclo[3.2.1]octan-
6-yl Sulfate. According to procedure (b) (5R)-6-hydroxy-1,6-
diazabicyclo[3.2.1]octan-7-one (190 mg, 1.33 mmol) was converted
to the desired compound as colorless oil (100 mg, 16% over 2 steps).
LC−MS Anal. Calcd for C6H9N2O5S, 221.0. Found [M] = 220.9. 1H
NMR (500 MHz, CDCl3): δ ppm 4.36 (s, 1H), 3.49−3.47 (m, 1H),
3.35−3.26 (m, 9H), 3.00−2.96 (1H, m), 2.84 (d, J = 11.5 Hz, 1H),
2.32−2.28 (m, 1H), 1.97−1.88 (m, 1H), 1.70−1.62 (m, 9H), 1.48−
1.41 (m, 9H), 1.01−0.99 (m, 12H).
Sodium (5R)-7-Oxo-1,6-diazabicyclo[3.2.1]octan-6-yl Sulfate.
According to procedure (c): (5R)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl sulfate (100 mg, 0.21 mmol) was converted to a white
solid, which was then purified by preparative HPLC [column:
KROMOSIL-C18 (150 × 25), 10 μ; mobile phase: H2O: acetonitrile;
gradient: (T % B): −0/1, 3/1, 3.1/98, 8/98, 8.1/1, 10/1; flow : 25
mL/min], and the pure fractions were lyophilized to afford the title
compound (15.0 mg, 28%) as an off-white solid. HRMS Anal. Calcd
for C6H9N2O5S [M], 221.0227. Found 221.0235. 1H NMR (500
MHz, DMSO-d6): δ ppm 3.97−3.96 (m, 1H), 3.10−3.05 (m, 2H),
2.98−2.92 (m, 2H), 1.93−1.92 (m, 1H), 1.72−1.65 (m, 2H), 1.50−
1.48 (m, 1H).
(2R,5R)-6-(Benzyloxy)-2-fluoro-1,6-diazabicyclo[3.2.1]octan-7-
one (A). LC−MS Anal. Calcd for C13H15FN2O2, 250.1. Found [M +
1
H] = 251.1. H NMR (500 MHz, CDCl3): δ ppm 7.43−7.34 (m,
5H), 5.45 (dd, J = 4.5, 46 Hz, 1H), 5.04 (d, J = 11.5 Hz, 1H), 4.89 (d,
J = 11.5 Hz, 1H), 3.36 (dd, J = 2.5, 5.5 Hz, 1H), 3.25 (d, J = 12 Hz,
1H), 3.02−2.98 (m, 1H), 2.13−1.98 (m, 2H), 1.92−1.86 (m, 1H),
1.73−1.66 (m, 1H). 19F NMR (470 MHz, CDCl3): δ ppm −155.49−
155.69 (m).
(2S,5R)-6-(Benzyloxy)-2-fluoro-1,6-diazabicyclo[3.2.1]octan-7-
one (B). LC−MS Anal. Calcd for C13H15FN2O2, 250.1. Found [M +
H] = 251.1. 1H NMR (500 MHz, CF3COOD): δ ppm 7.49−7.48 (m,
5H), 5.95 (ddd, J = 5, 10.5, 46 Hz, 1H, CHF), 5.19 (d, J = 11.5 Hz,
1H), 5.12 (d, J = 11.5 Hz, 1H), 4.10−4.04 (m, 1H), 3.71 (d, J = 3 Hz,
1H), 3.60 (d, J = 11.5 Hz, 1H), 2.69−2.64 (m, 1H), 2.45−2.42 (m,
1H), 2.22−2.15 (m, 1H), 2.03−1.96 (m, 1H). 19F NMR (376 MHz,
CDCl3): δ ppm −140.10−140.25 (m).
Please see the Supporting Information for the NMR experiments to
distinguish and characterize these two isomers.
(2R,5R)-2-Fluoro-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-7-one
Procedure (a). To a solution of (2R,5R)-6-(benzyloxy)-2-fluoro-1,6-
diazabicyclo[3.2.1]octan-7-one (300 mg, 1.19 mmol) in methanol (30
mL) was added 10% palladium on charcoal (300 mg). The reaction
mixture was hydrogenated at room temperature for 1 h using
hydrogen balloon pressure. The reaction mixture was filtered through
a Celite pad washing with MeOH (10 mL). The filtrate was
evaporated to give (2R,5R)-2-fluoro-6-hydroxy-1,6-diazabicyclo-
[3.2.1]octan-7-one (180 mg, 94%) as an off-white solid, which was
used in the next step without purification. LC−MS Anal. Calcd for
C6H9FN2O2, 160.1. Found [M + H] = 161.0.
Tetrabutylammonium (2R,5R)-2-Fluoro-7-oxo-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate Procedure (b). To a solution of (2R,5R)-2-
fluoro-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-7-one (180 mg, 1.12
mmol) in DCM (20 mL) was added Et3N (1.5 mL, 11.2 mmol),
followed by SO3.Pyr (1.07 g, 6.74 mmol) at 0 °C and stirred for 4 h.
Then, a solution of tetra(n-butyl)ammonium acetate (TBAA) (2.7 g,
8.99 mmol) in water (20 mL) was added and stirred at room
temperature for 2 h. The reaction mixture was diluted with DCM (50
mL) and the organic layer was separated. The organic layer was
washed with water (5 × 25 mL), dried over Na2SO4, filtered, and
evaporated. The resulting residue was chromatographed on silica
eluting with 0−100% EtOAc in hexane followed by 5% MeOH in
DCM affording colorless oil (220 mg, 40% over 2 steps). LC−MS
Anal. Calcd for C6H8FN2O5S, 239.0. Found [M] = 239.0.
Sodium (2S,5R)-7-Oxo-2-(trifluoromethyl)-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate (7). This was prepared using essentially
the same methodology as reported for avibactam (Ball, M. et al.
Organic Process Research and Development, 2016, 20, 1799.) except
starting from commercially-available (5S)-5-(trifluoromethyl)-2-pyr-
rolidinone.
tert-Butyl (5S)-2-Oxo-5-(trifluoromethyl)pyrrolidine-1-carboxy-
late. A solution of (5S)-5-(trifluoromethyl)-2-pyrrolidinone (5 g,
32.7 mmol) in DCM (60 mL) at 0 °C was treated with Et3N (5.5 mL,
39.2 mmol) and 4-dimethylaminopyridine (DMAP) (0.4 g, 0.1
mmol). Then, a solution of Boc2O (8.6 g, 39.2 mmol) in DCM (20
mL) was added dropwise over 10 min. After 0.5 h, the reaction
K
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mixture was partitioned between DCM and 10% aqueous citric acid.
The organic extract was washed with water, dried (Na2SO4), and
evaporated affording oil (9.4 g), which was chromatographed on silica
eluting with 0−25% EtOAc in toluene affording tert-butyl (S)-2-oxo-
5-(trifluoromethyl)pyrrolidine-1-carboxylate as oil (7.9 g, 96%). LC−
MS Anal. Calcd for C10H14F3NO3, 253.1. Found [M + Na] = 276.
(3R,6S)-N-(Benzyloxy)-6-(trifluoromethyl)piperidin-3-amine. For
a detailed explanation of this reaction sequence see Ball, M. et al.
Organic Process Research and Development, 2016, 20, 1799. These
steps, optimized for the manufacturing route of avibactam, were
carried out in a one-pot manner without full isolation/character-
ization.
Dimethyl sulfoxide (DMSO) (10 mL) was added to a mixture of
trimethylsulfoxonium iodide (1.6 g, 7.3 mmol) and potassium t-
butoxide (0.72 g, 6.4 mmol) in THF (7 mL). The reaction mixture
was stirred at room temperature for 1 h, then cooled to −12 °C
(internal temperature). A solution of tert-butyl (5S)-2-oxo-5-
(trifluoromethyl)pyrrolidine-1-carboxylate (1.5 g, 5.8 mmol) in
THF (4 mL) was added dropwise over 5 min and the mixture stirred
at −12 °C for 1 h. The reaction mixture was treated with 20%
aqueous ammonium chloride (13 mL), and the stirred mixture was
allowed to warm to room temperature and then extracted twice with
EtOAc. The combined extracts were washed with 10% aqueous NaCl
solution and then concentrated to approximately a 20 mL solution
which was used directly in the next stage.
then filtered, and concentrated to approximately 1.5 mL. This was
diluted with water and treated with a saturated aqueous sodium
bicarbonate solution (3 mL). The mixture was loaded onto a reverse
phase C18 cartridge (10 g size) and eluted with 0−40% ACN in
water. Evaporation of product-containing fractions gave a white solid
that was redissolved in water and rechromatographed using the same
chromatography conditions. The evaporation of product-containing
fractions gave a white solid that was redissolved in water and
rechromatographed using similar chromatography conditions except
that THF was used in place of ACN. Evaporation gave the title
compound as a white solid (86 mg, 51%). HRMS Anal. Calcd for
C7H8F3N2O5S [M], 289.0101. Found 289.0115. 1H NMR (400 MHz,
DMSO-d6): δ ppm 4.09−4.06 (m, 1H), 3.89−3.78 (m, 1H), 3.24 (d, J
= 12.0 Hz, 1H), 3.13−3.08 (m, 1H), 1.92−1.74 (m, 4H). 19F NMR
(376.4 MHz, DMSO-d6): δ ppm −74.7.
Sodium (2S,5R)-2-(Fluoromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl Sulfate (15). (2S,5R)-6-(Benzyloxy)-2-(hydroxymethyl)-
1,6-diazabicyclo[3.2.1]octan-7-one (3). A solution of ethyl (2S,5R)-
6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (5.0
g, 16.4 mmol) in THF (75 mL) and EtOH (75 mL) was treated at 0
°C with a solution of lithium borohydride in THF (4 M; 24 mL, 96
mmol). After 1 h at room temperature, the mixture was re-cooled to 0
°C, and a further portion of a solution of lithium borohydride in THF
(4 M; 12 mL, 48 mmol) was added. After a further 16 h, the mixture
was treated with saturated aqueous monopotassium phosphate (200
mL), and the mixture extracted several times with DCM. The
combined DCM extracts were washed with water then brine, dried
(Na2SO4), and evaporated affording brown oil (5 g). This was
chromatographed on silica eluting with 0−100% EtOAc in hexane
affording colorless oil (2.2 g, 47%). LC−MS Anal. Calcd for
The above EtOAc solution (20 mL) was treated with O-
benzylhydroxylamine hydrochloride, and the mixture heated at 60
°C for 2.75 h then allowed to cool to room temperature before
washing with a 10% aqueous NaCl solution. This solution was
reduced in volume to approximately 10 mL and used directly in the
next step.
1
C14H18N2O3, 262.1. Found [M + H] = 263.1. H NMR (400 MHz,
CDCl3): δ ppm 7.43−7.66 (m, 5H), 5.05 (d, J = 11.6 Hz, 1H), 4.90
(d, J = 11.6 Hz, 1H), 3.71−3.68 (m, 1H), 3.60−3.57 (m, 2H), 3.33
(s, 1H), 3.01−2.89 (m, 2H), 2.04−1.93 (m, 3H), 1.39−1.35 (m, 1H).
((2S,5R)-6-(Benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-
methyl Methanesulfonate. To a solution of (2S,5R)-6-(benzyloxy)-
2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (3 g, 11.4
mmol) in DCM (40 mL) was added Et3N (4.8 mL, 34.3 mmol) at
0 °C followed by DMAP (140 mg, 1.14 mmol). Then,
methanesulfonyl chloride (1.3 mL, 17.1 mmol) was added at 0 °C
and the reaction mixture was stirred for 2 h. The reaction mixture was
diluted with DCM (40 mL), washed with 1 N HCl (40 mL), water
(40 mL), and brine (20 mL). The organic layer was dried over
Na2SO4, filtered, and evaporated to obtain ((2S,5R)-6-(benzyloxy)-7-
oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)methyl methane sulfonate as oil
(3.2 g) which was taken through to the next step without further
purification. LC−MS Anal. Calcd for C15H20N2O5S, 340.1. Found [M
The above EtOAc solution (10 mL) was treated with
methanesulfonic acid (1.1 mL, 1.7 g, 17.4 mmol), and the mixture
heated at 45 °C for 1 h then allowed to cool to room temperature.
This was added to a solution of potassium bicarbonate (2.9 g, 29
mmol) in water (10 mL) and stirred at 45 °C for 3 h. After cooling,
the phases were separated and the EtOAc phase was washed with 10%
aqueous NaCl solution. The EtOAc solution was used as such in the
next stage.
The above EtOAc solution was cooled to −15 °C and concentrated
H2SO4 (0.6 g, 0.3 mL, 6 mmol) was added. Then, sodium
triacetoxyborohydride (0.5 g, 2.4 mmol) was added portionwise,
allowing the temperature to rise from −15 to −5 °C for over 1 h.
Water was added followed by concentrated aqueous ammonia (1
mL). The mixture was extracted with EtOAc, and the organic extract
washed with brine, dried, and evaporated, affording oil, which was
chromatographed on silica eluting with 15−40% EtOAc in DCM
affording (3R,6S)-N-(benzyloxy)-6-(trifluoromethyl)piperidin-3-
amine as oil (184 mg, 12% over 4 stages). LC−MS Anal. Calcd for
C13H17F3N2O, 274.1. Found [M + H] = 275.
(2S,5R)-6-(Benzyloxy)-2-(trifluoromethyl)-1,6-diazabicyclo-
[3.2.1]octan-7-one. A mixture of (3R,6S)-N-(benzyloxy)-6-
(trifluoromethyl)piperidin-3-amine (0.18 g, 0.67 mmol) and
potassium carbonate (0.53 g, 3.82 mmol) in DCM (20 mL) was
treated with triphosgene (0.2 g, 0.67 mmol) at −10 °C. After 0.5 h,
DMAP (3 mg, 0.03 mmol) was added. The reaction mixture was
stirred overnight and then washed with water, dried (Na2SO4), and
evaporated. The residue was chromatographed on silica eluting with
an EtOAc−DCM gradient affording a colorless solid (0.16 g, 81%).
LC−MS Anal. Calcd for C14H15F3N2O2, 300.1. Found [M + H] =
301.4.
Sodium (2S,5R)-7-Oxo-2-(trifluoromethyl)-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate. A solution of (2S,5R)-6-(benzyloxy)-2-
(trifluoromethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (163 mg, 0.54
mmol) in isopropyl alcohol (IPA) (5 mL) was treated with a sulfur
trioxide trimethylamine complex (85 mg, 0.61 mmol), Et3N (11 mg,
0.11 mmol), 10% palladium on charcoal (10 mg), and water (0.6
mL). The mixture was hydrogenated under balloon pressure for 4.5 h
and then more sulfur trioxide trimethylamine complex (82 mg, 0.3
mmol) was added. The mixture was stirred under nitrogen for 2 h,
1
+ H] = 341.1. H NMR (500 MHz, CDCl3): δ ppm 7.43−7.35 (m,
5H), 5.04 (d, J = 11.5 Hz, 1H), 4.89 (d, J = 11.5 Hz, 1H), 4.40 (dd, J
= 7.5, 11 Hz, 1H), 4.24 (dd, J = 5.5, 11 Hz, 1H), 3.72−3.64 (m, 1H),
3.34 (s, 1H), 3.09 (s, 3H), 3.00−2.99 (m, 2H), 2.03−1.98 (m, 2H),
1.59−1.56 (m, 2H).
(2S,5R)-6-(Benzyloxy)-2-(fluoromethyl)-1,6-diazabicyclo[3.2.1]-
octan-7-one. To a solution of ((2S,5R)-6-(benzyloxy)-7-oxo-1,6-
diazabicyclo[3.2.1]octan-2-yl)methyl methane sulfonate (3.2 g, 9.4
mmol) in ACN (20 mL) was added dropwise tetrabutyl ammonium
fluoride (1.0 M in THF, 8 mL, 15.9 mmol). The reaction mixture was
heated at 70 °C for 10 h and then concentrated under reduced
pressure, and the residue was dissolved in DCM (50 mL) and washed
with water (2 × 40 mL) and brine (20 mL). The organic phase was
dried over Na2SO4, filtered, and evaporated. The crude product was
purified by preparative HPLC [column: ATLANTIS-T3 (250 × 20),
5 μ; mobile phase: (A) 10 mM ammonium bicarbonate in H2O (B)
acetonitrile; flow rate: 20 mL/min, gradient (T % B): 0/40, 8/55, 10/
55, 10.1/98, 12/98, 12.1/40, 15/40] the pure fractions were
combined and lyophilized to obtain (2S,5R)-6-(benzyloxy)-2-
(fluoromethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (700 mg, 28%) as
an off-white solid. LC−MS Anal. Calcd for C14H17FN2O2, 264.1.
Found [M + H] = 265.1. 1H NMR (500 MHz, CDCl3): δ ppm 7.44−
7.35 (m, 5H), 5.05 (d, J = 11.5 Hz, 1H), 4.90 (d, J = 11.5 Hz, 1H),
L
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Article
4.72−4.55 (m, 2H), 3.62−3.50 (m, 1H), 3.33−3.16 (m, 1H), 3.17 (d,
J = 11.5 Hz, 1H), 2.98 (d, J = 11.5 Hz, 1H), 2.08−2.03 (m, 2H),
1.73−1.60 (m, 2H).
(2S,5R)-2-(Fluoromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]-
octan-7-one. According to procedure (a): (2S,5R)-6-(benzyloxy)-2-
(fluoromethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (500 mg, 1.89
mmol) was converted into the desired compound as an off-white
solid (329 mg, 100% yield), which was used immediately in the next
step without purification. LC−MS Anal. Calcd for C7H11FN2O2,
174.1. Found [M + H] = 175.1.
Tetrabutylammonium (2S,5R)-2-(Fluoromethyl)-7-oxo-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (b):
(2S,5R)-2-(fluoromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-7-
one (340 mg, 1.95 mmol) was converted into the desired compound
as colorless oil (201 mg, 21% over 2 steps). LC−MS Anal. Calcd for
C7H10FN2O5S, 253.0. Found [M] = 253.0. 1H NMR (400 MHz,
CDCl3): δ ppm 4.70−4.57 (m, 2H), 4.36−4.35 (m, 1H), 3.60−3.42
(m, 1H), 3.33−3.20 (m, 9H), 3.19−3.17 (m, 1H), 2.25−2.21 (m,
1H), 1.99−1.97 (m, 1H), 1.75−1.63 (m, 10H), 1.45−1.34 (m, 8H),
1.00 (t, J = 7.5 Hz, 12H).
Sodium (2S,5R)-2-(Fluoromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl Sulfate. According to procedure (c): (2S,5R)-2-
(fluoromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (200
mg, 0.40 mmol) was converted to the title compound as a white
solid (64 mg, 57%). HRMS Anal. Calcd for C7H10FN2O5S [M],
253.0289. Found 253.0301. 1H NMR (500 MHz, DMSO-d6): δ ppm
4.70−4.46 (m, 2H), 3.99−3.97 (m, 1H), 3.44−3.35 (m, 1H), 3.18 (d,
J = 11.5 Hz, 1H), 2.94−2.91 (m, 1H), 1.84−1.80 (m, 1H), 1.79−1.69
(m, 2H), 1.51−1.48 (m, 1H).
Sodium (2S,5R)-7-Oxo-2-(difluoromethyl)-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate (11). (2S,5R)-6-(Benzyloxy)-7-oxo-1,6-
diazabicyclo[3.2.1]octane-2-carbaldehyde (4). A solution of
(2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]-
octan-7-one (1.5 g, 5.7 mmol) in DCM (50 mL) was treated at 0 °C
with trichloroisocyanuric acid (1.9 g, 8.6 mmol) and TEMPO (90 mg,
0.6 mmol). The mixture was stirred at 0 °C for 2 h, then filtered
through Celite, and washed with DCM. The combined DCM filtrates
were washed with saturated aqueous sodium bicarbonate solution,
and brine, dried (Na2SO4), and evaporated to afford the known
aldehyde (4) (WO2015/136474) as oil (1.5 g, 100%), which was
used directly in the next step. LC−MS Anal. Calcd for C14H16N2O3,
260.1. Found [M + H] = 261.4. 1H NMR (500 MHz, CDCl3): δ ppm
9.73 (s, 1H), 7.44−7.36 (m, 5H), 5.07 (d, J = 11.5 Hz, 1H), 4.91 (d, J
= 11.5 Hz, 1H), 3.89 (d, J = 8.0 Hz, 1H), 3.27 (s, 1H), 3.14−3.12 (m,
1H), 2.56 (d, J = 12.0 Hz, 1H), 2.19−2.15 (m, 1H), 2.04−1.92 (m,
3H).
(2S,5R)-6-(Benzyloxy)-2-(difluoromethyl)-1,6-diazabicyclo[3.2.1]-
octan-7-one. A solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-
diazabicyclo[3.2.1]octane-2-carbaldehyde (1.5 g, 5.7 mmol) in
DCM (30 mL) at 0 °C was treated with DAST (1.5 mL, 12
mmol). The mixture was stirred for 4 h, then the solvent was removed
by purging with nitrogen. The residue was dissolved in EtOAc and
added to ice-cold water. The organic phase was separated, washed
with saturated aqueous sodium bicarbonate solution and brine, dried
(Na2SO4), and evaporated affording oil. This was chromatographed
on silica eluting with 20% EtOAc in hexane affording yellow oil (0.7 g,
44% over 2 steps). LC−MS Anal. Calcd for C14H16F2N2O2, 282.1.
Found [M + H] = 283.1. 1H NMR (500 MHz, CDCl3): δ ppm 7.43−
7.35 (m, 5H), 5.93 (td, J = 2, 55.5 Hz, 1H), 5.04 (d, J = 11.5 Hz, 1H),
4.89 (d, J = 11.5 Hz, 1H), 3.64−3.52 (m, 1H), 3.34 (t, J = 3.0 Hz,
1H), 3.12 (d, J = 12.0 Hz, 1H), 2.98 (d, J = 11.5 Hz, 1H), 2.07−2.03
(m, 1H), 1.94−1.89 (m, 2H), 1.68−1.65 (m, 1H).
1H), 3.65−3.63 (m, 1H), 3.33−3.28 (m, 1H), 3.17−3.14 (m, 1H),
2.99 (d, J = 12.0 Hz, 1H), 1.94−1.69 (m, 4H).
Tetrabutylammonium (2S,5R)-2-(Difluoromethyl)-7-oxo-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (b):
(2S,5R)-2-(difluoromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-
7-one (0.40 g, 2.1 mmol) was converted to the desired compound as
colorless oil (0.45 g, 42% over 2 steps). LC−MS Anal. Calcd for
1
C7H9F2N2O5S−, 271.0. Found [M] = 271.4. H NMR (400 MHz,
DMSO-d6): δ ppm 6.24 (td, J = 4.4, 55.5 Hz, 1H), 4.03−4.02 (m,
1H), 3.36−3.30 (m, 1H), 3.19−3.14 (m, 9H), 3.00 (d, J = 10.8 Hz,
1H), 1.88−1.83 (m, 1H), 1.77−1.71 (m, 3H), 1.60−1.52 (m, 8H),
1.35−1.28 (m, 8 H), 0.98−0.87 (m, 12H).
Sodium (2S,5R)-7-Oxo-2-(difluoromethyl)-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate. According to procedure (c): tetrabutylam-
monnium (2S,5R)-2-(difluoromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl sulfate (0.45 g, 0.88 mmol) was converted to the title
compound as a white solid (202 mg, 78%). HRMS Anal. Calcd for
C7H9F2N2O5S [M], 271.0195. Found 271.0208. 1H NMR (400 MHz,
DMSO-d6): δ ppm 6.24 (t, J = 4.4 Hz, 1H), 4.06 (m, 1H), 3.40 (m,
1H), 3.20 (m, 1H), 3.05 (m, 1H), 1.95−1.75 (m, 4H). 19F NMR
(470.59 MHz, DMSO-d6): δ ppm −122.87 (ddd, J = 13, 55, 283 Hz,
1F), −127.36 (ddd, J = 15, 55, 283 Hz, 1F).
Sodium (2S,5R)-2-(Chloromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl Sulfate (16). (2S,5R)-6-(Benzyloxy)-2-(chloromethyl)-1,6-
diazabicyclo[3.2.1]octan-7-one. To a solution of ((2S,5R)-6-
(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)methyl methane
sulfonate (2.6 g, 7.63 mmol) in ACN (30 mL) was added tetrabutyl
ammonium chloride (4.2 g, 15.3 mmol). The resulting reaction
mixture was heated at 70 °C for 16 h. The reaction mixture was
concentrated under reduced pressure, then the resulting residue was
dissolved in DCM (50 mL), washed with water (2 × 40 mL) and
brine (20 mL), and dried (Na2SO4). The filtrate was evaporated to
obtain oil, which was purified by chromatography on silica eluting
with 0−50% EtOAc in hexane as the eluent to obtain the desired
compound as an off-white solid (1.3 g, 59%). LC−MS Anal. Calcd for
C14H17ClN2O2, 280.1. Found [M + H] = 281.1. 1H NMR (500 MHz,
CDCl3): δ ppm 7.42−7.34 (m, 5H), 5.02 (d, J = 11.5 Hz, 1H), 4.90
(d, J = 11.5 Hz, 1H), 3.70−3.57 (m, 3H), 3.32 (d, J = 2.5 Hz, 1H),
2.97−2.90 (m, 2H), 2.05−2.01 (m, 2H), 1.69−1.57 (m, 2H).
(2S,5R)-2-(Chloromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]-
octan-7-one. According to procedure (a): (2S,5R)-6-(benzyloxy)-2-
(chloromethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (500 mg, 1.78
mmol) was converted to the desired compound as a white solid
(330 mg), which was used directly in the next step. LC−MS Anal.
Calcd for C7H11ClN2O2, 190.1. Found [M + H] = 191.1.
Tetrabutylammonium (2S,5R)-2-(Chloromethyl)-7-oxo-1,6
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (b):
(2S,5R)-2-(chloromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-7-
one (390 mg, 2.05 mmol) was converted to the desired compound as
colorless oil (110 mg, 10% over 2 steps). LC−MS Anal. Calcd for
1
C7H10ClN2O5S−, 269.0. Found [M] = 269.0. H NMR (500 MHz,
CDCl3): δ ppm 4.37−4.35 (m, 1H), 3.74−3.71 (m, 1H), 3.64−3.58
(m, 2H), 3.33−3.24 (m, 9H), 2.95 (d, J = 12 Hz, 1H), 2.20−2.15 (m,
1H), 2.00−1.91 (m, 1H), 1.71−1.63 (m, 10H), 1.48−1.41 (m, 8H),
1.00 (t, J = 7.5 Hz, 12H).
Sodium (2S,5R)-2-(Chloromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl Sulfate. According to procedure (c): tetrabutylammonium
(2S,5R)-2-(chloromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl
sulfate (110 mg, 0.21 mmol) was converted to the title compound as
an off-white solid (45 mg, 71%). HRMS Anal. Calcd for
C7H10ClN2O5S [M], 268.9993. Found 269.0006. 1H NMR (500
MHz, DMSO-d6): δ ppm 3.97−3.96 (m, 1H), 3.90−3.86 (m, 1H),
3.82−3.79 (m, 1H), 3.35−3.30 (m, 1H), 3.15 (d, J = 12 Hz, 1H),
2.85 (dt, J = 3.5, 12 Hz, 1H), 1.82−1.71 (m, 3H), 1.53−1.50 (m,
1H).
Sodium (2S,5R)-2-(Dichloromethyl)-7-oxo-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate (12). (2S,5R)-6-(Benzyloxy)-2-(dichloro-
methyl)-1,6-diazabicyclo[3.2.1]octan-7-one. A solution of (2S,5R)-
6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde
(2.2 g, 8.4 mmol) in DCM (100 mL) was treated with phosphorus
pentachloride (3.5 g, 16.9 mmol). After 16 h, the mixture was diluted
(2S,5R)-2-(Difluoromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]-
octan-7-one. According to procedure (a): (2S,5R)-6-(benzyloxy)-2-
(difluoromethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (0.60 g, 2.1
mmol) was converted to the desired compound as a white solid
(0.4 g, 100%), which was used directly in the next step. LC−MS Anal.
1
Calcd for C7H10F2N2O2, 192.1. Found [M + H] = 193.1. H NMR
(400 MHz, DMSO-d6): δ ppm 9.73 (s, 1H), 6.24 (td, J = 4.4, 55.5 Hz,
M
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with DCM and washed with ice-cold water and brine, dried
(Na2SO4), and evaporated. The residue was chromatographed on
silica eluting with 0−20% EtOAc in hexane affording a white solid
(130 mg, 5%).
converted to the title compound as a white solid (36 mg, 73%).
HRMS Anal. Calcd for C7H8F3N2O5S2 [M], 320.9821. Found
320.9836. H NMR (500 MHz, DMSO-d6): δ ppm 5.10 (dd, J =
3.5, 8 Hz, 1H), 4.07−4.06 (m, 1H), 3.50 (d, J = 12 Hz, 1H), 3.05 (d, J
= 12.5 Hz, 1H), 2.26−2.18 (m, 1H), 1.90−1.86 (m, 1H), 1.80−1.69
(m, 2H). 19F NMR (470 MHz, DMSO-d6): δ ppm −39.5.
1
LC−MS Anal. Calcd for C14H16Cl2N2O2, 314.1. Found [M + H] =
315.1. 1H NMR (400 MHz, CDCl3): δ ppm 7.42−7.33 (m, 5H), 5.84
(d, J = 4.8 Hz, 1H), 5.01 (d, J = 11.6 Hz, 1H), 4.86 (d, J = 11.6 Hz,
1H), 3.81−3.76 (m, 1H), 3.39−3.37 (m, 1H), 3.25 (d, J = 12.0 Hz,
1H), 3.04−3.00 (m, 1H), 2.11−1.97 (m, 3H), 1.75−1.65 (m, 1H).
(2S,5R)-2-(Dichloromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]-
octan-7-one. According to procedure (a): (2S,5R)-6-(benzyloxy)-2-
(dichloromethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (130 mg, 0.4
mmol) was converted to the desired compound an off-white solid
(85 mg, 92%), which was used immediately in the next stage. LC−MS
Anal. Calcd for C7H10Cl2N2O2, 224.0. Found [M + H] = 225.0.
Tetrabutylammonnium (2S,5R)-2-(Dichloromethyl)-7-oxo-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (b):
(2S,5R)-2-(dichloromethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-
7-one (85 mg, 0.37 mmol) was converted to the desired compound as
colorless oil (68 mg, 33%). LC−MS Anal. Calcd for C7H9Cl2N2O5S−,
303.0. Found [M] = 303.0. 1H NMR (500 MHz, CDCl3): δ 5.87 (d, J
= 5.0 Hz, 1H), 4.38 (br s, 1H), 3.71−3.70 (m, 1H), 3.31−3.26 (m,
10H), 2.09−2.02 (m, 3H), 1.80−1.70 (m, 1H), 1.69−1.63 (m, 8H),
1.48−1.40 (m, 8H), 1.01−0.98 (m, 12H).
Sodium (2S,5R)-2-(Dichloromethyl)-7-oxo-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate. According to procedure (c): tetrabutylam-
monnium (2S,5R)-2-(dichloromethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl sulfate (68 mg, 0.12 mmol) was converted to the title
compound as a white solid (36 mg, 88%). HRMS Anal. Calcd for
C7H9Cl2N2O5S [M], 302.9604. Found 302.9619. 1H NMR (400
MHz, DMSO-d6): δ ppm 6.25 (d, J = 0.8 Hz, 1H, CHCl2), 4.02 (m,
1H), 3.45 (m, 1H), 3.22 (d, 1H), 2.95 (m, 1H), 1.95−1.75 (m, 4H).
Sodium (2R,5R)-7-Oxo-2-[(trifluoromethyl)sulfanyl]-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate (22). (2R,5R)-6-(Benzyloxy)-
2-(trifluoromethylthio)-1,6-diazabicyclo[3.2.1]octan-7-one. A solu-
tion of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-
carboxylic acid (1 g, 3.62 mmol) in acetone (10 mL) was treated with
AgSCF3 (1.51 g, 7.24 mmol), Selectofluor (5.13 g, 14.5 mmol), and
2,6-lutidine (1.04 mL, 7.25 mmol). The reaction mixture was purged
with nitrogen and heated at 90 °C for 0.5 h in a sealed tube. The
reaction mixture was allowed to cool and then quenched with
saturated aqueous K2CO3. The mixture was extracted with diethyl
ether (2 × 50 mL), then the combined organic phases were washed
with brine (20 mL), dried (Na2SO4), filtered, and evaporated. The
crude material was purified by column chromatography, eluting with
20% EtOAc in hexane to afford a pale yellow solid (150 mg, 12%).
LC−MS Anal. Calcd for C14H15F3N2O2S, 332.1. Found [M + H] =
333.0. 1H NMR (400 MHz, CDCl3): δ ppm 7.43−7.32 (m, 5H), 5.18
(d, J = 8.0 Hz, 1H), 5.04 (d, J = 11.6 Hz, 1H), 4.90 (d, J = 11.6 Hz,
1H), 3.50 (d, J = 12.0 Hz, 1H), 3.34 (s, 1H), 3.00 (d, J = 12.0 Hz,
1H), 2.48−2.40 (m, 1H), 2.08−2.02 (m, 1H), 1.69−1.61 (m, 2H).
(2R,5R)-6-Hydroxy-2-(trifluoromethylthio)-1,6-diazabicyclo-
[3.2.1]octan-7-one. According to procedure (a): (2R,5R)-6-(benzyl-
oxy)-2-(trifluoromethylthio)-1,6-diazabicyclo[3.2.1]octan-7-one (200
mg, 0.602 mmol) was converted to the desired compound as an off
white solid (100 mg), which was used in the next step without
purification. LC−MS Anal. Calcd for C7H9F3N2O2S, 242.0. Found
[M + H] = 243.0.
Sodium (2R,5R)-7-Oxo-2-(trifluoromethylsulfonyl)-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate (25). (2R,5R)-6-(Benzyloxy)-
2-(trifluoromethylsulfonyl)-1,6-diazabicyclo[3.2.1]octan-7-one
(30). To a solution of (2R,5R)-6-(benzyloxy)-2-(trifluoromethylthio)-
1,6-diazabicyclo[3.2.1]octan-7-one (550 mg, 1.65 mmol) in CCl4 (5
mL), ACN (5 mL) and water (10 mL) was added sodium periodate
(1.4 g, 6.61 mmol) followed by ruthenium trichloride (34 mg, 0.16
mmol) at 0 °C. The reaction mixture was stirred for 3 h, then was
filtered through Celite, and washed with DCM (10 mL). The filtrate
was washed with water (10 mL), aqueous sodium thiosulfate (10
mL), and brine (10 mL). The organic layer was dried (Na2SO4),
filtered, and evaporated. The resulting crude material was triturated
with n-pentane followed by diethyl ether to give an off-white solid
(350 mg, 58%). LC−MS Anal. Calcd for C14H15F3N2O4S, 364.1.
Found [M + H] = 365.2. 1H NMR (400 MHz, CDCl3): δ ppm 7.40−
7.37 (m, 5H), 5.00 (d, J = 11.6 Hz, 1H), 4.89−4.86 (m, 2H), 3.52 (d,
J = 12.4 Hz, 1H), 3.44 (t, J = 4 Hz, 1H), 3.11 (d, J = 12.4 Hz, 1H),
2.37−2.28 (m, 1H), 2.23−2.17 (m, 2H), 1.91−1.85 (m, 1H).
(2R,5R)-6-Hydroxy-2-(trifluoromethylsulfonyl)-1,6-diazabicyclo-
[3.2.1]octan-7-one. According to procedure (a): (2R,5R)-6-(benzyl-
oxy)-2-(trifluoromethylsulfonyl)-1,6-diazabicyclo[3.2.1]octan-7-one
(350 mg, 0.96 mmol) was converted to the desired compound as an
off-white solid (250 mg, 95%), which was used in the next step
without purification. LC−MS Anal. Calcd for C7H9F3N2O4S, 274.0.
Found [M + H] = 275.2.
Tetrabutylammonium (2R,5R)-7-Oxo-2-(trifluoromethylsulfon-
yl)-1,6-diazabicyclo[3.2.1]octan-6-yl Sulfate. According to proce-
dure (b): (2R,5R)-6-hydroxy-2-(trifluoromethylsulfonyl)-1,6-
diazabicyclo[3.2.1]octan-7-one (250 mg, 0.91 mmol) was converted
to the desired compound as oil (200 mg). A portion of this material
(50 mg) was further purified by preparative HPLC [column:
KROMOSIL-C18 (150 × 25), 10 μ; mobile phase: (A) 10 mM
NH4OAc in H2O, (B) acetonitrile; flow rate: 25 mL/min; gradient (T
% B): 0/5, 8/40, 8.1/98, 10/98, 10.1/5, 12/5] to afford a white solid
−
(27 mg). LC−MS Anal. Calcd for C7H8F3N2O7S2 , 353.0. Found
1
[M] = 353.0. H NMR (500 MHz, DMSO-d6): δ ppm 5.35 (t, J = 9
Hz, 1H), 4.13−4.12 (m, 1H), 3.45 (d, J = 12.0 Hz, 1H), 3.20−3.14
(m, 9H), 2.28−2.23 (m, 1H), 2.13−2.11 (m, 1H), 1.97−1.84 (m,
2H), 1.59−1.53 (m, 8H), 1.34−1.27 (m, 8H), 0.95−0.92 (m, 12H).
Sodium (2R,5R)-7-Oxo-2-(trifluoromethylsulfonyl)-1,6-
diazabicyclo[3.2.1]octan-6-yl. According to procedure (c): tetrabu-
tylammonium (2R,5R)-7-oxo-2-(trifluoromethylsulfonyl)-1,6-
diazabicyclo[3.2.1]octan-6-yl sulfate (25 mg, 0.041 mmol) was
converted to the title compound as a white solid (13 mg, 86%).
HRMS Anal. Calcd for C7H8F3N2O7S2 [M], 352.9720. Found
1
352.9735. H NMR (500 MHz, DMSO-d6): δ ppm 5.37−5.33 (m,
1H), 4.13−4.12 (m, 1H), 3.45 (d, J = 12.0 Hz, 1H), 3.19 (d, J = 1.5,
10.5 Hz, 1H), 2.29−2.24 (m, 1H), 2.14−2.10 (m, 1H), 1.99−1.85
(m, 2H).
Sodium (2S,5R)-2-(1,1-Difluoroethyl)-7-oxo-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate (31). (2S,5R)-6-(Benzyloxy)-N-methoxy-N-
methyl-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide. To a
stirred solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo-
[3.2.1]octane-2-carboxylic acid (1.5 g, 5.43 mmol) in THF (50
mL) was added DIPEA (3.7 mL, 21.7 mmol), N,O-dimethylhydroxyl-
amine hydrochloride (794 mg, 8.15 mmol) at 0 °C followed by T3P
(50% in EtOAc, 6.9 mL, 10.8 mmol). The resulting reaction mixture
was stirred for 4 h, then diluted with EtOAc (100 mL), washed with
water (50 mL), sat. NaHCO3 solution (50 mL), and brine (50 mL).
The organic layer was dried (Na2SO4), filtered, and evaporated to give
oil. This was purified by silica chromatography eluting with 40%
EtOAc in hexane affording brown oil (900 mg, 53%). LC−MS Anal.
Tetrabutylammonium (2R,5R)-7-Oxo-2-(trifluoromethylthio)-
1,6-diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure
(b): (2R,5R)-6-hydroxy-2-(trifluoromethylthio)-1,6-diazabicyclo-
[3.2.1]octan-7-one (100 mg, 0.413 mmol) was converted to the
1
desired compound as colorless oil (80 mg, 34% over 2 steps). H
NMR (500 MHz, CDCl3): δ ppm 5.13 (d, J = 7.0 Hz, 1H), 4.40 (s,
1H), 3.54 (d, J = 12.0 Hz, 1H), 3.29−3.26 (m, 9H), 2.40−2.38 (m,
1H), 2.26−2.23 (m, 1H), 1.75−1.66 (m, 9H), 1.48−1.39 (m, 9 H),
1.09 (t, J = 7.5 Hz, 12H).
Sodium (2R,5R)-7-Oxo-2-((trifluoromethyl)thio)-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (c):
Tetrabutyl ammonium (2R,5R)-7-oxo-2-(trifluoromethylthio)-1,6-
diazabicyclo[3.2.1]octan-6-yl sulfate (80 mg, 0.24 mmol) was
1
Calcd for C16H21N3O4, 319.2. Found [M + H] = 320.1. H NMR
(400 MHz, CDCl3): δ ppm 7.44−7.34 (m, 5H), 5.04 (d, J = 11.6 Hz,
1H), 4.90 (d, J = 11.6 Hz, 1H), 4.53 (br s, 1H), 3.80 (s, 3H), 3.34 (s,
N
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1H), 3.26−3.10 (m, 4H), 2.91 (d, J = 11.2 Hz, 1H), 2.04−1.88 (m,
4H).
mmol) at 0 °C, and the reaction mixture was stirred for 4 h. The
reaction mixture was evaporated, and then DMSO (70 mL) and Ac2O
(16 mL) were added. The resulting mixture was stirred for 48 h, then
the reaction mixture was quenched with saturated aqueous NaHCO3
solution, and extracted with diethyl ether (2 × 100 mL). The organic
layer was washed with water (50 mL) and brine (50 mL), dried
(Na2SO4) filtered, and evaporated. The residue was chromatographed
on silica, eluting with 25% EtOAc in hexane to afford a yellow solid
(1.3 g, 24%). LC−MS Anal. Calcd for C17H17N3O3S, 343.1. Found
(2S,5R)-2-Acetyl-6-(benzyloxy)-1,6-diazabicyclo[3.2.1]octan-7-
one. To a stirred solution of (2S,5R)-6-(benzyloxy)-N-methoxy-N-
methyl-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide (900 mg,
2.81 mmol) in THF (20 mL) was added a solution of
methylmagnesium bromide in diethylether (3 M; 1.8 mL, 5.63
mmol) at −78 °C under a nitrogen atmosphere. The reaction mixture
was stirred at −78 °C for 1 h, then quenched with EtOAc (10 mL) at
−78 °C, followed by water (10 mL). The reaction mixture was
allowed to warm to room temperature, then diluted with EtOAc (100
mL) and washed with water (50 mL) and brine (50 mL). The organic
layer was dried (Na2SO4), filtered, and evaporated. The crude
material was purified by silica chromatography eluting with 25%
EtOAc in hexane to afford a white solid (500 mg, 64%). LC−MS
1
[M + H] = 344.1. H NMR (400 MHz, CDCl3): δ ppm 8.07 (d, J =
3.2 Hz, 1H), 7.69 (d, J = 3.2 Hz, 1H), 7.45−7.34 (m, 5H), 5.19 (d, J
= 6.4 Hz, 1H), 5.07 (d, J = 11.6 Hz, 1H), 4.92 (d, J = 11.6 Hz, 1H),
3.33 (d, J = 2.8 Hz, 1H), 3.11−3.00 (m, 2H), 2.29−2.18 (m, 1H),
2.14−2.06 (m, 2H), 1.81−1.68 (m, 1H).
(2S,5R)-6-(Benzyloxy)-2-(difluoro(thiazol-2-yl)methyl)-1,6-
diazabicyclo[3.2.1]octan-7-one (14). To a stirred solution of
(2S,5R)-6-(benzyloxy)-2-(thiazole-2-carbonyl)-1,6-diazabicyclo-
[3.2.1]octan-7-one (1.3 g, 3.79 mmol) in DCM (25 mL) was added
DAST (4.9 mL, 37.9 mmol) at 0 °C under a N2 atmosphere. The
resulting reaction mixture was stirred for 16 h. The reaction mixture
was diluted with DCM (50 mL), washed with saturated aqueous
NaHCO3 (25 mL), brine (25 mL), dried (Na2SO4), and evaporated.
The residue was purified by chromatography on silica eluting with
25% EtOAc in hexane to afford yellow oil (0.8 g, 57%). LC−MS Anal.
Calcd for C17H17F2N3O2S, 365.1. Found [M + H] = 366.0. 1H NMR
(400 MHz, CDCl3): δ ppm 7.92 (d, J = 1.6 Hz, 1H), 7.49 (d, J = 2.8
Hz, 1H), 7.41−7.33 (m, 5H), 4.99 (d, J = 11.6 Hz, 1H), 4.86 (d, J =
11.6 Hz, 1H), 4.20−4.04 (m, 1H), 3.38 (s, 1H), 3.25 (d, J = 12.0 Hz,
1H), 2.97 (d, J = 12.4 Hz, 1H), 2.14−2.02 (m, 3H), 1.77−1.65 (m,
1H).
1
Anal. Calcd for C15H18N2O3, 274.1. Found [M + H] = 275.1. H
NMR (500 MHz, CDCl3): δ ppm 7.43−7.35 (m, 5H), 5.06 (d, J =
11.5 Hz, 1H), 4.90 (d, J = 11.5 Hz, 1H), 3.92 (d, J = 7.5 Hz, 1H),
3.27 (d, J = 1.5 Hz, 1H), 3.03−3.00 (m, 1H), 2.48 (d, J = 11.5 Hz,
1H), 2.30 (s, 3H), 2.20−2.15 (m, 1H), 2.00−1.90 (m, 1H), 1.87−
1.82 (m, 1H), 1.57−1.55 (m, 1H).
(2S,5R)-6-(Benzyloxy)-2-(1,1-difluoroethyl)-1,6-diazabicyclo-
[3.2.1]octan-7-one. To a stirred solution of (2S,5R)-2-acetyl-6-
(benzyloxy)-1,6-diazabicyclo[3.2.1]octan-7-one (500 mg, 1.82 mmol)
in DCM (10 mL) was added DAST (2.4 mL, 18.2 mmol) at 0 °C
under a N2 atmosphere and stirred for 48 h. The volatile components
were removed by purging with nitrogen. The resulting residue was
partitioned between EtOAc (50 mL) and ice-cold water (50 mL).
The organic phase was separated, washed with saturated aqueous
NaHCO3 (25 mL) and brine (25 mL), dried (Na2SO4), and
evaporated. The crude material was purified by chromatography
eluting with 10% EtOAc in hexane to afford yellow oil (400 mg, 74%).
LC−MS Anal. Calcd for C15H18F2N2O2, 296.1. Found [M + H] =
297.1. 1H NMR (400 MHz, CDCl3): δ ppm 7.43−7.34 (m, 5H), 5.03
(d, J = 11.6 Hz, 1H), 4.88 (d, J = 11.6 Hz, 1H), 3.51−3.44 (m, 1H),
3.35−3.33 (m, 1H), 3.13 (d, J = 11.6 Hz, 1H), 2.94 (d, J = 12.0 Hz,
1H), 2.07−1.88 (m, 3H), 1.88−1.57 (m, 4H).
(2S,5R)-2-(Difluoro(thiazol-2-yl)methyl)-6-hydroxy-1,6-
diazabicyclo[3.2.1]octan-7-one. According to procedure (a):
(2S,5R)-6-(benzyloxy)-2-(difluoro(thiazol-2-yl)methyl)-1,6-
diazabicyclo[3.2.1]octan-7-one (800 mg, 2.19 mmol) was converted
to the desired compound as an off-white solid (301 mg, 49%). This
crude material was used in the next step without purification. LC−MS
1
Anal. Calcd for C10H11F2N3O2S, 275.1. Found [M + H] = 276.0. H
NMR (500 MHz, CDCl3): δ ppm 7.93 (d, J = 3.5 Hz, 1H), 7.51 (d, J
= 3.5 Hz, 1H), 4.25−4.15 (m, 1H), 3.81 (s, 1H), 3.36 (d, J = 12.0 Hz,
1H), 3.13 (d, J = 11.5 Hz, 1H), 2.23−2.15 (m, 2H), 2.08−2.03 (m,
1H), 1.89−1.80 (m, 1H).
Tetrabutylammonium (2S,5R)-2-(Difluoro(thiazol-2-yl)methyl)-
7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl Sulfate. According to pro-
cedure (b): (2S,5R)-2-(difluoro(thiazol-2-yl)methyl)-6-hydroxy-1,6-
diazabicyclo[3.2.1]octan-7-one (301 mg, 1.45 mmol) was converted
to the desired compound as colorless oil (250 mg, 38% over 2 steps).
1H NMR (500 MHz, CDCl3): δ ppm 7.90 (d, J = 3.0 Hz, 1H), 7.49
(2S,5R)-2-(1,1-Difluoroethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]-
octan-7-one. According to procedure (a): (2S,5R)-6-(benzyloxy)-2-
(1,1-difluoroethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (280 mg, 0.94
mmol) was converted to the desired compound as a white solid (175
mg), which was used directly in the next step. LC−MS Anal. Calcd for
1
C8H12F2N2O2, 206.1. Found [M + H] = 207.1. H NMR (500 MHz,
DMSO-d6): δ ppm 9.68 (s, 1H), 3.64−3.62 (m, 1H), 3.36−3.32 (m,
1H), 3.11 (d, J = 11.5 Hz, 1H), 3.00 (d, J = 11.5 Hz, 1H), 1.92−1.77
(m, 3H), 1.72−1.61 (m, 4H).
Tetrabutylammonium (2S,5R)-2-(1,1-Difluoroethyl)-7-oxo-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (b):
(2S,5R)-2-(1,1-difluoroethyl)-6-hydroxy-1,6-diazabicyclo[3.2.1]-
octan-7-one (170 mg, 0.82 mmol) was converted to the desired
compound as colorless oil (200 mg, 46%). LC−MS Anal. Calcd for
(d, J = 3.0 Hz, 1H), 4.38 (s, 1H), 4.18−4.12 (m, 1H), 3.33−3.23 (m,
10H), 2.21−2.09 (m, 2H), 1.99−1.91 (m, 1H), 1.86−1.80 (m, 1H),
1.68−1.62 (m, 8H), 1.47−1.39 (m, 8H), 1.01−0.98 (m, 12H).
Sodium (2S,5R)-2-(Difluoro(thiazol-2-yl)methyl)-7-oxo-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate. According to procedure (c):
tetrabutylammonium (2S,5R)-2-(difluoro(thiazol-2-yl)methyl)-7-oxo-
1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (100 mg, 0.16 mmol) was
converted to the title compound as a white solid (21 mg, 33%).
HRMS Anal. Calcd for C10H10F2N3O5S2 [M], 354.0024. Found
1
C8H11F2N2O5S−, 285.0. Found [M] = 285.2. H NMR (500 MHz,
CDCl3): δ ppm 4.35 (br s, 1H), 3.51−3.39 (m, 1H), 3.31−3.23 (m,
10H), 2.19−2.12 (m, 1H), 2.00−1.86 (m, 2H), 1.78−1.63 (m, 12H),
1.48−1.41 (m, 8H), 1.02−0.99 (m, 12H).
Sodium (2S,5R)-2-(1,1-Difluoroethyl)-7-oxo-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate. According to procedure (c): tetrabutylam-
monium (2S,5R)-2-(1,1-difluoroethyl)-7-oxo-1,6-diazabicyclo[3.2.1]-
octan-6-yl sulfate (50 mg, 0.09 mmol) was converted to the title
compound as a white solid (17 mg, 63%). HRMS Anal. Calcd for
C8H11F2N2O5S [M], 285.0351. Found 285.0365. 1H NMR (500
MHz, DMSO-d6): δ ppm 4.04−4.02 (m, 1H), 3.38−3.35 (m, 1H),
3.16 (d, J = 12 Hz, 1H), 3.02−3.00 (d, J = 11.5 Hz, 1H), 1.87−1.81
(m, 2H), 1.78−1.71 (m, 2H), 1.69 (t, J = 19.5 Hz, 3H).
1
354.0039. H NMR (500 MHz, DMSO-d6): δ ppm 8.04 (s, 2H),
4.04−3.97 (m, 2H), 3.21 (d, J = 12 Hz, 1H), 3.00 (dd, J = 10.5 Hz, J
= 1.0 Hz, 1H), 2.03−1.96 (m, 1H), 1.88−1.76 (m, 3H). 19F NMR
(470 MHz, DMSO-d6): δ ppm −96. 97 (dd, J = 260 Hz, J = 12 Hz),
−99. 25 (dd, J = 260 Hz, J = 16 Hz).
Sodium (2S,5R)-7-Oxo-2-[(trifluoromethoxy)methyl]-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate (17). (2S,5R)-6-(Benzyloxy)-
2-((trifluoromethoxy)methyl)-1,6-diazabicyclo[3.2.1]octan-7-one.
A solution of (2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-
diazabicyclo[3.2.1]octan-7-one (1 g, 3.81 mmol) in EtOAc (50 mL)
was treated with KF (0.88 g, 15.3 mmol), AgOTf (1.78 g, 11.4
mmol), Selectofluor (2.02 g, 5.72 mmol), 2-fluoro pyridine (1.21 mL,
11.4 mmol), and TMS-CF3 (1.45 mL, 11.4 mmol). The reaction
mixture was purged with nitrogen for 10 min and then heated at 80
Sodium (2S,5R)-2-[Difluoro(1,3-thiazol-2-yl)methyl]-7-oxo-1,6-
diazabicyclo[3.2.1]octan-6-yl Sulfate (10). (2S,5R)-6-(Benzyloxy)-
2-(thiazole-2-carbonyl)-1,6-diazabicyclo[3.2.1]octan-7-one (13).
To a stirred solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-
diazabicyclo[3.2.1]octane-2-carbaldehyde 4 (4.0 g, 12.5 mmol) in
DCM (40 mL) was added 2-(trimethylsilyl)thiazole (2.5 mL, 18.8
O
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Article
°C for 16 h in a sealed tube. The reaction mixture was allowed to cool
to room temperature, filtered, and washed with EtOAc. The
combined filtrates were washed with water (50 mL) and brine (50
mL), dried over anhydrous Na2SO4, filtered, and evaporated. The
crude material was purified by chromatography on silica eluting with
40% EtOAc in hexane to afford a yellow solid (200 mg, 16%). LC−
MS Anal. Calcd for C15H17F3N2O3, 330.1. Found [M + H] = 331.1.
1H NMR (500 MHz, CDCl3): δ ppm 7.43−7.34 (m, 5H), 5.03 (d, J =
without purification. LC−MS Anal. Calcd for C6H9ClN2O2, 176.0.
Found [M + H] = 177.0.
Tetrabutylammonium (2R,5R)-2-Chloro-7-oxo-1, 6-Diazabicyclo
[3.2.1] Octan-6-yl Sulfate. According to procedure (b): (2R,5R)-2-
chloro-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-7-one (150 mg, 0.849
mmol) was converted to the desired compound as colorless oil (130
mg, 30% over 2 steps). LC−MS Anal. Calcd for C6H8ClN2O5S−,
255.0. Found [M] = 254.9. 1H NMR (400 MHz, CDCl3): δ ppm 5.47
(d, J = 5.6 Hz, 1H), 4.42 (s, 1H), 3.64 (d, J = 12.0 Hz, 1H), 3.30−
3.22 (m, 9H), 2.38−2.34 (m, 1H), 2.21−2.10 (m, 1H), 1.96−1.80
(m, 2H), 1.70−1.62 (m, 8H), 1.49−1.39 (m, 8H), 1.02−0.98 (m,
12H).
11.5 Hz, 1H), 4.89 (d, J = 11.5 Hz, 1H), 4.13−4.05 (m, 2H), 3.67−
3.65 (m, 1H), 3.33 (s, 1H), 2.99 (s, 2H), 2.06−2.00 (m, 2H), 1.66−
1.61 (m, 2H).
(2S,5R)-6-Hydroxy-2-((trifluoromethoxy)methyl)-1,6-
diazabicyclo[3.2.1]octan-7-one. According to procedure (a):
(2S,5R)-6-(benzyloxy)-2-((trifluoromethoxy)methyl)-1,6-
diazabicyclo[3.2.1]octan-7-one (200 mg, 0.60 mmol) was converted
to the desired compound as an off-white solid (100 mg). LC−MS
Anal. Calcd for C8H11F3N2O3, 240.1. Found [M + H] = 241.0.
Tetrabutylammonium (2S,5R)-7-Oxo-2-((trifluoromethoxy)-
methyl)-1,6-diazabicyclo[3.2.1]octan-6-yl Sulfate. According to
procedure (b): (2S,5R)-6-hydroxy-2-((trifluoromethoxy)methyl)-1,6-
diazabicyclo[3.2.1]octan-7-one (120 mg, 0.5 mmol) was converted to
the desired compound as colorless oil (100 mg, 34% over 2 steps).
LC−MS Anal. Calcd for C8H10F3N2O6S−, 319.0. Found [M] = 319.0.
Sodium (2R,5R)-7-Oxo-2-(trifluoromethoxy)-1,6-diazabicyclo-
[3.2.1]octan-6-yl Sulfate. According to procedure (c): tetrabutylam-
monium (2S,5R)-7-oxo-2-((trifluoromethoxy)methyl)-1,6-
diazabicyclo[3.2.1]octan-6-yl sulfate (100 mg, 0.18 mmol) was
converted to the desired compound as a white solid. This was further
purified by preparative HPLC [column: X-SELECET-C18 (150 ×
19), 5 μ; mobile phase: (A) 10 mM ammonium bicarbonate in H2O,
(B) acetonitrile; flow rate: 18 mL/min; gradient (T % B): 0/5, 6/60,
6.1/98, 8/98, 8./5, 11/5]. The product-containing fractions were
lyophilized to afford the title compound as a white solid (31 mg,
51%). HRMS Anal. Calcd for C8H10F3N2O6S [M], 319.0206. Found
319.0220. 1H NMR (500 MHz, DMSO-d6): δ ppm 4.32 (dd, J = 10.5
Hz, J = 9.5 Hz, 1H), 4.16 (dd, J = 10.5 Hz, J = 5.5 Hz, 1H), 3.98 (d, J
= 3 Hz, 1H), 3.45−3.42 (m, 1H), 3.20 (d, J = 12.0 Hz, 1H), 2.91 (d, J
= 12.0 Hz, 1H), 1.85−1.69 (m, 3H), 1.50−1.45 (m, 1H). 19F NMR
(470 MHz, DMSO-d6): δ ppm −58.68.
Sodium (2R,5R)-2-Chloro-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-
yl Sulfate (23). (2R,5R)-6-(Benzyloxy)-2-chloro-1,6-diazabicyclo-
[3.2.1]octan-7-one. To a solution of (2S,5R)-6-(benzyloxy)-7-oxo-
1,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (1 g, 3.62 mmol) in
DMF (20 mL) and AcOH (4 mL) was added N-chlorosuccinimide
(4.83 g, 36.2 mmol). The reaction mixture was purged with nitrogen
gas for 5 min and then Pb(OAc)4 (2.4 g, 5.43 mmol) was added. The
reaction mixture was purged with nitrogen gas for further 5 min and
then heated at 60 °C for 4 h. The cooled mixture was treated with
saturated aqueous K2CO3 and extracted with diethyl ether (2 × 50
mL). The combined organic extracts were washed with brine (20
mL), dried over anhydrous Na2SO4, filtered, and evaporated. The
residue was chromatographed on silica eluting with 10% EtOAc in
hexane affording yellow oil (270 mg, 28%). LC−MS Anal. Calcd for
C13H15ClN2O2. 266.1. Found [M + H] = 267.0. 1H NMR (500 MHz,
CDCl3): δ ppm 7.43−7.36 (m, 5H), 5.49 (d, J = 5.5 Hz, 1H), 5.05 (d,
J = 11.5 Hz, 1H), 4.90 (d, J = 11.5 Hz, 1H), 3.55 (d, J = 12.0 Hz, 1H),
3.36 (dd, J = 5.5 Hz, J = 3.5 Hz, 1H), 2.98 (dd, J = 11.5 Hz, J = 3.0
Hz, 1H), 2.44−2.37 (m, 1H), 1.98−1.85 (m, 2H), 1.83−1.78 (m,
1H).
Sodium (2R,5R)-2-Chloro-7-oxo-1, 6-Diazabicyclo [3.2.1] Octan-
6-yl Sulfate. According to procedure (c): tetrabutylammonium
(2R,5R)-2-chloro-7-oxo-1, 6-diazabicyclo [3.2.1] octan-6-yl sulfate
(130 mg, 0.26 mmol) was converted to the desired compound as a
white solid (20 mg), which was further purified by preparative HPLC
[X-SELECT-C18 (150 × 19), 5 μ, mobile phase: H2O: MeCN]. The
collected fractions were freeze-dried to afford the title compound (5.0
mg, 7%) as an off-white solid. LC−MS Anal. Calcd for
1
C6H8ClN2O5S−, 255.0. Found [M] = 255.0. H NMR (500 MHz,
DMSO-d6): δ ppm 5.51 (dd, J = 6.0 Hz, J = 2.0 Hz, 1H), 4.10 (d, J =
3.0 Hz, 1H), 3.51−3.49 (obs, 1H), 3.07−3.05 (d, J = 12.0 Hz, 1H),
2.26−2.20 (m, 1H), 1.94−1.80 (m, 3H)..
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01535.
■
sı
Tested compounds including Smiles strings (CSV)
Molecular modeling for Figure 3A (PDB)
Molecular modeling for Figure 3B (PDB)
Includes synthesis and characterization of key com-
pounds, experimental procedures for enzyme inhibition
assays, in vitro antimicrobial susceptibility testing,
efficacy studies, protein crystallization, and collection
and processing of diffraction data (with resulting
statistics) (PDF)
Accession Codes
The coordinates and structure factors of the OXA-
48:ANT3310 (compound 21) complex were deposited to
the Protein Data Bank under accession code 6ZXI.
AUTHOR INFORMATION
Corresponding Author
■
David T. Davies − Antabio SAS, 31670 Labège, France;
orcid.org/0000-0001-7392-4886; Email: david.davies@
antabio.com
Authors
Simon Leiris − Antabio SAS, 31670 Labège, France
Magdalena Zalacain − Antabio SAS, 31670 Labège, France
Nicolas Sprynski − Antabio SAS, 31670 Labège, France;
orcid.org/0000-0001-8165-6949
́
̂
Jerome Castandet − Antabio SAS, 31670 Labège, France
Justine Bousquet − Antabio SAS, 31670 Labège, France
Clarisse Lozano − Antabio SAS, 31670 Labège, France
Agustina Llanos − Antabio SAS, 31670 Labège, France
Laethitia Alibaud − Antabio SAS, 31670 Labège, France
Srinivas Vasa − GVK Biosciences Pvt. Ltd., Hyderabad,
Telangana 500 076, India
NMR experiments showed coupling constants for the H atom on
the same carbon as the Cl of 5.5 Hz (coupling to the axial proton on
the adjacent carbon), thereby establishing that this H atom has an
equatorial disposition and the molecule has the stereochemistry as
shown. See the Supporting Information for related experiments in the
F analogue series.
(2R,5R)-2-Chloro-6-hydroxy-1,6-diazabicyclo[3.2.1]octan-7-one.
According to procedure (a): (2R,5R)-6-(benzyloxy)-2-chloro-1,6-
diazabicyclo[3.2.1]octan-7-one (220 mg, 0.824 mmol) was converted
to the desired compound as a white solid (150 mg), which was used
Ramesh Pattipati − GVK Biosciences Pvt. Ltd., Hyderabad,
Telangana 500 076, India; orcid.org/0000-0002-9925-
2704
P
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Article
Ravindar Valige − GVK Biosciences Pvt. Ltd., Hyderabad,
Telangana 500 076, India
(proposal MX21741) and the staff of beamline I04 for the
assistance.
Bhaskar Kummari − GVK Biosciences Pvt. Ltd., Hyderabad,
Telangana 500 076, India
ABBREVIATIONS
■
Srinivasu Pothukanuri − GVK Biosciences Pvt. Ltd.,
Hyderabad, Telangana 500 076, India; orcid.org/0000-
0002-0374-8060
Cyntia De Piano − International Health Management
Associates (IHMA), 1870 Monthey, Switzerland
Ian Morrissey − International Health Management Associates
(IHMA), 1870 Monthey, Switzerland
ACN, acetonitrile; AcOH, acetic acid; Ac2O, acetic anhydride;
AgNO3, silver nitrate; AgOTf, silver triflate; AIBN, azobisiso-
butyronitrile; Boc, tert-butoxycarbonyl; Boc2O, di-tert-butyl
decarbonate; Bu3SnH, tributyltin hydride; brine, saturated
aqueous sodium chloride solution; DAST, diethylaminosulfur
trifluoride; DCM, dichloromethane; DIPEA, N,N-diisopropy-
lethylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N-
dimethylformamide; dppf, 1,1′-bis(diphenylphosphino)-
ferrocene; EtOAc, ethyl acetate; EtOH, ethanol; Et3N,
triethylamine; IPA, isopropyl alcohol; MEM, meropenem;
MeOH, methanol; Na2SO4, sodium sulfate; Pb(OAc)4, lead
tetraacetate; SO3.pyr, pyridine sulfur trioxide complex;
Selectfluor, 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo-
[2.2.2]octane bis(tetrafluoroborate); TBAA, tetra(n-butyl)-
ammonium acetate; TEMPO, (2,2,6,6-tetramethylpiperidin-1-
yl)oxyl; TMSCF3, trimethyl(trifluoromethyl)silane; THF,
tetrahydrofuran; T3P, |propylphosphinic anhydride
Kirsty Holden − Evotec (UK) Ltd., Macclesfield, Cheshire
SK10 4TG, U.K.
Peter Warn − Evotec (UK) Ltd., Macclesfield, Cheshire SK10
4TG, U.K.
Francesca Marcoccia − Dipartimento di Biotecnologie
Mediche, University of Siena, Siena 53100, Italy
Manuela Benvenuti − Dipartimento di Biotecnologie, Chimica
e Farmacia, University of Siena, Siena 53100, Italy;
orcid.org/0000-0003-0709-2537
Cecilia Pozzi − Dipartimento di Biotecnologie, Chimica e
Farmacia, University of Siena, Siena 53100, Italy
Giusy Tassone − Dipartimento di Biotecnologie, Chimica e
Farmacia, University of Siena, Siena 53100, Italy;
orcid.org/0000-0002-2575-5528
Stefano Mangani − Dipartimento di Biotecnologie, Chimica e
Farmacia, University of Siena, Siena 53100, Italy;
orcid.org/0000-0003-4824-7478
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01535
Author Contributions
D.T.D. and S.L. designed the compounds and performed the
molecular modeling; C.P., R.E., S.V., R.P., R.V., B.K., and S.P.
synthesized the compounds; J.C., J.B., C.L., and A.L.
performed the enzymology and MIC experiments; M.Z.,
N.S., M.L., and M.E. devised the screening cascades and
biological strategies; M.Z., C.D.P., and I.M. devised and
performed the large-scale MIC profiling experiments; M.Z.,
K.H., and P.W. devised and performed the efficacy experi-
ments; F.M., M.B., C.P., S.M., G.T., and J.-D.D. performed the
protein crystallography and X-ray determination. All the
authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
(11) Lomovskaya, O.; Tsivkovski, R.; Nelson, K.; Rubio-Aparicio,
D.; Sun, D.; Totrov, M.; Dudley, M. N. Spectrum of β-lactamase
inhibition by the cyclic boronate QPX7728, an ultra-broad-spectrum
β-lactamase inhibitor of serine and metallo β-lactamases: Enhance-
ment of activity of multiple antibiotics against isogenic strains
ACKNOWLEDGMENTS
■
D.D. is grateful to Prof. Jim Anderson (University College
London) for helpful discussions. M.B., C.P., G.T., S.M., and J.-
D.D. would like to thank Diamond Light Source for beamtime
Q
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