Alternate "drug" Delivery Utilizing β-Lactam Cores: Syntheses and Biological Evaluation of β-Lactams Bearing Isocyanate Precursors

Article abstract of DOI:10.1021/acs.joc.6b02272

The synthesis of a small set of β-lactams containing isocyanate precursors is described. The release of the isocyanate precursor in model hydrolysis experiments was substantiated by trapping experiments, thus confirming that β-lactams can be designed that are capable of releasing alternatively reactive species. Preliminary biological assessments are also briefly discussed.

Full text of DOI:10.1021/acs.joc.6b02272

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Note
Alternate “Drug” Delivery Utilizing beta-Lactam Cores: Syntheses and
Biological Evaluation of beta-Lactams Bearing Isocyanate Precursors.
Mark W. Majewski, Patricia A. Miller, Allen G. Oliver, and Marvin J. Miller
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02272 • Publication Date (Web): 09 Dec 2016
Downloaded from http://pubs.acs.org on December 9, 2016
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Alternate “Drug” Delivery Utilizing βꢀLactam Cores: Syntheses and
Biological Evaluation of βꢀLactams Bearing Isocyanate Precursors.
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Mark W. Majewski, Patricia A. Miller, Allen G. Oliver, and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556,
USA
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Corresponding Author: mmiller1@nd.edu
Abstract: The synthesis of a small set of βꢀlactams containing isocyanate precursors is
described. The release of the isocyanate precursor in model hydrolysis experiments was
substantiated by trapping experiments, thus confirming that βꢀlactams can be designed that are
capable of releasing alternatively reactive species. Preliminary biological assessments are also
briefly discussed.
______________________________________________________________________________
Although serendipitously discovered over 80 years ago, the βꢀlactam family remains one
of the most widely prescribed classes of antibiotics.1 βꢀLactams inhibit the formation of
peptidoglycan crossꢀlinkages in the bacterial cell wall by acting as mimics of the terminal
Dꢀalaꢀ
D
ꢀala dipeptide of peptidoglycan. Unfortunately, bacterial resistance continues to be on the rise
and threatens modern healthcare. Bacteria have developed a myriad of mechanisms to stymie
successful antibiotic therapy, highlighted by the presence of βꢀlactamases. These rather
promiscuous enzymes catalyze the hydrolysis of βꢀlactam antibiotics and have received
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considerable attention.2 Many drug resistant strains of bacteria produce β–lactamases and more
than 900 unique forms of these enzymes have been identified.3
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There are a number of ways to remain one step ahead of bacterial resistance mechanisms.
One approach involves chemical modification of existing cores to make them less susceptible to
βꢀlactamase induced hydrolysis and other bacterial resistance mechanisms.4 More aggressive
methods involve coꢀadministration of an antibiotic and βꢀlactamase inhibitor, an example being
the combination of clavulanic acid (βꢀlactamase inhibitor) with amoxicillin (antibiotic), known
as Augmentin™.5 With this combination, the enzyme inhibitor protects the antibiotic from
competitive detrimental hydrolysis and inactivation. A third method of circumventing resistance
capitalizes on both the efficiency of bacterial enzymatic machinery and the novel ring opening
chemistry of βꢀlactams. Often, this involves the use of prodrugs.6 The βꢀlactam literature is
replete with examples in which the core has ultimately been used for this purpose. O’Callaghan
and Sykes reported a cephalosporin (1) that upon hydrolysis, generated 2ꢀmercaptopyridineꢀNꢀ
oxide, 2b, a species that exhibited separate antibacterial activity (Scheme 1).7
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Scheme 1. Utilization of the Cephalosporin Core for Drug Delivery.
This concept of enzymatically triggered release was subsequently extended to penicillins
by Smyth with the report of penicillin scaffolds, such as 3, that expelled a sulfonamide upon βꢀ
lactamase catalyzed ring opening (Scheme 2).8
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Scheme 2. Penam Core as Triggered Release System.
With the above precedence in mind, the aim of this work centered on the design and
syntheses of βꢀlactam scaffolds that exhibit a unique type of reactivity, specifically, release of a
broadly reactive isocyanate upon hydrolysis. As illustrated in Scheme 3, compounds such as 6
or 7, synthesized separately from starting material A or B respectively, may exert antibiotic
activity or βꢀlactamase deactivation through an enzymatically induced Lossen rearrangement9 to
generate an isocyanate (8). Although not a drug in itself, it is hypothesized that upon release the
isocyanate would react with nucleophiles in the vicinity and lead to either penicillin binding
protein (PBP) inhibition, βꢀlactamase inhibition, or other nonꢀspecific target modifications.
Utilizing βꢀlactam cores to fulfill this purpose is ideal, since it has been shown that cleavage of
the ring can result in the release of leaving groups.10 However, to the best of our knowledge, few
reports exist that have applied the concept of triggered release systems to monocyclic βꢀlactams
(monobactams).11 This concept of using an isocyanate produced from the Lossen rearrangement
for purposes of inhibition in biological systems was previously reported by Groutas.12 In that
study, succinimides were shown to be mechanismꢀbased inhibitors of a serine proteinase,
specifically human leukocyte elastase (HLE), and operated via an enzyme induced Lossen
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rearrangement in order to exert activity. Herein we report the syntheses and studies of βꢀlactams
with pendant hydroxamic acids that are appropriately substituted to be isocyanate precursors.
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Scheme 3. Hypothesis for Enzymatic Triggered “Drug” Release.
(BL = βꢀlactamase, PBP = Penicillin Binding Protein)
Syntheses of racemic monobactams devoid of Cꢀ3 functionality (see structure 6, Scheme
3 for numbering) are shown in Scheme 4. Starting with appropriately functionalized
hydroxamate 9, reaction with 4ꢀacetoxyꢀ2ꢀazetidinone under conditions first reported by Clauss13
gave hydroxamate containing monobactam 10 in good yield. The structure for 10 was confirmed
by Xꢀray crystallography to assure desired Nꢀalkylation (see supporting information).
Hydrogenolysis, followed by tosylation and mesylation gave intermediates 11aꢀb. Lastly,
nitrogen sulfonation was carried out using DMF•SO3 complex14 followed by conversion to the
tetrabutlyammonium (TBA) salts with tetrabutylammonium hydrogen sulfate (TBAHS). These
TBA salts were then either purified with normal phase silica gel to give 12a, or stirred in a
prepared potassium Dowex WX8 resin to give potassium salt 12b.
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Scheme 4. Racemic Syntheses of Monobactams 12aꢀb.
The next stage was to generate compounds with classic βꢀlactam sidechains, anticipating
that such side chains would likely be necessary to potentiate antiꢀbacterial activity. Beginning
with azetidinone 13 (Scheme 5), obtained from the degradation of penicillin V,15 reaction with 9
gave intermediate 14 with the expected anti stereochemistry, verified by NMR homo decoupling
experiments.15 Hydrogenolysis, followed by separate tosylation and mesylation gave compounds
15aꢀb and sulfonation gave final compounds 16aꢀb in modest yields.
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Scheme 5. Syntheses of Monobactams 16aꢀb.
To further expand structureꢀactivityꢀrelationship studies we were also interested in
incorporating the classic hydroxyethyl sidechain, common in many carbapenem antibiotics, into
our systems. Although incorporation of hydroxyethyl side chains onto the periphery of
monobactams does not impart antibiotic activity, related studies on βꢀlactamase susceptibilty
have not been reported. As shown in Scheme 6, the commercially available stereopure
azetidinone was reacted with hydroxamate 9 to give intermediate 17. Hydrogenolysis, followed
by tosylation and mesylation gave 18aꢀb and sulfonation produced penultimate compounds 19aꢀ
b. Removal of the silyl protecting group was accomplished with 1 M HCl to give monobactams
20aꢀb.
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Scheme 6. Syntheses of Monobactams 20aꢀb.
The syntheses of hydroxamate substituted cephalosporins started with commercially
available GCLE. As shown in Scheme 7, the silyl protected hydroxamate was directly installed
by means of Pd(0) chemistry to give 2116 in good yield. Desilyation with HF•pyridine gave
hydroxamic acid 22 and tosylation, followed by deprotection with TFA, gave final cephalosporin
23.
H
N
H
N
H
N
H
N
Ph
Ph
1. Pd(OAc)2
PPh3, 0 °C
S
S
OTBS
Ph
O
O
Cl
CO2PMB
N
O
2.
Ph
O
O
OTBS
CO2PMB
N
O
H
GCLE
21
NaH, 0°C, 59%
1. TsCl, Pyr.
MeCN, 0 °C to rt
30%
H
N
H
N
H
N
H
N
Ph
Ph
S
S
OTs
N
OH
N
HF•Pyridine
THF, 0 °C
84%
O
O
21
Ph
Ph
2. TFA, Anisole
DCM, 0 °C, 47%
O
O
COOH
CO2PMB
O
O
23
22
Scheme 7. Synthesis of Cephalosporin 23.
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Selected monobactams were then subjected to alkaline hydrolysis conditions followed by
LC/MS analysis of products in order to confirm whether hydrolysis of the βꢀlactam ring
ultimately produced products that were consistent with the Lossen rearrangement hypothesis
(Scheme 8). Attack by hydroxide was anticipated to expel the activated hydroxamate that would
spontaneously engage in the Lossen rearrangement to produce the transient isocyanate. This
species would then be trapped by hydroxide to give an unstable carbamic acid and subsequent
decarboxylation would produce aniline. Aniline, in turn, could then react with additional
isocyanate to ultimately give diphenylurea. Alternatively, 11a itself might be expected to trap
the isocyanate to give an adduct. Our studies indicated that 11aꢀb readily hydolyzed under these
conditions to produce products consistent with our proposed Lossen hypothesis (see supporting
information). Additionally, we also observed a competing reaction in which the leaving group
(i.e. tosyl or mesyl) was hydrolytically cleaved to give the a monobactam with free hydroxamic
acid. Studies on sulfonated compounds, specifically 16b, 19b, and 20b, showed that these
compounds were stable at pH 10 after 2ꢀ4 h of stirring. This is likely the result of the
incorporation of the ionizable group onto the βꢀlactam nitrogen, thus making the βꢀlactam less
prone to nonꢀenzymatic hydrolytic opening. After 24 h of stirring, these compounds showed
only slight degradation. Overall, these studies served as a proof of concept for the novel path of
reactivity using the monobactam core.
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Scheme 8. Hydrolysis of 11a and Expected Products, all Confirmed with LC/MS Monitoring.
With these encouraging results, compounds were screened against a panel of Gramꢀ
positive and Gramꢀnegative bacteria using agar diffusion assays, with final compounds also
tested for βꢀlactamase inhibition via synergy studies with known βꢀlactam antibiotics using
standard broth microdilution assays. While the monobactams were generally inactive, the
cephalosporins selectively targeted Gramꢀpositive bacteria, with notable zones of inhibition and
minimum inhibitory concentrations (MICs) observed (Table 1). Highlighted among the
compounds were the activities of compounds 21, 22, and 23, with MIC values of 0.4, 0.4, and
3.0 ꢁM against B. subtilis, respectively. Cephalosporins 21, 22, and 23 also targeted S. aureus,
with MIC values of 1.6, 0.8, and 12.6 ꢁM, respectively.
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Table 1. Zone of Growth Inhibition (mm) with MIC (ꢁM) in bold.
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B. subtilis
S. aureus
M. luteus
Cmpd.
ATCC 6633
SG511
ATCC 10240
18
0.4
30
0.4
25
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1.6
29
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0
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6.0
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3.0
12.6
21/25P
21
0
Ciprofloxacin
Compounds were dissolved in MeOH/DMSO at a concentration of 2
mM. Ciprofloxacin used in water at 1.66 ꢁg/mL.
In conclusion, the syntheses of functionally dense monobactams and cephalosporins
bearing isocyanate precursors have been reported. Hydrolysis experiments on model compounds
have substantiated our Lossen rearrangement hypothesis and thus a novel reactivity pathway.
EXPERIMENTAL PROCEDURES
General Comments
Commercial grade reagents and solvents were used without further purification except as
indicated below. Tetrahydrofuran (THF) was distilled from sodium and both DCM and MeCN
were distilled from calcium hydride. All reactions were carried out in oven or flameꢀdried
glassware under an atmosphere of dry argon only when specified in the experimental details. For
reactions in which a hydroxamic acid was made, all glassware used was washed with 6 M HCl
and dried prior to use to minimize contamination due to iron binding. All reactions were
magnetically stirred and monitored by analytical thinꢀlayer chromatography using aluminumꢀ
backed 0.2 mm silica gel 60 Fꢀ254 plates. Visualization was accomplished by UV light (254
nm), KMnO4, and vanillin spray. Column chromatography was performed with silica gel 60
1
13
(230–400 mesh). Both H NMR and C NMR spectra were recorded at ambient temperature
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with the residual solvent peaks as internal standards on a 300, 400, 500, or 600 MHz instrument.
All IR spectra were recorded on a FT IR by making thin films on NaCl plates. Highꢀresolution
mass spectra (HRMS, ESIꢀTOF) data were obtained with electrospray ionization and a timeꢀofꢀ
flight analyzer. Melting points were performed with a capillary melting point apparatus and are
uncorrected. All NMR spectra were processed with ACD/academic edition version 12 software
and reported using the software’s JOC report format.
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Nꢀ(Benzyloxy)ꢀNꢀ(2ꢀoxoazetidinꢀ4ꢀyl)benzamide (10): To a flame dried round bottomed flask
under argon was added Nꢀ(benzyloxy)benzamide (9, 1.19 g, 5.23 mmol). The flask was cooled
in an ice bath for 5 min and a solution of 0.8 M NaOEt in EtOH (10 mL, 8 mmol) was slowly
added. The reaction was stirred for 5 min, 4ꢀacetoxyꢀ2ꢀazetidinone (1.00 g, 7.74 mmol) in
anhydrous EtOH (5 mL) was added dropwise, and the reaction was stirred overnight warming to
room temperature. The reaction was diluted with EtOAc (75 mL) and then washed with H2O,
brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give crude product
that was triturated with hexanes and then purified with column chromatography (90/10
ether/hexanes) to give 10 (1.10 g, 3.71 mmol) as a white solid in 71% yield. mp = 124ꢀ126°C; 1H
NMR (300 MHz, CDCl3) δ = 7.28 ꢀ 7.73 (m, 8 H), 7.11 (dd, J = 7.22, 1.84 Hz, 2 H), 6.48ꢀ6.52
(br. s., 1 H), 5.99 ꢀ 6.05 (fine m, 1 H), 4.87 (d, J = 9.39 Hz, 1 H), 4.70 (d, J = 9.39 Hz, 1 H), 3.49
(dd, J = 14.9, 1.91 Hz, 1 H), 3.14 ꢀ 3.23 (m, 1 H); 13C NMR (125 MHz, CDCl3) δ = 170.7, 166.6,
133.9, 133.6, 131.7, 130.0, 129.4, 128.8, 128.7, 128.6, 80.5, 60.5, 42.4; IR (FTꢀIR) ν 1772, 1650
cmꢀ1; HRMS (ESIꢀTOF) calcd. for C17H16N2O3 (M + H+) 297.1234, found 297.1223.
Nꢀ(2ꢀOxoazetidinꢀ4ꢀyl)ꢀNꢀ(tosyloxy)benzamide (11a): To a flame dried round bottomed flask
was added 10 (202 mg, 0.681 mmol) and EtOAc (20 mL). Argon gas was bubbled through the
solution and then Pd/C (10%) was added (45 mg). The contents were once again purged with
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argon by immersing the needle directly into the solution. Once purged, the argon line was
removed and a vacuum line was inserted through the septum and held above the solution. A
balloon was filled with H2 gas and inserted through the rubber septum and into the solution
momentarily (~1 min) then held above the solution. The reaction was left to stir under an
atmosphere of H2. Upon completion (TLC: 100% EtOAc), the reaction was purged with argon
prior to filtration. The reaction mixture was filtered and the filter papers were washed with
MeOH. The filtrate was concentrated under reduced pressure to give crude material that was
then purified with ironꢀfree normal phase silica gel (90/10 EtOAc/hexanes) to give NꢀhydroxyꢀNꢀ
(2ꢀoxoazetidinꢀ4ꢀyl)benzamide (110 mg, 0.533 mmol) as a white solid in 78% yield. mp = 113ꢀ
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1
115 °C; H NMR (600 MHz, DMSOꢀd6) δ = 9.91 (s, 1 H), 8.31 (s, 1 H), 7.61 (d, J = 7.3 Hz, 2
H), 7.56 ꢀ 7.37 (m, 3 H), 5.87 (br. s., 1 H), 3.08 (dq, J = 2.1, 7.9 Hz, 1 H), 3.04 (dd, J = 1.5, 12.3
13
Hz, 1 H); C NMR (150 MHz, DMSOꢀd6) δ = 170.1, 166.7, 134.8, 131.0, 128.7, 128.4, 58.6,
41.4; HRMS (ESIꢀTOF) calcd. for C10H10NaN2O3 (M+ Na+) 229.0584, found 229.0579. To a
flame dried round bottomed flask under argon was added NꢀhydroxyꢀNꢀ(2ꢀoxoazetidinꢀ4ꢀ
yl)benzamide (102 mg, 0.494 mmol) and anhydrous MeCN (10 mL). TsCl (102 mg, 0.535
mmol) was added and the solution was cooled in an ice bath for 10 minutes. Triethylamine
(0.535 mmol, 0.0750 mL) was then added dropwise and the reaction was stirred for 3 h. The
solvent was removed under reduced pressure to give a yellowꢀwhite slurry that was dissolved in
CHCl3 (25 mL) and then washed with H2O, saturated NaHCO3, and brine. The organic layer was
collected, dried over MgSO4, filtered, and concentrated under reduced pressure to give crude
material that was purified with column chromatography (50/50 hexanes/EtOAc) to give 11a
1
(73.0 mg, 0.202 mmol) as a white solid in 42% yield. mp = 101ꢀ103 °C; H NMR (300 MHz,
CDCl3) δ = 7.65 ꢀ 7.17 (m, 9 H), 6.25 (br. s., 1 H), 5.79 (dd, J = 1.9, 4.8 Hz, 1 H), 3.44 (d, J =
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15.2 Hz, 1 H), 3.19 (dd, J = 2.5, 15.2 Hz, 1 H), 2.42 (s, 3 H); C NMR (75 MHz, CDCl3) δ =
171.2, 165.0, 146.7, 132.6, 132.0, 130.5, 130.1, 129.3, 128.9, 128.7, 64.0, 42.9, 21.9; HRMS
(ESIꢀTOF) calcd. for C17H16N2O5S (M + H+) 361.0853, found 361.0876.
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Nꢀ((Methylsulfonyl)oxy)ꢀNꢀ(2ꢀoxoazetidinꢀ4ꢀyl)benzamide (11b): To a flame dried round
bottom flask under argon was added NꢀhydroxyꢀNꢀ(2ꢀoxoazetidinꢀ4ꢀyl)benzamide (1.35 g, 6.50
mmol) and anhydrous MeCN (40 mL). The solution was cooled in an ice bath for 10 min and
MsCl (0.55 mL, 7.1 mmol) and Et3N (0.92 mL, 6.5 mmol) were added dropwise. The reaction
was stirred for 2 h and then the solvent was removed under reduced pressure to give a yellowꢀ
white slurry that was dissolved in CHCl3 (50 mL), washed with H2O, saturated NaHCO3 and
brine. The organic layer was collected, dried over MgSO4, filtered, and concentrated under
reduced pressure to give a yellow solid that was purified with column chromatography (60/40
EtOAc/hexanes) to give 11b (1.11 g, 3.90 mmol) as a white solid in 60% yield. mp = 112ꢀ113
°C; 1H NMR (300 MHz, CDCl3) δ = 7.68 ꢀ 7.48 (m, 5 H), 6.41 (br. s., 1 H), 5.79 (dd, J = 2.0, 4.7
13
Hz, 1 H), 3.42 (d, J = 14.8 Hz, 1 H), 3.25 ꢀ 3.16 (m, 1 H), 3.14 (s, 3 H); C NMR (75 MHz,
CDCl3) δ = 170.2, 164.7, 132.9, 132.1, 129.3, 128.6, 63.8, 42.9, 39.3; HRMS (ESIꢀTOF) calcd.
for C11H12N2O5S (M + H+) 285.0540, found 285.0528.
2ꢀOxoꢀ4ꢀ(Nꢀ(tosyloxy)benzamido)azetidineꢀ1ꢀsulfonic acid (12a): To a flame dried round
bottom flask under argon was added 11a (100 mg, 0.2 mmol) and anhydrous DCM (10 mL).
The solution was cooled in an ice bath for 5 min and DMF•SO3 (167 mg, 1.10 mmol) was added
in portions. The reaction was stirred for 1.5 h on ice and then diluted with DCM and 1 M
K2HPO4. Tetrabutylammonium hydrogen sulfate (TBAHS, 94.0 mg, 0.277 mmol) was added,
the separatory funnel shaken vigorously and vented, and the layers separated. The aqueous layer
was extracted with EtOAc and the pooled organic layers were dried over MgSO4, filtered, and
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concentrated under reduced pressure to give crude salt that was purified with normal phase silica
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38% yield. mp = 104ꢀ105 °C; H NMR (600 MHz, CD3OD) δ = 7.76 ꢀ 7.65 (br. s., 2 H), 7.55 ꢀ
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7.44 (m, 3 H), 7.39 ꢀ 7.27 (m, 4 H), 6.00 ꢀ 5.83 (br. s., 1 H), 3.55 (dd, J = 1.5, 14.1 Hz, 1 H), 3.33
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(d, J = 5.6 Hz, 1 H), 2.40 (s, 3 H); C NMR (150 MHZ, CD3OD) δ = 171.8, 163.4 (DMF ),
146.7, 132.4, 131.6, 130.2, 129.6, 129.1, 128.9, 127.8, 67.8 (broad), 41.1 (broad), 35.5 (DMF),
30.2 (DMF), 20.2; HRMS (ESIꢀTOF) calcd. for C17H16N2O8S2 (M ꢀ H+) 439.0275, found
439.0251.
Potassium 4ꢀ(Nꢀ((methylsulfonyl)oxy)benzamido)ꢀ2ꢀoxoazetidineꢀ1ꢀsulfonate (12b): To a
flame dried round bottom flask under argon was added 11b (200 mg, 0.7 mmol) and anhydrous
DCM (10 mL). The solution was cooled in an ice bath for 5 min and DMF•SO3 (324 mg, 2.10
mmol) was added in portions. The reaction was stirred for 2 h on ice and then diluted with DCM
and 1 M K2HPO4. TBAHS (240 mg, 0.703 mmol) was added, the separatory funnel was shaken
vigorously and vented, and the layers were separated. The aqueous layer was extracted with
EtOAc and the pooled organic layers were dried over MgSO4, filtered, and concentrated under
reduced pressure to give the crude TBA salt. The salt was stirred in a prepared Dowex resin (15
g, 50 WX8, 50ꢀ100 mesh, K+ form) for 3 h, the resin was filtered off, and the filtrate was
concentrated under reduced pressure to give 12b (88.0 mg, 0.218 mmol) as fine oil droplets in
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31% yield. H NMR (500 MHz, DMSOꢀd6) δ = 7.79 (br. s., 2 H), 7.68 ꢀ 7.57 (m, 2 H), 7.57 ꢀ
7.45 (m, 2 H), 5.72 (d, J = 4.8 Hz, 7 H), 3.49 ꢀ 3.39 (m, 3 H), 3.25 (dd, J = 5.6, 10.0 Hz, 2 H),
3.09 (dd, J = 2.2, 13.2 Hz, 2 H); 13C NMR (150 MHz, DMSOꢀd6) δ = 171.4, 162.6, 132.7, 129.6,
129.0, 128.4, 67.4, 40.4 (overlap with solvent), 38.2; IR (FTꢀIR) ν 1771, 1688 cmꢀ1; HRMS
(ESIꢀTOF) calcd. for C11H11N2O8S2 (M ꢀ H+) 362.9962, found 362.9985.
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Nꢀ(Benzyloxy)ꢀNꢀ((3S,4S)ꢀ2ꢀoxoꢀ3ꢀ(2ꢀphenoxyacetamido)azetidinꢀ4ꢀyl)benzamide (14): To a
flame dried round bottom flask under argon was added 9 (700 mg, 3. mmol). The flask was
cooled in an ice bath for 5 min and a solution of 0.8 M NaOEt in anhydrous EtOH (3.90 mL,
3.08 mmol) was added dropwise. The reaction was stirred for 5 min and then a solution of 4ꢀ
acetoxyꢀ2ꢀazetidinone (900 mg, 3 mmol) in anhydrous EtOH was added slowly. The reaction
was left to stir overnight under argon, warming to room temperature. The solvent was removed
under reduced pressure to give crude material that was dissolved in EtOAc (40 mL), washed with
H2O, brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give a
yellow oil that was purified with column chromatography (70/30 EtOAc/hexanes) to give 14
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(414 mg, 0.930 mmol) as a white solid in 30% yield. mp = 144ꢀ145°C; H NMR (300 MHz,
CDCl3) δ = 7.79 ꢀ 6.79 (m, 15 H), 6.15 (s, 1 H), 5.39 (d, J = 6.5, 1.6 Hz, 1 H), 4.79 (overlapping
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dd, J = 9.6, 13.2 Hz, 2 H), 4.44 (s, 2 H); C NMR (75 MHz, CDCl3) δ = 170.9, 169.7, 165.7,
157.1, 133.8, 133.3, 131.8, 130.1, 130.0, 129.4, 128.8, 128.7, 128.5, 122.4, 114.9, 80.4, 67.2,
66.5, 60.0; IR (FTꢀIR) ν 1762, 1652 cmꢀ1; HRMS (ESIꢀTOF) calcd. for C25H23N3NaO5 (M +
Na+) 468.1530, found 468.1552.
Nꢀ((3S,4S)ꢀ2ꢀOxoꢀ3ꢀ(2ꢀphenoxyacetamido)azetidinꢀ4ꢀyl)ꢀNꢀ(tosyloxy)benzamide (15a): To a
round bottom flask was added 14 (400 mg, 0.9 mmol) and THF (18 mL). The resulting solution
was subjected to hydrogenolysis using the same procedure as for the synthesis of 11a to give
crude NꢀHydroxyꢀNꢀ((3S,4S)ꢀ2ꢀoxoꢀ3ꢀ(2ꢀphenoxyacetamido)azetidinꢀ4ꢀyl)benzamide (343 mg,
0.965 mmol) as a yellow sticky solid. To a flame dried round bottomed flask under argon was
added NꢀhydroxyꢀNꢀ((3S,4S)ꢀ2ꢀoxoꢀ3ꢀ(2ꢀphenoxyacetamido)azetidinꢀ4ꢀyl)benzamide (220 mg,
0.61 mmol) and 20 mL of anhydrous THF. The resulting suspension was cooled in an ice bath
for 5 min, TsCl (165 mg, 0.867 mmol) and Et3N (0.103 mL, 0.743 mmol) were added, and the
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reaction was stirred for 2.5 h, warming to room temperature. The solvent was removed under
reduced pressure to give crude material that was dissolved in DCM, washed with H2O, saturated
NaHCO3, brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give
crude material that was purified with column chromatography (60/40 EtOAc/hexanes) to give
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15a (192 mg, 0.377 mmol) as a white solid in 55% yield. mp = 87ꢀ89 °C; H NMR (500 MHz,
CDCl3) δ = 7.65 ꢀ 6.83 (m, 15 H), 6.43 (s, 1 H), 5.91 (br. s., 1 H), 5.22 (d, J = 7.0 Hz, 1 H), 4.45
(s, 2 H), 2.40 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ = 170.8, 169.3, 156.9, 146.6, 132.3, 131.6,
129.94, 129.92, 129.73, 129.2, 128.8, 128.4, 122.1, 114.1, 70.1, 66.1, 60.8, 31.5, 21.7; HRMS
(ESIꢀTOF) calcd. for C25H24N3O7S (M + H+) 510.1329, found 510.1348.
Nꢀ((Methylsulfonyl)oxy)ꢀNꢀ((3S,4S)ꢀ2ꢀoxoꢀ3ꢀ(2ꢀphenoxyacetamido)azetidinꢀ4ꢀyl)benzamide
(15b): To a flame dried round bottom flask under argon was added crude NꢀhydroxyꢀNꢀ((3S,4S)ꢀ
2ꢀoxoꢀ3ꢀ(2ꢀphenoxyacetamido) azetidinꢀ4ꢀyl)benzamide (155 mg, 0.436 mmol) and anhydrous
MeCN (10 mL). The solution was cooled in an ice bath and then MsCl (0.047 mL, 0.61 mmol)
and Et3N (0.080 mL, 0.57 mmol) were added dropwise and the reaction was stirred for 2 h,
warming to room temperature. The solvent was removed under reduced pressure and the crude
material was dissolved in DCM, washed with H2O, saturated NaHCO3, and brine. The organic
layer was dried over MgSO4, filtered, and concentrated to give the crude product that was
purified twice with column chromatography (70/30 EtOAc/hexanes 1st column, 55/45
hexanes/EtOAc 2nd column) to give 15b (38 mg, 0.087 mmol) as a very sticky white foam in
20% yield. 1H NMR (600 MHz, CD3OD) δ = 7.67 ꢀ 7.57 (m, 3 H), 7.53 ꢀ 7.48 (m, 2 H), 7.28 (s,
2 H), 7.02 ꢀ 6.95 (m, 2 H), 6.93 (d, J = 8.2 Hz, 1 H), 5.81 (s, 1 H), 5.15 ꢀ 5.09 (s, 1 H), 4.48 (s, 2
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H), 3.32 ꢀ 3.28 (s, 3 H, overlap with CD3OD); C NMR (150 MHz, CD3OD) δ = 170.7, 165.4,
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157.6, 132.3, 132.1, 129.2, 128.7, 128.0, 121.4, 114.4, 114.3, 69.2, 66.5, 63.3, 37.5; HRMS
(ESIꢀTOF) calcd. for C19H19N3NaO7S (M + Na+) 456.0836, found 456.0824.
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(3S,4R)ꢀ2ꢀOxoꢀ3ꢀ(2ꢀphenoxyacetamido)ꢀ4ꢀ(Nꢀ(tosyloxy)benzamido)azetidineꢀ1ꢀsulfonic acid
(16a): To a flame dried round bottom flask under argon was added 15a (70.0 mg, 0.137 mmol)
and anhydrous DCM (10 mL). The solution was cooled in an ice bath for 10 min, DMF•SO3
(63.0 mg, 0.412 mmol) was added, and the reaction was stirred for 1.5 h. The reaction was then
diluted with DCM and 1 M K2HPO4. TBAHS (47.0 mg, 0.137 mmol) was added, the separatory
funnel was shaken vigorously and vented, and the layers separated. The aqueous layer was
extracted with EtOAc and the pooled organic layers was dried over MgSO4, filtered, and
concentrated under reduced pressure to give the crude salt that was purified with normal phase
silica gel (90/10 EtOAc/MeCN) to give 16a (31 mg, 0.050 mmol) as a white solid in 39% yield.
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mp = 112ꢀ114 °C; H NMR (600 MHz, CD3OD) δ = 7.63 ꢀ 7.48 (br. s. 2 H), 7.43 – 7.18 (m, 10
H), 7.07 ꢀ 6.96 (m, 3 H), 5.95 ꢀ 5.65 (very br. s., 1 H), 4.61 (s, 2 H), 2.38 (s, 3 H); 13C NMR (150
MHz, CD3OD) δ = 170.8, 170.5 (broad), 162.8, 157.7, 146.9, 132.0, 131.3, 129.7, 129.28,
129.23, 128.7, 127.4, 121.5, 114.5, 76.5 (broad), 66.6, 60.7, 48.4, 20.3; HRMS (ESIꢀTOF) calcd.
for C25H22N3O10S2 (M – H+) 588.0752, found 588.0737.
(3R, 4S)ꢀ4ꢀ(Nꢀ((Methylsulfonyl)oxy)benzamido)ꢀ2ꢀoxoꢀ3ꢀ(2ꢀphenoxyacetamido) azetidineꢀ1ꢀ
sulfonic acid (16b): To a flame dried round bottom flask under argon was added 15b (51.0 mg,
0.117 mmol) and anhydrous DCM (10 mL). The solution was cooled in an ice bath for 10 min,
DMF•SO3 (62.0 mg, 0.409 mmol) was added, and the reaction was stirred for 1.5 h. The
reaction was then diluted with DCM and 1 M K2HPO4. TBAHS (40.0 mg, 0.117 mmol) was
added, the separatory funnel was shaken vigorously and vented, and the layers separated. The
aqueous layer was extracted with EtOAc and the pooled organic layers were dried over MgSO4,
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filtered, and concentrated under reduced pressure to give the crude salt that was purified with
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normal phase silica gel (100% EtOAc to 90/10 EtOAc/MeCN) to give 16b (26 mg, 0.050 mmol)
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as a white solid in 43% yield. mp = 108ꢀ110 °C; H NMR (600 MHz, CD3OD) δ = 7.74 (d, J =
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7.3 Hz, 2 H), 7.56 (t, J = 7.6 Hz, 1 H), 7.45 (t, J = 7.9 Hz, 2 H), 7.33 ꢀ 7.27 (m, 2 H), 7.03 ꢀ 6.91
(m, 3 H), 6.04 (br. s., 1 H), 5.29 (d, J = 2.1 Hz, 1 H), 4.54 (s, 2 H), 3.36 ꢀ 3.31 (br. s., 3 H); 13C
NMR (150 MHz, CD3OD) δ = 171.4, 171.0, 162.8, 157.6, 132.2, 132.0, 129.2, 128.8, 128.4,
121.5, 114.4, 72.8, 66.5, 59.3, 37.0; HRMS (ESIꢀTOF) calcd. for C19H18N3O10S2 (M – H+)
512.0439, found 512.0415.
Nꢀ(Benzyloxy)ꢀNꢀ((3R,4S)ꢀ4ꢀ((R)ꢀ1ꢀ((tertꢀbutyldimethylsilyl)oxy)ethyl)ꢀ2ꢀoxoazetidinꢀ4ꢀ
yl)benzamide (17): To a flame dried round bottom flask under argon was added Nꢀ
(benzyloxy)benzamide (9, 4.25 g, 18.7 mmol). The flask was cooled in an ice bath for 10 min
and a solution of 0.8 M NaOEt in anhydrous EtOH (23.5 mL, 18.8 mmol) was added slowly.
The reaction was stirred for 5 min and (3R,4R)ꢀ3ꢀ((R)ꢀ1ꢀ((tertꢀbutyldimethylsilyl)oxy)ethyl)ꢀ2ꢀ
oxoazetidinꢀ4ꢀyl acetate (5.67 g, 19.8 mmol), dissolved in anhydrous EtOH (40 mL), was added
dropwise over 5 min. The reaction was left to stir overnight, warming to room temperature. The
solvent was removed under reduced pressure and the crude material was dissolved in EtOAc (75
mL), washed with H2O, brine, dried over MgSO4, filtered, and concentrated under reduced
pressure to give a yellow oil that was purified with column chromatography (85/15
DCM/EtOAc) to give 17 (5.17 g, 11.4 mmol) as a colorless oil in 61% yield. The product slowly
solidified into a waxy white solid at 0°C. mp = 79ꢀ80°C; 1H NMR (300 MHz, CDCl3) δ = 7.69 ꢀ
6.96 (m, 10 H), 6.00 ꢀ 5.91 (m, 1 H), 5.90 ꢀ 5.83 (m, 1 H), 4.81 (d, J = 9.3 Hz, 1 H), 4.67 (d, J =
9.6 Hz, 1 H), 4.24 (m, 1 H), 3.61 ꢀ 3.53 (dd, 2.2, 1.2 Hz, 1 H), 1.08 (d, J = 6.2 Hz, 3 H), 0.72 (s,
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9 H), ꢀ0.04 (s, 6 H); C NMR (125 MHz, CDCl3) δ = 170.5, 167.6, 134.1, 133.9, 131.6, 130.1,
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129.4, 128.94, 128.92, 128.8, 128.6, 128.4, 78.63, 64.3, 62.4, 25.8, 22.6, 18.0, ꢀ4.13, ꢀ4.86; IR
(FTꢀIR) ν 1751, 1669 cmꢀ1; HRMS (ESIꢀTOF) calcd. for C25H35N2O4Si (M + H+) 455.2361,
found 455.2351.
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Nꢀ((3R,4S)ꢀ4ꢀ((S)ꢀ1ꢀ((tertꢀButyldimethylsilyl)oxy)
ethyl)ꢀ2ꢀoxoazetidinꢀ4ꢀyl)ꢀNꢀ
(tosyloxy)benzamide (18a): To a flame dried round bottom flask was added 17 (3.58 g, 7.87
mmol) and EtOAc (25 mL). The resulting solution was subjected to hydrogenolysis under the
same conditions used in the synthesis of 11a to give crude Nꢀ((3R,4S)ꢀ4ꢀ((R)ꢀ1ꢀ((tertꢀ
butyldimethylsilyl) oxy)ethyl)ꢀ2ꢀoxoazetidinꢀ4ꢀyl)ꢀNꢀhydroxybenzamide (2.77 g, 7.59 mmol) as
an offꢀwhite sticky paste. 1H NMR (300 MHz, CDCl3) δ = 7.66 ꢀ 7.30 (m, 5 H), 7.00 ꢀ 6.89 (br.
s., 1 H), 5.89 (br. s., 1 H), 4.26 ꢀ 4.08 (m, 1 H), 3.60 (br. s., 1 H), 1.12 (d, J = 6.2 Hz, 3 H), 0.73
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(s, 9 H), ꢀ0.03 (d, J = 5.5 Hz, 6 H); C NMR (75 MHz, CDCl3) δ = 170.2, 168.9, 133.0, 131.5,
128.7, 128.2, 63.8, 63.2, 62.1, 25.8, 22.7, 17.9, ꢀ4.23, ꢀ4.95; IR (FTꢀIR) ν 1763, 1667 cmꢀ1;
HRMS (ESIꢀTOF) calcd. for C18H29N2O4Si (M + H+) 365.1891, found 365.1879. To a flame
dried round bottom flask under argon was added Nꢀ((3R,4S)ꢀ4ꢀ((R)ꢀ1ꢀ((tertꢀbutyldimethylsilyl)
oxy)ethyl)ꢀ2ꢀoxoazetidinꢀ4ꢀyl)ꢀNꢀhydroxybenzamide (1.30 g, 3.57 mmol) and anhydrous MeCN
(40 mL). The solution was cooled in an ice bath for 5 min, TsCl (880 mg, 4.6 mmol) and Et3N
(0.65 mL, 4.6 mmol, dropwise) were added, and the reaction was stirred, warming to room
temperature. After 4 h, the solvent was removed under reduced pressure and the crude material
was dissolved in DCM (100 mL). The reaction was washed with H2O, saturated NaHCO3, and
brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced
pressure to give crude material that was purified with column chromatography (70/30
hexanes/EtOAc) to give 18a (870 mg, 1.6 mmol) as a white solid in 47% yield. mp = 75ꢀ77°C;
1H NMR (300 MHz, CDCl3) δ = 7.79 ꢀ 7.16 (m, 9 H), 6.12 (br. s., 1 H), 5.79 (d, J = 1.7 Hz, 1 H),
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2.2 Hz, 6 H); C NMR (75 MHz, CDCl3) δ = 171.2, 166.0, 146.6, 132.8, 132.2, 130.8, 130.1,
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129.4, 128.9, 128.8, 66.1, 63.5, 62.8, 25.7, 22.8, 22.0, 17.9, ꢀ4.30, ꢀ4.96; HRMS (ESIꢀTOF)
calcd. for C25H35N2O6SSi (M + H+) 519.1974, found 519.1980.
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Nꢀ((3R,4S)ꢀ4ꢀ((S)ꢀ1ꢀ((tertꢀButyldimethylsilyl)oxy)ethyl)ꢀ2ꢀoxoazetidinꢀ4ꢀyl)ꢀNꢀ
((methylsulfonyl)oxy)benzamide (18b): To a flame dried round bottom flask under argon was
added
Nꢀ((3R,4S)ꢀ4ꢀ((R)ꢀ1ꢀ((tertꢀButyldimethylsilyl)oxy)ethyl)ꢀ2ꢀoxoazetidinꢀ4ꢀyl)ꢀNꢀ
hydroxybenzamide (300 mg, 0.8 mmol) and anhydrous MeCN. The solution was cooled in an ice
bath for 10 min, MsCl (0.0825 mL, 1.06 mmol) and Et3N (0.135 mL, 0.983 mmol, dropwise)
were added, and the reaction was left to stir for 2.5 h, warming to room temperature. The solvent
was removed under reduced pressure and the crude reaction was dissolved in DCM (50 mL).
The reaction was washed with H2O, saturated NaHCO3, and brine. The organic layer was dried
over MgSO4, filtered, and concentrated under reduced pressure to give crude material that was
purified with column chromatography (60/40 hexanes/EtOAc) to give 18b (255 mg, 0.576
mmol) as a white solid in 70% yield. 1H NMR (300 MHz, CDCl3) δ = 7.73 ꢀ 7.41 (m, 5 H), 6.54
(br. s., 1 H), 5.89 ꢀ 5.78 (fine m, 1 H), 4.25 ꢀ 4.10 (m, 1 H), 3.55 (br. s., 1 H), 3.19 (s, 3 H), 1.06
13
(d, J = 4.3 Hz, 3 H), 0.65 (s, 9 H), ꢀ0.07 (d, J = 2.9 Hz, 6 H); C NMR (75 MHz, CDCl3) δ =
170.2, 166.1, 132.9, 132.2, 129.3, 128.4, 65.8, 63.4, 63.0, 39.3, 25.7, 22.9, 17.8, ꢀ4.34, ꢀ4.99;
HRMS (ESIꢀTOF) calcd. for C19H31N2O6SSi (M + H+) 443.1667, found 443.1694.
(3S,4S)ꢀ3ꢀ((R)ꢀ1ꢀ((tertꢀButyldimethylsilyl)oxy)ethyl)ꢀ2ꢀoxoꢀ4ꢀ(Nꢀ(tosyloxy)
benzamido)azetidineꢀ1ꢀsulfonic acid (19a): To a flame dried round bottom flask under argon
was added 18a (125 mg, 0.241 mmol) and anhydrous DCM (10 mL). The solution was cooled in
an ice bath for 10 min, DMF•SO3 (107 mg, 0.722 mmol) was added, and the reaction was stirred
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for 2.5 h. The reaction was then diluted with DCM and 1 M K2HPO4. TBAHS (88.0 mg, 0.260
mmol) was added, the separatory funnel shaken vigorously and vented, and the layers were
separated. The aqueous layer was extracted with EtOAc and the pooled organic layers were
dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude salt that
was purified with normal phase silica gel (85/15 EtOAc/hexanes) to give 18a (108 mg, 0.181
mmol) as a white solid in 75% yield. mp = 116ꢀ118 °C; 1H NMR (600 MHz, CD3OD) δ = 7.88 ꢀ
7.68 (br. s., 2 H), 7.58 ꢀ 7.46 (m, 3 H), 7.33 (m, 4 H), 6.12 ꢀ 5.91 (br. s., 1 H), 4.32 (q, J = 6.2 Hz,
1 H), 3.87 (br. s., 1 H), 2.39 (s, 3 H), 1.36 (d, J = 6.5 Hz, 3 H), 0.75 (br. s., 9 H), 0.05 (s, 3 H),
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0.01 (br. s., 3 H); C NMR (150 MHz, CD3OD) δ = 172.0, 164.8, 146.7, 132.7, 131.5, 130.3,
129.6, 129.2, 128.7, 127.9, 69.1, 63.8, 61.0, 24.8, 21.7, 20.2, 17.2, ꢀ5.88, ꢀ6.06; HRMS (ESIꢀ
TOF) calcd. for C25H33N2O9S2Si (M ꢀ H+) 597.1402, found 597.1429.
(3S,4S)ꢀ4ꢀ((R)ꢀ1ꢀ((tertꢀButyldimethylsilyl)oxy)ethyl)ꢀ4ꢀ(Nꢀ((methylsulfonyl)oxy)
benzamido)ꢀ2ꢀoxoazetidineꢀ1ꢀsulfonic acid (19b): To a flame dried round bottom flask under
argon was added 18b (190 mg, 0.42 mmol) and anhydrous DCM (10 mL). The solution was
cooled in an ice bath for 5 min, DMF•SO3 (262 mg, 1.71 mmol) was added, and the reaction was
stirred for 1.5 h, warming to rt. The reaction was then diluted with DCM and 1 M K2HPO4.
TBAHS (145 mg, 0.190 mmol) was added, the separatory funnel was shaken vigorously and
vented, and the layers were separated. The aqueous layer was extracted with EtOAc and the
pooled organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure
to give the crude salt that was purified with normal phase silica gel (100% EtOAc to 90/10
EtOAc/MeCN) to give 19b (89 mg, 0.15 mmol) as a very hydroscopic white solid in 40% yield.
1
mp = 121ꢀ123 °C; H NMR (600 MHz, CD3OD) δ = 7.87 ꢀ 7.77 (m, 2 H), 7.60 (t, J = 7.3 Hz, 1
H), 7.51 (t, J = 7.9 Hz, 2 H), 6.11 ꢀ 6.02 (br. s., 1 H), 4.27 (dd, J = 4.4, 6.2 Hz, 1 H), 3.66 (t, J =
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2.2 Hz, 1 H), 3.48 ꢀ 3.34 (br. s., 3 H), 1.19 (d, J = 5.3 Hz, 3 H), 0.68 (s, 9 H), ꢀ0.03 (d, J = 17.6
Hz, 6 H); 13C NMR (150 MHz, CD3OD) δ = 171.9, 164.5, 132.6, 131.9, 128.7, 128.4, 68.8, 63.5,
60.6, 37.3, 24.8, 21.8, 17.1, ꢀ6.00, ꢀ6.11; HRMS (ESIꢀTOF) calcd. for C19H29N2O9S2Si (M ꢀ H+)
521.1089, found 521.1098.
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(3S,4S)ꢀ3ꢀ((R)ꢀ1ꢀHydroxyethyl)ꢀ2ꢀoxoꢀ4ꢀ(Nꢀ(tosyloxy)benzamido)azetidineꢀ1ꢀsulfonic acid
(20a): To a round bottom flask was added 19a (40.0 mg, 0.0660 mmol) and MeCN (5 mL). To
the resulting solution was added 1 M HCl (0.093 mL, 0.093 mmol) and the reaction was stirred
for 3 h. After 3 h, the reaction was not complete and additional 1 M HCl (1.22 mL, 1.22 mmol)
was added. After stirring for an additional 3 h, the reaction was neutralized to pH 7 with
dropwise addition of saturated NaHCO3 and then concentrated under reduced pressure to give
crude material that was purified with normal phase silica gel (75/25 EtOAc/MeCN) to give 20a
1
(13.0 mg, 0.0270 mmol) as a white solid in 40% yield. mp = 117ꢀ118 °C; H NMR (600 MHz,
CD3OD) δ = 7.85 ꢀ 7.67 (m, 2 H), 7.56 (d, J = 7.3 Hz, 2 H), 7.51 (t, J = 7.6 Hz, 1 H), 7.37 (t, J =
7.3 Hz, 2 H), 7.32 (d, J = 7.3 Hz, 2 H), 6.01 ꢀ 5.82 (br. s., 1 H), 4.15 ꢀ 4.08 (m, 1 H), 3.82 ꢀ 3.73
13
(br. s., 1 H), 2.40 (s, 3 H), 1.33 (d, J = 6.5 Hz, 3 H); C NMR (150 MHz, CD3OD) δ = 172.1,
164.5, 146.6, 132.5, 131.6, 130.3, 129.6, 129.2, 129.0, 127.8, 70.3, 63.2, 60.9, 20.5, 20.2; HRMS
(ESIꢀTOF) calcd. for C19H19N2O9S2 (M ꢀ H+) 483.0556, found 483.0537.
(3S,4S)ꢀ3ꢀ((R)ꢀ1ꢀHydroxyethyl)ꢀ4ꢀ(Nꢀ((methylsulfonyl)oxy)benzamido)ꢀ2ꢀoxoazetidineꢀ1ꢀ
sulfonic acid (20b): To a round bottom flask was added 19b (45 mg, 0.086 mmol) and MeCN (5
mL). To the resulting solution was added 1 M HCl (0.103 mL, 0.103 mmol) and the reaction
was stirred for 3 h. Additional 1 M HCl (1.6 mL, 1.6 mmol) was added and the reaction was
stirred for 2 h. The reaction was then neutralized to pH 7 with dropwise addition of saturated
NaHCO3 and concentrated under reduced pressure to give crude material that was purified with
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normal phase silica gel (65/35 EtOAc/MeCN) to give 20b (4.7 mg, 0.011 mmol) as a white solid
in 15% yield. 1H NMR (600 MHz, CD3OD) δ = 7.86 (d, J = 7.0 Hz, 2 H), 7.61 (s, 1 H), 7.51 (t, J
= 7.9 Hz, 2 H), 5.94 ꢀ 5.84 (m, 1 H), 4.08 ꢀ 3.97 (m, 1 H), 3.63 (dd, J = 2.3, 3.2 Hz, 1 H), 3.38
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(br. s., 3 H), 1.22 (d, J = 6.5 Hz, 3 H); C NMR (150 MHz, CD3OD) δ = 172.1, 164.4, 133.3,
132.2, 129.0, 128.4, 70.4, 63.0, 60.3, 37.0, 20.7; HRMS (ESIꢀTOF) calcd. for C13H15N2O9S2 (M ꢀ
H+) 407.0224, found 407.0206.
(6R,7R)ꢀ4ꢀMethoxybenzyl 3ꢀ((Nꢀhydroxybenzamido) methyl)ꢀ8ꢀoxoꢀ7ꢀ(2ꢀphenylacetamido)ꢀ
5ꢀthiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylate (22): To a flame dried round bottom flask
under argon was added 21 (170 mg, 0.24 mmol) and anhydrous THF (6 mL). HF•Pyridine (70%
HF, 2 drops) was added and the reaction was stirred for 2.5 h. The reaction was diluted with
EtOAc (30 mL), washed with 0.1 M HCl, dried over MgSO4, filtered, and concentrated under
1
reduced pressure to give 22 (120 mg, 0.20 mmol) as a sticky paste in 84%. H NMR (500 MHz,
CDCl3) δ = 7.57 ꢀ 6.80 (m, 14 H), 6.47 (br. s., 1 H), 5.72 (dd, J = 4.8, 9.0 Hz, 1 H), 5.07 (s, 2 H),
4.91 (d, J = 16.0 Hz, 1 H), 4.86 (d, J = 4.8 Hz, 1 H), 4.59 (d, J = 16.0 Hz, 1 H), 3.60 ꢀ 3.51
13
(overlapping signals, 3 H), 3.36 (d, J = 19.1 Hz, 1 H); C NMR (125 MHz, CDCl3) δ = 171.7,
169.2 (broad), 164.9, 162.0 (broad), 160.1, 133.9, 131.5, 130.9, 130.8, 129.6, 129.3, 128.77,
128.74, 128.4, 127.9, 126.7, 125.6, 114.2, 68.4, 59.4, 57.6, 55.5, 51.7 (very broad), 43.4, 26.8;
HRMS (ESIꢀTOF) calcd. for C31H29N3NaO7S (M + Na+) 610.1618, found 610.1613.
(6R,7R)ꢀ8ꢀOxoꢀ7ꢀ(2ꢀphenylacetamido)ꢀ3ꢀ((Nꢀ(tosyloxy)benzamido)methyl)ꢀ5ꢀthiaꢀ1ꢀ
azabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylic acid (23): To a flame dried round bottom flask under
argon was added 22 (100 mg, 0.2 mmol) and anhydrous MeCN (10 mL). The solution was
cooled in an ice bath for 10 min, TsCl (42.0 mg, 0.221 mmol) and anhydrous pyridine (0.0130
mL, 0.187 mmol) were added, and the reaction was stirred for 2 h, warming to room
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temperature. The reaction was then concentrated under reduced pressure at room temperature to
give crude material that was purified with column chromatography (80/20 ether/hexanes to 100%
ether) to give intermediate 4ꢀmethoxybenzyl (6R,7R)ꢀ8ꢀoxoꢀ7ꢀ(2ꢀphenylacetamido)ꢀ3ꢀ((Nꢀ
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(tosyloxy)benzamido)methyl)ꢀ5ꢀthiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylate
(38.0
mg,
1
0.0510 mmol) as a white solid in 30% yield. mp = 78ꢀ80°C; H NMR (500 MHz, CDCl3) δ =
7.48 ꢀ 7.23 (m, 14 H), 7.10 (d, J = 8.0 Hz, 2 H), 6.88 (d, J = 9.0 Hz, 2 H), 6.12 (d, J = 9.2 Hz, 1
H), 5.80 (dd, J = 4.2, 5.0 Hz, 1 H), 5.35 (d, J = 16.2 Hz, 1 H), 5.16 (s, 2 H), 4.95 (d, J = 4.8 Hz, 1
H), 4.82 (d, J = 16.2 Hz, 1 H), 3.80 (s, 3 H), 3.68 ꢀ 3.57 (overlapping signals, 3 H), 3.28 (d, J =
18.1 Hz, 1 H), 2.37 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ = 171.0, 170.9, 164.4, 161.2, 159.8,
146.4, 133.6, 131.8, 131.6, 130.7, 129.7, 129.6, 129.4, 129.19, 129.11, 128.4, 128.1, 127.7,
126.7, 126.6, 113.9, 68.1, 59.1, 57.4, 55.2, 52.8, 43.3, 26.9, 21.7 (apparent overlap of 1 aromatic
carbon); HRMS (ESIꢀTOF) calcd. for C38H35N3NaO9S2 (M + Na+) 764.1707, found 764.1692.
To a flame dried round bottom flask under argon was added 4ꢀmethoxybenzyl (6R,7R)ꢀ8ꢀoxoꢀ7ꢀ
(2ꢀphenylacetamido)ꢀ3ꢀ((Nꢀ(tosyloxy)benzamido)methyl)ꢀ5ꢀthiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀ
carboxylate (30.0 mg, 0.040 mmol) and anhydrous DCM (3 mL). The solution was cooled in an
ice bath for 10 min. Anisole (0.01 mL, 0.1 mmol) and TFA (1 mL) were added slowly, and the
reaction was stirred on ice for 1 h. The reaction was then concentrated under reduced pressure at
room temperature to give crude material that was purified with column chromatography (100%
DCM to 90/10 DCM/IPA w/ 0.3% AcOH) to give 23 (10.0 mg, 0.016 mmol) as an offꢀwhite
very fine powder in 47% yield. 1H NMR (600 MHz, CD3OD) δ = 7.57 (d, J = 8.2 Hz, 2 H), 7.50
ꢀ 7.46 (m, 1 H), 7.38 ꢀ 7.21 (m, 11 H), 5.72 (dd, J = 3.8, 4.7 Hz, 1 H), 5.26 (d, J = 15.8 Hz, 1 H),
5.08 (d, J = 4.7 Hz, 1 H), 4.79 (d, J = 15.8 Hz, 1 H), 3.67 (d, J = 17.6 Hz, 1 H), 3.58
(overlapping d, J = 14.2 Hz, 2 H), 3.37 (d, J = 17.3 Hz, 1 H), 2.40 (s, 3 H); 13C NMR (150 MHz,
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CD3OD) δ = 173.1, 171.6, 164.6, 162.9, 146.8, 134.9, 131.8, 131.6, 129.9, 129.7, 128.8, 128.7,
128.2, 128.1, 128.0, 127.2, 126.5, 124.0, 59.2, 57.6, 52.8, 41.7, 26.5, 20.2; HRMS (ESIꢀTOF)
calcd. for C30H26N3O8S2 (M – H+) 620.1167, found 620.1180.
ASSOCIATED CONTENT
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Supporting Information.
1
13
Agar diffusion assay procedure, MIC procedure, copies of H and C NMR spectra for new
compounds, and Xꢀray crystallographic data for compound 10 are included. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* mmiller1@nd.edu Tel: 1ꢀ574ꢀ631ꢀ7571 Fax:1ꢀ574ꢀ631ꢀ6652.
ACKNOWLEDGMENTS
We acknowledge the University of Notre Dame, and the NIH (NIHꢀ2R01ꢀAI054193) for partial
support of this work. We gratefully acknowledge VenatoRx Pharmaceuticals, Inc. for additional
antibacterial and βꢀlactamase inhibition testing. We acknowledge Nonka Sevova (Mass
Spectrometry and Proteomics Facility, UND) for mass spectroscopic analyses. M.W.M
acknowledges an ECK Global Health Fellowship (2014ꢀ2015, UND).
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