Syntheses and studies of new forms of N-sulfonyloxy β-lactams as potential antibacterial agents and β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.08.005

The synthesis of a small library of N-sulfonyloxy-2-azetidinones is reported and the preliminary results of the investigation of the biological activity of these molecules are discussed. These new multi-electrophilic β-lactams ('electrophilic bombs') disp

Full text of DOI:10.1016/j.bmc.2015.08.005

Accepted Manuscript
Syntheses and Studies of New Forms of N-Sulfonyloxy β-Lactams as Potential
Antibacterial Agents and β-Lactamase Inhibitors
Serena Carosso, Marvin J. Miller
PII:
S0968-0896(15)00672-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.005
BMC 12509
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 July 2015
30 July 2015
8 August 2015
Please cite this article as: Carosso, S., Miller, M.J., SynthesesandStudiesofNewFormsofN-Sulfonyloxyβ-Lactams
as Potential Antibacterial Agents and β-Lactamase Inhibitors, Bioorganic & Medicinal Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bmc.2015.08.005
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Syntheses and Studies of New Forms of N-Sulfonyloxy β-Lactams as Potential
Antibacterial Agents and β-Lactamase Inhibitors.
Serena Carosso and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556
*mmiller1@nd.edu
Abstract: The synthesis of a small library of N-sulfonyloxy-2-azetidinones is reported and the
preliminary results of the investigation of the biological activity of these molecules are discussed. These
new multi-electrophilic β-lactams (“electrophilic bombs”) display unexpected selectivity in their
antibacterial activity and β-lactamase inhibitory activity.
S
δ+
N
Br
δ+
N
δ+
N
Br
N
S
R
N
δ+
O
O SO2R(Ar)
N
N
N
O
OSO2Ar
O
OSO2Ar
O
OSO2Ar
"electrophilic bomb"?
1. Introduction
The production of β-lactamase enzymes is an important mechanism of bacterial
resistance to β-lactam antibiotics.1-5 These enzymes acylate the β-lactam generating an acyl-
enzyme species that undergoes subsequent hydrolysis with concomitant destruction of the
essential β-lactam ring. The enzyme is regenerated and the inactivated antibiotic molecule is
released. A temporarily successful approach to overcome the adverse action of these enzymes
has been the use of β-lactamase inhibitors in combination with classical β-lactam antibiotics,
such as penicillins.6-8 However, the development of variants of β-lactamases is rendering the
current inhibitors less effective. Three β-lactam derivatives are used clinically for the inhibition
of class A β-lactamases: clavulanic acid, sulbactam and tazobactam.9 Since these important
compounds have a similar mode of action, mutations in the targeted β-lactamase enzymes may
result in cross-resistance to all three compounds. No effective inhibitors have been developed for
metallo β-lactamase enzymes which are also of growing prevalence and threaten the utility of
1

our still essential β-lactam antibiotic arsenal. The development of new β-lactamase inhibitors
with different modes of action is therefore needed.
Our group previously reported a novel class of N-sulfonyloxy monocyclic β-lactams,
with the generalized structure 1 (Figure 1) and determined that many, including derivatives 2-4,
were efficient inhibitors of class A β-lactamases.10-14
R1
1
, Generalized structure of previously
reported N-sulfonyloxy substituted
R
O
β-lactams with potent
inhibitory activity.
β-lactamase
N
O SO2R(Ar)
1
OH
Me
O
O
N
N
N
O SO2Ar
O SO2Ar
O S
O
O
O
3
2
4
Figure 1. Previously reported β-lactamase inhibitors.
The mechanism of action of these N-tosyloxy-2-azetidinones is related to the ability of
the constituent sulfonyloxy group to act as a good leaving group (Scheme 1). Rapid acylation of
the active site of the serine β-lactamase is followed by an elimination step which generates an
acyl-enzyme species that is resilient to hydrolysis.
R1
O
R2
NH2
O
O
Ser
R1
7
R1
O
R2
R2
O
R1
O
O
N
R2
O
N
O
O
O
Ser
H
O S
O S
5
+
6
NH2
Ser
8
R1
O
R2
O
O
Ser
9
Scheme 1. Mechanism of action of N-tosyloxy-2-azetidinones.
2

Me
O
O
O
O
N
N
N
N
O
O
O
O
O
O S
O
O S
O
O S
O
O S
OCH3
10
11
12
13
Figure 2. Structures of previously reported β-lactamase inhibitors.
Syntheses of additional N-sulfonyloxy β-lactams 10-13 (Figure 2) with β-lactamase
inhibitory activity were reported in 1995.11 Studies on a model of the acyl-enzyme intermediate
in the active site of the crystal structure of the enzyme revealed that the sulfonate group was a
surrogate for the carboxylate group found in penicillin and other β-lactam antibiotics and also
indicated that aryl groups at the C4 position and in the sulfonyl group interacted with specific
hydrophobic pockets of the enzymes. However, recovery of the β-lactamase enzyme activity was
observed within 2-3 h. Further studies reported in 1999,13 led to the design, synthesis and study
of 3-hydroxyethyl substituted N-tosyloxy β-lactam 4 which was found to be an efficient inhibitor
of the TEM-1 enzyme from Escherichia Coli. The hydroxyethyl group was introduced in order
to improve the acyl-enzyme stability and, in fact, the compound resists deacylation from the
active site of the enzyme for several days. The relatively simple 4-methyl-N-tosyloxy-β-lactam
10 was also reported to inactivate the NMC-A β-lactamase from Enterobacter clocoae rapidly,
efficiently and irreversibly.12 This was of particular importance because NMC-A β-lactamases
display a broad substrate profile and are also able to hydrolyze carbapenems, which are now
considered to be antibiotics of last resort. In 2000, our group also reported the synthesis of a
variety of N-sulfonyloxy β-lactams of type 3 with different aryl groups (as in 11) other than tosyl
on the sulfonate moiety.14
As also illustrated in Scheme 1, the β-lactamase inhibitory activity of N-sulfonyloxy β-
lactams is initiated by the inherent electrophilic character of the N-sulfonyloxy β-lactam
carbonyl, followed by ring opening, loss of the sulfonate and subsequent generation of additional
electrophilic species (7-9) at or near the enzyme active site. Additional chemical reactivity
studies of N-sulfonyloxy β-lactams revealed that they also are electrophilic at the C3 position (α-
carbon) as shown in Scheme 2 (14 → 15). This is in stark contrast to classical N-unsubstituted or
3

N-alkyl β-lactams, which can be deprotonated at the α-carbon, thus making it nucleophilic
(Scheme 2, 16 → 17).
R1
Nuc
O
R1
H
δ+
Base, Nucleophile
Base, Electrophile
N
N
O
O SO2R(Ar)
14
15
R1
δ−
R1
E
N
N
O
R
O
R
16
17
Scheme 2. Reactivity of N-sulfonyloxy β-lactams vs N-unsubstituted or N-alkyl β-lactams.
Our early studies related to the syntheses of monocyclic β-lactams focused on developing
practical methods for making the β-lactam ring by N-C4 cyclization. Two processes relied on the
N-H acidity of hydroxamates to facilitate cyclization. The most widely used version relies on
Mitsunobu-mediated cyclization of α-hydroxy amino acid hydroxamates 18 to the N-alkoxy β-
lactams, 19, which were versatile intermediates for syntheses of oxamazins 20, monosulfactams
21 and monobactams 22, with a modified route still being used to synthesize the only clinically
used monobactam, aztreonam, 23 (Scheme 3). A second alternative relied on bromine-induced
cyclization of γ,δ-unsaturated hydroxamates (24 → 25).15-16 4-Halomethyl substituted β-lactams,
26 (Scheme 4), are versatile intermediates for syntheses of a variety of substituted monocyclic
and bicyclic β-lactams, including the highly functionalized monobactams (27) recently disclosed
by the Pfizer group.17 While the Mitsunobu process was used to make the N-sulfonyloxy β-
lactams described earlier,10-14 the halocyclization intermediates were never converted to N-
sulfonyloxy derivatives 28 (Scheme 4). These compounds possess multiple electrophilic sites
and therefore are extremely reactive species (“electrophilic bombs”).
4

OH
R1O
R3
R3
H
N
H
N
H
N
H
R1O
H H
H H
N
R2
R2
H H
N
R2
NH
R2
OR
Mitsunobu
O
O
O
O
N
O
N
H
O
OR4
O
OR
O
SO3H
19
20, oxamazins, R4 = CH2CO2H
21, monosulfactams, R4 = SO3H
22. monobactams
18
selectively acidic N-H
CO2H
R1
O
R1
O
Br
N
Br2
H
N
OCOR
N
N
H Me
Base
O
N
H
H
OCOR
S
H2N
O
25
N
24
O
SO3H
23
Scheme 3. Synthetic strategies toward monocyclic β-lactams.
CO2H
δ+
O
N
Br
δ+
δ+
X = OH, halo
H
N
H
N
R
N
H
C(O)R or S(O)2R
N
H
N
S
δ+
O
H2N
O
O
O SO2R(Ar)
N
N
O
H
O
SO3H
28
"electrophilic bomb"?
26
27
Scheme 4. Halomethyl substituted β-lactams as precursors of highly functionalized
monobactams.
Based on the growing need for new β-lactamase inhibitors and new forms of β-lactam
antibiotics, we decided to specifically prepare new N-sulfonyloxy β-lactams from the N-
hydroxy-4-bromomethyl derivatives with two initial focused goals: 1. Determination of
fundamental chemistry and potential bioactivity of the new multi-electrophilic β-lactams and 2.
Utilization of these reactive moieties as versatile intermediates for the syntheses of new
monobactam derivatives that would extend structure-activity relationship (SAR) studies of
heteroatom activated monocyclic β-lactams.18-23 Herein, we focus on the chemistry related to the
generation of and reactivity of 4-bromomethyl-N-sulfonyloxy β-lactams 28. The chemistry of
these compounds was expected to be especially interesting, since, based on the precedent already
noted, it could be anticipated that all the carbons of the core structure (28) would be electrophilic
(δ+). Not only did we find that the chemistry can be controlled and localized to carry out
extended syntheses but the initially anticipated random reactivity of these “electrophilic bombs”
was instead determined to induce remarkably selective, albeit moderate, antibiotic and β-
lactamase inhibitory activity.
5

2. Results and Discussion
The synthesis of the 4-halomethyl-N-sulfonyloxy β-lactams is summarized in Scheme 5
below. Similar to our previously reported method for halogen-induced β-lactam cyclization, we
first converted the simple γ,δ-unsaturated acid, vinyl acetic acid (29) to the corresponding
hydroxamic acid 31 via the intermediate acid chloride (30).15-16 Subsequent treatment of 31 with
CbzCl gave the O-protected hydroxamate 32 with the appropriately acidified N-H bond needed
for the halolactamization (32 → 33). Indeed, reaction of the protected hydroxamate with
bromine and aqueous base provided the desired 4-bromomethyl β-lactam 33 cleanly.
Hydrogenolytic removal of the Cbz group produced the free N-hydroxy β-lactam (34) that was
immediately reacted with a series of sulfonyl chlorides to generate a small library of N-
sulfonyloxy-4-bromomethyl β-lactams (28a-j) in moderate to good overall yields. Only
compounds 28g and 28h, with strong electron-withdrawing groups on the aromatic ring, were
obtained in low yields because of their high reactivity and instability during chromatographic
purification on silica gel.
6

Scheme 5. Syntheses of N-sulfonyloxy β-lactams 28a-j.
In addition to determining the biological activity (described later in this paper) of the
multi-electrophilic N-sulfonyloxy-4-bromomethyl β-lactams, we also wanted to determine if the
library of N-sulfonyloxy β-lactams could be expanded by reaction of intermediate 4-
bromomethyl derivatives like 33 (the OCbz compound) with nucleophiles to displace the halogen
without destruction of the β-lactam. With future goals of elaborating monobactams with
peripheral thiol, triazol and amine substitution, we reacted 33 with a representative thiol and
azide but found it impossible to isolate the expected substitution products. Suspecting that the
O-Cbz group of 33 was too labile or electronically activated the β-lactam to induce destruction,
we decided to replace it with a less activating allyl protecting group (Scheme 6). To this end, we
again hydrogenolytically removed the Cbz group of 33 to generate the intermediate N-hydroxy-
4-bromomethyl β-lactam (34) and, after solvent exchange, treated it directly with allyl bromide.
Interestingly, the allylation reaction proceeded in high yield to give 35 with no competitive
reaction of the free N-hydroxy group of the intermediate (34) with the bromomethyl group of
another molecule of N-hydroxy-4-bromomethyl β-lactam (34). This suggested that reactions of
the 4-bromomethyl group with other nucleophiles might not be facile. However, reaction of the
O-allyl-4-bromomethyl β-lactam (35) with a representative thiol (36, 2-mercapto-thiazoline)
proceeded to give the desired substitution product 37 in good yield. Catalytic Pd-mediated
deallylation produced the new free N-hydroxy β-lactam intermediate 38 which was immediately
reacted with p-toluenesulfonyl chloride to give the targeted thiosubstituted N-tosyloxy β-lactam
39.
H2
Pd/C 10%
Br
Br
Br
Br
DIPEA
CH3CN, 0 ˚C
N
N
N
MeOH
O
OCbz
O
OH
O
O
33
34
35
80%
N
K2CO3
CH3CN
68%
HS
S
36
N
N
N
Pd(OAc)2
PPh3
S
S
S
TsCl, DIPEA
S
S
S
CH3CN
0 ˚C
1,3-dimethyl
barbituric acid
MeOH
N
N
N
O
OTs
O
OH
38
O
O
65%
39
37
7

Scheme 6. Synthesis of N-sulfonyloxy β-lactam 39.
Similar reactions of 4-bromomethyl β-lactams with azide were anticipated to give the
corresponding azide derivatives that could serve as precursors to amine and triazole-substituted
β-lactams for extended structure-activity-relationship (SAR) studies. Tazobactam (40) is one of
the three clinically used β-lactam-based β-lactamase inhibitors. It contains a triazol substituent
and, similar to many of the N-sulfonyloxy β-lactams described in earlier schemes, it does not
have a substituent at the α-carbon of the β-lactam. The mechanism of action of tazobactam
(Scheme 7) involves ring opening by attack of a serine hydroxyl to generate an acyl enzyme
intermediate that, similar to the inhibitory mechanisms determined for our previously described
N-tosyloxy β-lactams (Scheme 1), undergoes further elimination chemistry to generate
additional electrophilic sites (an imine, 42, and enamines 43 and 44) that contribute to the
enzyme inhibitory chemistry.5 Furthermore, although a recent Pfizer patent disclosed alternative
routes to β-lactam azides and triazoles with antibacterial activity,17 there have been no reports
related to syntheses and studies of correspondingly substituted N-sulfonyloxy β-lactam
derivatives.
N
N
N
O
S
H
O
O
O
N
N
S
N
N
O
HN
O
CO2H
CO2H
O
Ser OH
Ser
40 tazobactam
41
N
N
N
HO2S
HN
N
N
N
N
N
N
HO2S
HN
HO2S
CO2H
O
O
O
N
CO2H
CO2H
O
O
O
Ser
Ser
Ser
43
44
42
Further reactions with β-lactamase enzymes
Scheme 7. Mechanism of action of tazobactam.
8

Again, reaction of sodium azide with the O-Cbz β-lactam derivative 33 resulted in
decomposition. However, hydrogenolytic removal of the Cbz group followed by alkylation of
the free N-hydroxy β-lactam with benzyl bromide gave the N-benzyloxy derivative 46 in 80%
yield, again with no competitive alkylation with the β-lactam bromomethyl group to give
dimeric-like structures (45, Scheme 8). Displacement of the bromide of the N-benzyloxy β-
lactam (46) with azide proceeded cleanly to give the targeted azide substituted β-lactam 47.
Click reactions24-26 of the azide with representative alkynes (48a-c) gave the triazoles (49a-c) as
expected. Reductive removal of the benzyl group gave the free N-hydroxy triazol-containing β-
lactams (50a-c), which, upon reaction with p-toluenesulfonyl chloride under standard conditions
produced a set of targeted N-tosyloxy-triazole containing β-lactams (51a-c) for study.
Br
H2
Pd/C 10%
Br
Br
N
O
O
N
N
MeOH, rt
O
OCbz
O
OH
N
O
OH
45
33
34
BnBr, DIPEA
CH3CN, 0 ˚C
80%
N3
Br
NaN3, NaI
N
N
DMF, rt
70%
O
O
Ph
O
O
Ph
47
46
R
sodium ascorbate
48a-c
CuSO4 5 H2O tBuOH/H2O
N
N
N
N
N
N
N
H2
Pd/C 10%
N
N
R
TsCl, DIPEA
CH3CN, 0 ˚C
R
R
N
N
N
MeOH, rt
O
O
Ph
O
OH
O
OTs
49a, 67%
49b, 77%
49c, 73%
50a-c
51a, 65%
51b, 70%
51c, 66%
CF3
H
N
H
N
a. R = CO2Et
b. R =
c. R =
O
O
Cl
Scheme 8. Syntheses of N-sulfonyloxy β-lactams 51a-c.
3. Biological activity
9

N-Sulfonyloxy β-lactams 28a-j, 39 and 51a-c were tested for antimicrobial activity using
agar diffusion assays. As displayed in Table 1, compounds 28a-i showed weak antibacterial
activity against Gram-positive bacteria but they were not active against Gram-negative bacteria.
Compound 28j, which contains a thiophene group instead of a phenyl ring on the sulfonyl
moiety, was not active. It is also worth noting the remarkable growth inhibition zone for
compound 28h against M. Vaccae. These results appeared to be in contrast with the electrophilic
properties of these molecules that present multiple electrophilic sites (“electrophilic bombs”) and
were anticipated to be extremely reactive and unselective. The reason for this selectivity is
unknown and, while classical β-lactam antibiotics work by PBP (penicillin binding protein)
interactions, details will be the subject of further investigations that will include consideration of
other targets. As also displayed in Table 1 compounds 39 and 51a-c, in which the bromine atom
at C4 had been displaced by a different functionality, were not active.
Zone of Growth Inhibition (mm)
B. Subtilis
S.
Aureus Luteus Vaccae Baumannii Dolosa
M.
M.
A.
B.
P.
Aeruginosa
E.
Coli
ATCC
6633
11V
SG511 ATCC IMET
10240 10670
ATCC
17961
h
K799/wt K799/61 DC0 DC2
h
h
17P
17P
16V
16
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
h
0
h
0
0
0
0
0
0
0
0
13V
15V
0
28a
28b
28c
28d
28e
28f
28g
28h
28i
11V
12
0
0
0
0
14P
13.5P
12*ppt
12ppt
14V
0
0
13
0ppt
0
14V
15V
14V
14V
15V
h ppt
16V
14V
0
13/17P
13
13
17
14V
19P
0
11V
11ppt
0
14
h
22
14P/23V
16V
19V
0
32
0
0
0
h
0
0
h
14Pppt
0
0
0
0
28j
39
0
0
H
0
15V
0
0
0
0
51a
51b
51c
0
0
0
0
13P
13P
0
13P
0
Table 1. Spectrum of antimicrobial activity in the Agar Diffusion Assay. All solutions were prepared by first
making a 20 mM solution in DMSO and diluting 10-fold with MeOH to give 2 mM solution. Plates were incubated
after inoculation at 37 ºC for 20 hours. Plates were photographed after 1 day. M. vaccae required a longer
incubation time, 48 hours and was photographed after 3 days. h: Indicates only a hint of growth inhibition
10

detectable. P: Indicates unclear inhibition zone. V: Indicates a very unclear inhibition zone. * Indicates a slightly
misshapen zone, most likely due to solubility issues. ppt: presence of precipitate.
Compounds 28c,e,f,i were also tested for β-lactamase inhibitory activity against selected
enzymes from classes A, B, C and D (Ambler classification). Also in this case an unusual
selectivity was observed (Table 2): while compound 28e was active only against the class A
enzyme SHV-12, compounds 28c,f inhibited enzymes from both classes A and D. Although
there are two β-lactamase inactivators that show activity against both classes A and C of β-
lactamases (the penem BRL 42715 and tazobactam), there are no examples of clinically used
inhibitors of both classes A and D. Also worth noting is the activity of 28c,f against the class D
enzyme Oxa-48, which is particularly relevant for its notable carbapenem-hydrolyzing activity
and its resistance to the clinically used β-lactamase inhibitors clavulanic acid, sulbactam and
tazobactam.
Ki (µM)
Enzyme
KPC-2
CTX-M-
14
Oxa-48
AmpC
SHV-
12
PAM
2035
C
NDM- VIM-1
1
Class
A
A
D
C
A
B
B
>160
>160
4.673
>160
0.198
>160
>160
N/A
28c
28e
28f
28i
>160
19.290
>160
>160
0.149
>160
>160
1.481
>160
>160
>160
>160
0.190
0.779
>160
>160
>160
>160
>160
>160
>160
>160
>160
N/A
Table 2. Ki values for compounds 28c,e,f,i against selected β-lactamase enzymes from classes A,B,C and
D.
4. Summary and Conclusion
The 4-bromomethyl substituted β-lactam 33 was synthetized using a bromine-induced
cyclization on a γ,δ-unsatured hydroxamate as the key step. Studies on the reactivity of this
compound were performed in order to evaluate the possibility to use it as an intermediate for the
syntheses of novel monocyclic and bicyclic β-lactams. The halocyclization intermediate was also
converted to N-sulfonyloxy derivatives 28a-j which displayed weak antibacterial activity against
11

Gram-positive bacteria. Compounds 28c,e,f also displayed selective β-lactamase inhibition
activity against enzymes from classes A and D. Further studies on the mode of action and
selectivity of these compounds are under consideration.
5. Experimental section
5.1. General experimental section
Unless otherwise indicated, chemicals and solvents were from commercial suppliers and
were used as received. All of the solvents were of analytical grade or were distilled prior to use.
Dichloromethane (CH2Cl2) and acetonitrile (CH3CN) were distilled from CaH2. Tetrahydrofuran
(THF) was distilled from a mixture of sodium metal and benzophenone ketyl.
Dimethylformamide (DMF) and diisopropylethylamine (DIPEA) were used from Acros Seal ®
1
anhydrous bottles. H NMR and 13C NMR spectra were obtained on a 500 MHz Bruker
spectrometer and FIDs were processed using ACD/SpecManager version 11. Chemical shifts (δ)
are given in parts per million (ppm) and are referenced to residual solvent peaks as internal
standards. Coupling constants (J) are reported in Hertz (Hz). High resolution, accurate mass
measurements were obtained with a Bruker micrOTOF II electrospray ionization time-of-flight
mass spectrometer in positive mode. Infrared spectra were recorded with a ThermoNicolet IR
200 Spectrometer and reported as cm-1. All reactions were conducted under Argon atmosphere
unless otherwise noted. Solvents were removed in vacuo on a rotary evaporator. Reactions were
monitored by thin layer chromatography (TLC) performed with aluminum-backed Merck 60-
F254 silica gel plates using a 254 nm lamp, ceric ammonium molybdate (CAM) stain, FeCl3
stain, KMnO4 stain or ninhydrin stain for visualization. Silica gel chromatography was
performed using Sorbent Technologies silica gel 60 (32-63 µm). Melting points were determined
in capillary tubes using a Thomas Hoover melting point apparatus and are uncorrected.
5.3. Procedures for Biological Assays
5.3.1. General Comments
All liquids and media were sterilized by autoclaving (121 ºC, 15 min) before use. All
aqueous solutions and media were prepared using distilled, deionized and filtered water
(Millipore Milli-Q Advantage A10 Water Purification System). Luria broth (LB) was purchased
12

from VWR. Mueller-Hinton No. 2 broth (MHII broth; cation adjusted) was purchased from
Sigma-Aldrich. McFarland BaSO4 turbidity standards were purchased from bioMérieux, Inc.
Sterile plastic petri dishes (145 mm x 20 mm; Greiner Bio-One) were purchased from VWR.
Ciprofloxacin was purchased from Sigma-Aldrich. The enzymes KPC-2, Oxa-48, AmpC and
NDM-1 were purchased from Emerald Bioscience and the enzymes CTX-14 and SHV-12 were
expressed and purified from E. coli at Rempex pharmaceuticals (San Diego, CA).
5.3.2. Procedures for Agar-diffusion Assays.
Antibacterial activity of the compounds was determined by a modified Kirby-Bauer27-28
agar-diffusion assay. Overnight cultures of test organisms were grown in LB broth for 18-24h
and standard suspensions of 1.5 x 106 CFU/mL were prepared in saline solution (0.9% NaCl)
according to a 0.5 BaSO4 McFarland Standard. This standardized suspension (0.1 mL) was
added to 34 mL of sterile, Mueller Hinton No. 2 agar tempered to 47-50 ºC. After gentle mixing,
the inoculated agar media was poured into a sterile plastic petri dish (145 mm x 20 mm) and
allowed to solidify near a flame with the lid cracked for 3̴ 0 min. Wells of 9.0 mm diameter were
cut from the petri dish agar and filled with exactly 50 µL of the test sample solution. For studies
using mixtures of compounds, both test compounds were diluted to equal concentrations and 50
µL of the mixed compound solution was added to the cut well. The petri dish was incubated at
37 ºC for 18-24 h and the inhibition zone diameters were measured (mm) with an electronic
caliper after 24-48 h.
5.3.3. Procedures for Enzyme Inhibition Assays
To the first row of a 2-mL deep 96-well block was added 1 mL of 50 mM sodium
phosphate buffer at pH 7 with 0.1 mg/mL BSA (bovine serum albumin). To the first row was
added an appropriate amount of a 10 mg/mL solution of the compound to be tested to make a
640 µM (4X) solution. Each of the other wells in the 96-well block were charged with 750 µL of
the aforementioned buffer. The rows were then repeatedly diluted 1:3 so that the final
concentrations of the individual 96-well plates would range in concentrations from 160 µM to 3
nM (8 dilutions). The master block was then used to add 50 µL to each corresponding well in the
96-well flat-bottom plate. An additional 50 µL of buffer was added to each well. Once the plates
13

were prepared, 50 µL of the enzyme to be tested against were added in buffer to each well [KPC-
2 (0.147 mg/mL), CTX-M-14 (0.102 mg/mL), Oxa-14 (0.331 mg/mL), AmpC (0.699 mg/mL),
SHV-12 (0.111 mg/mL), NDM-1 (0.110 mg/mL)]. The plates were incubated at rt for 10 min
before 50 µL of an appropriate indicator were added to each well and the optical density at 495
nm was measured over time. For each enzyme, other than NDM-1, nitrocefin was used.
Nitrocefin (10 µM for SHV-12, 50 µM for the other enzymes), was appropriate because when
cleaved by a β-lactamase, the color changes from yellow to red. For NDM-1, imipenem (10 µM)
was the indicator of choice. Rather than observe the growing presence of optical density at 495
nm, as with nitrocefin, the disappearance of imipenem was monitored. The inhibition curves
were monitored and used to perform enzyme kinetics according to Waley29 to determine the Ki
(µM) of each compound against the β-lactamases screened.
5.4. Chemistry: Protocols and analytical data
5.4.1. General procedure for the synthesis of compounds 28a-j from precursor 33. Cbz
protected β-lactam 33 (500 mg, 1.59 mmol)15-16 was dissolved in 14 mL of MeOH, at rt under an
argon atmosphere. Pd/C 10% (20 mg) was added and the mixture was flushed with hydrogen
gas. The reaction mixture was left to stir at rt under a hydrogen atmosphere for 2 h then it was
purged with argon and filtered through celite. The filtrate was evaporated under vacuum to give
crude N-hydroxy β-lactam 34 as a pink oil.
Compound 34 was dissolved in dry CH3CN (20 mL) and the solution was cooled to 0 ºC. DIPEA
(415 µL, 2.39 mmol) was added, followed by the sulfonyl chloride (2.39 mmol). The reaction
mixture was left to stir at 0 ºC for 10 min then the solvent was evaporated under vacuum. The
resulting residue was purified by column chromatography on silica gel (EtOAc/hexanes).
5.4.1.1. 4-(Bromomethyl)-2-oxoazetidin-1-yl-4-methylbenzenesulfonate (28a): yield 67%
1
(1.06 mmol, 356 mg); white solid; IR (KBr) 1792, 1649, 1596, 1380, 1178; m.p. 65-68 ºC; H
NMR (500 MHz, CDCl3) δ (ppm) = 7.90 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 4.29-4.25
(m, 1H), 3.75 (dd, J = 11.5, 3.1 Hz, 1H), 3.63 (dd, J = 11.5, 6.6 Hz, 1H), 2.95 (dd, J = 14.6, 6.1
13
Hz, 1H), 2.75 (dd, J = 14.6, 3.3 Hz, 1H), 2.47 (s, 3H). C NMR (125 MHz, CDCl3) δ (ppm) =
14

164.5, 146.9, 130.3, 129.6, 129.5, 58.5, 38.5, 31.5, 22.1. HRMS (ESI) m/z calcd for
C11H12BrNNaO4S [M+Na]+ 355.9568, found 355.9576.
5.4.1.2. 4-(Bromomethyl)-2-oxoazetidin-1-yl-4-methoxybenzenesulfonate (28b): yield 70%
(1.11 mmol, 390 mg); white solid; IR (KBr) 2844, 1797, 1595, 1497, 1460, 1379, 1266; m.p. 80-
83 ºC; 1H NMR (500 MHz, CDCl3) δ (ppm) = 7.93 (dt, J = 9.5, 2.5 Hz, 2H), 7.02 (dt, J = 9.5, 2.5
Hz, 2H), 4.26-4.23 (m, 1H), 3.90 (s, 3H), 5.72 (dd, J = 11.5, 3.0 Hz, 1H), 3.62 (dd, J = 11.5, 6.5
Hz, 1H), 2.94 (dd, J = 14.5, 6.0 Hz, 1H), 2.74 (dd, J = 14.5, 3.5 Hz, 1H); 13C NMR (125 MHz,
CDCl3) δ (ppm) = 165.2, 164.5, 131.9, 124.3, 114.9, 58.4, 56.0, 38.4, 31.6. HRMS (ESI) m/z
calcd for C11H12BrNNaO5S [M+Na]+ 371.9517, found 371.9526.
5.4.1.3. 4-(Bromomethyl)-2-oxoazetidin-1-yl-naphthalene-1-sulfonate (28c): yield 74% (1.18
1
mmol, 435 mg); thick colorless oil; IR (neat) 2969, 1803, 1731, 1593, 1507, 1379, 1183; H
NMR (500 MHz, CDCl3) δ (ppm) = 8.67 (dd, J = 8.5, 0.5 Hz, 1H), 8.37 (dd, J = 8.5, 1.5 Hz,
1H), 8.21 (d, J = 8.0 Hz, 1H), 7.98 (dt, J = 8.0, 0.5 Hz, 1H), 7.77 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H),
7.67 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 7.59 (dd, J = 8.0, 7.5 Hz, 1H), 4.16-4.14 (m, 1H), 3.59 (dd, J
= 11.0, 3.0 Hz, 1H), 3.44 (dd, J = 11.0, 7.0 Hz, 1H), 2.87 (dd, J = 14.7, 6.0 Hz, 1H), 2.65 (dd, J =
13
14.7, 3.5 Hz, 1H); C NMR (125 MHz, CDCl3) δ (ppm) = 164.1, 137.2, 134.3, 132.8, 129.5,
129.2, 129.0, 127.8, 125.0, 125.1, 124.2, 58.4, 38.5, 31.2. HRMS (ESI) m/z calcd for
C14H12BrNNaO4S [M+Na]+ 391.9568, found 391.9570
5.4.1.4.
4-(Bromoethyl)-2-oxoazetidin-1-yl
5-(dimethylamino)naphthalene-1-sulfonate
(28d): yield 72% (1.14 mmol, 473 mg); yellow solid; IR (KBr) 2834, 1804, 1571, 1455, 1377,
1183; m.p. 89-92 ºC; 1H NMR (500 MHz, CDCl3) δ (ppm) = 8.69 (d, J = 8.5 Hz, 1H), 8.35 (dd, J
= 7.5, 1.5 Hz, 1H), 8.30 (d, J = 8.5 Hz, 1H), 7.66 (dd, J = 8.5, 7.5 Hz, 1H), 7.56 (dd, J = 8.5, 7.5
Hz, 1H), 7.24 (d, J = 7.5 Hz, 1H), 4.14-4.10 (m, 1H), 3.54 (dd, J = 11.3, 3.0 Hz, 1H), 3.38 (dd, J
= 11.3, 7.5 Hz, 1H), 2.91-2.87 (m, 1H), 2.90 (s, 6H), 2.67 (dd, J = 14.5, 3.0 Hz, 1H); 13C NMR
(125 MHz, CDCl3) δ (ppm) = 164.1, 152.0, 133.4, 132.8, 130.5, 130.0, 129.6, 129.1, 123.2,
119.3, 116.1, 58.4, 45.7, 38.5, 31.1; HRMS (ESI) m/z calcd for C16H17BrN2NaO4S [M+Na]+
434.9990, found 434.9949.
15

5.4.1.5. 4-(Bromomethyl)-2-oxoazetidin-1-yl-naphthalene-2-sulfonate (28e): yield 67% (1.06
1
mmol, 394 mg); colorless oil; IR (neat) 2965, 1803, 1625, 1589, 1383, 1184; H NMR (500
MHz, CDCl3) δ (ppm) = 8.61 (d, J = 2.0 Hz, 1H), 8.01 (dd, J = 12.7, 8.5 Hz, 2H), 7.98-7.94 (m,
2H), 7.74-7.70 (m, 1H), 7.68-7.64 (m, 1H), 4.32-4.28 (m, 1H), 3.73 (dd, J = 11.5, 3.0 Hz, 1H),
3.66 (dd, J = 11.5, 6.5 Hz, 1H), 2.95 (dd, J = 15.0, 6.0 Hz, 1H), 2.74 (dd, J = 15.0, 3.5 Hz, 1H);
13C NMR (125 MHz, CDCl3) δ (ppm) = 164.6, 136.1, 132.1, 132.0, 130.3, 130.2, 130.0, 129.8,
128.4, 128.3, 123.2, 58.5, 38.5, 31.6. HRMS (ESI) m/z calcd for C14H12BrNNaO4S [M+Na]+
391.9568, found 391.9564.
5.4.1.6. 4-(Bromomethyl)-2-oxoazetidin-1-yl (E)-2-phenylethene-1-sulfonate (28f): yield 61%
1
(0.97 mmol, 336 mg); white solid; IR (KBr) 1798, 1613, 1375, 1172; m.p. 70-73 ºC; H NMR
(500 MHz, CDCl3) δ (ppm) = 7.75 (d, J = 15.0 Hz, 1H), 7.55-7.41 (m, 5H), 6.87 (d, J = 15.0 Hz,
1H), 4.34-4.32 (m, 1H), 3.76 (dd, J = 11.0, 3.5 Hz, 1H), 5.18 (dd, J = 11.0, 6.0 Hz, 1H), 3.01
(dd, J = 14.5, 6.0 Hz, 1H), 2.80 (dd, J = 14.5, 3.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ (ppm)
= 164.8, 149.3, 132.5, 131.7, 129.5, 129.3, 118.3, 58.7, 38.5, 31.7; HRMS (ESI) m/z calcd for
C12H13BrNO4S [M+H]+ 345.9749, found 345.9759.
5.4.1.7. 4-(Bromomethyl)-2-oxoazetidin-1-yl 4-nitrobenzenesulfonate (28g): yield 51% (0.81
1
mmol, 296 mg); white solid; IR (KBr) 1802, 1696, 1526, 1351, 1186; m.p. 50-55 ºC; H NMR
(500 MHz, CDCl3) δ (ppm) = 8.42 (dt, J = 9.1, 2.3 Hz, 2H), 8.24 (dt, J = 9.1, 2.3 Hz, 2H), 4.34-
4.32 (m, 1H), 3.78 (dd, J = 11.5, 3.4 Hz, 1H), 3.76 (dd, J = 11.5, 5.4 Hz, 1H), 3.49 (dd, J = 14.9,
13
6.4 Hz, 1H), 2.81 (dd, J = 14.9, 3.5 Hz, 1H); C NMR (125 MHz, CDCl3) δ (ppm) = 164.9,
151.7, 139.2, 131.0, 124.7, 58.6, 38.3, 31.5.
5.4.1.8. 4-(Bromomethyl)-2-oxoazetidin-1-yl 2,4-dinitrobenzenesulfonate (28h): yield 35%
(0.56 mmol, 228 mg); yellow solid; IR (KBr) 1804, 1638, 1529, 1348, 1225; m.p. 104.2-105.7
1
ºC; H NMR (500 MHz, CDCl3) δ (ppm) = 8.85 (d, J = 3.0 Hz, 1H), 8.48 (dd, J = 9.5, 3.0 Hz,
1H), 7.81 (d, J = 9.5 Hz, 1H), 4.49-4.45 (m, 1H), 3.72 (dd, J = 11.5, 4.5 Hz, 1H), 3.68 (dd, J =
13
11.5, 4.5 Hz, 1H), 3.21 (dd, J = 15.0, 3.5 Hz, 1H), 3.00 (dd, J = 15.0, 3.5 Hz, 1H); C NMR
(125 MHz, CDCl3) δ (ppm) = 167.0, 156.8, 142.8, 137.0, 129.7, 122.3, 117.7, 58.1, 38.1, 31.4.
16

5.4.1.9. 4-Bbromomethyl)-2-oxoazetidin-1-yl 2,4,6-triisopropylbenzenesulfonate (28i): yield
69% (1.10 mmol, 490 mg); white solid; IR (KBr) 2870, 1799, 1660, 1426, 1193; m.p. 96.6-97.9
ºC; 1H NMR (500 MHz, CDCl3) δ (ppm) = 7.10 (s, 2H), 4.31-4.27 (m, 1H), 4.08 (sept., J = 6.5
Hz, 2H), 3.72 (dd, J = 11.0, 3.2 Hz, 1H), 3.49 (dd, J = 11.0, 7.6 Hz, 1H), 2.98 (dd, J = 14.5, 5.9
Hz, 1H), 2.93 (sept., J = 6.9 Hz, 1H), 2.73 (dd, J = 14.5, 3.1 Hz, 1H), 1.29 (dd, J = 6.5, 4.0 Hz,
13
12H), 1.26 (d, J = 6.9 Hz, 6H); C NMR (125 MHz, CDCl3) δ (ppm) = 164.1, 155.6, 152.6,
127.4, 124.3, 58.7, 38.8, 34.5, 31.1, 30.4, 24.9, 23.7; HRMS (ESI) m/z calcd for C19H29BrNO4S
[M+H]+ 446.1001, found 446.0999.
5.4.1.10. 4-(Bromomethyl)-2-oxoazetidin-1-yl thiophene-2-sulfonate (28j): yield 75% (1.19
1
mmol, 389 mg); colorless oil; IR (neat) 2965, 1798, 1698, 1505, 1384, 1183; H NMR (500
MHz, CDCl3) δ (ppm) = 7.89 (dd, J = 4.0, 1.5 Hz, 1H), 7.85 (dd, J = 5.0, 1.5 Hz, 1H), 7.20 (dd, J
= 5.0, 4.0 Hz, 1H), 4.31-4.28 (m, 1H), 3.73 (dd, J = 11.5, 3.0 Hz, 1H), 3.63 (dd, J = 11.5, 6.5 Hz,
13
1H), 2.99 (dd, J = 14.7, 6.5 Hz, 1H), 2.79 (dd, J = 14.7, 3.0 Hz, 1H); C NMR (125 MHz,
CDCl3) δ (ppm) = 164.6, 137.6, 137.0, 128.4, 58.6, 38.5, 31.5; HRMS (ESI) m/z calcd for
C8H8BrNNaO4S2 [M+Na]+ 347.8976, found 347.8976.
5.4.2. 1-(Allyloxy)-4-(bromomethyl)azetidin-2-one (35):
Compound 33 (4.00 g, 12.7 mmol) was dissolved in MeOH (30 mL) at rt under an argon
atmosphere. Pd/C 10% (40 mg) was added and the mixture was flushed with hydrogen gas. The
reaction mixture was left to stir under an atmosphere of hydrogen, at rt, for 2.5 h then it was
purged with argon and filtered through celite. The solvent was evaporated under vacuum to give
crude N-hydroxy β-lactam 34 as a yellow oil.
Crude N-hydroxy β-lactam 34 was dissolved in dry CH3CN (40 mL) at rt, under an argon
atmosphere. K2CO3 (3.50 g, 25.5 mmol) was added, followed by allyl bromide (1.40 mL, 16.5
mmol). The reaction mixture was left to stir at rt for 15 h then water (20 mL) and Et2O (30 mL)
were added. The layers were separated and the aqueous layer was extracted with Et2O (20 mL).
The combined organic extracts were dried, filtered and evaporated under vacuum. Column
chromatography on silica gel (hexanes/EtOAc 7:3) afforded compound 35 in 80% yield (2.23 g,
1
10.2 mmol) as a yellow oil. IR (neat) 3082, 2955, 1777, 1644, 1420, 1343, 974; H NMR (500
MHz, CDCl3) δ (ppm) = 5.98 (dddd, J = 17.0, 10.0, 6.5, 6.5 Hz, 1H), 5.37 (ddd, J = 17.0, 2.8, 1.4
17

Hz, 1H), 5.32 (dddd, J = 10.0, 1.4, 1.0, 1.0 Hz, 1H), 4.48-4.39 (m, 2H), 4.08-4.06 (m, 1H), 3.63
(dd, J = 11.0, 4.2 Hz, 1H), 3.51 (dd, J = 11.0, 6.0 Hz, 1H), 2.83 (dd, J = 14.0, 5.3 Hz, 1H), 2.56
13
(dd, J = 14.0, 2.4 Hz, 1H); C NMR (125 MHz, CDCl3) δ (ppm) = 164.0, 132.3, 121.3, 77.8,
57.3, 37.9, 32.1; HRMS (ESI) m/z calcd for C7H11BrNO2 [M+H]+ 219.9973, found 219.9983.
5.4.3. 1-(Allyoxy)-4-(((4,5-dihydrothiazol-2-yl)thio)methyl)azetidin-2-one (37) Compound 35
(2.27 g, 10.31 mmol) was dissolved in dry CH3CN (50 mL) at rt under an argon atmosphere.
K2CO3 (2.85 g, 20.6 mmol) was added, followed by 2-thiazoline-2-thiol 36 (3.07 g, 25.8 mmol).
The resulting mixture was left to stir at rt for 24 h then the solvent was evaporated under
vacuum. The residue was purified by column chromatography on silica gel (hexanes/EtOAc 7:3)
to give compound 37 as a colorless oil in 68% yield (1.81 g, 7.01 mmol). IR (neat) 2932, 2852,
1768, 1644, 1572, 1418, 1349, 1305, 1202; 1H NMR (500 MHz, CDCl3) δ (ppm) = 5.96-5.90 (m,
1H), 5.35-5.27 (m, 2H), 4.41-4.33 (m, 2H), 4.17-4.09 (m, 3H), 3.53 (dd, J = 5.0, 1.5 Hz, 1H),
3.38 (dd, J = 8.0, 1.5 Hz, 2H), 3.27 (dd, J = 6.5, 1.5 Hz, 1H), 2.78 (dd, J = 5.5, 1.5 Hz, 1H), 2.49
13
(m, 1H); C NMR (125 MHz, CDCl3) δ (ppm) = 164.6, 164.5, 132.5, 121.2, 77.5, 64.2, 56.5,
37.7, 36.1, 34.0; HRMS (ESI) m/z calcd for C10H15N2O2S2 [M+H]+ 259.0575, found 259.0586.
5.4.4.
4-(((4,5-Dihydrothiazol-2-yl)thio)methyl)-2-oxoazetidin-1-yl
4-
methylbenzenesulfonate (39)
PPh3 (487 mg, 1.86 mmol) was added to a solution of Pd(OAc)2 (104 mg, 0.46 mmol) in dry
MeOH (7 mL) at rt, under an argon atmosphere. The mixture was left to stir at rt for 30 min then
1,3-dimethylbarbituric acid (726 mg, 4.64 mmol) was added followed by a solution of compound
37 (1.20 g, 4.64 mmol) in dry MeOH (10 mL). The reaction mixture was left to stir at rt for 2.5 h
then it was filtered through celite. The filtrate was evaporated under vacuum to afford crude N-
hydroxy-β-lactam 38 as an orange oil.
Crude 38 was dissolved in dry CH3CN (25 mL) and the resulting orange solution was cooled to 0
ºC. DIPEA (1.20 mL, 6.96 mmol) and TsCl (1.32 g, 6.96 mmol) were added sequentially and the
reaction mixture was left to stir at 0 ºC for 15 min. The solvent was evaporated under vacuum
and the resulting residue was purified by column chromatography on silica gel (hexanes/EtOAc
7:3 to 1:1). Compound 39 was obtained as a white solid in 65% yield (1.12 g, 3.02 mmol). IR
18

1
(neat) 2952, 1803, 1688, 1572, 1435, 1382, 1193; m.p. 72.9-75.0 ºC; H NMR (500 MHz,
CDCl3) δ (ppm) = 7.87 (d, J = 10.5 Hz, 2H), 7.37 (d, J = 10.5 Hz, 2H), 4.34-4.31 (m, 1H), 4.19-
4.15 (m, 2H), 3.74 (dd, J = 14.5, 3.6 Hz, 1H), 3.42 (dd, J = 8.0, 1.1 Hz, 1H), 3.40 (dd, J = 8.0,
2.0 Hz, 1H), 3.35 (dd, J = 14.5, 6.2 Hz, 1H), 2.87 (dd, J = 14.8, 6.2 Hz, 1H), 2.70 (dd, J = 14.8,
13
3.5 Hz, 1H), 2.46 (3H, s); C NMR (125 MHz, CDCl3) δ (ppm) = 165.6, 165.0, 146.7, 130.6,
130.3, 129.5, 64.1, 58.5, 37.6, 36.2, 33.4, 22.1; HRMS (ESI) m/z calcd for C14H17N2O4S3
[M+H]+ 373.0350, found 373.0353.
5.4.5. 1-(Benzyloxy)-4-(bromomethyl)azetidin-2-one (46)
Compound 33 (3.0 g, 9.5 mmol) was dissolved in 25 mL of MeOH at rt, under an argon
atmosphere. Pd/C 10% (50 mg) was added and the reaction mixture was flushed with hydrogen.
The mixture was left to stir ar rt, under hydrogen atmosphere for 2.5 h then it was purged with
argon and filtered through celite. The filtrate was evaporated under vacuum to give the crude N-
hydroxy β-lactam 34 as a pink oil.
Crude compound 34 was dissolved in dry CH3CN (40 mL) at rt, under an argon atmosphere.
K2CO3 (2.64 g, 19.1 mmol) was added, followed by benzyl bromide (1.48 mL, 12.4 mmol). The
reaction mixture was left to stir at rt for 12 h then the solvent was evaporated under vacuum. The
resulting residue was purified by column chromatography on silica gel (hexanes/EtOAc 7:3) to
give the produce in 80% yield ( 2.0 g, 7.6 mmol) as a colorless oil. IR (neat) 3063, 3031, 2957,
2882, 1772, 1648, 1497, 1454, 1360, 1214; 1H NMR (500 MHz, CDCl3) δ (ppm) = 7.41-7.37 (m,
5H), 4.98 (d, J = 11.0 Hz, 1H), 4.94 (d, J = 11.0 Hz, 1H), 3.77-3.74 (m, 1H), 3.33 (dd, J = 11.0,
4.0 Hz, 1H), 3.22 (dd, J = 11.0, 6.5 Hz, 1H), 2.77 (dd, J = 13.7, 5.5 Hz, 1H), 2.50 (dd, J = 13.7,
13
2.5 Hz, 1H); C NMR (125 MHz, CDCl3) δ (ppm) = 164.0, 135.4, 129.5, 129.4, 128.9, 78.8,
57.5, 38.1, 31.9; HRMS (ESI) m/z calcd for C11H13BrNO2 [M+H]+ 270.0130, found 270.0101.
5.4.6. 4-(Azidomethyl)-1-(benzyloxy)azetidin-2-one (47):
Compound 46 (3.0 g, 11.1 mmol) was dissolved in 40 mL of dry DMF at rt, under an argon
atmosphere. NaN3 (2.90g, 44.4 mmol) was added, followed by NaI (167 mg, 1.11 mmol). The
reaction mixture was left to stir at rt for 48 h then it was partitioned between water (30 mL) and
EtOAc (40 mL). The aqueous layer was extracted with EtOAc (30 mL) and the combined
organic extracts were dried (Na2SO4), filtered and evaporated under vacuum. The residue was
19

purified by column chromatography on silica gel (hexanes/EtOAc 7:3) to give the product in
70% yield (1.80 g, 7.77 mmol) as a colorless oil; IR (neat) 2928, 2104, 1774, 1673, 1498, 1386;
1H NMR (500 MHz, CDCl3) δ (ppm) = 7.41-7.37 (m, 5H), 4.98 (d, J = 11.0 Hz, 1H), 4.94 (d, J =
11.0 Hz, 1H), 3.77-3.74 (m, 1H), 3.33 (dd, J = 11.0, 4.0 Hz, 1H), 3.22 (dd, J = 11.0, 6.5 Hz, 1H),
2.77 (dd, J = 13.7, 5.5 Hz, 1H), 2.50 (dd, J = 13.7, 2.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ
(ppm) = 164.4, 135.5, 129.5, 129.3, 128.9, 78.6, 56.1, 51.2, 35.6. HRMS (ESI) m/z calcd for
C11H12N4NaO2 [M+Na]+ 255.0858, found 255.0828.
5.4.7. Ethyl 1-((1-(benzyloxy)-4-oxoazetidin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxylate
(49a):
Compound 47 (350 mg, 1.50 mmol) was dissolved in a tBuOH/H2O 4:1 mixture (10 mL). Ethyl
propiolate (152 µL, 1.50 mmol) was added, followed by CuSO4·5H2O (7.49 mg, 0.03 mmol) and
sodium ascorbate (30 mg, 0.15 mmol). The reaction mixture was left to stir at rt for 48 h then it
was partitioned between EtOAc (6 mL) and water (8 mL). The aqueous layer was extracted with
EtOAc (3 x 8 mL) and the combined organic extracts were dried (Na2SO4), filtered and
evaporated under vacuum. The residue was purified by column chromatography on silica gel
(from hexanes/EtOAc 1:1 to EtOAc) to give the product in 67% yield (0.33 g, 1.01 mmol) as a
white solid; IR (KBr) 1776, 1732, 1654, 1543, 1370; m.p. 93-95 ºC; 1H NMR (500 MHz, CDCl3)
δ (ppm) = 8.01 (bs, 1H), 7.41-7.34 (m, 5H), 4.93 (s, 2H), 4.48 (dd, J = 14.6, 4.7 Hz, 1H), 4.44-
4.38 (m, 3H), 3.83-3.80 (m, 1H), 2.78 (dd, J = 14.0, 5.3 Hz, 1H), 2.43(dd, J = 14.0, 2.4 Hz, 1H),
13
1.40 (t, J = 7.1 Hz, 3H); C NMR (125 MHz, CDCl3) δ (ppm) = 163.6, 160.6, 140.8, 135.1,
129.7, 129.6, 129.2, 128.7, 78.8, 61.6, 56.1, 50.5, 36.2, 14.5; HRMS (ESI) m/z calcd for
C16H19N4O4 [M+H]+ 331.1406, found 331.1381.
5.4.8. Ethyl 1-((4-oxo-1-(toxyloxy)azetidin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxylate
(51a)
Compound 49a (136 mg, 0.41 mmol) was dissolved in MeOH (10 mL) at rt, under an argon
atmosphere. Pd/C 10% (35 mg) was added and the mixture was flushed with hydrogen and left to
stir at rt for 4 h. The reaction mixture was purged with argon and filtered through celite and the
solvent was evaporated under vacuum to give N-hydroxy β-lactam 50a as a colorless oil.
20

Crude 50a was dissolved in dry CH3CN (10 mL) at rt, under an argon atmosphere. The solution
was cooled to 0 ºC and DIPEA (100 µL, 0.61 mmol) was added, followed by TsCl (117 mg, 0.61
mmol). The reaction mixture was left to stir at 0 ºC for 10 min then the solvent was evaporated
under vacuum. The resulting residue was purified by column chromatography on silica gel
(hexanes/EtOAc 1:1 to EtOAc) to give the product in 65% yield (105 mg, 0.27 mmol) as a white
1
solid; IR (KBr) 2983, 1807, 1727, 1596, 1544, 1379, 1194; m.p. 117-120 ºC; H NMR (500
MHz, CDCl3) δ (ppm) = 8.55 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 4.94
(dd, J = 15.0, 3.1 Hz, 1H), 4.87 (dd, J = 15.0, 3.8 Hz, 1H), 4.45 (dd, J = 14.2, 7.1 Hz, 2H), 4.47-
4.43 (m, 1H), 2.99 (dd, J = 15.5, 6.9 Hz, 1H), 2.64 (dd, J = 14.5, 4.0 Hz, 1H), 2.44 (s, 3H), 1.43
13
(dd, J = 7.1, 7.1 Hz, 3H); C NMR (125 MHz, CDCl3) δ (ppm) = 165.7, 160.7, 147.2, 140.6,
138.8, 130.4, 129.9, 129.3, 61.7, 57.5, 49.6, 36.1, 22.1, 14.6; HRMS (ESI) m/z calcd for
C16H19N4O6S [M+H]+, 395.1025, found 395.1037.
5.4.9. N-(Prop-2-yn-1-yl)benzamide (48b):
A catalytic amount of dry DMF (one drop) was added to a mixture of benzoic acid (1.0 g, 8.18
mmol) and (COCl)2 (1.40 mL, 16.4 mmol) in dry CH2Cl2 (50 mL) at 0 ºC, under an argon
atmosphere. The reaction mixture was left to stir at 0 ºC for 30 min then the solvent was
evaporated under vacuum. The residual HCl was eliminated by co-evaporation with CHCl3
(10mL) and toluene (10 mL). The crude acid chloride was dried under vacuum and then
dissolved in dry CH2Cl2 (20 mL). The solution was cooled to -78 ºC and DIPEA (3.56 mL, 20.4
mmol) was added, followed by propargyl amine (0.79 mL, 12.3 mmol). The reaction mixture
was left to stir overnight being allowed to warm to rt during this period. The solvent was
evaporated under vacuum and the residue was purified by column chromatography on silica gel
(hexanes/EtOAc 1:1 to EtOAc) to give the product in 89% yield (1.16 g, 7.28 mmol) as a white
solid; IR (KBr) 3300, 2928, 2116, 1642, 1538; m.p. 108.5-109.5 ºC; 1H NMR (500 MHz, CDCl3)
δ (ppm) = 7.80-7.78 (m, 2H), 7.48-7.45 (m, 1H), 7.40-7.36 (m, 2H), 6.94 (bs, 1H), 4.20 (dd, J =
7.6, 2.5 Hz, 2H), 2.24 (dd, J = 2.5, 2.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ (ppm) = 167.6,
133.9, 131.9, 128.8, 127.4, 79.9, 71.9, 29.9; HRMS (ESI) m/z calcd for C10H10NO [M+H]+
160.0762, found 160.0756.
21

5.4.10.
N-((1-((1-Benzyloxy)-4-oxoazetidin-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)benzamide (49b): Compound 47 (350 mg, 1.50 mmol) was added to a mixture of
compound 48b (239 mg, 1.50 mmol), CuSO4·5H2O (7.49 mg, 0.03 mmol) and sodium ascorbate
(30.0 mg, 0.15 mmol) in tBuOH/H2O 4:1 (8 mL). The reaction mixture was left to stir at rt for 48
h then it was partitioned between water (15 mL) and EtOAc (15 mL). The aqueous layer was
extracted with EtOAc (15 mL) and the combined organic extracts were dried (Na2SO4), filtered
and evaporated under vacuum. The residue was purified by column chromatography on silica gel
(hexanes/EtOAc 1:1 to EtOAc) to give the product in 77% yield (452 mg, 1.16 mmol) as a white
1
solid; IR (KBr) 3061, 1780, 1638, 1523, 1431, 1298; m.p. 124.7-126.8 ºC; H NMR (500 MHz,
CDCl3) δ (ppm) = 7.78-7.76 (m, 2H), 7.67-7.66 (m, 1H), 7.43-7.40 (m, 1H), 7.34-7.31 (m, 5H),
4.87-4.82 (m, 2H), 4.86 (d, J = 12.0 Hz, 1H), 4.84 (d, J = 12.0 Hz, 1H), 4.65 (dd, J = 15.0, 5.7
Hz, 1H), 4.58 (dd, J = 15.0, 5.7 Hz, 1H), 4.38 (dd, J = 14.5, 4.5 Hz, 1H), 4.28 (dd, J = 14.5, 5.0
Hz, 1H), 3.81-3.78 (m, 1H), 2.68 (dd, J = 14.1, 5.2 Hz, 1H), 2.39 (dd, J = 14.1, 2.2 Hz, 1H); 13C
NMR (125 MHz, CDCl3) δ (ppm) = 167.7, 163.9, 145.4, 135.2, 134.2, 131.8, 129.6, 129.5,
129.0, 128.7, 127.3, 123.9, 78.7, 56.4, 50.3, 36.4, 35.5; HRMS (ESI) m/z calcd for C21H22N5O3
[M+H]+ 392.1723, found 392.1738.
5.4.11.
2-((4-(Benzamidomethyl)-1H-1,2,3-triazol-1-yl)methyl)-4-oxoazetidin-1-yl4-
methylbenzenesulfonate (51b): Compound 49b (78 mg, 0.2 mmol) was dissolved in MeOH (5
mL) at rt, under an argon atmosphere. Pd/C 10% (10 mg) was added and the mixture was flushed
with hydrogen and left to stir at rt for 4 h. The reaction mixture was filtered through celite and
the solvent was evaporated under vacuum to give N-hydroxy β-lactam 50b as a colorless oil.
Crude 50b was dissolved in dry CH3CN (20 mL) at rt, under an argon atmosphere. The solution
was cooled to 0 ºC and DIPEA (51 µL, 0.3 mmol) was added, followed by TsCl (56 mg, 0.3
mmol). The reaction mixture was left to stir at 0 ºC for 10 min then the solvent was evaporated
under vacuum. The resulting residue was purified by column chromatography on silica gel
(hexanes/EtOAc 1:1 to EtOAc) to give the product in 70% yield (63.8 mg, 0.14 mmol) as a white
1
solid; IR (KBr) 2120, 1789, 1642, 1488, 1390, 1297; m.p. 71.0-73.0 ºC; H NMR (500 MHz,
CDCl3) δ (ppm) = 7.96 (bs, 1H), 7.86-7.83 (m, 2H), 7.81-7.79 (m, 2H), 7.53-7.49 (m, 1H), 7.45-
7.41 (m, 2H), 7.35-7.33 (m, 2H), 6.84 (bs, 1H), 4.86 (dd, J = 14.7, 3.5 Hz, 1H), 4.79-4.74 (m,
3H), 4.45-4.41 (m, 1H), 2.97 (dd, J = 15.2, 6.5 Hz, 1H), 2.70 (dd, J = 15.2, 3.5 Hz, 1H), 2.41 (s,
22

13
3H); C NMR (125 MHz, CDCl3) δ (ppm) = 167.7, 165.4, 147.0, 134.2, 131.9, 130.4, 130.3,
129.3, 128.8, 127.3, 124.6, 57.8, 49.6, 36.6, 35.6, 22.0; HRMS (ESI) m/z calcd for C21H22N5O5S
[M+H]+ 456.1342, found 456.1349.
5.4.12. 2-Chloro-N-(prop-2-yn-1-yl)-5-(trifluoromethyl)benzamide (48c):
A catalytic amount of dry DMF (one drop) was added to a mixture of 2-chloro-5-
(trifluoromethyl)benzoic acid (1.0 g, 4.45 mmol) and (COCl)2 (0.77 mL, 8.90 mmol) in dry
CH2Cl2 (50 mL) at 0 ºC, under an argon atmosphere. The reaction mixture was left to stir at 0 ºC
for 30 min then the solvent was evaporated under vacuum. The residual HCl was removed by co-
evaporation with CHCl3 (10mL) and toluene (10 mL). The crude acid chloride was dried under
vacuum then it was dissolved in dry CH2Cl2 (20 mL). The solution was cooled to –78 ºC and
DIPEA (1.86 mL, 10.7 mmol) was added, followed by propargyl amine (0.43 mL, 6.67 mmol).
The reaction mixture was left to stir overnight being allowed to warm to rt during this period.
The solvent was evaporated under vacuum and the residue was purified by column
chromatography on silica gel (hexanes/EtOAc 1:1 to EtOAc) to give the product in 77% yield
(897 mg, 3.43 mmol) as a white solid; IR (KBr) 1650, 1546, 1434, 1332, 1266; m.p. 115-117 ºC;
1H NMR (500 MHz, CDCl3) δ (ppm) = 7.92 (m, 1H), 7.60 (dd, J = 8.0, 2.5 Hz, 1H), 7.52 (dd, J
= 8.0, 0.5 Hz, 1H), 6.63 (bs, 1H), 4.25 (dd, J = 5.2, 2.5 Hz, 2H), 2.29 (dd, J = 2.5, 2.5 Hz, 1H);
13C NMR (125 MHz, CDCl3) δ (ppm) = 164.9, 135.1, 134.8, 131.2, 130.1, 129.8, 128.3, 127.6,
78.7, 72.5, 30.1; HRMS (ESI) m/z calcd for C11H8ClF3NO [M+H]+ 262.0247, found 262.0216.
5.4.13.
N-((1-((1-(Benzyloxy)-4-oxoazetidin-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-2-
chloro-5-(trifluoro-methyl) benzamide (49c):
Compound 47 (280 mg, 1.20 mmol) was added to a mixture of compound 48c (315 mg, 1.20
mmol), CuSO4·5H2O (6.0 mg, 0.024 mmol) and sodium ascorbate (23.8 mg, 0.12 mmol) in
tBuOH/H2O 4:1 (8 mL). The reaction mixture was left to stir at rt for 48 h then it was partitioned
between water (15 mL) and EtOAc (15 mL). The aqueous layer was extracted with EtOAc (15
mL) and the combined organic extracts were dried (Na2SO4), filtered and evaporated under
vacuum. The residue was purified by column chromatography on silica gel (hexanes/EtOAc 1:1
to EtOAc) to give the product in 73% yield (433 mg, 0.88 mmol) as a white solid; IR (KBr)
1761, 1646, 1536, 1377, 1298, 1128; m.p. 38-41 ºC; 1H NMR (500 MHz, CDCl3) δ (ppm) = 7.75
23

(bs, 1H), 7.69 (dd, J = 5.5, 5.5 Hz, 1H), 7.65 (s, 1H), 7.52-7.49 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H),
7.31-7.29 (m, 5H), 4.86 (s, 2H), 4.60 (d, J = 6.0 Hz, 2H), 4.40 (dd, J = 14.5, 4.5 Hz, 1H), 4.31
(dd, J = 14.5, 4.5 Hz, 1H), 3.79-3.76 (m, 1H), 2.65 (dd, J = 14.0, 5.0 Hz, 1H), 2.33 (dd, J = 14.0,
13
2.5 Hz, 1H); C NMR (125 MHz, CDCl3) δ (ppm) = 165.8, 163.8, 144.7, 136.0, 135.1, 135.0,
130.9, 129.5, 129.3, 129.0, 127.8, 126.9, 124.6, 124.1, 122.4, 78.6, 56.3, 50.0, 36.0, 35.5; HRMS
(ESI) m/z calcd for C22H20ClF3N5O3 [M+H]+ 494.1207, found 494.1191.
5.4.14.
2-((4-((2-Chloro-5-(trifluoromethyl)benzamido)methyl)-1H-1,2,3-triazol-1-
yl)methyl)-4-oxoazetidin-1-yl 4-methyl benzenesulfonate (51c):
Compound 49c (340 mg, 0.69 mmol) was dissolved in MeOH (10 mL) at rt, under an argon
atmosphere. Pd/C 10% (30 mg) was added and the mixture was flushed with hydrogen and left to
stir at rt for 4 h. The reaction mixture was filtered through celite and the solvent was evaporated
under vacuum to give N-hydroxy β-lactam 50c as a colorless oil.
Crude 50c was dissolved in dry CH3CN (20 mL) at rt, under an argon atmosphere. The solution
was cooled to 0 ºC and DIPEA (179 µL, 1.04 mmol) was added, followed by TsCl (197 mg, 1.04
mmol). The reaction mixture was left to stir at 0 ºC for 10 min then the solvent was evaporated
under vacuum. The resulting residue was purified by column chromatography on silica gel
(hexanes/EtOAc 1:1 to EtOAc) to give the product in 66% yield (257 mg, 0.46 mmol); white
solid; IR (KBr) 2960, 1808, 1655, 1541, 1383, 1194; m.p. 50-54 ºC. 1H NMR (500 MHz, CDCl3)
δ (ppm) = 8.03 (s, 1H), 7.91-7.90 (m, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.60-7.58 (m, 1H), 7.50 (d, J
= 8.5 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.16 (bs, 1H), 4.86 (dd, J = 14.5, 3.5 Hz, 1H), 4.80-4.70
(m, 3H), 4.46-4.42 (m, 1H), 2.95 (dd, J = 15.3, 6.5 Hz, 1H), 2.62 (dd, J = 15.3, 4.0 Hz, 1H), 2.42
(s, 3H); 13C NMR (125 MHz, CDCl3) δ (ppm) = 165.4, 165.3, 147.1, 144.5, 135.6, 134.9, 131.1,
130.4, 130.1, 129.7, 129.3, 128.2, 128.1, 127.4, 124.5, 57.8, 49.6, 36.4, 35.8, 22.0; HRMS (ESI)
m/z calcd for C22H20ClF3N5O5S [M+H]+ 558.0826, found 558.0831.
ASSOCIATED CONTENT
Supporting information
24

The NMR spectra of the compounds are available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding author *e-mail: mmiller1@nd.edu
Author Contributions
§The authors contributed equally to the manuscript.
ACKNOWLEDGMENTS
We gratefully acknowledge Patricia Miller and Kyle D. Watson (UND) for biological assays,
Nonka Sevova and Dr. Bill Boggess (Mass Spectrometry and Proteomics Facility, UND) for
mass spectroscopic analyses and Dr. Jaroslav Zajicek (Lizzadro Magnetic Resonance Research
Center, UND) for NMR assistance. We acknowledge the University of Notre Dame, Drs. Scott
Hecker and Tomasz Glinka at Rempex Pharmaceuticals, Inc., and NIH (1R21AI098689) for
support of this work. KDW also acknowledges a fellowship from the Notre Dame CBBI training
grant (NIH T32 GM075762).
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