Novel dithiocarbamate carbapenems with anti-MRSA activity

Article abstract of DOI:10.1016/S0968-0896(98)00069-8

A new series of 1β-methyl carbapenems, in which a disubstituted-aminothiocarbonylthio moiety was attached to the C-2 position of the carbapenem nucleus, were prepared and evaluated for anti-MRSA activity. These derivatives showed good in vitro antibacterial activity against high-level MRSA, and the finding of good affinity for PBP-2' supported these results. Some of the compounds having favorable protein-binding affinity showed excellent in vivo anti-MRSA activity. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00069-8

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1089±1101
Novel Dithiocarbamate Carbapenems with
Anti-MRSA Activity
Norikazu Ohtake,* Hideaki Imamura, Hideki Jona, Hideo Kiyonaga,
Aya Shimizu, Minoru Moriya, Hiroki Sato, Masato Nakano,
Ryosuke Ushijima and Susumu Nakagawa
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-2611, Japan
Received 23 January 1998; accepted 6 April 1998
AbstractÐA new series of 1b-methyl carbapenems, in which a disubstituted-aminothiocarbonylthio moiety was
attached to the C-2 position of the carbapenem nucleus, were prepared and evaluated for anti-MRSA activity. These
derivatives showed good in vitro antibacterial activity against high-level MRSA, and the ®nding of good anity for
PBP-20 supported these results. Some of the compounds having favorable protein-binding anity showed excellent in
vivo anti-MRSA activity. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
With the thiazolothio structural feature in mind, we
envisioned a dithiocarbamate moiety at the C-2 side
chain as in (7), which was arrived at by cleaving the 1±5
bond of the thiazole ring as shown in Figure 2. The
targeted dithiocarbamate carbapenems (7)7 were pre-
pared via the coupling reaction of the carbapenem
diphenylphosphate (3)9 and dithiocarbamic acid salts
(2). Compared with imipenem, they showed potent in
vitro and in vivo anti-MRSA activity, and good anity
for PBP-20.
Nosocomial infections caused by methicillin-resistant
Staphylococcus aureus (MRSA) are becoming dicult to
treat because there are few therapeutic agents that are
e€ective against MRSA. Vancomycin,1 a potent anti-
MRSA agent, is used as a ®rst-line therapeutic agent, but
its adverse e€ects restrict clinical use. Therefore, new
and potent anti-MRSA agents with better safety pro®les
are highly desired.
It was demonstrated that MRSA strains produced a
new penicillin-binding protein, PBP-20 (PBP-2a),2 which
showed low anity to various types of b-lactams such
as penicillins, cephems, and carbapenems marketed so
far, resulting in high resistance. However, it was recently
disclosed that new b-lactams such as CP0467 (I),3 LY-
206763 (II),4 SM-17466 (III),5 and a Merck compound
(IV)6 showed good anti-MRSA activity and high anity
for the PBP-20 protein of MRSA (see Figure 1). Coin-
cidentally, these b-lactams had a thiazolothio moiety at the
C-3 position of the cephem (or carbacephem) nucleus or
at the C-2 position of the carbapenem nucleus, and this
moiety was speculated to play an important role in
improving the anti-MRSA activity of these drugs.
A preliminary account of this work8 has been presented
previously, and herein we describe the synthesis and
structure±activity relationships of these novel dithio-
carbamate carbapenems and present, in depth, the bio-
logical pro®les of the represented compounds.
Results and Discussion
Chemistry
Dithiocarbamate carbapenems (7a±7z) were prepared
as shown in Scheme 1. The coupling reaction of the
carbapenem diphenylphosphates (3, 4)9 and triethyl-
ammonium 1-pyrrolidinyldithiocarbamate (2a), easily
derived from carbon disul®de and pyrrolidine (1a), did
not proceed e€ectively in THF or DMF, probably due
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00069-8

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N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
to the low nucleophilicity of 2a. After adding lithium
chloride to this system,6 the reaction proceeded
smoothly to give the desired products (5a, 6a) in good
yields. We speculated that lithium dithiocarbamate gen-
erated in situ during this reaction accelerated the pro-
duction of the coupling products. The other coupling
products (5b±5z, 6b) were obtained by the above
method. Deprotection of 5 and 6 was usually carried out
by catalytic hydrogenation in the presence of NaHCO3.
In the case of the compounds 7e, 7u, 7w, which have
double bonds in the side chains, deprotection was car-
ried out by Zinc reduction method.10 The crude carba-
penem products (7, 8) were puri®ed by reversed-phase
column chromatography followed by lyophilization to
a€ord the ®nal carbapenems.
The thiocarbamate carbapenems (11a, 11b) were pre-
pared via the acylation of 2-mercaptocarbapenem (9),11
Figure 1. The structures of the anti-MRSA b-lactams.
Figure 2. Dithiocarbamate structure envisioned from the thiazolothio moiety.
Scheme 1. Preparation of the dithiocarbamate carbapenems.

N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1091
derived from 3 according to the published method as
shown in Scheme 2.
1b-methyl analogues (8a, 8b), and their thiocarbamate
derivatives (11a, 11b) together with those of imipenem
and vancomycin, against S. aureus including MRSA,
Escherichia coli, and Pseudomonas aeruginosa. Table 1
also shows dehydropeptidase-I (DHP-1) susceptibilities.
The dithiocarbamate carbapenems (7a, 7b) showed
good antibacterial activity against not only MSSA but
also against high-level MRSA. Their anti-MRSA activity,
which was superior to that of imipenem by 16 to 32-fold,
was attributed to their good PBP-20 anity. The IC50s
of 7a, 7b and imipenem were 5.6, 9.6 and 125 mg/mL,
respectively. The dithiocarbamate carbapenems also
exhibited good-to-moderate activity against Staphy-
lococcus epidermidis, Enterococcus faecalis, and E. coli,
but were inactive against P. aeruginosa. It is evident that
the des 1b-methylation of 7a and 7b a€ected the
anti-staphylococcal activity and DHP-I susceptibility by
a factor of 1/2 to 1/4, and that replacement of the thio-
carbonyl moiety by a carbonyl moiety also reduced the
activity to 1/4 to 1/8. The above ®ndings indicate that
the 1b-methyl substitution and thiocarbonyl moiety of
the side chain are important to anti-MRSA activity.
Other dithiocarbamate carbapenems (7u-2±7u-4, 7z-2±
7z-4), including quaternized ammonium compounds,
were prepared as shown in Scheme 3. The iodide (12),
derived from the intermediate (5u-1) by selective propane-
sulfonylation of the allylic alcohol moiety and sub-
sequent substitution with sodium iodide, was
quaternized with appropriate amines such as triethyl-
amine and pyridine. The quaternized compounds were
subsequently deprotected by zinc reduction to give 5u-
2±5u-4.
Quaternization of the coupling products (5z-1, 5z-2)
with iodomethane followed by deprotection by catalytic
hydrogenation gave 7z-2, 7z-3.
Biological properties
Table 1 shows the comparative antibacterial activities of
the dithiocarbamate carbapenems (7a, 7b), their des
Scheme 2. Preparation of the thiocarbamate carbapenems.

1092
N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
These results prompted us to investigate further deriva-
tization of dithiocarbamate carbapenem to identify a
new anti-MRSA agent. Tables 2 and 3 show the anti-
bacterial activities, DHP-I susceptibility, PBP-20 anity,
and human protein-binding anity of various types of
dithiocarbamate carbapenems. All of the compounds
listed in the Tables showed good anti-MRSA activity
and PBP-20 anity as expected. The in vitro anti-MRSA
activity improved as the hydrophobicity of the side
chain moiety increased (7b!7h, 7k!7n). However, the
resulting derivatives (7h, 7n) showed high protein-bind-
ing anity [7b (88%)!7h (99%), 7k (80%)!7n
(99%)], which a€ected their in vivo activities (see
Table 4).4 Therefore, we introduced an appropriate
hydrophilic group into the side chain in order to reduce
this protein-binding anity. Introducing amine (7l!7z-
1) and quaternized amine (7z-1!7z-3, 7m!7u-3)
groups is one of the most e€ective methods available to
reduce protein-binding. In addition, the introduction of
the hydroxyl (7c!7o) and amide (7a!7v, 7e!7w,
7l!7y) moieties was found to reduce the protein-bind-
ing anity. The piperidine derivative (7z-4), having
both the amine and amide moieties, showed favorable
protein-binding anity without any loss of anti-MRSA
activity.
As for DHP-I susceptibility, the dithiocarbamate carba-
penems, except for the piperidinium derivatives (7z-2,
7z-3) and amide derivatives (7v, 7w, 7x), were generally
more susceptible than imipenem. Interestingly, the
introduction of the amide group improved DHP-I sus-
ceptibility. Quaternization of the piperidine derivative
improved the DHP-I stability greatly. However, the
stability of other quaternary ammonium derivatives (7u-
2, 7u-3, 7u-4) was not improved.5c
Among the synthesized dithiocarbamate carbapenems, 8
compounds (7b, 7h, 7o,12 7t, 7w, 7y, 7z-2, 7z-4) were
selected as representatives and further evaluated for
their in vitro and in vivo anti-MRSA activity, epilepto-
genic potential by rat intraventricular assay, and
pharmacokinetic parameters in mice, as shown in Table 4.
Except for 7h, these compounds showed good in vivo
anti-MRSA activity that was comparable or superior to
that of vancomycin against MRSA, re¯ecting their good
in vitro anti-MRSA activity. The compound 7h was
inactive in vivo due to its high protein-binding anity
(99%). The compounds 7b and 7o showed better Cmax
and AUC values than the rest. On the contrary, the
quaternary ammonium and amine derivatives (7z-2, 7z-
4) with relatively low protein-binding anities, and 7h
Scheme 3. Preparation of other dithiocarbamate carbapenems.

N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1093
with high protein-binding anity, showed low Cmax and
AUC values. The epileptogenicity of the selected com-
pounds at 100 mg/rat head, as measured by rat intra-
ventricular assay, suggested that 7z-4 had no
epileptogenic potential, although the quaternary
ammonium derivative (7z-2) showing high epileptogeni-
city. Other derivatives (7u-2±7u-4, 7z-3) having cationic
moiety on the side chain also showed high epileptogen-
icity.13 This ®nding seems to be compatible with the
results reported by Sunagawa et al.14
Varian GEM-300 spectrometer (300 MHz), in the
designated solvent, using tetramethylsilane (TMS) or
residual DOH (d 4.80) as an internal standard. Mass
spectra (MS) were measured using a JEOL JMS-
SX102A spectrometer. UV spectra were taken on a
SHIMADZU SPD-10A spectrometer in 0.1 M 3-morpho-
linopropanesulfonate (MOPS) bu€er (pH 7.0). TLC was
performed with Merck Kieselgel F254 precoated plates.
The silica gel for column chromatography was WAKO
gel C-300. Reversed-phase column chromatography was
carried out on YMC-gel ODS-AQ 120±S50.
In conclusion, we have prepared a new series of dithio-
carbamate carbapenems which showed good in vitro anti-
MRSA activity. The biological properties of the selected
compounds indicated that dithiocarbamate carba-
penems deserve attention as potential anti-MRSA agents.
General procedure for the preparation of the
dithiocarbamate carbapenems
The experimental procedure of 7a was described as a
representative example.
Experimental
General methods
(1) Triethylammonium 1-pyrrolidinyldithiocarbamate (2a).
To an ice-cooled solution of pyrrolidine (5.00 mL,
59.9 mmol) in THF (200 mL) was added triethylamine
(25.0 mL, 180 mmol) and carbon disul®de (5.40 mL,
89.9 mmol), and the mixture was stirred for 30 min at
IR absorption spectra were recorded on a Horiba FT-
1
200 spectrometer. H NMR spectra were recorded on a
Table 1. Comparative antibacterial activities (MIC, mg/mL) and DHP-I susceptibilities of the dithiocarbamate carbapenems (7a, 7b)
and their analogues
Compd
7a
8a
11a
7b
8b
11b
R1:
R2:
Me
H
Me
Me
H
Me
Organism
IPM
VCM
S. aureus 209P NIHJ JC1
S. aureus BB6294a
S. aureus CSa1009a,b
S. epidermidis MB5181a
E. faecalis MB4966
E. coli NIHJ JC2
0.012
3.13
6.25
3.13
12.5
6.25
0.025
6.25
0.05
0.012
3.13
6.25
6.25
0.006
12.5
0.05
<0.006
100
0.39
1.56
1.56
1.56
1.56
25
25
25
25
>25
>25
25
>25
100
12.5
12.5
12.5
12.5
0.2
100
6.25
6.25
>25
0.65
12.5
6.25
25
2.12
12.5
0.78
>25
0.62
0.78
1.56
>25
11.7
0.10 >25
1.56 >25
1.00
P. aeruginosa MB5002
DHP-I susceptibilityc
>25
3.18
>25
9.99
Ð
aMethicillin-resistant strain.
bb-Lactamase-producing strain.
cRelative rate of hydrolysis to imipenem, porcine renal DHP-I.

1094
N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
Table 2. In vitro antibacterial activities (MIC, mg/mL) and biological properties of dithiocarbamate carbapenems
Compd
7c
7d
7e
7f
7g
7h
7i
7j
R:
organism
S. aureus 209P NIHJ JC1
S. aureus BB6294a
S. aureus CSa1009a,b
S. epidermidis MB5181a
E. faecalis MB4966
E. coli NIHJ JC2
0.025
3.13
3.13
3.13
6.25
6.25
0.025
3.13
3.13
3.13
6.25
0.012
3.13
3.13
3.13
6.25
12.5
0.025
3.13
3.13
3.13
0.05
1.56
3.13
1.56
6.25
0.012
1.56
1.56
3.13
1.56
12.5
<0.006
0.78
1.56
<0.006
1.56
3.13
0.39
1.56
0.78
3.13
12.5
25
25
25
25
>25
>25
25
>25
12.5
P. aeruginosa MB5002
>25
>25
>25
>25
DHP-I susceptibilityc
2.26
3.4
2.71
2.18
2.0
2.51
2.29
1.94
1.8
4.41
2.05
PBP±20 anity (IC50, mg/mL)
Serum protein-binding (%)d
NT
95
5.4
94
2.9
99
NT
99
0.2
99
88
99
99
Compd
7k
7l
7m
7n
7o
7p
7q
7r
R:
organism
S. aureus 209P NIHJ JC1
S. aureus BB6294a
S. aureus CSa1009a,b
S. epidermidis MB5181a
E. faecalis MB4966
E. coli NIHJ JC2
0.025
3.13
6.25
6.25
6.25
3.13
0.025
0.025
1.56
3.13
1.56
6.25
12.5
0.012
1.56
1.56
1.56
6.25
0.025
3.13
6.25
6.25
6.25
0.78
0.025
6.25
6.25
3.13
6.25
6.25
0.025
6.25
6.25
0.025
6.25
6.25
1.56
3.13
3.13
6.25
3.13
3.13
6.25
12.5
25
>25
>25
>25
0.78
0.78
P. aeruginosa MB5002
>25
>25
>25
>25
>25
>25
DHP-I susceptibilityc
3.03
3.10
2.63
6.2
3.09
1.65
3.8
2.38
2.32
2.21
PBP±20 anity (IC50, mg/mL)
Serum protein-binding (%)d
12.0
80
2.0
98
NT
99
NT
84
8.0
69
NT
65
98
47
*
MIC was determined by an agar dilution method using Mueller±Hinton medium (Difco).
aMethicillin-resistant strain.
bb-Lactamase-producing strain.
cRelative rate of hydrolysis to imipenem, porcine renal DHP-I.
dBinding rate for human serum.
NT; not tested.

N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1095
Table 3. In vitro antibacterial activities (MIC, mg/mL) and biological properties of dithiocarbamate carbapenems
Compd
7s
7t
7u-1
7v
7w
7x
7y
R2:
organism
S. aureus 209P NIHJ JC1
S. aureus BB6294a
S. aureus CSa1009a,b
S. epidermidis MB5181a
E. faecalis MB4966
E. coli NIHJ JC2
0.025
6.25
6.25
6.25
6.25
3.13
0.012
3.13
3.13
3.13
3.13
0.012
0.025
3.13
6.25
3.13
6.25
0.2
0.025
6.25
12.5
3.13
3.13
3.13
0.012
3.13
6.25
3.13
3.13
12.5
0.025
3.13
6.25
6.25
6.25
0.39
3.13
3.13
3.13
6.25
6.25
12.5
>25
P. aeruginosa MB5002
>25
>25
25
>25
>25
>25
DHP-I susceptibilityc
2.23
2.88
1.9
3.43
2.8
0.82
2.5
0.64
2.9
0.46
1.9
1.05
2.6
PBP±20 anity (IC50, mg/mL)
Serum protein-binding (%)d
NT
69
85
94
51
90
98
64
Compd
R2:
7u-2
7u-3
7u-4
7z-1
7z-2
7z-3
7z-4
Y:
X: +NHMe
Organism
S. aureus 209P NIHJ JC1
S. aureus BB6294a
S. aureus CSa1009a,b
S. epidermidis MB5181a
E. faecalis MB4966
E. coli NIHJ JC2
0.012
3.13
3.13
3.13
6.25
0.78
25
<0.006
3.13
3.13
0.025
0.025
3.13
6.25
0.012 <0.006
0.006
6.25
3.13
6.25
1.56
1.56
3.13
12.5
3.13
6.25
1.56
1.56
1.56
3.13
3.13
1.56
3.13
0.39
3.13
6.25
3.13
6.25
3.13
1.56
0.1
1.56
1.56
P. aeruginosa MB5002
>25
>25
>25
>25
>25
25
DHP-I susceptibilityc
1.94
2.19
1.40
1.53
0.11
1.0
0.15
1.34
1.1
PBP±20 anity (IC50, mg/mL)
Serum protein-binding (%)d
2.2
87
NT
54
NT
51
2.1
91
NT
60
30
58
*MIC was determined by an agar dilution method using Mueller±Hinton medium (Difco).
aMethicillin-resistant strain.
bb-Lactamase-producing strain.
cRelative rate of hydrolysis to imipenem, porcine renal DHP-I.
dBinding rate for human serum.
NT; not tested.

1096
N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
Table 4. Anti-MRSA activities and other biological properties of the representative dithiocarbamate carbapenems
Compd
7b
7h
7o
4.36
7t
7w
7y
7z-2
5.50
7z-4
3.84
In vitro anti-MRSA activitya G-mean MIC (mg/mL)
In vivo anti-MRSA activityb ED50 (mg/mL)
Anity to PBP-20c IC50 (mg/mL)
DHP-I susceptibility
Serum protein-binding (%)d
4.59
5.91 >50
1.97
3.55
2.88
1.9
6.41
4.90
2.9
6.25
2.74
2.6
4.80
3.8
7.30
1.0
2.19
1.1
9.6
2.12
1.8
1.94
99(99)
0/5
1.65
2.88
85(92) 90(77) 64(48) 30(24) 58(34)
0.64
1.05
0.11
0.75
88(69)
0/5
47(58)
0/5
Epileptogenicity (100 mg/rat head, n=5)
0/5
0/5
0/5
5/5
0/5
Pharmacokinetic parameters in micee
Cmax (mg/mL)
T1/2 (min)
21.6
27.6
1061
38
5.1
12.5
134
24.5
22.1
13.1
18.8
11.6
13.7
10.4
13.1
5.6
9.5
4.4
12.4
AUC (mg/min/mL)
Urinary recovery (%)
1026
38
551
26
431
37
287
25
146
48
123
29
1.1
aHigh-level MRSA (27 strains); geometric-mean MIC (mg/mL) of methicillin, imipenem, and vancomycin were 3000, 115 and 1.33,
respectively.
bED50s (mg/kg) of imipenem and vancomycin were >200 and 5.56, respectively.
cIC50 (mg/mL) of imipenem was 125.
dBinding rate for human (mouse) serum at a carbapenem concentration of 10 mg/mL.
eThe drugs were administered intraperitoneally in combination with 40 mg/kg of cilastatin.
the same temperature. The resulting precipitate was
collected by ®ltration, washed with diisopropyl ether,
and dried under reduced pressure to yield 2a (13.5 g,
91%): 1H NMR (300 MHz, D2O) d 1.26 (9H, d,
J=7.0 Hz), 1.97 (4H, m), 3.18 (6H, q, J=7.0 Hz), 3.75
(4H, m); IR (KBr) cm 1 1375, 1165, 1001, 943.
10% Pd-C (300 mg), and the mixture was stirred over-
night at room temperature under a hydrogen atmos-
phere. The reaction mixture was passed through a pad
of celite and the ®ltrate was concentrated under reduced
pressure to ca. 20 mL. After the insoluble of the aqueous
layer was removed by ®ltration, the ®ltrate was sub-
jected to reversed-phase column chromatography, which
was eluted with H2O and then 20% MeOH±H2O. The
fractions detected by HPLC were combined and lyo-
(2) p-Nitrobenzyl (1R,5S,6S)-6-[(R)-1-hydroxyethyl]-2-[(1-
pyrrolidinyl)thiocarbonylthiol-1-methyl-1-carbapen-2-em-
3-carboxylate 5a. To a stirred solution of p-nitrobenzyl
(1R,5S,6S)-2-diphenoxyphosphoryloxy-6-[(R)-1-hydroxy-
ethyl]-1-methyl-1-carbapen-2-em-3-carboxylate 3 (2.00 g,
3.36 mmol) in THF (50 mL) were added 2a
(1.00 g, 4.03 mmol) and lithium chloride (171 mg,
4.03 mmol), and the mixture was stirred overnight at
room temperature under a nitrogen atmosphere. The
reaction mixture was poured into H2O and extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo. Pur-
i®cation of the residue by silica gel column chroma-
tography gave 5a (1.21 g, 74%): 1H NMR (300 MHz,
CDC13) d 1.16 (3H, d, J=7.5 Hz), 1.36 (3H, d,
J=6.3 Hz), 1.90±2.20 (4H, m), 3.37 (1H, dd, J=3.0,
6.3 Hz), 3.73 (2H, m), 3.87 (2H, d, J=3.0, 9.9 Hz), 5.25
(1H, J=14.0 Hz), 5.48 (1H, d, J=14.0 Hz), 7.64 (2H, d,
1
philized to give 7a (117 mg, 51%): H NMR (300 MHz,
D2O) d 1.11 (3H, d, J=7.0 Hz), 1.28 (3H, d, J=6.0 Hz),
1.90±2.20 (4H, m), 3.53 (1H, dd, J=3.0, 6.0 Hz),
3.60±3.90 (5H, m), 4.25 (1H, m), 4.36 (1H, dd, J=3.0,
1
10.0 Hz); IR (KBr) cm
1749, 1608, 1437, 1389;
FAB-HRMS m/z calcd for C15H20N2O4S2Na
(M+Na)+: 379.0763, found 379.0742; UV lmax 276 (e
15400).
The following compounds (7b±7d, 7f±7l, 7n±7t, 7v±7y,
7z-1, 7z-4, 8a, 8b) were prepared as described for the
preparation of 7a.
7b. 1H NMR (300 MHz, D2O) d 1.10 (3H, d, J=7.3 Hz),
1.28 (3H, d, J=6.3 Hz), 3.47 (3H, s), 3.48 (3H, s), 3.53
(1H, dd, J=3.0, 6.0 Hz), 3.71 (1H, m), 4.26 (1H, m),
1
1
J=9.0 Hz), 8.22 (2H, d, J=9.0 Hz); IR (KBr) cm
1772, 1522, 1437, 1346.
4.36 (1H, dd, J=3.0, 10.0 Hz); IR (KBr) cm 1749,
1603, 1387, 1248, 1147; FAB-HRMS m/z calcd for
C13H17N2O4S2Na2 (M+2Na-H)+: 375.0425, found
375.0430; UV lmax 277 (e 13700).
(3) Sodium (IR,5S,6S)-6-[(R)-1-hydroxyethyl]-2-[(1-
pyrrolydinyl)thiocarbonylthio]-1-methyl-1-carbapen-2-em-
3-carboxylate 7a. To
0.61 mmol) in THF (30 mL) and EtOH (5 mL) were
added NaHCO3 (51 mg, 0.61 mmol) in H2O (30 mL) and
a
solution of 5a (300 mg,
7c. 1H NMR (300 MHz, D2O) d 1.11 (3H, d, J=6.9 Hz),
1.21 (3H, d, J=6.9 Hz), 1.28 (3H, d, J=6.3 Hz), 3.43
(3H, br s), 3.50±3.55 (1H, m), 3.65±3.79 (1H, m), 3.80±

N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1097
4.12 (2H, m), 4.20±4.32 (1H, m), 4.33±4.40 (1H, m); IR
1
7n. 1H NMR (300 MHz, D2O) d 1.10 (3H, d, J=7.0 Hz),
1.26 (3H, d, J=6.0 Hz), 1.55 (4H, m), 1.83 (4H, m), 3.51
(1H, dd, J=2.5, 6.0 Hz), 3.73 (1H, m), 3.90±4.15 (4H,
m), 4.24 (1H, m), 4.35 (1H, dd, J=2.5, 10.0 Hz); IR
(KBr) cm 1760, 1602; FAB-MS m/z 429 (M+2Na-
H)+; UV lmax 282 (e 16400).
(KBr) cm 3425, 1757, 1603, 1389, 1282; FAB-MS m/z
389 (M+2Na-H)+; UV lmax 278 (e 13000).
1
1
7d. H NMR (300 MHz, D2O) d 0.89, 0.93 (3H, each d,
J=7.3 Hz), 1.10 (3H, d, J=7.3 Hz), 1.28 (3H, d,
J=6.5 Hz), 1.62±1.84 (2H, m), 3.44 (3H, s), 3.49±3.54
(1H, m), 3.66±3.79 (1H, m), 3.80±4.06 (2H, m), 4.20±
1
7o. H NMR (300 MHz, D2O) d 1.15, 1.17 (3H, each d,
1
4.31 (1H, m), 4.32±4.39 (1H, m); IR (KBr) cm 3407,
1757, 1604, 1389, 1267; FAB-MS m/z 403 (M+2Na-
H)+; UV lmax 279 (e 15900).
J=7.3 Hz), 1.33 (3H, d, J=6.3 Hz), 3.01±3.11 (1H, m),
3.55 (1H, d, J=2.3 Hz), 3.91±3.98 (2H, m), 4.09±4.15
(1H, m), 4.20±4.37 (2H, m), 4.38±4.45 (1H, m); IR
1
(KBr) cm 3430, 1749, 1608, 1506; FAB-HRMS m/z
calcd for C14H19N2O5S2Na2 (M+2Na-H)+: 405.0531,
7f. 1H NMR (300 MHz, D2O) d 1.16 (3H, d, J=7.3 Hz),
1.26±1.43 (9H, m), 3.61 (1H, m), 3.73±4.18 (5H, m),
found 405.0523; UV lmax 277 (e 13,700).
1
4.26±4.38 (1H, m), 4.39±4.45 (1H, m); IR (KBr) cm
1768, 1606, 1558, 1379; FAB-MS m/z 403 (M+2Na-
H)+; UV lmax 280 (e 15200).
1
7p. H NMR (300 MHz, D2O) d 1.07, 1.09 (3H, each d,
J=5.9 Hz), 1.22, 1.31 (3H, each d, J=7.2 Hz), 1.26
(3H, d, J=6.4 Hz), 3.51 (1H, m), 3.65±3.80 (1H, m),
3.85±4.18 (6H, m), 4.24 (1H, dq, J=6.2, 6.2 Hz), 4.31±
7g. 1H NMR (300 MHz, D2O) d 1.01 (3H, m), 1.25 (3H,
d, J=6.4 Hz), 3.46 (1H, m), 3.61 (1H, m), 3.75 (3H, s),
1
4.39 (1H, m); IR (KBr) cm 3347, 1753, 1604, 1386;
FAB-MS m/z 419 (M+2Na-H)+; UV lmax 281 (e
17100).
1
4.22 (2H, m), 7.50 (5H, m); IR (KBr) cm 1749, 1603,
1363; FAB-MS m/z 437 (M+2Na-H)+; UV lmax 281 (e
17800).
7q. 1H NMR (300 MHz, D2O) d 1.10 (3H, d, J=7.4 Hz),
1.25 (3H, d, J=6.3 Hz), 3.52 (1H, dd, J=2.9, 5.7 Hz),
3.70 (1H, m), 4.00±4.30 (3H, m), 4.34 (1H, m), 4.40±
4.80 (3H, m); IR (KBr) cm 3400, 1755, 1604, 1489,
1390; FAB-MS m/z 381 (M+Na)+; UV lmax 279 (e
15800).
1
7h. H NMR (300 MHz, D2O) d 1.02, 1.12 (3H, each d,
J=7.3 Hz), 1.27 (3H, d, J=6.3 Hz), 3.44, 3.47 (3H, each
s), 3.52 (1H, m), 3.74 (1H, m), 4.25 (1H, m), 4.35 (1H,
m), 5.00±5.40 (2H, m), 7.20±7.50 (5H, m); IR (KBr)
1
1
cm 1749, 1608, 1387; FAB-MS m/z 451 (M+2Na-
H)+; UV lmax 280 (e 17300).
7r. 1H NMR (300 MHz, D2O) d 1.12 (3H, d, J=7.0 Hz),
1.29 (3H, d, J=6.0 Hz), 1.65 (2H, m), 2.02 (2H, m), 3.54
(1H, dd, J=3.0, 6.0 Hz), 3.65±3.90 (3H, m), 4.06 (1H,
m), 4.26 (1H, m), 4.37 (1H, dd, J=3.0, 10.0 Hz); IR
7i. 1H NMR (300 MHz, D2O) d 0.64±0.92 (3H, m),
1.09±1.15 (3H, m), 3.04±3.74 (5H, m), 4.11±4.25 (2H,
m), 4.83±5.63 (2H, m), 6.96±7.80 (7H, m); IR (KBr)
1
1
cm
3406, 1753, 1603, 1389; FAB-MS m/z 489
(KBr) cm 1749, 1604, 1434, 1386; FAB-MS m/z 395
(M+Na)+; UV lmax 277 (e 16100).
(M+Na)+; UV lmax 284 (e 24000).
7j. 1H NMR (300 MHz, D2O) d 1.08 (3H, d, J=7.3 Hz),
1.27 (3H, d, J=6.3 Hz), 3.38±3.56 (4H, m), 3.72 (1H,
m), 4.18±4.42 (1H, m), 5.15±5.55 (2H, m), 7.02 (1H, m),
7.16 (1H, m), 7.41 (1H, m); IR (KBr) cm 1747, 1612,
1392; FAB-MS m/z 435 (M+Na)+; UV lmax 281 (e
16300).
7s. 1H NMR (300 MHz, D2O) d 1.08 (3H, d, J=7.0 Hz),
1.25 (3H, d, J=6.5 Hz), 1.62 (2H, m), 2.00 (2H, m), 3.50
(1H, dd, J=2.5, 5.5 Hz), 3.60±3.85 (4H, m), 4.03 (1H,
m), 4.23 (1H, m), 4.35 (2H, m); IR (KBr) cm
1749,1602, 1430, 1386; FAB-MS m/z 409 (M+Na)+;
1
1
UV lmax 281 (e 16400).
7k. 1H NMR (300 MHz, D2O) d 1.10 (3H, d, J=7.3 Hz),
1.27 (3H, d, J=6.6 Hz), 2.37 (4H, m), 3.5 3 (1H, dd,
J=3.0, 6.0 Hz), 3.72 (1H, m), 4.20±4.40 (6H, m); IR
(KBr) cm 1751, 1601, 1489, 1394; FAB-MS m/z 387
(M+2Na-H)+; UV lmax 280 (e 15600).
7t. 1H NMR (300 MHz, D2O) d 1.08 (3H, d, J=7.0 Hz),
1.25 (3H, d, J=6.5 Hz), 1.55±2.30 (6H, m), 3.51 (1H,
m), 3.71 (1H, m), 3.80±4.15 (4H, m), 4.23 (1H, m), 4.34
1
1
(1H, dd, J=3.0, 9.5 Hz); IR (KBr) cm 3400, 1745,
1691, 1612, 1390; FAB-HRMS m/z calcd for
C17H24N2O5S2Na
(M+Na)+:
423.1024,
found
7l. 1H NMR (300 MHz, D2O) d 1.11 (3H, d, J=7.3 Hz),
1.28 (3H, d, J=6.5 Hz), 1.71 (6H, m), 3.52 (1H, dd,
J=3.0, 5.9 Hz), 3.69 (1H, m), 3.90±4.30 (5H, m), 4.36
(1H, dd, J=3.0, 9.8 Hz); IR (KBr) cm 1749, 1603,
1227; FAB-MS m/z 393 (M+Na)+; UV lmax 280 (e
18000).
423.1018; UV lmax 280 (e 17700).
1
7v. H NMR (300 MHz, D2O) d 1.12, 1.16 (3H, each d,
J=7.4 Hz), 1.32 (3H, d, J=6.3 Hz), 3.56 (3H, s), 3.52±
3.60 (1H, m), 3.67±3.80 (1H, m), 4.25±4.35 (1H, m), 4.41
(1H, dd, J=3.0, 9.7 Hz), 4.46±4.48 (2H, m); IR (KBr)
1

1098
N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1
cm 3391, 1751, 1680, 1605, 1390; FAB-HRMS m/z
calcd for C14H19N3O5S2Na (M+Na)+: 396.0664, found
396.0668; UV lmax 280 (e 8350).
in DMF (5.9 mL) were added sodium hydrosul®de-
hydrate (67.0 mg, 1.20 mmol) in DMF (2.0 mL) and
diisopropylethylamine (0.41 mL, mmol) at 40 ꢀC under
a nitrogen atmosphere. After the mixture was stirred for
20 min at the same temperature, dimethylcarbamyl
chloride (0.22 mL, 2.40 mmol) was added, and the
resulting mixture was further stirred overnight at 4 ꢀC.
The reaction mixture was concentrated in vacuo and the
residue was puri®ed by silica gel column chromato-
graphy to yield 10b (180 mg, 40%): 1H NMR (300 MHz,
7w. 1H NMR (300 MHz, D2O) d 1.02±1.16 (3H, m), 1.26
(3H, d, J=6.4 Hz), 3.48±3.58 (1H, m), 3.60±3.75 (1H,
m), 4.19±4.30 (1H, m), 4.30±4.40 (1H, m), 4.42±4.73
(3H, m), 5.15±5.38 (2H, m), 5.75±5.98 (1H, m); IR
(KBr) cm 1749, 1683, 1604, 1394; FAB-MS m/z 422
(M+Na)+; UV lmax 281 (e 14800).
1
CDCl3)
d 1.17 (3H, d, J=7.4 Hz), 1.34 (3H, d,
7x. 1H NMR (300 MHz, D2O)
d
1.04 (3H, d,
J=6.3 Hz), 3.01 (3H, brs), 3.08 (3H, brs), 3.37 (1H, dd,
J=3.0, 6.3 Hz), 4.05±4.13 (1H, m), 4.28 (1H, m), 4.37
(1H, d, J=3.5, 10.3 Hz), 5.25 (1H, J=13.5 Hz), 5.49
(1H, d, J=13.5 Hz), 7.65 (2H, d, J=8.9 Hz), 8.22 (2H,
d, J=8.9 Hz); IR (KBr) cm 1772, 1668, 1522, 1348;
FAB-MS m/z (M+H)+ 449.
J=7.3 Hz), 1.25 (3H, d, J=6.4 Hz), 3.49±3.51 (1H, m),
3.64±3.73 (1H, m), 4.21±4.25 (1H, m), 4.34 (1H, d,
J=9.6 Hz), 4.53±4.64 (2H, m), 5.17 (2H, s), 7.32±7.45
1
1
(5H, m); IR (KBr) cm 1764, 1751, 1681, 1608, 1392;
FAB-MS m/z 494 (M+2Na-H)+; UV lmax 281 (e
16600).
(2) To a solution of 10b (170 mg, 0.38 mmol) in THF
(5.1 mL) and EtOH (0.85 mL) were added NaHCO3
(32 mg, 0.38 mmol) in H2O (5.1 mL) and 10% Pd-C
(50 mg), and the mixture was stirred for 4.5 h at room
temperature under a hydrogen atmosphere. The reac-
tion mixture was passed through a pad of celite and the
®ltrate was concentrated in vacuo to ca. 5 mL. After the
insoluble of the aqueous layer was removed by ®ltra-
tion, the ®ltrate was subjected to reversed-phase column
chromatography, which was eluted with H2O and then
10% CH2CN±H2O. The fractions detected by HPLC
were combined and lyophilized to give 11b (40 mg,
31%): 1H NMR (300 MHz, D2O) d 1.11 (3H, d,
J=7.0 Hz), 1.28 (3H, d, J=6.0 Hz), 1.90±2.20 (4H, m),
3.53 (1H, dd, J=3.0, 6.0 Hz), 3.60±3.90 (5H, m), 4.25
7y. 1H NMR (300 MHz, D2O) d 1.09 (3H, d, J=7.3 Hz),
1.26 (3H, d, J=6.4 Hz), 3.42±3.58 (3H, m), 3.60±3.78
(1H, m), 4.15±4.32 (2H, m), 4.34±4.42 (1H, m); IR
(KBr) cm 1751, 1670, 1394; FAB-HRMS m/z calcd
for C15H19N3O5S2Na (M+Na)+: 375.0425, found
1
375.0430; UV lmax 279 (e 15300).
7z-1. 1H NMR (300 MHz, D2O)
d 1.12 (3H, d,
J=7.3 Hz), 1.29 (3H, d, J=6.4 Hz), 2.32 (3H, s), 2.64
(4H, m), 3.55 (1H, dd, J=3.0, 5.9 Hz), 3.69 (1H, m),
1
3.90±4.50 (6H, m); IR (KBr) cm 1757, 1604, 1389,
1288; FAB-MS m/z 408 (M+Na)+; UV lmax 281 (e
16100).
7z-4. 1H NMR (300 MHz, D20)
d
1.17 (3H, d,
(1H, m), 4.36 (1H, dd, J=3.0, 10.0 Hz); IR (KBr) cm
1
J=7.2 Hz), 1.34 (3H, d, J=6.5 Hz), 2.84 (3H, s), 3.61
(1H, dd, J=2.8, 5.8 Hz), 3.68±3.85 (5H, m), 4.21 (2H, s),
4.20±4.42 (4H, m), 4.33 (1H, quint, J=6.1 Hz), 4.44
(1H, dd, J=2.8, 9.8 Hz), 4.66±4.72 (2H, m), 5.73±5.81
(1H, m); IR (KBr) cm 1760, 1660, 1600, 1380, 1220;
FAB-HRMS m/z calcd for C18H27N4O5S2 (M+H)+:
1749, 1608, 1437, 1389; FAB-HRMS m/z calcd for
C13H17N2O5SNa2 (M+2Na-H)+: 359.0654, found
359.0666; UV lmax 286 (e 8260).
1
The compound 11a was prepared as described for the
preparation of 11b.
443.1423, found 443.1434; UV lmax 281 (e 19400).
11a. 1 NMR (300 MHz, D2O) d 1.12 (3H, d, J=7.0 Hz),
1.28 (3H, d, J=6.0 Hz), 1.90 (4H, m), 3.30±3.70 (6H,
8a. 1H NMR (300 MHz, D2O) d 1.25 (3H, d, J=6.5 Hz),
1.95±2.10 (4H, m), 2.99 (1H, dd, J=9.5, 17.0 Hz), 3.45±
3.52 (2H, m), 3.65±3.85 (4H,m), 4.15±4.35 (2H, m); IR
1
m), 4.25 (2H, m); IR (KBr) cm 3400, 1790, 1640,
1610, 1370; FAB-HRMS m/z calcd for C15H20N2O5SNa
(M+Na)+: 363.0991, found 363.0973; UV lmax 287 (e
6480).
1
(KBr) cm 1764, 1600, 1430; FAB-MS m/z (M+Na)+
365; UV lmax 275 (e 12100).
8b. 1H NMR (300 MHz, D2O) d 1.24 (3H, d, J=6.5 Hz),
2.97 (1H, dd, J=9.0, 15.0 Hz), 3.25±3.50 (8H, m), 4.15±
4.35 (2H, m); IR (KBr) cm 1801, 1600, 1373; FAB-
MS m/z (M+Na)+ 339; UV lmax 275 (e 12100).
Sodium (1R,5S,6S)-2-(N-allyl-N-methylaminothiocarbon-
ylthio)-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-em-
3-carboxylate (7e). To a solution of 5e (400 mg,
0.81 mmol) in THF (8.0 mL) and 0.35 M phosphate
bu€er (pH 6.0, 15 mL) was added zinc dust (1.2 g,
mmol), and the mixture was stirred vigorously for 2 h.
The reaction mixture was passed through a pad of celite
and the ®ltrate was concentrated in vacuo to ca. 20 mL.
1
Sodium (1R,5S,6S)-2-[dimethylaminocarbonylthio]-6-[(R)-
1-hydroxyethyl]-methyl-1-carbapen-2-em-3-carboxylate
(11b). (1) To a stirred solution of 3 (590 mg, 0.99 mmol)

N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1099
After the insoluble of the aqueous layer was removed by
®ltration, the ®ltrate was subjected to reversed phase
column chromatography, which was eluted with 3%
CH3CN±H2O. The fractions detected by HPLC were
(2) To a solution of 12 (700 mg, 1.08 mmol) in CH3CN
(26 mL) was added pyridine (90 mg, 1.14 mmol), and the
mixture was stirred overnight at room temperature. The
reaction mixture was concentrated in vacuo, and the
residue was deprotected by a similar method for the
preparation of 7e to give 7u-3 (17%): 1H NMR
(300 MHz, D2O) d 1.07 (3H, d, J=7.3 Hz), 1.26 (3H, d,
J=6.4 Hz), 2.16±2.31 (2H, m), 3.51 (1H, dd, J=3.1,
6.0 Hz), 3.60±3.75 (1H, m), 4.00±4.20 (2H, m), 4.24 (1H,
quint, J=6.3 Hz), 4.33 (1H, dd, J=2.5, 9.5 Hz), 4.39±
4.71 (2H, m), 5.24 (2H, s), 5.88±5.98 (1H, m), 8.08 (2H,
dd, J=6.6, 8.8 Hz), 8.57 (1H, t, J=7.8 Hz), 8.83 (2H, d,
J=5.5 Hz); IR (KBr) cm 1 1756,1604, 1425, 1380; FAB-
MS m/z 460 (M+H)+; UV lmax 280 (e 17300).
1
combined and lyophilized to give 7e (132 mg, 42%): H
NMR (300 MHz, D2O) d 1.13, 1.15 (3H, each d,
J=6.9 Hz), 1.32 (3H, d, J=6.3 Hz), 3.48, 3.50 (3H, each
s), 3.52±3.60 (1H, m), 3.69±3.83 (1H, m), 4.24±4.35 (1H,
m), 4.36±4.44 (1H, m), 4.52±4.65, 4.72±4.78 (2H, each
m), 5.17±5.40 (2H, m), 5.80±6.01 (1H, m); IR (KBr)
1
cm 3431, 1759, 1606, 1367, 1267; FAB-MS m/z 401
(M+2Na-H)+; UV lmax 279 (e 16600).
The following compounds (7m, 7u-1) were prepared as
described for the preparation of 7e.
The following compounds (7u-2, 7u-4) were prepared as
described for the preparation of 7u-3.
7m. 1H NMR (300 MHz, D2O)
d 1.18 (3H, d,
J=6.9 Hz), 1.35±1.37 (3H, m), 2.35±2.50 (2H, 3.59±3.62
(1H, m), 3.77 (1H, dq, J=6.8, 7.3 Hz), 4.18±4.24, 4.60±
4.7 (3H, each m), 4.28±4.37 (1H, m), 5.74±5.90 (1H, m),
7u-2. 1H NMR (300 MHz, D2O) d 1.17 (3H, d,
J=7.2 Hz), 1.34 (3H, d, J=6.3 Hz), 2.52±2.82 (2H, m),
3.17 (9H, s), 3.61 (1H, dd, J=3.0, 5.9 Hz), 3.71±3.83
(1H, m), 4.04 (2H, s), 4.10±4.48 (4H, m), 4.61±4.99 (2H,
m), 6.21±6.32 (1H, m); IR (KBr) cm 1756,1604, 1419,
1384; FAB-MS m/z 440 (M+H)+; UV lmax 279 (e
15100).
1
6.03±6.13 (1H, m); IR (KBr) cm
3396, 1753,1386;
FAB-MS m/z 413 (M+2Na-H)+; UV lmax 281 (e
18100).
1
7u-1. 1H NMR (300 MHz, D2O) d 1.15±1.21 (3H, m),
1.35 (3H, d, J=6.4 Hz), 2.32±2.46 (2H, m), 3.61 (1H,
dd, J=3.0, 5.9 Hz), 3.76 (1H, dq, J=7.6, 9.8 Hz),
4.10±4.23 (4H, m), 4.28±4.33 (1H, quint, J=6.1 Hz),
7u-4. 1H NMR (300 MHz, D2O) d 1.14 (3H, d,
J=7.2 Hz), 1.31 (3H, d, J=6.0 Hz), 2.52±2.75 (2H, m),
3.21 (3H, s), 3.42±3.50 (2H, m), 3.53±3.65 (3H, m), 3.71
(1H, dd, J=7.8, 9.8 Hz), 4.02±4.20 (9H, m), 4.30 (1 H,
quint, J=3.0 Hz), 4.41 (1H, dd, J=3.0, 9.9 Hz), 6.23±
4.43 (1H, dd, J=2.9, 9.6 Hz), 4.66±4.72 (2H, m),
1
5.72±5.82 (1H, m); IR (KBr) cm
1754,1604, 1388;
FAB-MS m/z 443 (M+2Na-H)+; UV lmax 280 (e
17700).
1
6.29 (1H, m); IR (KBr) cm 1749, 1271; FAB-MS m/z
482 (M+H)+; UV lmax 274 (e 19400).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-1-methyl-2-[4-(pyr-
idinium)methyl-1,2,3,6-tetrahydropyridin-1-yl]thiocarbon-
ylthio]-1-carbapen-2-em-3-carboxylate (7u-3). (1) To a
solution of 5u-1 (5.90 g, 11.1 mmol) in TUF (47 mL)
were added diisopropylethylamine (7.51 mL, 14.4 mmol)
and n-propanesulfonylchloride (1.37 mL, 12.2 mmol),
and the mixture was stirred for 2 h at 0 ꢀC. The reaction
mixture was poured into H2O and extracted with
EtOAc. The organic layer was washed with 0.1 N HCl,
5% aqueous NAHCO3, and brine, dried over MgSO2,
and concentrated in vacuo. To a solution of the residue
in acetone (30 mL) was added sodium iodide (4.99 g,
3.33 mmol) at 0 ꢀC, and the mixture was stirred for 2 h
at room temperature. The reaction mixture was poured
into H2O and extracted with EtOAc. The organic layer
was washed with 5% aqueous Na2S2O3, and brine, dried
over MgSO4, and concentrated in vacuo to give 12
(1R,5S,6S)-2-[(4,4-Dimethylpiperadinio)thiocarbonylthio]-
6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-em-3-car-
boxylate (7z-2). To an ice-cooled solution of 5z-1
(500 mg, 0.96 mmol) in acetone (10 mL) and THF
(10 mL) was added iodomethane (0.3 mL, 4.8 mmol) and
the mixture was stirred for 2 h at the same temperature.
After being stirred for additional 2 h at room tempera-
ture, the reaction mixture was concentrated in vacuo.
To a solution of the residue in THF (25 mL) and EtOH
(5.0 mL) were added NaHCO3 (80 mg, 0.95 mmol) in
H2O (25 mL) and 10% Pd-C (500 mg), and the mixture
was stirred overnight at room temperature under a
hydrogen atmosphere. The reaction mixture was passed
through a pad of celite and the ®ltrate was concentrated
in vacua to ca. 20 mL. After the insoluble of the aqueous
layer was removed by ®ltration, the ®ltrate was sub-
jected to reversed-phase column chromatography, which
was eluted with H2O and then 10% MeOH±H2O. The
fractions detected by HPLC were combined and lyo-
philized to give 7z-2 (48 mg, 13%): 1H NMR (300 MHz,
D2O) d 1.09 (3H, d, J=7.3 Hz), 1.27 (3H, d, J=6.8 Hz),
3.28 (6H, s), 3.50±3.70 (7H, m), 4.25 (1H, m), 4.38 (1H,
1
(6.33 g, 89%): H NMR (300 MHz, CDCl3) d 1.15 (3H,
d, J=7.3 Hz), 1.36 (3H, d, J=6.3 Hz), 2.38±2.53 (2H,
m), 3.37 (1H, m), 3.92 (2H, s), 3.95±4.35 (3H, m), 4.42±
4.65 (2H, m), 5.24 (1H, d, J=13.8 Hz), 5.48 (1H, d,
J=13.8 Hz), 7.63 (2H, d, J=8.2 Hz), 8.21 (2H, d,
1
J=8.2 Hz); IR (KBr) cm 1771,1522, 1435.

1100
N. Ohtake et al./Bioorg. Med. Chem. 6 (1998) 1089±1101
1
dd, J=3.0, 10.0 Hz), 4.50 (3H, brs); IR (KBr) cm
1749, 1603, 1387; FAB-MS m/z 400 (M+H)+; UV lmax
280 (e 13500).
40 mg/kg. The levels of carbapenems were determined
by biological assay with a paper disk method using
Bacillus subtilis ATCC 12432 as the indicator organism.
The inoculated agar plates (antibiotic medium No. 1;
Difco) were incubated at 37 ꢀC for 16 h. The contents of
the disk were calculated from a standard curve.
The compound 7z-3 was prepared as described for the
preparation of 7z-2.
7z-3. 1H NMR (300 MHz, D2O)
J=7.3 Hz), 1.26 (3H, d, J=6.3 Hz), 3.15 (3H, s), 3.46±
d 1.08 (3H, d,
Acknowledgements
3.80 (6H, m), 4.08±4.42 (4H, m), 7.45±7.62 (5H, m); IR
1
We are grateful to Ms. T. Ahmed and Ms. A. Dobbins,
Co., for their critical reading of this
(KBr) cm 1766, 1456; FAB-MS m/z 476 (M+H)+;
Merck
manuscript.
&
UV lmax 280 (e 15200).
Determination of MIC. MIC was determined by agar
dilution method using Mueller±Hinton medium (Difco).
The bacterial suspension was inoculated at ca. 104 cells
per spot on the agar plates. The MIC was de®ned as the
lowest concentration which inhibited visible growth
after incubation at 37 ꢀC for 18 h. In the case of methi-
cillin-resistant staphylococci (MRS), 2% NaCl was
supplemented in the medium, and inoculated agar plates
were incubated at 35 ꢀC for 48 h. MRS strains used in
this study were isolated recently at di€erent hospitals in
Japan.
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