5,6-cis-Penems: Broad-spectrum anti-methicillin-resistant Staphylococcus aureus β-lactam antibiotics

Article abstract of DOI:10.1021/jm9703348

5,6-cis-Penem derivatives have been synthesized and evaluated as anti- MRSA antibiotics. The cis-penems 5 and 6 showed potent activities against not only MRSA but also a wide variety of bacteria including β-lactamase- producing microorganisms. These compo

Full text of DOI:10.1021/jm9703348

2126
J . Med. Chem. 1997, 40, 2126-2132
Expedited Articles
5,6-cis-P en em s: Br oa d -Sp ectr u m An ti-Meth icillin -Resista n t Sta ph ylococcu s
a u r eu s â-La cta m An tibiotics
Masaji Ishiguro,* Rie Tanaka, Koushi Namikawa, Takaaki Nasu, Hidekazu Inoue, Takashi Nakatsuka,
Yoshiaki Oyama, and Seiichi Imajo
Suntory Institute for Bioorganic Research and Suntory Ltd. Institute for Biomedical Research, 1-1 Wakayamadai, Shimamoto,
Mishima, Osaka 618, J apan
Received May 20, 1997X
5,6-cis-Penem derivatives have been synthesized and evaluated as anti-MRSA antibiotics. The
cis-penems 5 and 6 showed potent activities against not only MRSA but also a wide variety of
bacteria including â-lactamase-producing microorganisms. These compounds were designed
to have high affinity to the penicillin-binding protein 2a of MRSA and to form stable acyl
intermediates with â-lactamases by blocking the deacylating water molecule.
In tr od u ction
Among known resistance mechanisms of bacteria
against antibiotics, alteration of the target site struc-
tures is a major concern in the field of â-lactam-
interacting enzymes, such as â-lactamases1,2 and peni-
cillin-binding proteins (PBPs).3 â-Lactamases change
their active site structures to gain higher affinities to
various â-lactam antibiotics for degradation of the
â-lactam structure, while the penicillin-binding protein
of methicillin-resistant Staphylococcus aureus (MRSA)
has an altered active site structure having a reduced
affinity toward â-lactam antibiotics. Since amino acid
F igu r e 1. Illustration of a putative binding of penicillin
sequences for the mutant â-lactamases have been
derivative at the active site of class A â-lactamase. Putative
elucidated from their gene sequences, it is possible to
examine a plausible contribution of the mutated amino
hydrogen bondings between penicillin and â-lactamase are
shown by broken lines. Ser70 is the active center for the
catalysis. R: acyl substituent at C-6.
acids for the resistance through three-dimensional
structures of â-lactamases.4,5 Recently, a three-dimen-
sional structure of transpeptidase PBP2x, a high mo-
lecular weight penicillin-binding protein of Klebsiella
pneumoniae, has been reported,6 and it has been sug-
gested that the high molecular weight transpeptidases
have active site residues similar to the class A â-lacta-
mases. Thus, the reaction of â-lactam antibiotics on
PBPs such as the PBP2a of MRSA and the PBP2 of
Escherichia coli would be analogous to that of â-lactam
antibiotics with class A â-lactamases in Michaelis
complex formation and acylation.
the carboxylate of â-lactam antibiotics with the Ser130
residue (Ambler numbering for class A â-lactamases is
used for residue number unless otherwise specified)9 of
class A â-lactamases,10 the substituents at C-2 and C-6
may play a crucial role in adequate disposition of the
C-3 carboxylate for initiation of the acylation. Hence,
alteration of amino acid residues proximal to those
binding sites of the â-lactam-interacting enzymes would
interrupt or facilitate the interaction of the carboxylate
for the acylation.
Recently developed penem and carbapenem antibi-
otics are essentially inactive against MRSA. Those
antibiotics have the characteristic 5,6-trans-hydroxy-
ethyl side chain at C-6 and a variety of C-2 side chains.
A recent crystallographic structure of an acyl intermedi-
ate of TEM-1 â-lactamase with 6R-(hydroxymethyl)-
penicilloic acid7 indicated that the hydroxyethyl moiety
should reside in the binding site similar to that of the
C-6 acyl moiety of penicillins (Figure 1). On the other
hand, the crystallographic structure analysis on penem
derivatives has inferred that the side chains at C-2 of
penem derivatives are required to have a specific
conformation at another side of the binding site.8
Taking account of the importance of the interaction of
It has been reported that through introduction of a
fairly large substituent at C-2 such as carbolinyl,11
thiazolobenzimidazoylmethylthio,12 and 4-arylthiaz-
oylthio13 moieties, carbapenem derivatives show an
improved activity against MRSA, suggesting the alter-
ation of the binding pocket for C-2 substituents. On the
other hand, except the trans-hydroxyethyl group, C-6
side chains effective for anti-MRSA activity have not
been reported for penem or carbapenem derivatives.
Antimicrobial activity of 5,6-cis-penems bearing the
hydroxyethyl at C-6 (e.g., compound 22) indicated that
the S configuration of the hydroxyethyl group is prefer-
able for the interaction with PBPs, although both
isomers were not stable to â-lactamases.14 The suscep-
tibility of the cis-penems to â-lactamases implies insta-
bility of an acyl intermediate against attack of a
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0022-2623(97)00334-8 CCC: $14.00 © 1997 American Chemical Society

5,6-cis-Penems
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2127
Sch em e 1. Synthesis of 5,6-cis-Penem Bearing Carbon
at C-2a
F igu r e 2. 5,6-trans- and -cis-penem derivatives.
trans-penem 12 was isomerized to a mixture of the 5,6-
cis-penem 13 and the starting trans-penem 12 (1:2 ratio)
by photoirradiation.18 After separation of the cis-penem
13 from the mixture by silica gel chromatography,
removal of the TBS group of 13 and subsequent depro-
tection of 14 provided the desired cis-penem 3.
The cis-penems 5 and 6 were synthesized by another
route, starting from the (3R,4S)-azetidinone 8 (Scheme
2). The N-alkylation of the azetidinone 8 with p-
nitrobenzyl iodoacetate gave the N-alkylazetidinone 15.
Treatment of the azetidinone 15 with lithium diisopro-
pylamide and successive reactions with carbon disulfide
followed by treatment with benzoyl chloride afforded the
ketene dithioacetal derivative 16. After generation of
the crude chloride 17 by treatment with sulfuryl chlo-
ride, the chloride 17 was reacted with morpholine and
methyl iodide to provide the 5,6-cis-penem 18.19 Oxi-
dation of the methylthio group to the sulfoxide 19 and
then substitution of the sulfoxide moiety with pyrro-
lidinylthiol derivatives furnished the protected penem
20. Deprotection of the hydroxyl group and carbox-
ylic acid gave the desired 5,6-cis-penem derivative 5
(or 6).
a
(a) Chlorosulfonyl isocyanate, i-Pr2O then CH3COSH; (b)
Cu2O, AcOH; (c) RCOSH (R ) (2R)-tetrahydrofuranyl), NaOH; (d)
ClCOCO2allyl, Et3N; (e) P(OEt)3, toluene, reflux; (f) hν; (g) Bu4NF,
AcOH; (h) Pd(OAc)2.
hydrolyzing water molecule, a crucial problem to be
overcome for design of broad spectrum cis-penem anti-
biotics. Herein, we report that novel 5,6-cis-penem
derivatives show a potent antimicrobial activity against
MRSA and note that the structure-activity relationship
of the cis-penems against â-lactamase-producing bac-
teria shows a significant influence of the C-2 side chain
structures for the deacylation in the acyl enzymes.
Resu lts a n d Discu ssion
Ch em istr y
Hydroxypropyl derivatives 2-6 were synthesized to
examine an effect on the antimicrobial activity by the
alteration of the bulkiness of the C-6 side chain. The
cis-penem derivatives 3 and 4 retained the antimicrobial
activity against Gram-positive bacteria including MRSA
as shown in Table 1. The methyl group of the hydroxy-
propyl moiety did not reduce the activity of the cis-
penem but rather enhanced the activity against MRSA
(compounds 3 and 22). The corresponding trans-penem
2 greatly reduced the antimicrobial activity against all
organisms tested. This result implies that the hydroxy-
propyl moiety of the cis-penems interacts at a less
hindered site, whereas the hydroxyethyl moiety of the
trans-penems binds at a limited space which does not
accommodate the additional methyl group of the hy-
droxypropyl moiety.
The cis-penem 3 was synthesized as shown in Scheme
1. Starting from commercially available ethyl (S)-3-
hydroxypentanoate, the vinyl sulfide 7 was prepared by
the same method as previously described for the prepa-
ration of (R)-3-[(tert-butyldimethylsilyl)oxy]-1-(phenylth-
io)-1-pentene.15 The coupling of the vinyl sulfide 7 with
chlorosulfonyl isocyanate in diisopropyl ether at room
temperature afforded the (3R,4S)-azetidinone 8 as a
major product (3:1 mixture of the diastereomeric 3,4-
trans isomers), which was then converted to the ac-
etoxyazetidinone 9 in acetic acid using cupric acetate
at 100 °C.16 The penem skeleton was built by a
conventional method in three steps: reaction of the
acetoxyazetidinone 9 with sodium acylthiolates (R )
(2R)-tetrahydrofuranoyl) to afford the ester 10; forma-
tion of the oxamate 11 by reaction with allyl chloroox-
alate; penem ring formation by intramolecular Wittig
reaction of the phosphorane generated in situ by treat-
ment of 11 with triethyl phosphite.17 Then, the 5,6-
The cis-penems 3 and 4 were unstable against the
â-lactamase-producing bacteria. A study on hydrolysis
of the cis-penem 22 by class A â-lactamases indicated

2128 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Ishiguro et al.
Sch em e 2. Synthesis of 5,6-cis-Penem Bearing Sulfur
substituents at C-2 should bind firmly at the binding
site and block the deacylating water molecule. Thus,
the present result would become good evidence for the
deacylation mechanism proposed by us.10 Although
another process of the deacylation proposed by Herzberg23
can explain the instability of the cis-penems 3 and 4, it
does not account for the stability of the alkylthio-
substituted cis-penems 5 and 6. Recently, Strynadka
et al. have reported a crystal structure of TEM-1
â-lactamase complexed with a borane derivative and
postulated that the complex structure is a transition-
state mimic based on Herzberg’s assumption.24 How-
ever, the present finding suggests that it will be
necessary to investigate if the complex structure rep-
resents the true transition state for the deacylation.
at C-2a
The alkylthio-bearing cis-penems retained the activity
against MRSA. Conformational analysis25 of the hy-
droxypropyl (or hydroxyethyl) group indicated that the
cis-penem has a single local energy minimum at the
torsion angle (160°) for C7-C6-C8-O11, while the trans-
penem has two local minima at -160° and -50° where
the individual conformations are found in the crystal
structures.8 This infers that in the Michaelis complex
formation of the cis-penems, the conformationally con-
strained substituents at C-6 and the carbonyl oxygen
of â-lactam are sufficient to determine the disposition
of the C-3 carboxylate group suitable for the interaction
with the Ser130 residue irrespective of the C-2 side
chains. For the trans-penems, not only the hydroxy-
ethyl group at C-6 but also the side chains at C-2 would
play an important role in locating the carboxylate
proximal to the Ser130 residue due to the conforma-
tional flexibility of the hydroxyethyl group. Although
it is hard to explore the binding site model for PBP2a
through model building due to the lack of the structural
details of the PBPs, the present results imply that the
PBP2a of MRSA has a larger binding pocket at the C-2
substituent-binding region of PBP2a than the other
PBPs such as the PBP2 of E. coli.
a
(a) BrCH2CO2PNB, K2CO3; (b) LiN(i-Pr)2, CS2 then benzoyl
chloride; (c) SO2Cl2; (d) morpholine then MeI; (e) m-ClPBA; (f) 1-
(p-nitrobenzyl)-3-mercaptopyrrolidine; (g) TBAF; (h) H2/Pd-C.
that 22 was hydrolyzed more rapidly (>1000 times) than
the corresponding trans-penem 1,15 opening the tet-
rahydrofuran ring to give rise to the hydroxybutylidene
derivative 23 as observed in the hydrolysis of the trans-
penem (Scheme 3).20 This indicates that the acyl
enzymes formed by the cis-penems are not resistant to
the hydrolytic attack of the deacylating water, while
trans-penem derivatives form stable acyl enzymes with
class A and C â-lactamases.21
Since after formation of the acyl enzyme, the (6R,8S)-
hydroxyethyl or -hydroxypropyl moiety of the cis-pen-
ems would loosely reside at the binding site, it would
allow the acyl moiety to have a more mobile conforma-
tion which will permit the deacylating water molecule
to enter at the site for the deacylation in a similar mode
proposed for the penicillin hydrolysis.10 On the other
hand, the fir mLy bound hydroxyethyl moiety of the
trans-penems would keep the conformation of the rest
of the acyl group unchanged to block the attack of the
deacylating water molecule.
In conclusion, it will be conceivable that the trans-
penems lose the effective interaction at the C-2 sub-
stituent-binding site of PBP2a and the resulting enzyme-
substrate complex would not have the suitable interaction
between the carboxylate and the Ser130 residue for the
acylation. On the other hand, the carboxylate of the
cis-penems would be located at the suitable position for
the acylation only through the interaction of the C-6
substituents and the â-lactam carbonyl oxygen at the
oxyanion hole. The stability of the acyl enzymes of
â-lactamases is largely dependent on not only the C-6
substituents but also the C-2 moieties. The putative
deacylating water which enters at the binding site for
the C-2 moieties clearly explains the distinct stability
between 3 and 5. Thus, the cis-penems 5 and 6 showed
potent activities against a wide variety of bacteria such
as MRSA and Pseudomonas aeruginosa including â-lac-
tamase-producing microorganisms. The present find-
ings should provide a better understanding about the
interaction of â-lactam antibiotics with penicillin-
interacting enzymes as well as the hydrolytic mecha-
nism of â-lactamases and thus afford important infor-
mation for design of anti-MRSA and â-lactamase-
resistant â-lactam antibiotics.
The cis-penems 5 and 6 showed good activity against
â-lactamase-producing bacteria as well as MRSA (Table
1). We assume that thioalkyl moieties (SR in compound
24) would be converted to the R-configuration via
â-lactamase-catalyzed tautomerization of the ∆2- to ∆3-
dihydrothiazole (Scheme 4), as observed in the carba-
penems bearing a sulfur atom at C-2.22 The R-oriented

5,6-cis-Penems
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2129
Ta ble 1. In Vitro Antibacterial Activity (MIC, µg/mL)a of Penem Derivatives
compound no.
microorganisms
1
2
3
4
5
6
22
0.78
25
0.39
0.05
0.78
IPMo
S. a.c
S. a.d
E. f.e
0.05
>100
0.78
<0.025
0.1
0.78
0.39
0.78
12.5
0.20
0.20
25
0.1
0.1
3.13
0.78
<0.025
0.2
0.78
6.25
0.1
1.56
0.39
0.78
6.25
<0.025
>100
0.78
<0.025
0.2
>100
6.25
0.39
0.05
3.13
3.13
1.56
0.05
0.2
0.78
6.25
0.39
3.13
0.78
0.78
25
B. s.f
0.78
100
12.5
>100
12.5
>100
>100
25
E. c.g
E. cb,h
C. f.b,i
K. p.j
S. m.k
E. c.b,l
P. v.m
P. s.n
0.1
25
0.2
6.25
1.56
3.13
>100
>100
1.56
50
25
>100
0.78
25
100
1.56
>100
0.1
0.39
0.2
0.78
0.2
3.13
1.56
>100
6.25
50
25
>100
>100
1.56
1.56
>100
>100
>100
100
12.5
a
Minimum inhibitory concentration was determined in heart infusion agar medium. Inoculum size: 106 cells/mL. Incubation: 24 h
at 37 °C. â-Lactamase-producing organism. c Staphylococcus aureus 209P J C-1. Staphylococcus aureus (MRSA). e Enterococcus faecalis
b
d
ATCC 19433. f Bacillus subtilis ATCC 6633. Escherichia coli NIHJ J C-2. Escherichia coli KC-14/RGN 823. Citrobacter freundii GN
g
h
i
j
k
l
m
7391. Klebsiella pneumoniae PCI 602. Serratia marcescens IAM 1136. Enterobacter cloacae NTCC9394. Proteus vulgaris GN 7919.
Pseudomonas aeruginosa PAO-1. o Imipenem.
n
trophotometer: only selected absorptions are reported. For
analysis of carboxylic acids or their salts, reverse phase HPLC
analysis was carried out using a TSK ODS-80Tm column (4.6
mm i.d. × 150 mm) and a Shimazu liquid chromatograph 6A
system. An acetonitrile/water (including 0.1 v/v % trifluoro-
acetic acid) was employed to elute compounds from the column
(flow rate 1 mL/min). Eluting materials were also detected
by measuring their UV absorbance with a Waters 991J
photodiode array detector. For purification of carboxylic acids
or their salts, reverse phase column chromatography on an
ODS Chromatorex (100-200 mesh; (Fuji Davison Chemical
Ltd.) or preparative HPLC separations on a YMC SH-343-5
S-5 120A AM ODS column (20 mm i.d. × 250 mm), elution
with acetonitrile/water (including 0.1 v/v % acetic acid, flow
rate 10 mL/min), was carried out.
Sch em e 3. Degradation Product of 5,6-cis-Penem
Sch em e 4. Plausible Tautomerization (∆2 f ∆3) of Acyl
Enzyme
(3R,4S)-3-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-
4-(p h en ylth io)a zetid in -2-on e (8). To a solution of chloro-
sulfonyl isocyanate (6.6 mL, 75.8 mmol) in diisopropyl ether
(117ml) was added 7 (15.8 g, 51.2 mmol) in diisopropyl ether
(31 mL) under Ar at room temperature. The reaction mixture
was stirred at room temperature for 4 h. The mixture was
cooled to -50 °C and treated with thiophenol (13.0 mL, 127
mmol) and then pyridine (10.2 mL, 127 mmol) followed by
stirring at -20 °C for 30 min. The mixture was poured into
saturated KHSO4 (100 mL) and extracted with AcOEt (100
mL). The organic phase was washed with saturated NaHCO3
(100 mL) and then saturated Na2SO4 (100 mL), dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
Purification of the residue by chromatography on silica gel
provided 7.67 g (29%) of a mixture of 8 and (3S,4R)-3-[(S)-1-
[(tert-butyldimethylsilyl)oxy]propyl]-4-(phenylthio)azetidin-2-
one in a 3:1 ratio. 8 (white solid recrystallized from n-hexane-
toluene): mp 123-124 °C; IR (cm-1) (KBr) 3158 (NH), 1762
(â-lactam CdO); 1H NMR (CDCl3) δ 7.42-7.52 (2H, m), 7.33-
7.42 (3H, m), 6.03 (1H, brs), 5.09 (1H, d, J ) 2.6 Hz), 4.00-
4.08 (1H, m), 3.14 (1H, t, J ) 2.6 Hz), 1.45-1.67 (2H, m), 0.87
(9H, s), 0.06 (3H, s), 0.05 (3H, s, SiMe). Anal. (C18H29NO2-
SSi) C, H, N.
Exp er im en ta l Section
Melting points (uncorrected) were determined in open
capillary tubes with a Buchi 535 apparatus. The NMR spectra
were recorded on J EOL J NM-GX270 and Brucker ARX 400
FT NMR spectrometers in either CDCl3 solution with tetra-
methylsilane as an internal standard or D2O solution with
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) as
an internal standard. Chemical shifts are reported in ppm
relative to the standards. Positive ion fast atom bombardment
mass spectra (FABMS) were recorded on a J EOL J MS-AX5000
instrument, and high-resolution mass spectra (HRMS) were
recorded on a J EOL J MS-HX/HX110A instrument.
All reactions were performed under a positive atmosphere
of argon. Organic solutions obtained after workup were dried
over anhydrous Na2SO4 unless otherwise specified. Flash
column chromatography was conducted with Merck silica gel
60 Art 9385 (230-400 mesh), and preparative TLC of Merck
Kieselgel 60F254 Art 13895 (1 mm) was used. Compounds
showed satisfactory purity by TLC on Merck Kieselgel 60F254
Art 5714 plates (visualized by UV light at 254 nm and/or by
6.3 w/v % phosphomolybdic acid in ethanol). FT IR spec-
(3R,4S)-3-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-
4-a cetoxya zetid in -2-on e (9). To a solution of 8 (4.92 g, 14.0
mmol) in AcOH (30 mL) was added Cu(OAc)2‚H2O (2.00 g, 10.0
mmol) at room temperature, and the mixture was heated at
100 °C for 75 min. The reaction mixture was filtered through
Celite, and the solvent was removed under reduced pressure.
The residue was diluted with AcOEt (100 mL) and poured into
water (100 mL). The organic phase was washed with satu-
rated NaHCO3 (100 mL) and then saturated Na2SO4 (100 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. Purification of the residue by chromatography on
silica gel provided 3.37 g of 9 as a white solid in 80% yield. 9:
mp 72-74 °C; IR (cm-1) (KBr) 3170, 1780, 1748; 1H NMR
(CDCl3) δ 6.46 (1H, brs), 5.84 (1H, s), 4.01-4.10 (1H, m), 3.31
(1H, t, J ) 4.3 Hz), 2.11 (3H, s), 1.53-1.68 (2H, m), 0.87 (9H,

2130 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Ishiguro et al.
s), 0.07 (3H, s), 0.06 (3H, s); HRMS (FAB) m/ z (M + 23) calcd
for C14H27NO4NaSi 324.1607, found 324.1605.
in 77% yield as a pale yellow oil. 14: IR (cm-1) (neat) 2955,
1790, 1704; H NMR (CDCl3) δ 5.86-6.03 (1H, m), 5.58 (1H,
1
d, J ) 4.0 Hz), 5.22-5.47 (3H, m), 4.77 (1H, dd, J ) 5.3, 13.9
Hz), 4.63 (1H, dd, J ) 5.3, 13.9 Hz), 4.12-4.27 (1H, m), 3.95-
4.06 (1H, m), 3.82-3.95 (2H, m), 2.39-2.55 (1H, m), 1.76-
2.10 (5H, m), 1.03 (3H, t, J ) 7.3 Hz).
(3R,4S)-3-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-
4-((R)-t et r a h yd r o-2-fu r yl)a zet id in -2-on e (10). (R)-Tet-
rahydro-2-furanthiolcarboxylic acid (1.19 g, 9.0 mmol) was
buffered at pH 9-10 with 1 N NaOH. To this solution was
added 9 (1.81 g, 6.0 mmol) in acetone (6 mL) at room
temperature. The reaction mixture was stirred at 50 °C for
10 min and buffered at pH 8-9 with 1 N NaOH followed by
stirring for 2 h. The residue was diluted with AcOEt (30 mL)
and poured into water (30 mL). The organic phase was washed
with saturated NaHCO3 (30 mL) and then saturated Na2SO4
(30 mL), dried over anhydrous Na2SO4, and concentrated
under reduced pressure. Purification of the residue by chro-
matography on silica gel provided 1.79 g as a colorless oil in
80% yield. 10: IR (cm-1) (KBr) 3158, 1770, 1698; 1H NMR
(CDCl3) δ 6.28, 6.27 (1H, brs), 5.24, 5.21 (1H, d, J ) 2.6 Hz),
4.43-4.52 (1H, m), 3.91-4.16 (3H, m), 3.30 (1H, dd, J ) 2.6,
2.6 Hz), 2.18-2.37 (1H, m), 1.85-2.18 (3H, m), 1.42-1.68 (2H,
m), 0.89 (9H, s), 0.08 (6H, s); HRMS (FAB) m/ z (M + 1) calcd
for C17H32NO4SSi 374.1821, found 374.1822.
(5S,6R)-6-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-
2-((R)-tetr a h yd r o-2-fu r yl)p en em -3-ca r boxylic Acid Allyl
Ester (12). To a solution of 10 (1.49 g, 4.0 mmol) in CH2Cl2
(2 mL) were added monoallyl oxalyl chloride (0.99 g, 6.7 mL)
in CH2Cl2 (1.6 mL) and then triethylamine (0.69 g, 6.8 mmol)
in CH2Cl2 (1.6 mL) at -20 °C. The reaction mixture was
stirred at the same temperature for 1.5 h. The mixture was
poured into H2O (30 mL) and extracted with CH2Cl2 (30 mL).
The organic phase was washed with saturated NaHCO3 (30
mL) and then saturated Na2SO4 (30 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. After
azeotropic drying with toluene, to the residue was added
triethyl phosphite (5.5 mL, 32.0 mmol) at room temperature.
The reaction mixture was stirred at 70 °C for 1 h. After
removal of the excess of triethyl phosphite in vacuo, the
reaction mixture was stirred under xylene (20 mL) reflux for
3 h. The mixture was poured into H2O (30 mL) and extracted
with n-hexane (30 mL). The organic phase was dried over
anhydrous MgSO4 and concentrated under reduced pressure.
Purification of the residue by chromatography on silica gel
provided 1.04 g of the product 12 as a yellow oil (58% yield).
12: IR (cm-1) (neat) 2955, 1790, 1707; 1H NMR (CDCl3) δ
5.84-6.02 (1H, m), 5.58 (1H, d, J ) 1.3 Hz), 5.32-5.49 (2H,
m), 5.24 (1H, d, J ) 10.6 Hz), 4.59-4.82 (2H, m), 4.00-4.11
(1H, m), 3.89-4.00 (1H, m), 3.73-3.89 (2H, m), 2.33-2.50 (1H,
m), 1.87-2.05 (2H, m), 1.69-1.87 (1H, m), 1.45-1.69 (2H, m),
0.89 (9H, s), 0.08 (3H, s), 0.07 (3H, s); CD (CH3CN) λmax 251
nm (θ ) -0.84 × 105).
(5R,6R)-6-((S)-1-H yd r oxyp r op yl)-2-((R)-t et r a h yd r o-2-
fu r yl)p en em -3-ca r boxylic Acid (3). To a solution of 14 (20
mg, 0.059 mmol) in CH2Cl2 (0.5 mL) were added Pd(PPh3)4
(0.7 mg, 0.0006 mmol), PPh3 (0.8 mg, 0.003 mmol), and sodium
2-ethylhexanoate (11 mg, 0.067 mmol) at room temperature,
and the mixture was stirred at the same temperature for 2 h.
After completion of reaction, the reaction mixture was diluted
with AcOEt (5 mL) and extracted with H2O (5 mL) twice. The
combined water phase was lyophilized, and the residue was
purified with HPLC (HPLC conditions: column, YMC D-
ODS-5 120A AM type i.d. 20 × 250 mm; eluent A, H2O:CH3-
CN:AcOH ) 950:50:1; eluent B, H2O:CH3CN:AcOH ) 50:950:
1; A:B ) 100:0 to 0:60 linear gradient for 60 min; flow rate, 9
mL/min; temperature, room temperature; detected at 254 nm);
9 mg of 3 was obtained as an off-white powder after lyophiliza-
tion of the fraction containing the desired product. 3: IR
1
(cm-1) (KBr) 3401, 1771; H NMR (CDCl3) δ 5.60 (1H, d, J )
4.0 Hz), 5.34 (1H, t, J ) 7.3 Hz), 4.12-4.25 (1H, m), 3.93-
4.07 (1H, m), 3.81-3.93 (2H, m), 2.40-2.59 (1H, m), 1.78-
2.12 (4H, m), 1.48-1.63 (1H, m), 1.04 (3H, t, J ) 7.3 Hz). Anal.
(C13H16NO5SNa) C, H, N.
2-[(3R,4S)-3-[1(S)-[(ter t-Bu tyldim eth ylsilyl)oxy]pr opyl]-
4-(ph en ylth io)-2-oxoazetidin -1-yl]acetic Acid p-Nitr oben -
zyl Ester (15). To a solution of (3R,4S)-3-[1(S)-[(tert-butyl-
dimethylsilyl)oxy]propyl]-4-(phenylthio)-2-azetidinone (8; 1.53
g, 4.35 mmol) in anhydrous DMF (6.7 mL) were added
iodoacetic acid p-nitrobenzyl ester (1.66 g, 4.78 mmol) and K2-
CO3 (1.82 g, 13.2 mmol). The reaction mixture was heated at
50-55 °C for 4.5 h. The reaction mixture was diluted with
H2O (50 mL) and extracted with CH2Cl2 (100 mL and then 50
mL). The combined organic phase was washed with saturated
NaCl (100 mL) and dried, followed by concentration under
reduced pressure. Purification of the residue by chromatog-
raphy on silica gel (38 g, AcOEt:n-hexane ) 1:8) provided 2.20
g of 15 as a pale yellow oil (93% yield). 15: 1H NMR (CDCl3)
δ 8.22 (2H, d, J ) 8.7 Hz), 7.47 (2H, d, J ) 8.7 Hz), 7.40-7.50
(2H, m), 7.25-7.35 (3H. m), 5.30 (1H, d, J ) 2.1 Hz), 5.22 (1H,
d, J ) 13.2 Hz), 5.16 (1H, d, J ) 13.2 Hz), 4.25 (1H, d, J )
17.8 Hz), 4.05-4.10 (1H, m), 3.93 (1H, d, J ) 17.8 Hz), 3.17
(1H, dd, J ) 2.1, 2.8 Hz), 1.55-1.70 (2H, m), 0.91 (3H, t, J )
7.5 Hz), 0.86 (9H, s), 0.06 (3H, s), 0.01 (3H, s).
2-[Bis(ben zoylth io)m eth yliden e]-2-[(3R,4S)-3-[1(S)-[(ter t-
bu tyld im eth ylsilyl)oxy]p r op yl]-4-(p h en ylth io)-2-oxoa ze-
tid in -1-yl]a cetic Acid p-Nitr oben zyl Ester (16). To a
solution hexamethyldisilazane (1.65 g, 10.2 mmol) in THF (25
mL) was added 1.71 N n-BuLi in n-hexane (5.3 mL, 9.06 mmol)
at room temperature. The reaction mixture was stirred at
room temperature for 30 min and then cooled to -78 °C. To
this solution was added a solution of 15 (2.50 g, 4.59 mmol) in
THF (5 mL) at -78 °C. The reaction mixture was stirred at
the same temperature for 10 min, and to this solution was
added CS2 (0.55 mL, 9.14 mmol) followed by a solution of
benzoyl chloride (1.6 mL, 13.8 mmol) in THF (5 mL). The
reaction mixture was stirred for 10 min, and to this solution
was added acetic acid (0.45 mL, 7.84 mmol). The reaction
mixture was diluted with AcOEt (200 mL) and washed with
saturated NaCl (100 mL) saturated NaHCO3 (100 mL), and
saturated NaCl (100 mL). The organic phase was dried, and
the solvent was removed in vacuo. Purification of the residue
by chromatography on silica gel (50 g, AcOEt:n-hexane ) 1:3)
provided 3.37 g of 16 as a pale yellow oil (88% yield). 16: 1H
NMR (CDCl3) δ 7.99 (2H, d, J ) 8.7 Hz), 7.92 (2H, d, J ) 8.7
Hz), 7.80 (2H, d, J ) 7.1 Hz), 7.69 (2H, d, J ) 7.1 Hz), 7.50-
7.60 (2H, m), 7.40-7.50 (4H, m), 7.35-7.40 (2H, m), 7.20-
7.35 (3H, m), 5.85 (1H, d, J ) 2.8 Hz), 5.30 (1H, d, J ) 12.9
Hz), 5.24 (1H, d, J ) 12.9 Hz), 3.95-4.00 (1H, m), 3.12 (1H,
dd, J ) 2.5, 2.8 Hz), 1.50-1.65 (2H, m), 0.90 (3H, t, J )
7.5 Hz), 0.83 (9H, s), 0.02 (3H, s) -0.01 (3H, s). Anal.
(C42H44N2O8S3Si) C, H, N.
(5R,6R)-6-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-
2-((R)-tetr a h yd r o-2-fu r yl)p en em -3-ca r boxylic Acid Allyl
Ester (13). A solution of 12 (343 mg, 1.0 mmol) in AcOEt
(200 mL) was irradiated by use of a high-pressure UV lamp
(200 W; Ishiirikaseisakusho) through a Pyrex filter for 1 h.
The solvent was removed, and purification of the residue by
chromatography on silica gel provided 106 mg of 13 in 32%
yield as a colorless oil together with 191 mg of unchanged
material in 56% yield. 13: mp 80-83 °C; IR (cm-1) (neat)
3500, 1790, 1704; 1H NMR (CDCl3) δ 5.85-6.02 (1H, m), 5.55
(1H, d, J ) 4.0 Hz), 5.20-5.48 (3H, m), 4.58-5.82 (2H, m),
4.31-4.42 (1H, m), 3.79-4.09 (3H, m), 2.34-2.53 (1H, m),
1.71-2.10 (5H, m), 0.97 (3H, t, J ) 7.9 Hz), 0.87 (9H, s), 0.11
(3H, s), 0.12 (3H, s); HRMS (FAB) m/ z (M + 23) calcd for
C22H35NO5SNaSi 476.1903, found 476.1889.
(5R,6R)-6-((S)-1-H yd r oxyp r op yl)-2-((R)-t et r a h yd r o-2-
fu r yl)p en em -3-ca r boxylic Acid Allyl Ester (14). To a
solution of 13 (138 mg, 0.30 mmol) in THF (0.61 mL) were
added AcOH (0.069 mL, 1.2 mmol) and then 1.0 M tetra-n-
butylammonium fluoride in THF (0.89 mL, 0.89 mmol) at room
temperature. The reaction mixture was stirred at 50 °C for 6
h. The reaction mixture was poured into H2O (10 mL) and
extracted with AcOEt (10 mL). The organic phase was washed
with saturated KHSO4 (10 mL), saturated NaHCO3 (10 mL),
and saturated Na2SO4 (10 mL), dried over anhydrous Na2SO4,
and concentrated under reduced pressure. Purification of the
residue by chromatography on silica gel provided 79 mg of 14
(5R,6R)-6-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-

5,6-cis-Penems
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2131
2-(m eth ylth io)p en em -3-ca r boxylic Acid p-Nitr oben zyl
Ester (18). To a solution of 16 (2.43 g, 2.93 mmol) in CH2Cl2
(40 mL) was added sulfuryl chloride (0.44 mL, 4.41 mmol) at
-5 °C. The reaction mixture was stirred at the same tem-
perature for 15 min, and to this solution was added allyl
acetate (1.6 mL, 14.8 mmol) followed by diphenyl disulfide (639
mg, 2.93 mmol). The reaction mixture was stirred at room
temperature for 5 min. After cooling with an ice bath for 20
min, to the solution were added a solution of morpholine (0.77
mL, 8.80 mmol) in CH2Cl2 (4 mL) and triethylamine (0.60 mL,
4.30 mmol) and after 10 min a mixture of methyl iodide (0.70
mL, 11.2 mmol) and triethylamine (0.40 mL, 2.87 mmol). The
reaction mixture was stirred at room temperature for 1 h. The
mixture was diluted with AcOEt (200 mL) and washed with
saturated KHSO4 (100 mL), saturated NaHCO3 (100 mL), and
saturated NaCl (100 mL). The organic phase was dried, and
the solvent was removed in vacuo. Purification of the residue
by chromatography on silica gel (50 g, AcOEt:n-hexane ) 1:8)
provided 1.07 g of 18 as a pale yellow powder (70% yield). 18:
1H NMR (CDCl3) δ 8.21 (2H, d, J ) 8.7 Hz), 7.61 (2H, d, J )
8.7 Hz), 5.73 (1H, d, J ) 4.0 Hz), 5.47 (1H, d, J ) 13.8 Hz),
5.21 (1H, d, J ) 13.8 Hz), 4.35 (1H, dt, J ) 4.5, 9.5 Hz), 4.09
(1H, dd, J ) 4.0, 9.5 Hz), 2.56 (3H, s), 1.75-1.90 (2H, m), 0.99
(3H, t, J ) 7.5 Hz), 0.88 (9H, s), 0.12 (3H, s), 0.12 (3H, s).
Anal. (C23H32N2O6S2Si) C, H, N.
(5R,6R)-6-[(S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]p r op yl]-
2-(m eth ylsu lfin yl)p en em -3-ca r boxylic Acid p-Nitr oben -
zyl Ester (19). To a solution of 18 (1.068 g, 2.03 mmol) in
CH2Cl2 (20 mL) was added m-chloroperbenzoic acid (393
mg, 2.28 mmol) at -30 °C. The reaction mixture was stirred
for 1 h. The mixture was diluted with AcOEt (200 mL) and
washed with 0.01 N sodium thiosulfate (30 mL), saturated
NaHCO3 (100 mL), and saturated NaCl (100 mL). The organic
phase was dried, and the solvent was removed in vacuo.
Purification of the residue by chromatography on silica gel (50
g, AcOEt:n-hexane ) 1:1) provided 793 mg of 19 as a pale
yellow powder (72% yield). 19: 1H NMR (CDCl3) δ 8.24 (2H,
d, J ) 8.7 Hz), 7.60 (0.8H, d, J ) 8.7 Hz), 7.58 (1.2H, d, J )
8.7 Hz), 5.93 (0.6H, d, J ) 4.2 Hz), 5.78 (0.4H, d, J ) 4.2 Hz),
5.44 (0.6H, d, J ) 13.5 Hz), 5.24 (0.4H, d, J ) 13.5 Hz), 5.23
(0.6H, d, J ) 13.5 Hz), 4.35-4.45 (1H, m), 4.18 (1H, dt, J )
4.2, 10.2 Hz), 2.95 (3H, s), 1.75-1.85 (2H, m), 1.00 (3H, t, J )
7.4 Hz), 0.88 (9H, s), 0.13 (6H, s). Anal. (C23H32N2O7S2Si) C,
H, N.
(5R,6R)-2-[[(S)-1-(p-Nitr oben zyl)p yr r olid in -3-yl]th io]-
6-[(S)-1-[(ter t-b u t yld im et h ylsilyl)oxy]p r op yl]p en em -3-
ca r boxylic Acid p-Nitr oben zyl Ester (20). To a solution
of 19 (179 mg, 0.33 mmol) in dimethylformamide (6 mL) were
added a solution of diisopropylamine (75 mL, 0.43 mmol) in
dimethylformamide (2 mL) and a solution of (S)-3-mercapto-
1-(p-nitrobenzyl)pyrrolidine (84 mg, 0.43 mmol) in dimethyl-
formamide (2 mL). After 20 min the reaction mixture was
diluted with ethyl acetate (100 mL), and the solution was
washed with saturated KHSO4 (50 mL), saturated NaHCO3
(50 mL), and saturated NaCl (50 mL) successively. The
organic phase was dried, and the solvent was removed in
vacuo. Purification of the residue by chromatography on silica
gel (10 g, AcOEt:n-hexane ) 1:8) provided 110 mg of 20 as a
pale yellow oil (50% yield). 20: 1H NMR (CDCl3) δ 8.21 (2H,
d, J ) 8.7 Hz), 7.61 (2H, d, J ) 8.7 Hz), 5.69 (1H, d, J ) 4.0
Hz), 5.46 (1H, d, J ) 13.8 Hz), 5.20 (1H, d, J ) 13.8 Hz), 4.3-
4.4 (1H, m), 4.08 (1H, dd, J ) 4.0, 9.8 Hz), 3.75-3.9 (1H, m),
3.63 (2H, s), 3.1-3.2 (1H, m), 2.65-2.75 (1H, m), 2.45-2.55
(1H, m), 2.3-2.45 (1H, m), 1.7-1.95 (3H, m), 0.99 (3H, t, J )
7.4 Hz), 0.87 (9H, s), 0.11 (3H, s), 0.10 (3H, s).
(5R,6R)-2-[[(S)-1-(p-Nitr oben zyl)p yr r olid in -3-yl]th io]-
6-((S)-1-h yd r oxyp r op yl)p en em -3-ca r boxylic Acid p-Ni-
tr oben zyl Ester (21, R ) P NB). To a solution of 20 (140
mg, 0.20 mmol) in THF (0.5 mL) were added AcOH (0.045 mL,
0.8 mmol) and then 1.0 M tetra-n-butylammonium fluoride in
THF (0.55 mL, 0.55 mmol) at room temperature. The reaction
mixture was stirred at 50 °C for 6 h. The reaction mixture
was poured into H2O (10 mL) and extracted with AcOEt (10
mL). The organic phase was washed with saturated KHSO4
(10 mL), saturated NaHCO3 (10 mL), and saturated Na2SO4
(10 mL), dried over anhydrous Na2SO4, and concentrated
under reduced pressure. Purification of the residue by chro-
matography on silica gel provided 110 mg of 21 in 90% yield
as a pale yellow powder. 21: IR (cm-1) (NaCl) 3447, 1785,
1
1684, 1552; H NMR (CDCl3) δ 8.22 (2H, d, J ) 8.8 Hz), 8.21
(2H, d, J ) 8.7 Hz), 7.61 (2H, d, J ) 8.5 Hz), 7.51 (2H, d, J )
8.5 Hz), 5.78 (1H, d, J ) 4.0 Hz), 5.60 (2H, s), 5.47 (1H, d, J
) 13.7 Hz), 5.20 (1H, d, J ) 13.8 Hz), 4.07-4.18 (1H, m), 3.87-
4.00 (2H, m), 3.45-3.71 (4H, m), 2.32-2.48 (1H, m), 1.94-
2.15 (2H, m), 1.50-1.70 (1H, m), 1.07 (3H, t, J ) 7.5 Hz). Anal.
(C27H28N4O8S2) C, H, N.
(5R,6R)-2-[((S)-1-P h en a cylp yr r olid in -3-yl)th io]-6-((S)-
1-h yd r oxyp r op yl)p en em -3-ca r boxylic Acid p-Nitr oben -
zyl Ester (21, R ) p h en a cyl). In the manner described
above, compound 21 (R ) phenacyl) was prepared from the
sulfinyl derivative 19 in 79% yield. 21: IR (cm-1) (NaCl) 3400,
1
1782, 1690; H NMR (CDCl3) δ 8.21 (2H, d, J ) 8.8 Hz), 7.96
(2H, d, J ) 7.2 Hz), 7.54-7.70 (3H, m), 7.37-7.54 (2H, m),
5.75 (1H, d, J ) 4.0 Hz), 5.46 (1H, d, J ) 13.7 Hz), 5.21 (1H,
d, J ) 13.7 Hz), 4.07-4.20 (1H, m), 4.00 (2H, s), 3.85-3.96
(1H, m), 3.94 (1H, dd, J ) 4.1, 10.5 Hz), 3.49 (1H, dd, J ) 7.3,
10.0 Hz), 2.90-3.01 (1H, m), 2.62-2.85 (2H, m), 1.40-1.53
(1H, m), 1.87-2.09 (2H, m), 1.51-1.70 (1H, m), 1.06 (3H, t, J
) 7.5 Hz).
(5R,6R)-2-[((S)-P yr r olid in -3-yl)th io]-6-((S)-1-h yd r oxy-
p r op yl)p en em -3-ca r boxylic Acid (5). A solution of the ester
21 (R ) PNB; 68 mg, 0.11 mmol) in tetrahydrofuran (5.5 mL)
was added to a suspension of palladium/carbon (10%, 110 mg)
in phosphate buffer (pH 7.0, 0.1 M, 5.5 mL) under hydrogen
atmosphere. After vigorous stirring at room temperature for
3 h, the catalyst was removed by filtration. The resulting
solution
was evaporated under
reduced pressure
and then lyophilized. The residue was purified by use of
reverse phase ODS-HPLC using water-acetonitrile (1 mM
ammonium formate) as mobile phase. Pure product 5 was
obtained as a white powder (22 mg, 58% yield). 5: IR (cm-1
)
(KBr) 3420, 1764, 1596; 1H NMR (D2O) δ 5.81 (1H, d, J ) 3.6
Hz), 4.03-4.22 (3H, m), 3.78 (1H, dd, J ) 6.5, 12.8 Hz), 3.39-
3.61 (4H, m), 2.48-2.61 (1H, m), 1.80-1.94 (1H, m), 1.49-
1.65 (1H, m), 1.00 (3H, t, J ) 7.4 Hz). Anal. (C13H17N2O4S2Na)
C, H, N.
(5R,6R)-2-[((S)-1-P h en a cylp yr r olid in -3-yl)th io]-6-((S)-
1-h yd r oxyp r op yl)p en em -3-ca r boxylic Acid (6). A solution
of the ester 21 (R ) phenacyl; 59 mg, 0.1 mmol) in tetrahy-
drofuran (2.5 mL) was added to a suspension of palladium/
carbon (10%, 100 mg) in phosphate buffer (pH 7.0, 0.1 M, 1.6
mL) under hydrogen atmosphere. After vigorous stirring at
room temperature, the catalysis was removed by filtration. The
resulting solution was lyophilized, and the residue was puri-
fied by use of reverse phase ODS-HPLC using water-aceto-
nitrile (1 mM ammonium formate) as mobile phase. Pure
product 6 was obtained as a white solid (9.0 mg, 20% yield).
6: IR (cm-1) (NaCl) 3430, 1772, 1694, 1595, 1376; 1H NMR
(CD3OD) δ 8.00 (2H, d, J ) 7.0 Hz), 7.66 (1H, t, J ) 7 Hz),
7.53 (2H, t, J ) 7 Hz), 5.72 (1H, d, J ) 3.0 Hz), 4.58 (2H, s),
3.90-4.08 (3H, m), 3.62-3.70 (1H, m), 3.20-3.30 (3H, m),
2.50-2.60 (1H, m), 1.95-2.05 (1H, m), 1.85-1.95 (1H, m),
1.50-1.60 (1H, m), 1.03 (3H, t,
(C21H23N2O5S2Na) C, H, N.
J ) 7.5 Hz). Anal.
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