Synthesis and human leukocyte elastase inhibitory evaluation of phosphate triesters and acyl phosphates of penam sulfides and sulfones

Article abstract of DOI:10.1016/S0968-0896(01)00139-0

The synthesis of 6,6-dibromo-3α-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3a), 6α-chloro-3α-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3b), benzyl 6α-(diphenyl-phosphate)oxypenicillanate sulfone (4) and 6,6-dibromo-3α-(methylphosphate)carbonyl-2,2-dimethylpenam sulfone (12) are reported. When tested as inhibitors of human leukocyte elastase, the compound 4 proved to be the most active.

Full text of DOI:10.1016/S0968-0896(01)00139-0

Bioorganic & Medicinal Chemistry 9 (2001) 2113–2117
Synthesis and Human Leukocyte Elastase Inhibitory
Evaluation of Phosphate Triesters and Acyl Phosphates
of Penam Sulfides and Sulfones
Marıa Laborde,a German Pezzenati,a Patricia Yovaldi,a Oreste A. Mascaretti,a,*
Rolando C. Rossib and Juan Pablo Rossib,*
a
´
Instituto de Quımica Organica de Sıntesis, Casilla de Correo 991, 2000 Rosario, Argentina
´
´
Departamento de Quımica Biologica-IQUIFIB, Facultad de Farmacia y Bioquımica Universidad de Buenos Aires,
Junin 956, 1113 Buenos Aires, Argentina
b
´
´
´
Received 16 August 2000; accepted 9 April 2001
Abstract—The synthesis of 6,6-dibromo-3a-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3a), 6a-chloro-3a-(diphe-
nylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3b), benzyl 6a-(diphenyl-phosphate)oxypenicillanate sulfone (4) and 6,6-
dibromo-3a-(methylphosphate)carbonyl-2,2-dimethylpenam sulfone (12) are reported. When tested as inhibitors of human leuko-
cyte elastase, the compound 4 proved to be the most active. # 2001Elsevier Science Ltd. All rights reserved.
Introduction
tetrahedral intermediates. Examples include inhibitors
of serine proteinases such as human leukocyte elastase,6
and active-site serine b-lactamases of classes A7 and
C.8,9
Mechanistic understanding of enzymatic processes is
exerting an increasingly important influence on the
design of enzyme inhibitors. Transition-state analogues
which are among the most effective types of enzyme
inhibitors arise from this approach.1 The family of pep-
tide-bond-cleaving hydrolases, the peptidases (=pro-
teases, EC 3.4), are sub-grouped, according to the
reactive group at the active site involved in catalysis.
Serine proteinases2 represent one of the four main clas-
ses of proteolytic enzymes, the other three being
aspartyl proteinases, cysteine proteinases and metallo-
proteinases. The serine proteinase family includes many
well-studied enzymes, such as elastases. Human leuko-
cyte elastase (HLE, EC 3.4.21.37) is a serine proteinase
found in the azurophilic granules of polymorphonuclear
leukocytes.3 Several b-lactam compounds have been
found to accomplish HLE inactivation through acyla-
tion of the hydroxyl group in the enzyme’s active site.4,5
Some years ago, Pratt et al. demonstrated that the
phosphonate monoesters monoanion and phosphona-
midates of structure 1 (Fig. 1) (where L is a leaving
group) inhibit active-site serine b-lactamases of classes
A7 and C,8,9 by phosphorylation of the active-site serine
hydroxyl group. Song and Kluger10 reported the synth-
esis of benzylpenicillin methyl phosphate and found that
this compound is a substrate and inactivator of Escher-
ichia coli RTEM b-lactamase. These authors proposed
that the compound acylate the E-amino group of Lys-
234 of the enzyme. More recently, Li and Pratt11 have
demonstrated that acyl phosph(on)ates monoanion of
structure 2a–c inhibits the class C b-lactamase of
Enterobacter cloacae P99 and compound 2c reacts with
the class A TEM-b-lactamase to form an acyl enzyme.
Clearly, the compounds 1 and 2a–c have the potential to
form both the acyl and phosphor(on)yl enzyme species.
Four co-ordinate tetrahedral phosphorous derivatives
are known to be inhibitors of proteolytic enzymes by
acting as phosphorylating agents of the serine hydroxyl
group and thereby produce transition-state analogues of
What distinguishes HLE from active site serine b-lacta-
mases (classes A, C, and D according to Ambler’s clas-
sification) is the mechanism to effect peptide or b-lactam
bond cleavage. From the point of view of their
mechanism, an active-site serine b-lactamase is not an
active-site serine proteinase.
*Corresponding author. fax; +54-341-437-0477; e-mail: masca@
citynet.net.ar
0968-0896/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00139-0

2114
M. Laborde et al. / Bioorg. Med. Chem. 9 (2001) 2113–2117
Figure 1.
We envisioned that phosphate triesters of the penam
sulfides and sulfones as well as acyl-phosphates of
penam sulfides and sulfones could potentially resemble
the tetrahedral intermediate or the transition state for
the formation and/or breakdown of the intermediate
and, therefore, act as inhibitor of HLE as well as serine
b-lactamases.
sence of catalytic amount of pyridine, at room tem-
perature. Subsequent oxidation of 6 with potassium
permanganate in acetic acid afforded the corresponding
sulfone 3a. The 6a-chloro-3a-hydroxymethyl-2,2-dim-
ethyl-penam sulfone (7)15 was phosphorylated by the
phosphochloridate methodology. The compound 3b
was obtained in 60% yield after purification by column
chromatography.
The synthesis of benzyl 6a-(diphenylphosphate) oxype-
nicillanate sulfone (4), was performed by phosphoryla-
tion of benzyl 6a-hydroxypenicillanate (8)16 using the
phosphochloridate methodology,14 and subsequent
oxidation (Scheme 2). The structure of compounds 3a,b
Results and Discussion
We describe here the synthesis of a series of prototypical
phosphate triesters of penan sulfides (structures 6 and 9)
sulfones (structures 3a,b and 4) and acyl-phosphonate
of penam sulfide (structure 11) and their sulfone (struc-
ture 12). These new phosphate triesters and acyl-phos-
phate of penam sulfides and sulfones were evaluated as
inhibitors of human leukocyte elastase. This enzyme has
been the subject of extensive studies, both in terms of its
biological role in numerous diseases12 and of the devel-
opment of suitable therapeutic inhibitors to supplement
the body’s elastase inhibitory capacity and thereby shift
the proposed proteinase/antiproteinase imbalance in
pathogenic conditions.
1
and 4 were ascertained by 31P, 13C, and H NMR spec-
troscopy.
Synthesis of 6,6-dibromo-3ꢀ-(methylphosphate)carbonyl-
2,2-dimethylpenam sulfone
The starting material was the ready available 6,6-
dibromo-3a-chlorocarbonyl-2,2-dimethylpenam (10), pre-
viously reported by us.17 Conversion of the acid chloride
10 into its corresponding 6,6-dibromo-3a-(methylpho-
sphate)carbonyl-2,2-dimethylpenam (11) was achieved
with bis(tetrabutylammonium) methylphosphate using
the methodology reported by Song and Kluger.10 Oxi-
dation of the penam sulfide 11 with potassium perman-
ganate in acetic acid gave the corresponding sulfone (12)
(Scheme 3). The compounds 11 and 12 were unstable at
30 ꢀC under atmospheric conditions.
Chemistry
Synthesis of penam and penicillanate sulfone phosphate
triesters
The synthesis of 6,6-dibromo-3a-(diphenylphos-
phate)oxymethyl-2,2-dimethyl penam sulfone (3a) is
shown in Scheme 1. The starting materials was 6,6-
dibromo 3a-hydroxymethyl-2,2-dimethylpenam (5).13
Conversion of 5 into the 6,6-dibromo-3a-(diphenylphos-
phate)oxymethyl-2,2-dimethyl penam (6) in 75% iso-
lated yield was accomplished by phosphorylation with
diphenylphosphochloridate14 in acetonitrile in the pre-
In vitro HLE inhibition
Activity was measured following the increase in absor-
bance at 410 nm due to the release of p-nitroaniline by
hydrolysis of the substrate N-methoxysuccinyl-Ala-Ala-
Pro-Val-p-nitroanilide. Measurements were made at
20 ꢀC in 1mL of assay medium containing 100 mM
KH2PO4 (pH 7.0, at 20 ꢀC), 0.05 IU of human leukocyte
elastase and concentrations of substrate and inhibitors
Scheme 1.
Scheme 2.

M. Laborde et al. / Bioorg. Med. Chem. 9 (2001) 2113–2117
2115
Scheme 3.
as indicated. Time courses of product formation where
tested for linearity and only the initial, linear parts where
considered to calculate the rates. Inhibitors were dissolved
in dimethyl sulfoxide (DMSO). In all cases, final con-
centration of DMSO in the incubation media was 2% in
volume, to minimize inhibitory effects of this solvent.
HLE. The stability of all compounds was tested by
measuring their spectra under identical conditions as
during enzymatic determinations but omitting the addi-
tion of substrate. Spectra remained unchanged after 1h
of incubation.
We assayed six different compounds. Compound 4 is
near 10-fold more potent for the inhibition of human
elastase than compounds 3b, 6, and 3a, and near 100-
fold than compounds 11 and 12 (Table 1).
Conclusion
The structure–activity relationship studies around the
phosphate triesters group at C-3a and C-6a positions as
well as acyl-phosphate at C3a position of the penam
nucleus as inhibitors of HLE have been studied. It will
be interesting to find whether specifically more potent
analogues at C-6a with better leaving groups at the
phosphate esters and also of phosphate-penam monoe-
sters can be obtained by structural variation.
All the compounds inhibit through interaction with the
substrate according to a simple competitive mechanism,
with the following equation for steady-state activity:
Where Vmax is the activity at substrate concentration,
[S], tending to infinity, KM is the substrate concentration
for half-maximal activity in the absence of inhibitor and
Ki is the equilibrium constant for the dissociation of
inhibitor, I, from the enzyme.
Experimental
Proton and carbon magnetic resonance spectra (1H and
13C NMR) were taken on a Bruker AC 200 spectro-
meter operating at 200 and 50.0 MHz, respectively, with
samples referenced to internal tetramethylsilane (CDCl3
solutions). Phosphorous magnetic resonance spectra
(31P NMR) were recorded on a Bruker 250 MHz Four-
ier-transform instrument operating at 101.25 MHz with
samples referenced to external (capillary) 85% H3PO4.
Mass spectra were obtained from the Mass Spectro-
metry Facility at Ohio State University Chemical
Instrument Center. Infra-red spectra (IR) were recorde-
don a Bruker IFS 25 FT-IR spectrometer. Melting point
were taken on a Ernst Leitz melting point apparatus
and are uncorrected. Analytical thin-layer chromato-
graphy (TLC) was carried out with silica gel 60 F254 pre-
coated aluminium sheets (Merck); column chromato-
graphy was performed on silica gel grade 60 (Merck).
Since the mechanism of inhibition is due to the compe-
tition between the substrate N-methoxysuccinyl-Ala-
Ala-Pro-Val-p-nitroanilide and the different compounds
assayed, it seems reasonable that the molecule which is
more likely to compete with the substrate is also the
most potent. The comparison of compounds 6 and 3a, 4
and 9 indicate that substitution in position 1 (sulfide
and sulfone) is not relevant for the inhibition. Compar-
ison among compounds 4 with 3b and 3a, indicates that
substitution of the phosphate triester in position 6 with
orientation a, for a chlorine or bromine atom modifies
the potency of the inhibitor. Differences between the
triester phosphate compound 3a and the acyl-phosphate
12 significantly change the potency for the inhibition of
Table 1. Values of Ki=KM[I]/[([S]+KM) (vo/viÀ1)], were obtained by
measuring activity either in the absence (vo) or in the presence (vi) of
inhibitors.a
Human Leukocyte Elastase was purchased from Sigma
Chemical Co. A stock solution (1mg mL À1) was pre-
pared in sodium acetate 50 mM (pH 5.5), and frozen at
À20 ꢀC until used. N-Methoxysuccinyl-Ala-Ala-Pro-
Val-p-nitroanilide (Sigma Chemical Co.) was dissolved
in dimethyl sulfoxide (DMSO). Enzyme activity was
assayed in potassium phosphate 100 mM, pH 7 (final
volume 1mL). The reaction was started by addition of
10–30 mL of the enzyme stock solution. The release of p-
nitroaniline was followed spectrophotometrically at
410 nm in a Beckman spectrophotometer DU 640
(30 ꢀC). The inhibitors were dissolved in DMSO. Con-
trol experiments were run in all the cases with equal
amounts of DMSO.
Compound
KiÆSD (mM)
4
9
1.3Æ0.5
1.5Æ0.5
11.0Æ2
3b
6
3a
12
11
12.0Æ3
14.0Æ2
79.0Æ25
142.0Æ30
Measurements of vo versus [S] allowed to calculate a value of
KM=1.8Æ0,15 mM
aMeasurements of vi were performed in media with either 3.125 mM
(inhibitors 4, 9, 3b, 6 and 3a) or 1 25mM (inhibitors 12 and 11) of each
inhibitor, for two concentrations of the substrate: 1.25 and 2.5 mM.

2116
M. Laborde et al. / Bioorg. Med. Chem. 9 (2001) 2113–2117
6,6-Dibromo-3ꢀ-(diphenylphosphate)oxymethyl-2,2-di-
methyl penam (6). Diphenylphosphochloridate (0.31mL,
1.48 mmol) was added very slowly to a solution of
170 mg, (0.49 mmol) of 6,6-dibromo 3a-hydroxy-
methyl-2,2-dimethylpenam (5) dissolved in a mixture of
dry acetonitrile (1.5 mL) and pyridine (0.12 mL) at room
temperature in a nitrogen atmosphere. After being stir-
red for 2 h, the mixture was diluted with chloroform
(10 mL) and washed with water (2Â5 mL). The organic
phase was dried (Na2SO4) and concentrated under
reduced pressure. The products were purified by column
chromatography (eluant: hexane/ethyl acetate 75ꢀ:25) to
give compound 6 as white crystals, mp 67.5–69.0 C. 1H
NMR (CDCl3) d 1.41 (3H, s, a-CH3), 1.51 (3H, s, b-
CH3), 4.18–4.20 (3H, m 10-H and 3-H), 5.49 (1H, s, 5-
H), 7.19–7.39 (10H, m, Ar–H); 13C NMR (CDCl3) d
24.14 (C-8), 33.49 (C-9), 58.94 (C-6), 62.91 (C-2), 64.79
(d, JP,C=5.5 Hz, C-10), 67.18 (d, JP,C=7.0 Hz, C-3),
78.57 (C-5), 119.84 (d, JP,C=4.3 Hz, Ph C-2), 125.50 (Ph
C-4), 129.78 (Ph C-3), 150.13 (d, JP,C=7.4 Hz, Ph C-1)
and 165.26 (C-7); 31P NMR (CDCl3) d À12.7. EI mass
spectrum: m/z (M+) 579 (0.6%), 577 (1.1), 575 (0.6),
498 (20.6), 418 (8.7), 317 (5.2), 284 (66.7), 251 (100) and
170 (28.7). Found: M+, 576.91453. calcd for
C20H20SO5PN79Br81Br: M+, 576.91447.
+ b-CH3), 4.08–4.32 (3H, m, 10-H and 3-H), 4.47 (1H,
d, J=1.53 Hz, 6-H), 5.13 (1H, d, J=1.53 Hz. 5-H),
7.18–7.41 (10H, m, Ar–H); 13C NMR (CDCl3) d 18.55
and 18.82 (C-8 and C-9), 55.44 (C-6), 60.62 (d,
JP,C=4.7 Hz, C-3), 62.97 (C-2), 65.49 (d, JP,C=5.3 Hz,
C-10), 68.84 (C-5), 119.80 (dd, JP,C=4.1Hz, Ph C-2),
125.68 (Ph C-4), 129.84 (Ph C-3), 150.00 (d, JP,C=8.5 Hz,
Ph C-1), 166.67 (C-7). LR-MS (EI) m/z (M+) 485.04
(0.20), 487.02 (0.14), 105.04 (100). HR-MS (ES). Found
(M+Na) 508.0362. Calcd for C20H21SO7PN35Cl Na
(M+Na) 508.0363.
Benzyl
6ꢀ-(diphenylphosphate)oxypenicillanate
(9).
According to the procedure previously described, the
benzyl 6a-hydroxypenicillanate (8)16 was phosporylated
to afford the compound 9 in 73% yield, as a colorless
1
oil. H NMR (CDCl3) d 1.36 (3H, s, a-CH3), 1.53 (3H,
s, b-CH3), 4.52 (1H, s, 3-H), 5.17 (2H, d, AB
J=1.53 Hz.-CH2-Ph), 5.29 (1H, d, J5,6H=1.37 Hz, 5-H)
2
5.35 (1H, dd, J5,6H=1.37 Hz, JP,H=10 Hz, 6-H), 7.19–
7.38 (15H, m, Ar–H); 13C NMR (CDCl3) d 25.18 (C-8),
33.70 (C-9), 64.05 (C-2), 67.31(– CH2–Ph), 69.33 (C-5),
69.40 (C-3), 85.52 (d, JP,C=7.8 Hz, C-6), 119.90 (d,
JP,C=4.3 Hz,–O–Ph C-2), 125.64 (–O–Ph C-4), 128.44,
128.52, 134.45 (–CH2–Ph–C–), 128.48 (–O–Ph C-3),
150.03 (d, JP,C=8.5 Hz,–O–Ph C-1), 165.25 (d,
JP,C=8.6 Hz, C-7), 166.60 (–C–CO2–CH2–); 31P NMR
(CDCl3) d À14.48. Found M+, 539.11923. Calcd for
C27H26SO7PN: M+, 539.11659.
6,6-Dibromo-3ꢀ-(diphenylphosphate)oxymethyl-2,2-di-
methyl penam sulfone (3a). To a solution of 6,6-
dibromo-3a-(diphenylphosphate)oxymethyl-2,2-dime-
thyl penam 6 (143 mg. 0.20 mmol) in a mixture of acetic
acid/water (62.5:37.5) (4.0 mL) was added powdered
potassium permanganate (68 mg. 0.43 mmol) and the
mixture was stirred for 10 min. The reaction mixture
was then quenched with drops of H2O2 until dis-
appearance of color, diluted with CH2Cl2 (5 mL) and
water (5 mL). The layers were separated and the aqu-
eous layer extracted with CH2Cl2 (2Â5 mL). The com-
bined organic layers were washed with 5% aqueous
sodium bicarbonate solution (2Â10 mL) and water
(1Â6 mL), dried (Na2SO4) and the solvent removed
under reduced pressure to yield 3a (118 mg, 98%) as a
Benzyl 6ꢀ-(diphenylphosphate)oxypenicillanate sulfone
(4). According to a similar procedure to that used for
the synthesis of 3a, compound 9 was converted to 4
(77% yield) as colorless oil. IR (film) gmax 1804 (b-lac-
tam), 1754 (ester) 1322, and 1127 cmÀ1 (sulfone). 1H
NMR (CDCl3) d 1.23 (3H, s, a-CH3), 1.51 (3H, s, b-
CH3), 4.42 (1H, s, 3-H), 4.65 (1H, d, J5,6H=1.38 Hz, 5-
H), 5.22 (2H, m.–CH2–Ph), 5.75 (1H, J5,6H=1.32 Hz,
2JP,H=10.39 Hz, 6-H), 7.18–7.40 (15H, m, Ar–H); 13C
NMR (CDCl3) d 18.37 (C-8), 19.64 (C-9), 62.82 (C-3),
63.25 (C-2), 67.90 (C-5). 68.26 (C-10), 78.11 (d,
JP,C=7.7 Hz, C-6), 119.90 (d, JP,C=4.0 Hz, Ph C-2),
125.89 (Ph C-4), 128.72, 128.96, 129.94, 134.11 (–CH2–
Ph C-), 128.75 (Ph C-3), 149.85 (d, JP,C=8.0 Hz, Ph C-
1), 164.78 (d, J=8.0 Hz, C-7) and 165.75 (–C–CO2–
CH2–).
1
colorless oil. H NMR (CDCl3) d 1.38 and 1.41 (each
3H, s, a+b-CH3), 4.13–4.39 (3H, m, 10-H and 3-H),
4.86 (1H, s, 5-H), 7.17–7.40 (10H, m, Ar–H); 13C NMR
(CDCl3) d 18.18 and 19.18 (C-8 and C-9), 44.02 (C-6),
60.92 (d, JP,C=9.3 Hz, C-3), 64.17 (C-2), 64.99 (t,
JP,C=5.5 Hz, C-10), 73.32 (C-5), 119.80 (dd,
JP,C=4.5 Hz, Ph C-2), 125.70 (Ph C-4), 129.86 (Ph C-3),
150.02 (d, JP,C=7.3 Hz, Ph C-1), 164.94 (C-7). 31P
NMR (CDCl3) d À13.17. EI mass spectrum: m/z (M+)
612 (0.49), 610 (1.16), 608 (0.48), 464 (7.01), 389 (8.25),
318 (3.24), 251 (85.06), 250 (100), 216 (38.84), 214
(40.68), 170 (46.49). Found M+, 608.92256. calcd for
C20H20SO7PN79Br81Br: M+, 608.90429.
6,6-Dibromo-3ꢀ -(methylphosphate)carbonyl-2,2-dime-
thylpenam (11). To a stirred suspension of 6,6-dibromo-
penicillanic acid (2.8 mmol) in a mixture of anhydrous
benzene (8 mL) and anhyd DMF (0.15 mL, 1.9 mmol) at
20 ꢀC, oxalyl chloride (1.29 mL, 3.3 mmol) was added
dropwise while a slow stream of nitrogen was passed
through the mixture. After 50 min the mixture was cooled
at À23 ꢀC, then a solution of bis(tetrabutylammonium)
methylphosphate10 (1.98 g, 3.33 mmol) in dry CH2Cl2
(16 mL) was added. The mixture was stirred at À23 ꢀC
for 3 h. It was then warmed to room temperature, and
stirred 2 h. The CH2Cl2 phase was washed with H2O, sat
CuSO4, H2O, 5% NaHCO3, H2O, dried over Na2SO4
and concentrated under reduced pressure. The products
were purified by column chromatography (eluant:
CHCl3/methanol 97:3) to give in 66% yield compound 11
6ꢀ-Chloro-3ꢀ-(diphenyl-phosphate)oxymethyl-2,2-dime-
thyl penam sulfone (3b). According to the procedure
previously described, the 6a-chloro-3a-hydroxymethyl-
2,2-dimethyl-penam sulfone (7) was phosphorylated to
afford the compound 3b in 60% yield, as a white solid.
The product was characterized by mp 77.0–79.0 ꢀC. IR
(film) gmax 1808 (b-lactam), 1325 and 1186 cmÀ1 (sul-
1
fone). H NMR (CDCl3) d 1.39 and 1.41 (each 3H, s, a

M. Laborde et al. / Bioorg. Med. Chem. 9 (2001) 2113–2117
2117
as white crystals. The product was characterized by mp
99.0–101.0 ꢀC. IR (KBr) gmax 1804 (b-lactam), and
Perrone, E. Bioorg. Med. Chem. Lett. 1995, 5, 687. (c) Finke,
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J. J.; MacCoss, M.; Westler, W. M.; Knight, W. B. Biochem-
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Bioorg. Med. Chem. Lett. 1996, 6, 823. (f) Cvetovich, R. J.;
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1
1754 cmÀ1 (ester). H NMR (CDCl3) d 1.46 (3H, s, a-
CH3), 1.62 (3H, s, b-CH3), 3.73 (3H, s, –OCH3), 4.55
(1H, s, 3-H), 5.80 (1H, s, 5-H); 13C NMR (CDCl3) d
25.77 (C-8), 33.33 (C-9), 52.38 (–OCH3), 56.49 (C-6),
64.45 (C-2), 69.55 (C-3), 80.56 (C-5), 164.34 (C-7),
166.97 (–C–CO2–P–).
6,6-Dibromo-3ꢀ -(methylphosphate)carbonyl-2,2-dime-
thylpenam sulfone (12). According to a similar proce-
dure to that used for the synthesis of 3a compound 11
was converted to 12 (98% yield) as a white solid. Mp
181.0–183.0 ꢀC. IR (KBr) gmax 1808 (b-lactam), 1749
(acyl phosphate) 1325, and 1127 cmÀ1 (sulfone). 1H
NMR (CDCl3) d 1.41 (3H, s, a-CH3), 1.62 (3H, s, b-
CH3), 3.85 (3H, s, –OCH3), 4.52 (1H, s, 3-H), 5.01 (1H,
s, 5-H); 13C NMR (CDCl3) d 18.46 and 19.58 (C-8 and
C-9), 43.33 (C-6), 53.19 (O-CH3), 62.90 (C-3), 64.52 (C-
2), 73.30 (C-5), 164.00 (C-7), and 165.84 (C–CO2–P–).
6. Durette, P. L.; Chabin, R. M.; Fletcher, D. S.; Green, B. G.;
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7. (a) For the crystal structure of serine b-lactamase of Sta-
phylococcus aureus inactivated by
a methyl-phosphonate
monoester monoanion inhibitor, see: Chen, C. C. H.; Rahil, J.;
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8. (a) For inhibition of the class C serine-b-lactamase of
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Acknowledgements
The authors thank Professor Ming-Daw Tsai (The
Department of Chemistry, The Ohio State University)
for recording 31P NMR and mass spectra and Profesor
Manuel G. Sierra and Dr. Ernesto G. Mata (IQUIOS,
1
Rosario) for recording H and 13C NMR. The authors
would also like to thank CONICET (Consejo Nacional
de Investigaciones Cientıficas y Tecnicas) and Agencia
Nacional de Promocion Cientıfica
(Argentina) for financial support.
y Tecnologica
9. (a) For inhibition of the class C serine-b-lactamase of
Enterobacter cloacae P99 by phosphonoamidates see: Laws,
A. P.; Page, M. I.; Slater, M. J. Bioorg. Med. Chem. Lett.
1993, 3, 2317. (b) Rahil, J.; Pratt, R. F. Biochemistry 1993, 32,
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J. E.; Payne, D. J.; Tolson, D. A.; Walker, G. Bioorg. Med.
Chem. Lett. 1994, 4, 1667.
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