Dicationic dithiocarbamate carbapenems with anti-MRSA activity

Article abstract of DOI:10.1016/S0968-0896(01)00044-X

A new class of β-methylcarbapenems bearing a doubly quaternarized 1,4-diazabicyclooctane (DABCO) substituted dithiocarbamate moiety at the C-2 side chain was prepared, and the biological profiles of the compounds, including in vitro and in vivo anti-MRSA activity and DHP-I susceptibility, were evaluated to identify a carbapenem derivative that was superior to BO-3482 (1). As a result, we discovered a 1@β-methyl-2-[4-(4-carbamoylmethyl-1,4-diazabicyclo[2,2,2octanediium-1-yl)methyl-1,2,3,6-tetrahydropyridinylthiocarbonylthio]carbapenem, 14a showing greater than 2-fold better anti-MRSA activity in a mouse infection model and 3-fold better DHP-I susceptibility as compared with BO-3482 (1). Copyright

Full text of DOI:10.1016/S0968-0896(01)00044-X

Bioorganic & Medicinal Chemistry 9 (2001) 1571–1578
Dicationic Dithiocarbamate Carbapenems with
Anti-MRSA Activity
Hideaki Imamura,* Norikazu Ohtake, Hideki Jona, Aya Shimizu, Minoru Moriya,
Hiroki Sato, Yuichi Sugimoto, Chinatsu Ikeura, Hideo Kiyonaga, Masato Nakano,
Rie Nagano, Shinnosuke Abe, Koji Yamada, Terutaka Hashizume
and Hajime Morishima
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, Okubo-3, Tsukuba 300-2611, Ibaraki, Japan
Received 7 December 2000; accepted 31 January 2001
Abstract—A new class of 1b-methylcarbapenems bearing a doubly quaternarized 1,4-diazabicyclooctane (DABCO) substituted
dithiocarbamate moiety at the C-2 side chain was prepared, and the biological profiles of the compounds, including in vitro and in
vivo anti-MRSA activity and DHP-I susceptibility, were evaluated to identify a carbapenem derivative that was superior to BO-
3482 (1). As a result, we discovered a 1b-methyl-2-[4-(4-carbamoylmethyl-1,4-diazabicyclo[2,2,2]octanediium-1-yl)methyl-1,2,3,6-
tetrahydropyridinylthiocarbonylthio]carbapenem, 14a showing greater than 2-fold better anti-MRSA activity in a mouse infection
model and 3-fold better DHP-I susceptibility as compared with BO-3482 (1). # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
MRSA activity. Ultimately, we selected BO-3482 (1) for
further evaluation based on its balanced in vitro and in
vivo anti-MRSA activity together with its low seizure
potential. However, the in vivo anti-MRSA activity and
susceptibility of BO-3482 (1) to renal dehydropeptidase-I
(DHP-I) remain to be improved.
Methicillin-resistant Staphylococcus aureus (MRSA) has
been recognized as one of the major pathogens causing
nosocomial infections. In spite of severe side effects,
vancomycin,1 a glycopeptide antibiotic, has been used
as a first-line therapeutic agent for infections caused by
MRSA. Alternative therapeutic agents with fewer side
effects have been clinically desired. During the past
decade, extensive synthetic efforts have been made to
confer anti-MRSA activity on b-lactams such as a
cephalosporin or a carbapenem. As a result, some
cephalosporin2 and carbapenem3 derivatives with
potent in vitro anti-MRSA activity were identified by
introducing hydrophobic functional groups into the C-3
or C-7 side chain of the cephalosporin nucleus or the
C-2 side chain of the carbapenem nucleus. However,
clinical application of these anti-MRSA b-lactams has
not been achieved because they may have unfavorable
biological and physicochemical properties due to their
increased hydrophobicity.
Derivatization of dithiocarbamate carbapenems by
introducing hydrophobic side chains into the C-2 posi-
tion resulted in excellent in vitro anti-MRSA activity,
whereas in vivo anti-MRSA activity was unexpectedly
reduced, probably due to high serum protein binding
rates. These hydrophobic dithiocarbamate carbapenems
showed higher susceptibility to DHP-I than did imi-
penem. Introduction of a quaternary ammonium moiety
into the C-2 side chain is one way to reduce the protein
binding rate and to improve DHP-I susceptibility. In
fact, quaternarization of amine moieties of some
dithiocarbamate carbapenems resulted in decreasing the
protein binding rates to some extent and improving
DHP-I susceptibility. Unfortunately, these quaterna-
rized carbapenems displayed significant epileptogenicity
in an intracerebroventricular rat head assay.
In a previous paper,4 we reported that dithiocarbamate
carbapenems showed potent in vitro and in vivo anti-
Recently, Merck scientists discovered two novel anti-
MRSA carbapenems, L-742,7285 and L-786,392,6 which
showed excellent in vitro and in vivo anti-MRSA
activity together with good DHP-I stability. In particular,
*Corresponding author. Tel.:+81-298-77-2000; fax: +81-298-77-
2027; e-mail: imamrahk@banyu.co.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00044-X

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H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
L-786,392 possessed extended antibacterial activity
against vancomycin-resistant Enterococci (Fig. 1). We
speculated that these carbapenems with highly hydro-
phobic C-2 side chains, such as a fluorenone and naph-
tylsultam group, became sufficiently hydrophilic after
the introduction of a doubly quaternarized 1,4-diazabi-
cyclooctane (DABCO) moiety, resulting in excellent in
vivo anti-MRSA activity. On the based of this specula-
tion, we tried to introduce a DABCO group into the
side chains of representative dithiocarbamate carbape-
nems (2, 3 and 4, see Fig. 2) to improve in vivo anti-
MRSA activity. In this paper, we describe the synthesis
Result and Discussion
Chemistry
Preparation of 10a was outlined in Scheme 1 as a gen-
eral procedure for dicationic dithiocarbamate carbape-
nems (Table 1). The benzyl alcohol moiety of a
carbapenem intermediate 7, derived from 5,7 was selec-
tively activated with n-propanesulfonyl chloride and
subsequently treated with sodium iodide to give an
iodide 8. Iodide 8 was substituted with N-substituted
DABCO triflate salt 9a5a to afford a protected doubly
quaternarized carbapenem, which was deprotected by
catalytic hydrogenation in a mixture of THF and sodium
3-morpholinopropanesulfonate buffer (MOPS, pH 7.0) at
room temperature, giving the crude carbapenem. Pur-
ification and ion-exchange of the crude material was
of dithiocarbamate carbapenems with
a DABCO
moiety and their biological properties, including in
vitro and in vivo anti-MRSA activity and DHP-I
susceptibility.
Figure 1. Chemical structure of Merck compounds.
Figure 2. Dithiocarbamate carbapenems.
Scheme 1. Preparation of the dithiocarbamate carbapenem 10a. Reagents: (a) i-Pr2NEt, LiCl, THF, rt, (b) (i) 1-propanesulfonyl chloride, i-Pr2NEt,
THF, 0ꢀC; (ii) Nal, acetone, 0ꢀC, (c) (i) CH3CN, rt; (ii) 10% Pd/C, H2, THF–H2O, rt.

H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
1573
Table 1. Dicationic dithiocarbamate carbapenems
concurrently performed by reversed-phase column
chromatography to produce 10a as an amorphous
powder after lyophilization.
human serum protein binding rates and epileptogenic
potential, of the dicationic dithiocarbamate carba-
penems (10a–c, 11, 12, 13 and 14a–c) and the original
compounds (2, 3 and 4). Both imipenem (IPM) and
vancomycin (VCM) were used as reference drugs.
A 100-g scale preparation of the selected compound 14a
is highlighted in Scheme 2. Protected doubly qua-
ternarized carbapenem 17a was prepared by reacting an
iodide 16 with 9a in 98% yield. Deprotection of 17a was
carried out by the Zinc reduction method8 to give the
crude carbapenem, which was purified, ion-exchanged
by reversed-phase column chromatography, and crystal-
lized from 80% EtOH–H2O to produce 14a as colorless
prisms.
First, we looked at the effects of the DABCO moiety in
the dicationic derivatives (10a, 11, 12, 13, and 14a) on
antibacterial activity against MRSA and methicillin-
resistant Staphylococcus epidermidis (MRSE). Gen-
erally, these derivatives showed potent in vitro anti-
MRSA activity and high affinity to PBP (penicillin-
binding protein)-20, similar to the effects of the original
compounds (2, 3 and 4). It should be noted that a
DABCO moiety was effective in enhancing antibacterial
activity against the MRSE strain in the case of
the phenyl (10a) and 1,2,3,6-tetrahydropyridyl (14a)
derivatives.
Biological properties
Table 2 shows the in vitro antibacterial activity and
other biological properties, such as DHP-I susceptibility,
Scheme 2. Preparation of the dithiocarbamate carbapenem 14a. Reagents: (a) (i) 1-propanesulfonyl chloride, i-Pr2NEt, THF, 0ꢀC; (ii) Nal, acetone,
0ꢀC; (b) 9a, CH3CN, rt, (c) Zu dust, THF, phosphate buffer (pH 6.5), rt.

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H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
Table 2. In vitro activitya and biological properties of dithiocarbamate carbapenems
Organism
10a
10b
10c
11
2
IPM
VCM
S. aureus 209P NIHJ JC1
S. aureus BB6294b
ꢁ0.006
1.56
1.56
0.78
3.13
ꢁ0.006
3.13
3.13
0.78
3.13
0.012
6.25
6.25
3.13
6.25
ꢁ0.006
3.13
6.25
0.39
1.56
0.012
1.56
1.56
3.13
1.56
ꢁ0.006
100
100
100
0.78
1.56
0.39
1.56
1.56
1.56
1.56
S. aureus CSa1009b
S. epidermidis MB5181b
E. faecalis MB4966
P. aeruginosa MB5002
>25
>25
>25
12.5
>25
>25
DHP-I susceptibilityc
1.76
2.3
85
1.67
1.7
81
1.71
12
56
0/5
2.55
2.1
70
1.94
1.8
99
1.00
>25
<5.0
5/5
—
—
41
—
PBP-20 affinity (IC50, mg/mL)
Serum protein-binding (%)d
Epileptogenicity (200 mg/rat head, n=5)
1/5
2/5
0/5
0/5
12
13
3
14a
14b
14c
4
S. aureus 209P NIHJ JC1
S. aureus BB6294b
ꢁ0.006
1.56
3.13
0.39
1.56
ꢁ.006
1.56
1.56
0.78
0.78
ꢁ0.006
0.78
1.56
0.39
1.56
ꢁ0.006
3.13
3.13
0.78
3.13
ꢁ0.006
3.13
6.25
0.78
3.13
ꢁ0.006
3.13
6.25
0.78
3.13
0.025
1.56
3.13
1.56
6.25
S. aureus CSa1009b
S. epidermidis MB5181b
E. faecalis MB4966
P. aeruginosa MB5002
>25
>25
>25
>25
>25
>25
>25
DHP-I susceptibilityc
2.37
1.9
90
2.45
1.6
93
4.41
—
99
0/5
0.51
1.1
55
0.57
2.7
—
0.46
—
59
0/5
2.63
6.2
98
PBP-20 affinity (IC50, mg/mL)
Serum protein-binding (%)d
Epileptogenicity (200 mg/rat head, n=5)
0/5
0/5
0/5
0/5
0/5
aMIC (mg/ml) determined by agar dilution method.
bMethicillin-resistant.
cRelative rate of hydrolysis to imipenem, porcine renal DNP-I.
dBinding rate for human serum.
Since the monocationic derivatives exhibited significant
epileptogenicity,4c we were concerned that this class of
dicationic dithiocarbamate carbapenems could also
have epileptogenic potential. Fortunately, these dica-
tionic derivatives showed far less epileptogenicity than
did the monocationic derivatives in the rat head assay,
suggesting that the dicationic DABCO moiety may not
induce seizures as does the monocationic ammonium
group.
corresponding dicationic derivatives (10a, 13 and 14a)
and vancomycin showed ED50 values of 6.0, 7.7, 1.9 and
5.5 mg/kg, respectively, suggesting that the DABCO
moiety may play a key role in improving in vivo anti-
MRSA activity. We assumed that the good in vivo anti-
MRSA activity was related to the significant decrease in
protein binding rates. Since the protein binding rate
(99%) of 2, for example, was improved up to 85% in the
case of 10a, the plasma concentration of the free carba-
penem of 10a was approximately 15-fold higher than
that of 2, so that 10a showed approximately 10-fold
improvement in in vivo efficacy in comparison with 2.
Next, the effect of the DABCO moiety on the serum
protein binding rate was examined. As expected, the
DABCO moiety effectively contributed to reduce serum
protein binding rates in the phenyl and 1,2,3,6-tetra-
hydropyridyl derivatives (11=70%, 14a=55%). How-
ever, the naphtyl derivatives (12 and 13) still showed
high protein binding rates (12=90%, 13=93%) in spite
of the introduction of the DABCO moiety.
Optimization of the carbamoylmethyl group on the
quaternarized DABCO moiety of 10a and 14a was car-
ried out by replacement with a hydroxyethyl (10b and
10c) or hydroxypropyl group (14b and 14c). In the
phenyl derivatives (10b and 10c), in vitro anti-MRSA
activity was decreased compared with that of 10a, while
the 1,2,3,6-tetrahydropyridyl derivatives (14b and 14c)
retained the in vitro anti-MRSA activity to some extent
(Table 2). Taken together, it was concluded that 14a
With respect to DHP-I susceptibility, the 1,2,3,6-tetra-
hydropyridyl derivative (14a) showed approximately
5-fold improvement as compared with the original
compound 4. The other dicationic derivatives (10a, 11,
12 and 13) displayed DHP-I susceptibility comparable
to that of the original compounds (2 and 3).
with
a
[4-(4-carbamoylmethyl-1,4-diazabicyclo[2,2,2]
octanediium-1-yl)methyl-1,2,3,6-tetrahydropyridinyl-
thiocarbonylthio] moiety seemed optimal in this class.
To examine the effects of the DABCO moiety on in vivo
anti-MRSA activity, we evaluated the in vivo anti-
MRSA activity of representative compounds (10a, 13
and 14a) in a mouse systemic infection model by using
homotypic MRSA BB6221 and compared our findings
with the activity of the original compounds. In this
infection model, the original hydrophobic dithiocarba-
mate carbapenems (2, 3 and 4) showed ED50 values of
>50, >50 and 7.5 mg/kg, respectively. By contrast, the
Table 3 shows the biological properties of the dicationic
derivative 14a, its original compound 4, and BO-3482
(1). It is obvious that 14a was superior to the original
compound 4 in all respects. In addition, 14a showed
considerable improvement in vivo anti-MRSA activity
and DHP-I susceptibility as compared with 1. 14a
also possessed an excellent safety profile in a 48-h
rabbit nephrotoxicity study (225 mg/kg), similar to
that of 1.

H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
Table 3. Anti-MRSA activities and other biological properties of 14a, 4 and 1
1575
Compound
14a
4
1
In vitro anti-MRSA activitya G-mean MIC (mg/mL)
In vivo anti-MRSA activityb ED50 (mg/kg)
Affinity to PBP-20c IC50 (mg/mL)
5.36
1.86
1.1
2.02
7.50
6.2
2.63
98
4.36
4.80
3.8
DHP-I susceptibilityd
0.51
1.65
Serum protein-binding (%)e
Epileptogenicity (200 mg/rat head, n=5)
48-Hour nephrotoxicity (225 mg/kg, single iv, n=3)
55
0/5
No change
47
0/5
No change
0/5
NTf
aHigh level MRSA (27 strains); geometric-mean MIC methicilin, 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.
dRelative rate of hydrolysis to imipenem, porcine renal DHP-I.
eBinding rate for human serum at a carbapenem concentration of 10 mg/mL.
fNot tested.
In summary, we prepared a new class of dicationic
dithiocarbamate carbapenems in pursuit of a carbape-
nem derivative with improved in in vivo anti-MRSA
activity and DHP-I susceptibility. As a result, 14a was
identified as having excellent in vivo anti-MRSA activ-
ity, favorable DHP-I susceptibility, and a good safety
profile. Further biological properties of 14a such as
pharmacokinetic parameters will be reported in the near
future.
membrane isolated from MRSA BB6294 strain. The
membrane fraction was incubated with carbapenems at
30 ꢀC for 10 min. Binding affinity was expressed as the
inhibitory concentration for [14C]benzylpenicillin binding
by 50% (IC50), which was determined by Bio-Imaging
Analyzer (BAS2000, Fuji Photo Film Co., Ltd., Tokyo,
Japan) after exposure of dried gel film to the imaging
plate.
Systemic infection
Male ICR mice, 4 weeks of age, were infected intra-
peritoneally with homotypic MRSA BB6221 suspended
in 5% gastric mucin. Each agent was administered sub-
cutaneously at 1 h after infection in combination with
cilastatin at a dose of 40 mg/kg. The ED50 values were
calculated by the Probit method.
Experimental
Determination of MIC
MICs were determined by a 2-fold serial agar dilution
method with Mueller–Hinton medium (Difco Labora-
tories, Detroit, MI). An overnight culture was diluted to
give a final concentration of approximately 106CFU/
mL. A portion of the dilution was delivered onto a
drug-containing agar surface with an inoculum appara-
tus (Microplanter: Sakuma Seisakusho Co., Ltd.,
Tokyo, Japan). The final inoculum size was approxi-
mately 104CFU per spot. The MIC was defined as the
lowest concentration of antibiotics that completely pre-
vented visible growth after incubation at 37 ꢀC for 20 h.
Determination of epileptogenicity
Male SD rats, 7 weeks of age, were cannulated in the
right cerebroventricle before 1 week of drug adminis-
tration. The carbapenems were dissolved in saline,
adjusted to pH 7.0, and an aliquot of 10 mL/head was
intracerebroventricularly injected (n=5). Convulsant
behavior was monitored for 30 min after administration.
Determination
dehydropeptidase-1 (DHP-I)
of
susceptibility
to
renal
Generalmethods
Melting points were measured on a Yanaco MP micro-
melting point apparatus and were not corrected. The 1H
NMR spectra were recorded on a Varian VXR-300
spectrometer with tetramethylsilane (TMS) as an inter-
nal standard. IR absorption spectra were recorded with
a Horiba FT-200 spectrometer. Specific rotations were
measured with a Jasco DIP-370 polarimeter. UV spectra
were taken on a SHIMAZU SPD-10A spectrometer in
0.1 M 3-morpholinopropanesulfonate (MOPS) buffer
(pH 7.0). Mass spectra (MS) were measured on a JEOL
JMS-SX102A spectrometer. TLC was performed with
Merck Kieselgel F254 precoated plates. The silica gel
used for column chromatography was WAKO gel
C-300. Reversed-phase column chromatography was
carried out on YMC-gel ODS-AQ 120-S50. All reac-
tions involving air-sensitive reagents were performed
under nitrogen using syringe-septum cap techniques.
The relative hydrolysis rate of carbapenems by porcine
renal DHP-I was determined, taking the initial hydro-
lysis rate of imipenem as 1.0. Partially purified porcine
DHP-I (final concentration, 0.3 U/mL) was incubated
with 50 mM carbapenem at 35 ꢀC in 50 mM MOPS buf-
fer, pH 7.0. The initial hydrolysis rate was monitored by
the spectrophotometric method. One unit of activity
was defined as the amount of enzyme hydrolyzing 1 mM
of glycyldehydrophenylalanine per min when the sub-
strate, 50 mM, was incubated at 35 ꢀC in 50 mM MOPS
buffer, pH 7.0.
Affinity for PBP-20
The affinity for PBP-20 of MRSA was determined by
competition assay with [14C]benzylpenicillin using

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H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
Generalprocedure for the preparation of cationic
zwitterionic dithiocarbamate carbapenems
(18H, m), 5.21 (2H, m), 7.49 (4H, m); FAB-HRMS m/z
calcd for C28H38N5O5S2 [M]+: 588.2314, found
588.2300; UV lmax 300 (e 9920).
The experimental procedure for 10a is described as a
representative example.
The following compounds (10b, 10c, 11, 12, 13 14b and
14c) were prepared as described for the preparation of 10a.
p-Nitrobenzyl(1 R,5S,6S)-6-[(R)-1-hydroxyethyl]-2-[N-(4-
hydroxymethylbenzyl)-N-methyl]aminothiocarbonylthio]-
1-methyl-1-carbapen-2-em-3-carboxylate 7. To a stirred
solution of p-nitrobenzyl (1R,5S,6S)-2-diphenoxyphos-
phoryloxy-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-
em-3-carboxylate 5 (5.0 g, 8.4 mmol) in THF (120 mL)
were added 6 (3.27 g, 10 mmol) and lithium chloride
(428 mg, 10 mmol), and the mixture was stirred over-
night at room temperature. The reaction mixture was
poured into H2O and extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4,
and evaporated under reduced pressure. The residue
was purified by silica gel column chromatography to
give 7 (2.1 g, 42%): IR nmax (KBr) 1778, 1546, 1468
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[N-[4-[4-(2-hydro-
xyethyl)-1,4-diazabicyclo[2.2.2]octanediium-1-ylmethyl]-
benzyl]-N-methylamino]thiocarbonylthio]-1-methyl-1-car-
bapen-2-em-3-carboxylate chloride 10b. IR nmax (KBr)
3423, 1749, 1691, 1606, 1387, 1214, 1101 cmꢂ1 1H
;
NMR (300 MHz, D2O) d 1.06 (3H, m), 1.23 (3H, d,
J=6.3 Hz), 3.37–3.74 (7H, m), 3.97–4.35 (18H, m),
5.05–5.45 (2H, m), 7.40–7.57 (4H, m); FAB–HRMS m/z
calcd for C28H39N4O5S2 [M]+: 575.2362, found
575.2353; UV lmax300 (e 8640).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[N-[4-[4-(3-hydroxy-
propyl)-1,4-diazabicyclo[2.2.2]octanediium-1-ylmethyl]-
benzyl]-N-methylamino]thiocarbonylthio]-1-methyl-1-car-
bapen-2-em-3-carboxylate chloride 10c. IR nmax (KBr)
cmꢂ1 1H NMR (300 MHz, CDCl3) d 1.16 (3H, d,
;
J=7.5 Hz), 1.27 (3H, d, J=6.4 Hz), 2.43 (1.6H, s), 2.49
(1.4H, s), 3.42 (2H, s), 3.88 (2H, m), 4.18 (3H, m), 4.51
(2H, m), 4.43 (1H, m), 7.47 (2H, d, J=8.3 Hz), 7.78 (4H,
m), 8.24 (2H, d, J=8.3 Hz); FAB-HRMS m/z calcd for
C27H30N3O7S2 [M+H]+: 572.1525, found 572.1523.
3398, 1745, 1606, 1390, 1203, 1105 cmꢂ1 1H NMR
;
(300 MHz, D2O) d 0.78–1.23 (6H, m), 1.95–2.05 (2H,
m), 2.57–3.01 (2H, m), 3.30–3.74 (9H, m), 3.90–4.19
(14H, m), 4.85–5.70 (2H, m), 7.35–7.53 (4H, m); FAB-
HRMS m/z calcd for C29H41N4O5S2 [M]+: 589.2518,
Found: 589.2504; UV lmax300 (e 6420).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[N-[4-(4-carbamoyl-
methyl-1,4-diazabicyclo[2.2.2]octanediium-1-ylmethyl)-
benzyl]-N-methylamino]thiocarbonylthio]-1-methyl-1-car-
bapen-2-em-3-carboxylate chloride 10a. To a solution of
7 (2.5 g, 4.4 mmol) in THF (40 mL) were added diiso-
propylamine (2.34 mL, 13.4 mmol) and n-propanesulfo-
nylchloride (1.47 mL, 13.1 mmol) at 0 ꢀC, and the
mixture was stirred for 2 h. The reaction mixture was
poured into H2O and extracted with EtOAc. The
organic layer was successively washed with 0.1 N HCl,
5% aqueous NaHCO3 and brine, dried over MgSO4,
and evaporated under reduced pressure. To a solution
of the residue in acetone (40 mL) was added sodium
iodide (2.6 g, 17.5 mol) at 0 ꢀC, and the mixture was
stirred for 2 h at room temperature. The reaction mix-
ture was poured into H2O and extracted with EtOAc.
The organic layer was washed with 5% aqueous
Na2S2O3 and brine, dried over MgSO4, and evaporated
under reduced pressure to give iodide 8. To a solution of
8 in CH3CN (57 mL) was added 9a (2.09 g, 6.6 mmol) at
0 ꢀC, and the mixture was stirred for 12 h at room tem-
perature. The reaction mixture was evaporated under
reduced pressure. To a solution of the residue in THF
(113 mL) and 0.5 M sodium 3-morpholinopropane-
sulfonate buffer (pH 7.0, 113 mL) was added 10% pal-
ladium carbon catalyst (4.24 g), and the mixture was
stirred overnight at room temperature under a hydrogen
atmosphere. The catalyst was removed by filtration and
the filtrate was subjected to reversed-phase column
chromatography filled with saturated NaCl solution.
Eluant was monitored by HPLC. The fractions eluted
with 30% MeOH/H2O containing the desired com-
pound were combined and lyophilized to give 10a
(603 mg, 22%): IR nmax (KBr) 3731, 2970, 1757, 1693,
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[N-[4-(4-carbamoyl-
methyl-1,4-diazabicyclo[2.2.2]octanediium-1-ylethyl)benzyl]-
N-methylamino]thiocarbonylthio]-1-methyl-1-carbapen-2-
em-3-carboxylate chloride 11. IR nmax (KBr) 3423, 1753,
1695, 1387, 1126, 1093 cmꢂ1; 1H NMR (300 MHz, D2O)
d 1.00 (1.5H, d, J=7.3 Hz), 1.09 (1.5H, d, J=7.3 Hz),
1.27 (3H, d, J=6.3 Hz), 3.18–3.30 (2H, m), 3.42 (1.5H,
s), 3.46 (1.5H, s), 3.38–3.55 (1H, m), 3.63–3.93 (3H, m),
4.05–4.42 (14H, m), 4.49 (2H, s), 5.20 (2H, m), 7.29–
7.44 (4H, m); FAB–HRMS m/z calcd for C29H40N5O5S2
[M]+: 602.2471, found 602.2446; UV lmax 300 (e 9550).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[N-[4-(4-carbamoyl-
methyl-1,4-diazabicyclo[2.2.2]octanediium-1-ylmethyl)-1-
naphtylmethyl]-N-methylamino]thiocarbonylthio]-1-methyl-
1-carbapen-2-em-3-carboxylate chloride 12. IR nmax
1
(Nujol) 1754, 1697, 1598 cmꢂ1; H NMR (300 MHz,
D2O) d 0.92 (3H, m), 1.11 (3H, m), 3.03–4.22 (18H, m),
4.98–6.02 (4H, m), 7.03–8.41 (4H, m); FAB–HRMS m/z
calcd for C32H40N5O5S2 [M]+: 638.2471, found:
638.2452; UV lmax 287 (e 17,800).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[N-[6-(4-carbamoyl-
methyl-1,4-diazabicyclo[2.2.2]octanediium-1-ylmethyl)-2-
naphtylmethyl]-N-methylamino]thiocarbonylthio]-1-methyl-
1-carbapen-2-em-3-carboxylate chloride 13. IR nmax
(Nujol) 1754, 1699, 1600 cmꢂ1 1H NMR (300 MHz,
;
D2O) d 0.67 (1.2H, d, J=7.4 Hz), 0.81 (1.8H, d,
J=7.4 Hz), 0.94 (1.8H, d, J=6.4 Hz), 1.03 (1.2H, d,
J=6.3 Hz), 3.17 (1H, m), 3.23 (1.8H, s), 3.26 (1.2H, s),
3.42 (1H, m), 3.83–4.26 (16H, m), 5.01 (4H, m), 7.30
(1H, m), 7.41 (1H, m), 7.52 (1H, m), 7.83 (3H, m); FAB-
HRMS m/z calcd for C32H40N5O5S2 [M]+: 638.2471,
found 638.2507; UV lmax 300 (e 8920).
1
1385, 1209, 1113 cmꢂ1; H NMR (300 MHz, D2O) d
1.04 (3H, m), 1.23 (3H, d, J=6.5 Hz), 3.58 (5H, m), 4.18

H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
1577
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[1,2,3,6-tetrahydro-
4-[4-(2-hydroxyethyl)-1,4-diazabicyclo[2.2.2]octanediium-
1-ylmethyl]-1-pyridyl]thiocarbonylthio-1-methyl-1-carba-
pen-2-em-3-carboxylate chloride 14b. IR nmax (KBr)
3.83 (8H, m), 4.06 (8H, m), 4.19 (3H, m), 4.30 (3H, m),
4.59 (1H, m), 5.12 (1H, m), 5.33 (1H, d, J=14.0 Hz),
5.46 (1H, d, J=14.0 Hz), 6.18 (1H, m), 7.68 (2H, d,
J=8.8 Hz), 8.22 (2H, d, J=8.8 Hz); FAB–HRMS m/z
calcd for C32H42N6O7S2 [M]+: 686.2556, found
686.2547.
1751, 1604, 1419, 1390 cmꢂ1 1H NMR (300 MHz,
;
D2O) d 1.08 (3H, d, J=7.3 Hz), 1.25 (3H, d, J=6.3 Hz),
2.40–2.70 (2H, m), 3.48–3.80 (4H, m), 3.98–4.18 (12H,
m), 4.19–4.42 (5H, m), 5.55–4.70 (1H, m), 6.20–6.35
(1H, m); FAB–HRMS m/z calcd for C25H37N4O5S2
[M]+: 537.2205, found: 537.2233; UV lmax 300 (e
8620).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[1,2,3,6-tetrahydro-
4-(4-carbamoylmethyl-1,4-diazabicyclo[2.2.2]octanediium-
1-ylmethyl)-1-pyridyl]thiocarbonylthio-1-methyl-1-carba-
pen-2-em-3-carboxylate chloride 14a. To a solution of
17 (488 g, 507 mmol) in THF (7 L) and 0.7 M phosphate
buffer (pH 6.0, 10 L) was added zinc dust (2 kg), and the
reaction mixture was stirred vigorously for 40 min. The
reaction mixture was passed through a pad of Celite and
the filtrate was evaporated under reduced pressure to
about 3 L. The aqueous layer was subjected to reversed-
phase column chromatography in which the column
was filled with saturated NaCl solution. Eluant was
monitored by HPLC. The fractions eluted with 5%
CH3CN/H2O containing the desired compound were
combined, lyophilized and crystallized from 80%
EtOH/H2O to give 14a as colorless prisms (101.5 g,
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[1,2,3,6-tetrahydro-
4-[4-(3-hydroxypropyl)-1,4-diazabicyclo[2.2.2]octanediium-
1-ylmethyl]-1-pyridyl]thiocarbonylthio-1-methyl-1-carba-
pen-2-em-3-carboxylate chloride 14c. IR nmax (KBr)
1747, 1699, 1650, 1538 cmꢂ1; 1H NMR (300 MHz, D2O)
d 1.09 (3H, d, J=7.3 Hz), 1.26 (3H, d, J=6.3 Hz), 1.95–
2.12 (2H, m), 2.40–2.72 (2H, m), 3.53 (1H, dd, J=5.8,
2.9 Hz), 3.58–3.78 (5H, m), 3.88–4.12 (13H, m), 4.20–
4.48 (5H, m), 6.22–6.38 (1H, m); FAB–HRMS m/z
calcd for C26H39N4O5S2 [M]+: 551.2362, found
551.2360; UV lmax 300 (e 9180).
1
34%): IR nmax (KBr) 1749, 1695, 1608, 1388 cmꢂ1; H
p-Nitrobenzyl(1 R,5S,6S)-6-[(R)-1-hydroxyethyl]-2-[(1,2,
3,6-tetrahydro-4-iodomethyl)-1-pyridyl]thiocarbonylthio-
1-methyl-1-carbapen-2-em-3-carboxylate 16. To a solu-
tion of 15 (215 g, 403 mmol) in THF (1700 mL) were
added diisopropylamine (91.3 mL, 524 mmol) and
n-propanesulfonylchloride (49.9 mL, 443 mmol) at 0 ꢀC,
and the mixture was stirred for 2 h. The reaction mix-
ture 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 MgSO4, and
evaporated under reduced pressure. To a solution of
residue in acetone (1100 mL) was added sodium iodide
(181 g, 1.21 mol) 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 evaporated under
reduced pressure to give 16 (236.7 g, 91%) as a solid: IR
NMR (300 MHz, D2O) d 1.11 (3H, d, J=7.2 Hz), 1.28
(3H, d, J=6.3 Hz), 2.56 (2H, m), 3.56 (1H, dd, J=5.6,
2.9 Hz), 3.70 (1H, q, J=7.6 Hz), 4.08 (6H, m), 4.26
(12H, m), 4.38 (1H, dd, J=9.7, 2.7 Hz), 4.45 (2H, s),
4.68 (1H, m), 6.42 (1H, m); FAB-HRMS m/z calcd for
C25H36N5O5S2 [M]+: 550.2158, found 550.2140. Anal.
calcd for C25H36N5O5S+ Clꢂ 3H2O: C, 46.90; H, 6.61;
N, 10.94; S, 10.02, found C, 46.91; H, 6.77; N, 10.95; S,
9.99; UV lmax 300 (e 6370).
1-Carbamoylmethyl-4-aza-1-azoniabicyclo[2.2.2]-octane
trifluoromethanesulfonate 9a5a
To a solution of DABCO (659 g, 5.88 mol) in CH3CN
(7.0 L) was added a solution of 2-chloroacetamide (500
g, 5.35 mol) in CH3CN (7.0 L) at 0 ꢀC, and the reaction
mixture was stirred for 16 h at room temperature. The
resulting precipitates were collected, washed with
CH3CN and dried to give the chloride salt (943 g, 86%).
To a solution of the chloride salt (943 g, 4.59 mol) in
MeOH (6.0 L) was added a solution of AgOTf (1.12 kg,
4.36 mol) in CH3CN (4.0 L) at room temperature. After
being stirred for 30 min, the mixture was filtered and the
filtrate was evaporated under reduced pressure. The
residue was crystallized from EtOH to give 9a (898 g,
65%): 1H NMR (300 MHz, D2O) d 3.22 (6H, t,
J=7.5 Hz), 3.65 (6H, t, J=7.5 Hz), 4.02 (2H, s). Anal.
calcd for C9H16N3O4F3S: C, 33.85; H, 5.05; N, 13.16,
found C, 34.02; H, 5.02; N, 13.14.
1
nmax (KBr) 1771, 1522, 1435cmꢂ1; H NMR (300 MHz,
CDCl3) d 1.15 (3H, d, J=7.3Hz), 1.36 (3H, d, J=6.3Hz),
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,
J=8.2 Hz); FAB–HRMS m/z calcd for C24H27N3O6S2I
[M+H]+: 644.0386, found 644.0369.
p-Nitrobenzyl(1 R,5S,6S)-6-[(R)-1-hydroxyethyl]-2-[1,2,3,6-
tetrahydro-4-(4-carbamoylmethyl-1,4-diazabicyclo[2.2.2]oc-
tanediium-1-ylmethyl)-1-pyridyl]thiocarbonylthio-1-methyl-
1-carbapen-2-em-3-carboxylate iodide trifluoromethane-
sulfonate 17a. To a solution of 16 (236 g, 367 mmol) in
CH3CN (960 mL) was added 9a (128 g, 385 mmol) at
0 ꢀC, and the mixture was stirred for 30 min at room
temperature. The reaction mixture was evaporated
under reduced pressure and the resulting precipitates
were washed with EtOAc/THF/CH3CN (10:3:1) to give
17 (349.2 g, 98%) as a solid: IR nmax (KBr) 3403, 1772,
9b and 9c were prepared as described above by using
2-bromoethanol or 3-bromopropanol instead of chloro-
acetamide.
9b. 1H NMR (300 MHz, D2O) d 3.14 (6H, m), 3.40 (8H,
m), 3.65 (2H, t, J=7.6 Hz).
1
1693, 1608, 1523, 1467, 1029, 850, 638 cmꢂ1; H NMR
9c. 1H NMR (300 MHz, D2O) d 1.96 (2H, m), 3.15 (6H,
m), 3.38 (8H, m), 3.63 (2H, m).
(300 MHz, DMSO-d6) d 1.13 (6H, m), 3.46 (3H, m),

1578
H. Imamura et al. / Bioorg. Med. Chem. 9 (2001) 1571–1578
5. (a) Yasuda, N.; Huffman, M. A.; Ho, G.; Xavier, L. C.;
Acknowledgements
Yang, C.; Emerson, K. M.; Tsay, F.; Li, Y.; Kress, M. H.;
Rieger, D. L.; Karady, S.; Sohar, P.; Abramson, N. L.;
DeCamp, A. E.; Mathre, D.; Douglas, A. W.; Dolling, U. H.;
Grabowski, E. J. J.; Reider, P. J. J. Org. Chem. 1998, 63, 5438.
(b) Greenlee, M. L; Laub, J. B.; Rouen, G. P.; DiNinno, F.;
Hammond, M. L.; Huber, J. L.; Sundelof, J. G.; Hammond,
G. G. Bioorg. Med. Chem. Lett. 1999, 9, 3225.
We are grateful to Ms. Anne Dobbins, Merck & Co.,
for her critical reading of this manuscript.
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