Structure-activity relationships of 1β-methyl-2-(5-phenylpyrrolidin-3-ylthio)carbapenems

Article abstract of DOI:10.1016/S0968-0896(01)00430-8

Structure-activity relationship studies of 1β-methyl-2-[(3S,5R)-5-(4-aminomethylphenyl)pyrrolidin-3- ylthio]carbapenems, especially those pertaining to the relationship between antibacterial activity and side-chain structure were conducted. These studies suggested that the trans-(3S,5R)-5-phenylpyrrolidin-3-ylthio side-chain and the aminomethyl group at the 4-position of the phenyl ring play a key role in enhancing the antibacterial activity against the MRSA and Pseudomonas aeruginosa strains. In particular, the basicity of a substituent at the 4-position of the phenyl ring were shown to greatly contribute to the antibacterial activity against MRSA and methicillin-resistant Staphyloccocus epidermidis strains. In contrast, the amidine group was shown to lead to potent antibacterial activity against P. aeruginosa strains comparable to that of imipenem, however, a good correlation between the basicity of the 4-substituent and antipseudomonal activity was not observed. In conclusion, the 4-aminomethyl or methylaminomethyl group on the phenyl ring was the best substituent for antipseudomonal activity.

Full text of DOI:10.1016/S0968-0896(01)00430-8

Bioorganic & Medicinal Chemistry 10 (2002) 1595–1610
Structure–Activity Relationships of
1ꢀ-Methyl-2-(5-phenylpyrrolidin-3-ylthio)carbapenems
Hiroki Sato, Hiroki Sakoh, Takashi Hashihayata, Hideaki Imamura,*
Norikazu Ohtake, Aya Shimizu, Yuichi Sugimoto, Shunji Sakuraba,
Rie Bamba-Nagano, Koji Yamada, Terutaka Hashizume
and Hajime Morishima
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd.,
Okubo-3, Tsukuba 300-2611, Ibaraki, Japan
Received 27 September 2001; accepted 30 November 2001
Abstract—Structure–activity relationship studies of 1b-methyl-2-[(3S,5R)-5-(4-aminomethylphenyl)pyrrolidin-3-ylthio]carbape-
nems, especially those pertaining to the relationship between antibacterial activity and side-chain structure were conducted. These
studies suggested that the trans-(3S,5R)-5-phenylpyrrolidin-3-ylthio side-chain and the aminomethyl group at the 4-position of the
phenyl ring play a key role in enhancing the antibacterial activity against the MRSA and Pseudomonas aeruginosa strains. In par-
ticular, the basicity of a substituent at the 4-position of the phenyl ring were shown to greatly contribute to the antibacterial activity
against MRSA and methicillin-resistant Staphyloccocus epidermidis strains. In contrast, the amidine group was shown to lead to
potent antibacterial activity against P. aeruginosa strains comparable to that of imipenem, however, a good correlation between the
basicity of the 4-substituent and antipseudomonal activity was not observed. In conclusion, the 4-aminomethyl or methylamino-
methyl group on the phenyl ring was the best substituent for antipseudomonal activity. # 2002 Elsevier Science Ltd. All rights
reserved.
Introduction
identified 1b-methyl-2-[(3S,5R)-5-(4-aminomethylphe-
nyl)pyrrolidin-3-ylthio]carbapenem 1a, which is excep-
The emergence of multidrug-resistant bacteria, espe-
cially methicillin-resistant Staphyloccocus aureus
(MRSA), has caused serious concern in clinics world-
wide. Due to the lack of better therapeutic agents, van-
comycin has been used for the treatment of infections
caused by MRSA despite severe side effects. Recently,
the emergence of vancomycin-resistant MRSA has
raised an alarm about the overuse of vancomycin.1
Therefore, the development of new therapeutic agents
with potent anti-MRSA activity, as well as good safety
profiles, has been urgently demanded. Carbapenem
antibiotics such as imipemem2 and meropenem3 possess
antibacterial activity against a broad range of pathogens
including S. aureus and Pseudomonas aeruginosa and
have good safety profiles, however, their antibacterial
activity against MRSA is still insufficient for clinical use.
tionally broad spectrum antibiotic having anti-MRSA
activity as well as activity against P. aeruginosa (Fig. 1).4
We assumed that its ambidextrous antibacterial activity
would be due to the basicity of an 4-aminoalkyl sub-
stituent on the phenyl ring and a 5-phenylpyrrolidi-
nylthio group with a trans-(3S,5R)-configuration. To
investigate the above assumption, further modifications
of the pyrrolidinyl side chain were performed. In this
paper, we describe the synthesis of 1b-methyl-2-[5-(sub-
stituted -phenyl)pyrrolidin -3 -ylthio]carbapenems and
related compounds, and the detailed structure–activity
relationships (SARs) between the side-chain structures
and the ambidextrous antibacterial activity against
MRSA and P. aeruginosa.
Previously, we reported that in the course of developing
a novel carbapenem with unique biological profiles, we
*Corresponding author. Tel.: +81-298-77-2000; fax: +81-298-77-
2027; e-mail: imamrahk@banyu.co.jp
Figure 1. Chemical structure of 1a.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00430-8

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H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
Scheme 1. Synthesis of 9g. Reagents: (a) NaBH4, MeOH, 0 ꢀC; (b) (i) MsCI, Et3N, CH2CI2, ꢁ30 ꢀC; (ii) MeNH2/MeOH, rt; (iii) Alloc–Cl, 0 ꢀC;
(c) (i) HCl–MeOH, reflux; (ii) Alloc–Cl, 0 ꢀC; (d) (i) MsCI, Et3N, CH2CI2, ꢁ40 ꢀC; (ii) AcSK, DMF, 60 ꢀC.
Scheme 2. Synthesis of 1a–1m. Reagents: (a) (for 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, lj, 1k and 1m) (i) NaOH, MeOH, 0 ꢀC; (ii) 10, i-Pr2NEt, CH3CN;
(iii) (PPh3)2PdCl2, n-Bu3SnH, H2O, CH2CI2, 0 ꢀC, (b) (for 1l) (i) NaOH, MeOH, 0 ꢀC; (ii) 11, i-Pr2NEt, CH3CN; (iii) H2,Pd/C, rt.
Results and discussion
Similarly, the protected thiols, 9a–9f and 9h–9m, were
converted to the carbapenems, 1a–1f and 1h–1m,
respectively (Scheme 2).7
Chemistry
The synthesis of 1g is highlighted in Schemes 1 and 2 as
a representative example of an improved synthetic
method of producing 1b-methyl-2-(5-phenylpyrrolidin-
3-ylthio)carbapenems. The pyrrolidine aldehyde 5 which
is easily derived from commercially available (R)-4-
hydroxy-2-pyrrolidone, was used as the starting mat-
erial.5 Reduction of 5 with NaBH4 in MeOH at 0 ꢀC
afforded the alcohol 6 in 90% yield. Mesylate, which
was derived from 6, was substituted with methylamine
(40% MeNH2/MeOH); subsequently, the obtained
amine was protected as an allyloxycarbamate to provide
7 in 68% yield. Deprotection of 7 under acidic condi-
tions (HCl/MeOH, reflux) and subsequent protection of
the pyrollidine nitrogen with allyl chloroformate gave
the alcohol, 8, in quantitative yield, which was con-
verted to the thioacetate, 9g, in 88% yield by the treat-
ment of the corresponding mesylate with potassium
thioacetate. The thiol obtained by the hydrolysis of 9g
(NaOH/MeOH, 0 ꢀC) was coupled with the carbapenem
enolphosphate, 10, in the presence of N,N-diisopropyl-
ethylamine in CH3CN to produce an adduct in 53%
yield.
The synthesis of 2 is outlined in Scheme 3. The alde-
hyde, 12,8 was converted to the a,b-unsaturated ester,
13, in 94% yield using Horner–Emmons reaction.
Michael addition of a diethyl malonate anion to 13 did
not proceed, probably due to the steric hindrance
around the b-carbon of the conjugated ester caused by
the bulky cis-configurated TBSO group. When the
TBSO group of 13 was inverted to a trans-configura-
tion, Michael addition of this compound to a diethyl
malonate anion proceeded smoothly to give triester 15,
which was converted to the diol, 17, via deethoxy-
carbonylation9 and subsequent reduction of the result-
ing diester 16. Cyclization of the diol 17 to cyclohexane
18 was achieved in 75% yield by a modified Mitsunobu
reaction using phenylsulfonylacetonitrile and 1.10-(azo-
dicarbonyl)dipiperidine.10 Removal of the phenylsulfo-
nyl group of 18 by the treatment with HgCl2–Mg gave
19; the cyano moiety of 19 was hydrogenated with
Raney Ni and subsequently protected with allyl chloro-
formate to produce 20.
Transformation of 20 to 21 was carried out in a similar
manner as that described above (80% yield). The sec-
ondary alcohol, 21, was converted to 22 by inversion using
Mitsunobu reaction11 and subsequent basic hydrolysis.
The alcohol 22 was then converted to thioacetate 23 by
Mitunobu reaction using thioacetic acid. Finally, con-
version of 23 to compound 2 was achieved by a method
similar to that described for the preparation of 1g.
Finally, deprotection of the adduct using the method
reported by Guibe et al.6 and subsequent purification by
reversed-phase column chromatography furnished
compound 1g as an amorphous power after lyophiliza-
tion in 41% yield. Protected thiols (9a–9f and 9h–9m)
were prepared according to our previous method start-
ing from commercially available d-malic acid.4e

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1597
Scheme 3. Synthesis of 23. Reagents: (a) triethyl phosphonoacetate, NaH, THF, 0 ꢀC; (b) (i) tetra-n-butylammonium fluoride, THF, 0 ꢀC; (ii) PPh3,
DIAD, AcOH, THF; 0 ꢀC; (iii) NaOH, MeOH, rt; (iv) TBS–Cl, imidazole, DMF, rt; (c) (i) diethyl malonate, tetra-n-butylammonium bromide,
NaH, THF, 50 ꢀC; (d) NaCI, DMSO, 160 ꢀC; (e) LiAIH4, THF, ꢁ20 ꢀC; (f) PPh3, PhSO2CH2CN, 1,10-(azodicarbonyl)dipiperidine, THF, 50 ꢀC;
(g) HgCl2, Mg, THF, MeOH, 18 ꢀC; (h) (i) Raney Ni, rt; (ii) Alloc–CI, i-Pr2NEt, 0 ꢀC; (i) (i) HCl/MeOH, rt; (ii) Alloc–CI, NaOH, 1,4-dioxane, H2O;
(j) (i) PPh3, DIAD, AcOH, THF, 0 ꢀC; (ii) NaOH, MeOH, rt; (k) PPh3, DIAD, thioacetic acid, THF, 0 ꢀC.
Scheme 4. Synthesis of 28. Reagents: (a) (i) 25, Na(OAc)3BH, AcOH, THF, rt; (ii) PNZ–Cl, NaOH, 1,4-dioxane, H2O; (b) (i) Ac2O, pyndine,
DMAP, THF, rt; (ii) tetra-n-butylammonium fluoride, THF, rt; (iii) MsCl, i-Pr2NEt, THF, 0 ꢀC; (iv) NaN3, DMF, rt; (v) PPh3, H2O, THF, rt; (vi)
PNZ–Cl, NaOH, 1,4-dioxane, H2O; (c) (i) NaOH, MeOH, rt; (ii) PPh3, DIAD, thioacetic acid, THF, 0 ꢀC.
The synthesis of 3 is outlined in Scheme 4. Reductive
amination of the 2-aminoethanol 24 with substituted
benzaldehyde 25, PNZ protection of resultant amine
afforded alcohol 26. The primary hydroxyl group of 26
was acetylated, and subsequent desilylation, azidation
of resultant benzilic hydroxyl group with NaN3 to
afford azide. The azide was reduced with triphenylphos-
phine and H2O to provide an amino group which was
protected with an PNZ group to furnish 27. The sec-
ondary alcohol, after deacetylation of 27, was converted
to thioacetate 28 by inversion using Mitsunobu reac-
tion11 Thioacetate 28 was hydrolized to the corre-
sponding thiol which was in turn coupled with
carbapenem diphenylphosphate 11 in the presence of
N,N-diisopropylethylamine in CH3CN. Deprotection of
the coupling adduct was accomplished by hydrogena-
tion with H2 and Pd/C, and the resulting crude carba-
penem was purified by reversed-phase column
chromatography to furnish the final compound 3 as an
amorphous powder after lyophilization.
Biological properties
The carbapenems obtained above were evaluated for in
vitro antibacterial activity against S. aureus, including
an MRSA (pMS520/Smith), a methicillin-resistant Sta-
phylococcus epidermidis (MRSE, MB5181), Escherichia
coli (NIHJ JC2), and P. aeruginosa including a ceftazi-
dime-resistant P. aeruginosa (AKR17), and were eval-
uated for porcine renal dehydropeptidase-I (DHP-I)
susceptibility (Tables 1–4). Standard serial dilution
techniques were employed for the MIC determinations.
Both imipenem and vancomycin were used as reference
drugs.

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H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
Table 1. In vitro activitya and biological properties of 1a and related compounds
Organism
Compounds
(1a)
(2)
(3)
(4a)
VCM
0.39
IPM
S. aureus 209P NIHJ JC1
S. aureus pMS52O/Smithb
S. epidermidis MB5l8lb
E. coli NIHJ JC2
P. aeruginosa AK109
P. aeruginosa AKR17c
ꢂ0.006
0.78
1.56
ꢂ0.006
6.25
6.25
ꢂ0.006
12.5
6.25
ꢂ0.006
3.13
6.25
ꢂ0.006
50
50
0.78
1.56
0.025
0.39
3.13
0.05
0.78
12.5
0.20
3.13
>25
0.025
6.25
6.25
>100
>100
>100
0.1
1.56
3.13
DHP-I susceptibilityd
0.29
0.11
0.05
0.05
—
1.0
aMlC(mg/mL) determined by agar dilution method.
bMethicillin-resistant.
cCeftazidime-resistant.
dRelative rate of hydrolysis to imipenem, porcine renal DHP-I.
Table 2. In vitro activitya and biological properties of la and related compounds
Organism
R
(1a)
(1b)
(4b)
(4c)
S. aureus 209P NIHJ JC1
S. aureus pMS520/Smithb
S. epidermidis MB5181b
E. coli NIHJ JC2
P. aeruginosa AK109
P. aeruginosa AKR1
ꢂ0.006
0.78
1.56
0.025
0.39
3.13
ꢂ0.006
1.56
3.13
0.025
0.78
6.25
ꢂ0.006
ꢂ0.006
25
>25
25
>25
0.025
6.25
25
0.025
3.13
6.25
DHP-I susceptibilityd
Organism
0.29
0.12
<0.05
<0.05
VCM
IPM
R
(4d)
(4e)
S. aureus 209P NIHJ JC1
S. aureus pMS52O/Smithb
S. epidermidis MB5181b
E. coli NIHJ JC2
P. aeruginosa AK109
P. aeruginosa AKR17c
ꢂ0.006
3.13
12.5
0.025
3.13
3.13
ꢂ0.006
3.13
12.5
0.10
6.25
0.39
0.78
1.56
ꢂ0.006
50
50
0.1
1.56
3.13
>100
>100
>100
6.25
DHP-l susceptibilityd
—
—
—
1.0
aMIC(mg/mL) determined by agar dilution method.
bMethicillin-resistant.
cCeftazidime-resistant.
dRelative rate of hydrolysis to imipenem, porcine renal DHP-l.

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1599
To examine which part(s) of the C-2 side chain in 1a is/
are essential for broad spectrum antibacterial activity,
the following three compounds were prepared and their
antibacterial activities were compared with that of 1a
(Table 1): compound 2; the phenyl group of 1a being
replaced with a cyclohexyl group, compound 3; the
pyrrolidine ring being replaced with an aminoethyl
group, and compound 4a; the 4-aminomethyl group on
the phenyl ring being removed. The cyclohexyl ana-
logue, 2, was 8-fold and 4-fold less active against
MRSA (pMS520/Smith) and ceftazidime resistant P.
aeruginosa strains, respectively, than 1a, suggesting that
the phenyl ring of 1a plays a key role in enhancing the
ambidextrous antibacterial activity. Comparison of the
antibacterial activity of 3 and 1a showed that the pyr-
rolidine ring was also essential to the broad spectrum
antibacterial activity of 1a. Moreover, the des-amino-
methyl analogue 4a exhibited 4-fold and 16-fold less
antibacterial activity against the MRSA (pMS520/
Smith) and P. aeruginosa (AKR17) strains, respectively,
compared with 1a. The above results suggest that both
the aminomethylphenyl group and the (3S,5R)-con-
figurated pyrrolidine ring contributed greatly to the
ambidextrous antibacterial activity.
gated further by comparing the antibacterial activity of
compounds 4b–4e with that of a non-basic functional
group, such as a carboxylic acid or hydroxyl group in
place of the aminomethyl group (Table 2). Replacement
with a carboxyl group (4b) resulted in a 32-fold or more
decrease in the antibacterial activity against the MRSA,
MRSE, and P. aeruginosa strains. It is interesting to
note that the cis-carboxyl analogue, 4c, was more active
against the P. aeruginosa strains than was the trans-
analogue, 4b. Replacement with a hydroxymethyl group
(4d) resulted in an 8-fold reduction in the antibacterial
activity against the MRSE and P. aeruginosa (AKR109)
strains. These results suggest that a basic functional
group on the phenyl ring of 1a is necessary for the
antibacterial activity against the gram-positive bacteria.
Table 3 shows the antibacterial activity of the regio-
isomers (1c and 1e) of 1a with respect to the amino-
methyl part, and shows the antibacterial activity of the
corresponding cis-isomers (1d and 1f). It is evident that
the 4-aminomethyl analogue, 1a, showed the best
antibacterial activity against the MRSA, MRSE and
P. aeruginosa strains among the three regio-isomers (1a,
1c and 1e). As expected, the trans-isomers (1a, 1c, 1e)
showed better antibacterial activity against the MRSA
and P. aeruginosa strains than the corresponding cis-
isomers (1b, 1d, 1f). Only the para-isomer, 1a, showed
Subsequently, the effects of the aminomethyl group of
the phenyl ring on the antibacterial activity was investi-
Table 3. In vitro activitya and biological properties of 1a and related compounds
Organism
R
(1a)
(1b)
(1c)
(1d)
S. aureus 209P NIHJ JC1
S. aureus pMS520/Smithb
S. epidermidis MB5181b
E. coli NIHJ JC2
P. aeruginosa AK109
P. aeruginosa AKR1
ꢂ0.006
0.78
1.56
0.025
0.39
3.13
ꢂ0.006
1.56
3.13
0.025
0.78
6.25
ꢂ0.006
1.56
3.13
0.10
0.78
ꢂ0.006
3.13
3.13
0.05
0.78
6.25
12.5
DHP-I susceptibilityd
Organism
0.29
0.12
0.16
<0.05
VCM
IPM
R
(1e)
(1f)
S. aureus 209P NIHJ JC1
S. aureus pMS520/Smithb
S. epidermidis MB5181b
E. coIi NlHJ JC2
P. aeruginosa AK1 09
P. aeruginosa AKR17c
ꢂ0.006
6.25
3.13
0.10
3.13
ꢂ0.006
6.25
6.25
0.10
6.25
25
0.39
0.78
1.56
ꢂ0.006
50
50
0.1
1.56
3.13
>100
>100
>100
12.5
DHP-I susceptibilityd
<0.05
<0.05
—
1.0
aMlC(mg/mL) determined by agar dilution method.
bMethicillin-resistant.
cCeftazidime-resistant.
dRelative rate of hydrolysis to imipenem, porcine renal DHP-l.

1600
H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
antibacterial activity against MRSA and MRSE strains
comparable to that of vancomycin and more potent
activity against P. aeruginosa strains than imipenem.
bacterial activity against the MRSA and MRSE strains
comparable to that of 1a, while showing an 8-fold
decrease in activity against the P. aeruginosa strain
(AKR17). We considered that installment of a strong
basic group into the phenyl ring would be necessary for
enhancing the antibacterial activity against the P. aeru-
ginosa strain (AKR17). The amidine analogue, 1l, which
has a more basic group than the primary amine, showed
a 4-fold reduction in the antibacterial activity against
the P. aeruginosa strain (AKR17) compared with 1a.
The diamine analogue, 1m, was 2-fold less active than 1l
against the P. aeruginosa strain (AK109). These results
suggest that the basicity of the substituent on the phenyl
ring is necessary for the antipseudomonal activity but
dose not correlated well with the antibacterial activity
against P. aeruginosa strain (AKR17). In contrast, the
antibacterial activity of the compounds with a basic
group on the phenyl ring against the MRSA and MRSE
strains was comparable to that of 1a, suggesting that a
substituent with some degree of basicity on the phenyl
ring is likely to have good antibacterial activity against
these gram-positive bacteria.
Finally, we investigated the relationship between the
antibacterial activity and the basicity of a substituent
installed on the 4-position of the phenyl ring in this class
of compounds (Table 4).
Previously reported, N-methylation (1g) of the primary
amine of 1a maintained broad spectrum antibacterial
activity, while installment of other N-substituents
brought about a large reduction in the antibacterial
activity against the P. aeruginosa strain (AKR17). The
cyclic analogue, 1h, with a pyrroline group almost
maintained the broad antibacterial activity. When the
primary amine in 1a was replaced with the weaker basic
group, O-methylhydroxylamine, the resulting compound,
1i, showed significantly decreased activity against the
MRSA, MRSE and P. aeruginosa strains. Similarly,
replacement of the pyrroline ring in 1h with a weaker
basic tetrahydropyridazine ring (1j) resulted in a large
reduction in the antibacterial activity against the
MRSA, MRSE and P. aeruginosa strains. In contrast,
1k, which has basic thioamidine moiety, showed anti-
In summary, considering the structure of the C-2 side
chain and antibacterial activity we have derivatized a
Table 4. In vitro activitya of trans-3,5-disubstituted pyrrolidinylthio 1b-methylcarbapenems
Organism
R
(1a)
(1g)
(1 h)
(1i)
S. aureus 209P NIHJ JC1
S. aureus pMS520/Smithb
S. epidermidis MB5181b
E. coIi NIHJ JC2
P. aeruginosa AK109
P. aeruginosa AKR17c
ꢂ0.006
0.78
1.56
0.025
0.39
1.56
ꢂ0.006
0.78
1.56
0.025
0.39
1.56
ꢂ0.006
1.56
1.56
0.025
0.39
3.13
ꢂ0.006
12.5
12.5
0.10
25
>25
DHP-l susceptibilityd
Organism
0.29
0.25
0.36
0.16
R
(1j)
(1k)
(1l)
(1m)
S. aureus 209P NIHJ JC1
S. aureus pMS520/Smithb
S. epidermidis MB5181b
E. coli NIHJ JC2
P. aeruginosa AK109
P. aeruginosa AKR17c
ꢂ0.006
12.5
12.5
0.10
12.5
25
ꢂ0.006
1.56
1.56
0.05
1.56
ꢂ0.006
1.56
0.78
0.05
0.78
ꢂ0.006
0.78
1.56
0.05
1.56
12.5
6.25
6.25
DHP-I susceptibilityd
0.14
0.30
0.25
0.26
aMIC(mg/mL) determined by agar dilution method.
bMethicillin-resistant.
cCeftazidime-resistant.
dRelative rate of hydrolysis to imipenem, porcine renal DHP-I.

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1601
novel class of 1b-methyl-2-(5-phenylpyrrolidin-3-ylthio)-
General methods
carbapenems. Our SAR studies suggest that atrans-
(3S,5R)-disubstituted pyrrolidine skeleton with a 4-
aminomethylphenyl moiety as the C-2 side chain is
essential to the ambidextrous antibacterial activity
against MRSA as well as P. aeruginosa. Various basic
substituents on the phenyl ring showed potent anti-
bacterial activity against the MRSA and MRSE strains
comparable to that of vancomycin, while the neutral or
acidic substituents were ineffective in the MRSE strain.
In contrast, the 4-aminomethyl, 4-N-methylamino-
methyl and amidine groups on the phenyl ring led to
potent antipseudomonal activity comparable or super-
ior to that of imipenem, however the antipseudomonal
activity of the analogues was not correlated with the
basicity of their 4-subsituent.
Melting points were measured on a Yanaco MP micro-
melting point apparatus and were not corrected. 1H
NMR spectra were recorded on a Varian VXR-300
spectrometer, a JEOL JNM-EX270 spectrometer, and a
JEOL JNM-A500 spectrometer with tetramethylsilane
(TMS) as an internal standard. 13C NMR spectra were
recorded on a JEOL JNM-A500 spectrometer and a
JEOL JNM-EX270 spectrometer. 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-morpholinopropanesulfo-
nate (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 chroma-
tography was carried out on YMC gel ODS-AQ 120-
S50. All reactions involving air-sensitive reagents were
performed under nitrogen atmosphere using syringe-
septum cap techniques.
Experimental
Antibiotics and strains
Imipenem is a product of Banyu Pharmaceutical Co.,
Ltd., Japan. Vancomycin was purchased from Sigma
Chemical Co., St. Louis, MO, USA. The antibiotics
were dissolved in 10 mM 3-morpholino-propanesulfo-
nate (MOPS) buffer, pH 7.0 on the day of use. The
strains used in the study were our collections. Methi-
cillin-resistant S. aureus pMS520/Smith was a generous
gift from M. Inoue, Kitasato University, School of
Medicine, Japan.
General procedure for the preparation of 5-aryl
pyrrolidinylthio-1ꢀ-methylcarbapenems
(2R,4R)-1-tert-Butoxycarbonyl-4-tert-butyldimethylsiloxy-
2-[4-(hydroxymethyl)phenyl]pyrrolidine 6. To a solution
of 5 (143 g, 35.5 mmol) in MeOH (3000 mL) was added
NaBH4 (14.8 g, 390 mmol) at 0 ꢀC, and the mixture was
stirred for 30 min at 0 ꢀC. The reaction mixture was
quenched by adding 10% aqueous NH4Cl and was
poured into H2O. The whole was extracted with EtOAc,
and the organic layer was washed with brine, dried over
MgSO4, and evaporated under reduced pressure. The
residue was purified by silica gel column chromato-
graphy (n-hexane/EtOAc=5/1ꢃ4/1) to give 6 (130.1 g,
90.0%) as a colorless oil. [a]2d0 +34.6 (c 1.0, CHCl3); IR
Determination of MIC
MICs were determined by the 2-fold serial agar dilution
method with Mueller-Hinton medium (Difco Labora-
tories, Detroit, MI, USA). An overnight culture was
diluted in the corresponding broth with phosphate-buf-
fered saline supplemented with 0.01% gelatin to give a
final concentration of approximately 106 CFU/mL. A
portion of the dilution was delivered onto a drug-con-
taining agar surface with an inoculum apparatus
(Microplanter: Sakuma Seisakusho, Co., Ltd., Tokyo,
Japan). The final inoculum size was approximately 104
CFU per spot. The plates were incubated at 37 ꢀC for
20 h. The MIC was defined as the lowest concentration
of antibiotics at which visible growth was inhibited.
n
max (KBr) 3404, 1700,1365, 1253, 1159, 1091 cmꢁl1; 1H
NMR (300 MHz, CDCl3) d 0.08 (6H, s), 0.80 (9H, s),
1.16 (6H, s), 1.44 (3H, s), 1.87 (1H, m), 2.50 (1H, m),
3.40 (1H, m), 3.86 (1H, m), 4.37 (1H, m), 4.66 (2H, s),
[4.72 (0.7H, m), 4.87 (0.3H, m), each rotamer], 7.26 (4H,
s); 13C NMR (67.5 MHz, CDCl3, major signals) d ꢁ5.0,
ꢁ4.9, 17.8, 25.6, 28.0, 44.6, 54.6, 60.0, 64.6, 69.8, 79.4,
125.9, 126.5, 139.3, 143.8, 154.4; FAB-HRMS calcd for
C22H38NO4Si (M+H)+: 408.2570, found 408.2572.
Determination of susceptibility to renal dehydropeptidase-1
(DHP-I)
(2R,4R)-2-[4-(N-Allyloxycarbonyl-N-methylaminomethyl)-
phenyl]-1-tert-butoxycarbonyl-4-tert-butyldimethylsiloxy-
pyrrolidine 7. To a solution of 6 (130.1 g, 320 mmol) in
CH2Cl2 (2600 mL) were added triethylamine (64.8 g,
640 mmol) and methanesulfonyl chloride (27.2 mL, 352
mmol) at ꢁ30 ꢀC. The reaction mixture was poured into
H2O, and the whole was extracted with EtOAc. The
organic layer was washed with brine, and dried over
MgSO4. After the removal of MgSO4 by filtration, 40%
methylamine/MeOH (1488 mL) was added to the fil-
trate at 0 ꢀC and the mixture was stirred for 30 min at
the same temperature. The mixture was evaporated
under reduced pressure, and the residue was dissolved in
1,4-dioxane (3000 mL) and H2O (500 mL). To this
The relative hydrolysis rate of carbapenems was deter-
mined by porcine renal DHP-I, in which the initial
hydrolysis rate of imipenem was considered to be 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 3-morpholinopropanesulfonate (MOPS)
buffer, 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.

1602
H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
solution was added allyl chloroformate (46.3 g, 384
mmol); the pH was maintained at 9.0 with 5 M aqueous
NaOH at 5 C. The reaction mixture was poured into
H2O, and the whole was 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 (n-hex-
ane/EtOAc=5/1) to give 7 (110.5 g, 68.5%) as a color-
less oil. [a]d20 +30.4 (c 1.0, CHCl3); IR nmax (Nujol)
(1H, m), 2.63 (1H, m), 2.80 (3H, s), 2.91 (3H, s), 3.71
(1H, m), 3.86 (1H, m), 4.50 (5H, m), 4.61 (2H, d, J=5.8
Hz), 4.94 (2H, m), 5.29 (3H, m), 5.83 (2H, m), 7.20 (2H,
d, J=8.8 Hz), 7.24 (2H, d, J=8.8 Hz). To the above
solution of the above (91.1 g, 201 mmol) in DMF (3000
mL) was added potassium thioacetate (46.1 g, 403
mmol) at room temperature; this solution was stirred
for 3 h at 60 ꢀC. The reaction mixture was poured into
H2O, and the whole was 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 (n-hex-
ane/EtOAc=2/1) to give 9g as a colorless oil (83.9 g,
96.3%). [a]d25 +50.6 (c 1.0, CHCl3); IR nmax (Nujol)
1
1704, 1394, 1253, 1139, 1091, 837, 775 cmꢁ1; H NMR
(300 MHz, CDCl3) d 0.02 (3H, s), 0.06 (3H, s), 0.78 (9H,
s), 1.16 (6H, s), 1.42 (3H, s), 1.87 (1H, m), 2.49 (1H, m),
[2.82 (1.4H, s), 2.83 (1.6H, s) each rotamer], 3.40 (1H,
m), 3.83 (1H, m), 4.36 (1H, m), 4.42 (2H, s), 4.62 (2H,
m), 4.70 (0.7H, m), 4.89 (0.3H, m), 5.27 (2H, m), 5.95
(1H, m), 7.15 (2H, d, J=8.2 Hz), 7.20 (2H, d, J=8.2
Hz); 13C NMR (67.5 MHz, CDCl3, major signals) d
ꢁ4.6, ꢁ4.5, 18.1, 25.9, 28.4, 34.3, 44.9, 52.3, 55.1, 60.3,
66.3, 70.3, 79.7, 117.4, 126.5, 127.4, 133.4, 135.7, 144.5,
154.7, 156.8; FAB-HRMS m/z calcd for C27H44N2O5-
SiNa (M+Na)+: 527.2917, found 527.2918.
1700, 1658, 1147, 760 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 2.26 (1H, m), 2.32 (3H, s), 2.36 (1H, m), [2.86
(1.5H, s), 2.87 (1.5H, s) each rotamer], 3.62 (1H, m),
4.09 (2H, m), 4.46 (2H, s), 4.56 (1H, m), 4.64 (2H, d,
J=5.5 Hz), 4.94 (2H, m), 5.36 (4H, m), 5.78 (2H, m),
7.21 (4H, s); 13C NMR (67.5 MHz, CDCl3, major sig-
nals) d 30.2, 34.0, 38.7, 39.8, 51.6, 52.5, 59.9, 65.3, 65.7,
77.4, 116.2, 116.9, 125.4, 127.2, 132.8, 136.1, 141.1,
141.7, 154.0, 156.1, 194.5; FAB-HRMS m/z calcd for
C22H29N2O5S (M+H)+: 433.1797, found 433.1776.
(2R,4R)-2-[4-(N-Allyloxycarbonyl-N-methylaminomethyl)-
phenyl]-1-allyloxycarbonyl-4-hydroxypyrrolidine 8. To a
solution of 7 (110.5 g, 219 mmol) in MeOH (1200 mL)
was added concd HCl (83 mL), and the mixture was
stirred for 30 min at reflux temperature. The mixture
was evaporated under reduced pressure, and the residue
was dissolved in 1,4-dioxane (1000 mL) and H2O (200
mL). To this solution was added allyl chloroformate
(46.3 mL, 384 mmol); the pH was maintained at 9.0
with 5 M aqueous NaOH at 5 ꢀC. The reaction mixture
was poured into H2O, and the whole was 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 chroma-
tography (n-hexane/EtOAc=1/2ꢃ1/4) to give 8 (82.0 g,
100%) as a colorless oil. [a]2d0 +49.2 (c 1.0, CHCl3); IR
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-[4-(N-
methylaminomethyl)phenyl]pyrrolidin-3-ylthio]-1-methyl-
1-carbapen-2-em-3-carboxylic acid dihydrochloride 1g.
To an ice-cooled solution of 9g (83.9 g, 194 mmol) in
MeOH (850 mL) was added 1 M aqueous NaOH (214
mL). After being stirred for 30 min at the same tem-
perature, the reaction mixture was adjusted to pH 7.0
with 1 M aqueous HCl and was concentrated under
reduced pressure. The mixture was poured into H2O,
and the whole was extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and
evaporated under reduced pressure. To a stirred solu-
tion of the residue and allyl (1R,5S,6S)-2-diphenylphos-
phoryloxy-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylate 10 (123 g, 247 mmol) in CH3CN
(1000 mL) was added N,N-diisopropylethylamine (42.5
mL, 252 mmol) in dropwise fashion at 0 ꢀC. After being
stirred overnight at 6 ꢀC, the mixture was poured into
H2O and the whole was 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
(n-hexane/EtOAc=1/4) to give the adduct, allyl
(1R,5S,6S)-2-[(3S,5R)-1-allyloxycarbonyl-5-[4-(N-allyl-
oxycarbonyl -N -methylaminomethyl)phenyl]pyrrolidin-
3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-
em-3-carboxylate (66.8 g, 53.7%) as a foam. [a]2d5 +82.8
(c 1.0, CHCl3); IR nmax (KBr) 3367, 2937, 2875, 1754,
n
max (Nujol) 3440, 1693, 1286, 1143, 1081, 769 cmꢁ1; 1H
NMR (300 MHz, CDCl3) d 2.01 (1H, m), 2.59 (1H, m),
2.84 (3H, br s), 3.62 (1H, dd, J=11.8, 3.6 Hz), 3.91 (1H,
m), 4.48 (5H, m), 4.62 (2H, d, J=5.5 Hz), 4.95 (2H, m),
5.27 (3H, m), 5.90 (2H, m), 7.20 (2H, d, J=9.3 Hz), 7.25
(2H, d, J=9.3 Hz); 13C NMR (67.5 MHz, CDCl3, major
signals) d 34.0, 42.9, 52.0, 54.9, 59.8, 65.4, 65.9, 69.4,
116.3, 117.0, 125.8, 127.5, 132.7, 135.5, 142.5, 143.0,
154.7, 156.3; FAB-HRMS m/z calcd for C20H27N2O5
(M+H)+: 375.1920, found 375.1929.
(2R,4S)-4-Acetylthio-2-[4-(N-allyloxycarbonyl-N-methyl-
aminomethyl)phenyl]-1-allyloxycarbonylpyrrolidine 9g.
To a solution of 8 (81.9 g, 219 mmol) in CH2Cl2 (1600
mL) were added triethylamine (61.0 g, 438 mmol) and
methanesulfonyl chloride (18.6 mL, 241 mmol) at
ꢁ40 ꢀC. The reaction mixture was poured into H2O, and
the whole was extracted with EtOAc. The organic layer
was washed with brine and dried over MgSO4. Subse-
quently the mixture was evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc=3/2) to give (2R,4R)-
2-[4-(N-allyloxycarbonyl-N-methylaminomethyl)phenyl]-
1-allyloxycarbonyl-4-mesyloxypyrrolidine as a colorless
1
1700, 1403, 769 cmꢁ1; H NMR (300 MHz, CDCl3) d
1.22 (3H, d, J=7.2 Hz), 1.36 (3H, d, J=6.3 Hz), 2.26
(1H, m), 2.40 (1H, m), 2.87 (3H, s), 3.23 (1H, dd,
J=7.1, 2.6 Hz), 3.30 (1H, m), 3.73 (2H, m), 4.03 (1H,
m), 4.24 (2H, m), 4.46 (2H, s), 4.58 (1H, m), 4.64 (2H,
m), 4.69 (1H, m), 4.83 (1H, dd, J=13.5, 5.5 Hz), 4.93
(1H, m), 5.12 (1H, m), 5.34 (6H, m), 5.93 (3H, m), 7.14
(4H, m); 13C NMR (67.5 MHz, CDCl3, major signals) d
16.1, 21.1, 32.9, 39.5, 40.6, 43.1, 51.2, 51.4, 54.2, 55.7,
59.0, 59.6, 65.0, 65.2, 65.4, 65.4, 115.8, 116.5, 177.7,
1
oil (91.1 g, 92.0%). H NMR (300 MHz, CDCl3) d 2.22

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1603
124.8, 127.3, 130.8, 132.2, 135.7, 140.7, 141.3, 147.3,
148.5, 153.5, 155.4, 159.6, 172.0; FAB-HRMS m/z calcd
for C33H42N3O8S (M+H)+: 640.2693, found 640.2691.
hydroxyethyl]-1-methyl-1-carbapen-2-em-3-carboxylate
as a foam (222 mg, 65.4%). IR nmax (KBr) 3373, 2966,
1751, 1587, 1392, 1086 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 1.22 (3H, d, J=7.0 Hz), 1.36 (3H, d, J=6.2
Hz), 2.24 (1H, m), 2.41 (1H, m), 3.21 (1H, dd, J=7.2,
2.4 Hz), 3.30 (1H, m), 3.78 (2H, m), 4.03 (1H, m), 4.24
(2H, m), 4.49 (5H, m), 4.63 (2H, m), 4.64 (1H, m), 4.75
(1H, m), 5.04 (2H, m), 5.26 (4H, m), 5.43 (1H, m), 5.94
(2H, m), 7.20 (4H, m); FAB-HRMS m/z calcd for
C32H40N3O8S (M+H)+: 626.2536, found: 626.2534.
To an ice-cooled solution of the adduct (66.3 g, 103.6
mmol) in CH2Cl2 (2072 mL) were successively added
H2O (9.32 mL), bis(triphenylphosphine)palladium(II)
dichloride (3.61 g, 5.18 mmol), and tributyltin hydride
(100.4 mL, 373 mmol). After being stirred for 30 min at
the same temperature, the reaction mixture was con-
centrated under reduced pressure to ca. 300 mL and
poured into H2O. The separated aqueous layer was con-
centrated under reduced pressure to ca. 500 mL. After
removal of the insoluble matter by filtration, the con-
centrated aqueous layer was subjected to reversed-phase
column chromatography. The eluent was monitored by
HPLC, and the fractions eluted with 15% CH3CN/H2O
were combined and adjusted to pH 6.0 with 1 M aqu-
eous HCl. The combined fractions were concentrated
under reduced pressure and lyophilized to give 1g as an
amorphous powder (20.2 g, 41.7%). The amorphous
powder was crystallized from 85% EtOH/H2O to yield
1g as dihydrochloride salt. [a]2d0 +9.0 (c 1.0, H2O); IR
To the ice-cooled solution obtained above (210 mg, 0.34
mmol) in CH2Cl2 (10 mL) were successively added H2O
(30 mL), bis(triphenylphosphine)palladium(II) dichlor-
ide (11.8 mg, 0.017 mmol), and tributyltin hydride (329
mL, 1.22 mmol) under a nitrogen atmosphere. After
being stirred for 20 min at the same temperature, the
reaction mixture was poured into H2O. The separated
aqueous layer was washed with CH2Cl2 and con-
centrated under reduced pressure to ca. 3 mL. After
removal of the insoluble matter by filtration, the con-
centrated aqueous layer was subjected to reversed-phase
column chromatography. The eluent was monitored by
HPLC, and the fractions eluted with 15% CH3CN/H2O
were combined and adjusted to pH 6.4 with 0.05 M
aqueous HCl. The combined fractions were con-
centrated under reduced pressure and lyophilized to give
1a as an amorphous powder (110 mg, 72.9%). IR nmax
(KBr) nmax 3373, 1751, 1587, 1392, 1086 cmꢁ1 1H
;
NMR (500 MHz, D2O, as monohydrochloride) d 1.02
(3H, d, J=7.3 Hz), 1.08 (3H, d, J=6.4 Hz), 2.33 (1H, dd,
J=14.0, 6.7 Hz), 2.52 (3H, s), 2.57 (1H, m), 3.17 (1H, dq,
J=9.1, 7.3 Hz), 3.27 (2H, m), 3.70 (1H, dd, J=12.8, 5.8
Hz), 4.04 (5H, m), 4.88 (1H, dd, J=11.0, 6.7 Hz), 7.35
(4H, m); 13C NMR (125MHz, D2O, as mono-
hydrochloride) d 15.4, 19.7, 31.9, 35.9, 40.7, 42.1, 51.4,
51.9, 55.6, 58.5, 61.2, 64.7, 128.1, 130.2, 131.8, 134.2,
135.2, 136.7, 167.3, 176.3; FAB-HRMS m/z calcd
for C22H30N3O4S (M+H)+: 432.1957, found 432.1950;
;
(KBr) 3421, 1749, 1646, 1558 cmꢁ1 1H NMR
(300 MHz, D2O) d 1.22 (3H, d, J=7.0 Hz), 1.27 (3H, d,
J=6.5 Hz), 2.51 (1H, m), 2.73 (1H, m), 3.40 (3H, m),
3.86 (1H, dd, J=12.5, 6.0 Hz), 4.25 (5H, m), 5.03 (1H,
dd, J=10.5, 7.0 Hz), 7.20 (4H, m); FAB-HRMS m/z
calcd for C21H28N3O4S (M+H)+: 418.1801, found:
418.1800; UV lmax298 (e 9520).
.
.
Anal. calcd for C22H29N3O4S 2HCl H2O: C, 50.57; H,
6.37; N, 8.04; S, 6.14; found: C, 50.87; H, 6.45; N, 7.83;
S, 6.02.
(1R,5S,6S)-2-[(3S,5S)-5-(4-Aminomethylphenyl)pyrroli-
din-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylic acid hydrochloride 1b. IR nmax (KBr)
3421, 1749, 1652, 1558 cmꢁ1; 1H NMR (300 MHz, D2O)
d 1.22 (3H, d, J=7.0 Hz), 1.27 (3H, d, J=6.0 Hz), 2.14
(1H, m), 3.00 (1H, m), 3.37 (1H, m), 3.46 (2H, m), 3.80
(1H, m), 4.14 (1H, m), 4.20 (2H, s), 4.24 (1H, m), 7.52
(2H, d, J=8.0 Hz), 7.55 (2H, d, J=8.0 Hz); FAB-
HRMS m/z calcd for C21H28N3O4S (M+H)+:
418.1801, found: 418.1793; UV lmax 298 (e 9910).
(1R,5S,6S)-2-[(3S,5R)-5-(4-Aminomethylphenyl)pyrroli-
din-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylic acid hydrochloride 1a. To an ice-
cooled solution of (2R,4S)-4-acetylthio-2-[4-(N-allyloxy-
carbonylaminomethyl)phenyl]-1-allyloxycarbonyl-
pyrrolidine 9a (227 mg, 0.54 mmol) in MeOH (8 mL)
was added 1 M aqueous NaOH (540 mL) under a nitro-
gen atmosphere. After being stirred for 20 min at 0 ꢀC,
the reaction mixture was adjusted to pH 7.0 with 1 M
aqueous HCl and concentrated under reduced pressure.
The mixture was poured into H2O, and the whole was
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated under
reduced pressure. To a stirred solution of the residue
and 10 (271 mg, 0.54 mmol) in CH3CN (10 mL) was
added N,N-diisopropylethylamine (153 mL, 0.87 mmol)
in a dropwise fashion at 0 ꢀC. After being stirred over-
night at 4 ꢀC, the mixture was poured into H2O and the
whole was extracted with EtOAc. The organic layer was
washed with brine, dried over MgSO4, and evapo-
rated under reduced pressure. The residue was pur-
ified by silica gel column chromatography (n-hexane/
EtOAc=2/3) to give the adduct, allyl (1R,5S,6S)-2-
[(3S,5R)-1-allyloxycarbonyl-5-[4-(N-allyloxycarbonyl-
aminomethyl)phenyl]pyrrolidin -3 -ylthio] -6 -[( R) - 1 -
(1R,5S,6S)-2-[(3S,5R)-5-(3-Aminomethylphenyl)pyrroli-
din-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylic acid hydrochloride 1c. IR nmax (KBr)
1749, 1648, 1558 1540 cmꢁ1; 1H NMR (300 MHz, D2O)
d 1.21 (3H, d, J=7.3 Hz), 1.25 (3H, d, J=6.4 Hz), 2.51
(1H, m), 2.74 (1H, m), 3.41 (3H, m), 3.84 (1H, m), 4.22
(5H, s), 5.03 (1H, m), 7.52 (4H, m); FAB-HRMS m/z
calcd for C21H28N3O4S (M+H)+: 418.1801, found:
418.1818; UV lmax 299 (e 10,700).
(1R,5S,6S)-2-[(3S,5S)-5-(3-Aminomethylphenyl)pyrroli-
din-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylic acid hydrochloride 1d. IR nmax (KBr)
1747, 1650, 1558, 1540 cmꢁ1; 1H NMR (300 MHz, D2O)
d 1.21 (3H, d, J=7.3 Hz), 1.26 (3H, d, J=6.3 Hz), 2.16
(1H, m), 3.02 (1H, m), 3.35 (1H, m), 3.43 (2H, m), 3.78

1604
H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
(1H, m), 4.20 (5H, m), 7.55 (4H, m); FAB-HRMS m/z
calcd for C21H28N3O4S (M+H)+: 418.1801, found:
418.1794; UV lmax 299 (e 9920).
To an ice-cooled solution of diastereomer I (219 mg,
0.38 mmol) in CH2Cl2 (7.6 mL) were successively added
H2O (34 mL), bis(triphenylphosphine)palladium(II)
dichloride (13.4 mg, 0.019 mmol), and tributyltin
hydride (370 mL, 1.36 mmol) under a nitrogen atmo-
sphere. After being stirred for 30 min at the same tem-
perature, the reaction mixture was poured into H2O.
The separated aqueous layer was washed with CH2Cl2
and concentrated under reduced pressure. After
removal of the insoluble matter by filtration, the con-
centrated aqueous layer was subjected to reversed-phase
column chromatography. The eluent was monitored by
HPLC, and the fractions eluted with 15% CH3CN/H2O
were combined and adjusted to pH 6.0 with 0.05 M
aqueous HCl. The combined fractions were con-
centrated under reduced pressure and lyophilized to give
1 h as an amorphous powder (29.4 mg, 16.6%). IR nmax
(1R,5S,6S)-2-[(3S,5R)-5-(2-Aminomethylphenyl)pyrroli-
din-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylic acid hydrochloride 1e. IR nmax (KBr)
1
1753, 1588, 1395 cmꢁ1; H NMR (300 MHz, D2O) d
1.20 (3H, d, J=7.3 Hz), 1.25 (3H, d, J=6.3 Hz), 2.16
(1H, m), 2.44 (1H, m), 3.27 (3H, m), 3.52 (1H, dd,
J=12.3, 6.8 Hz), 4.15 (4H, m), 4.57 (1H, m), 7.55 (4H,
m); FAB-HRMS m/z calcd for C21H28N3O4S (M+H)+:
418.1801, found: 418.1812.
(1R,5S,6S)-2-[(3S,5S)-5-(2-Aminomethylphenyl)pyrroli-
din-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-
2-em-3-carboxylic acid hydrochloride 1f. IR nmax (KBr)
1751, 1576, 1394, 1286 cmꢁ1; 1H NMR (300 MHz, D2O)
d 1.20 (3H, d, J=7.3 Hz), 1.25 (3H, d, J=6.3 Hz), 1.99
(1H, m), 2.83 (1H, m), 3.29 (3H, m), 3.56 (1H, dd,
J=12.3, 6.8 Hz), 4.15 (4H, m), 4.63 (1H, m), 7.55 (4H,
m); FAB-HRMS m/z calcd for C21H28N3O4S (M+H)+:
418.1801, found: 418.1796; UV lmax 301 (e 9480).
(KBr) 1737, 1650, 1558 cmꢁ1 1H NMR (300 MHz,
;
D2O) d 1.25 (3H, d, J=7.3 Hz), 1.29 (3H, d, J=6.3 Hz),
2.54 (1H, m), 2.78 (1H, m), 3.41 (3H, m), 3.89 (1H, dd,
J=12.6, 5.6 Hz), 4.26 (3H, m), 5.09 (1H, dd, J=11.1,
7.0 Hz), 7.50 (3H, s); FAB-MS m/z 430 (M+H)+; UV
lmax 298 (e 9160).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-[4-(5-
isoindoliniy)pyrrolidin-3-ylthio]-1-methyl-1-carbapen-2-
em-3-carboxylic acid hydrochloride 1 h. To an ice-
cooled solution of (4S)-4-acetylthio-4-[5-(N-allyloxy-
carbonyl)isoindolinyl]-1-allyloxycarbonylpyrrolidine 9h
(435 mg, 1.09 mmol) in MeOH (10 mL) was added
1 M aqueous NaOH (1.09 mL) under a nitrogen atmo-
sphere. After being stirred for 10 min at 0 ꢀC, the reac-
tion mixture was adjusted to pH 7.0 with 1 M aqueous
HCl and concentrated under reduced pressure. The
mixture was poured into H2O, and the whole was
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated under
reduced pressure. To a stirred solution of the residue
and 10 (600 mg, 1.20 mmol) in CH3CN (10 mL) was
added N,N-diisopropylethylamine (284 mL, 1.63 mmol)
in a dropwise fashion at 0 ꢀC. After being stirred over-
night at 4 ꢀC, the mixture was poured into H2O and the
whole was 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 (n-hexane/EtOAc=
1/4ꢃEtOAc) to give both allyl (1R,5S,6S)-2-[(3S,5R)-5-
[5-(N-allyloxycarbonyl)isoindolinyl]-1-allyloxycarbonyl-
pyrrolidin-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-
carbapen-2-em-3-carboxylate (diastereomer I, 238.7 mg,
38.0%) and allyl (1R,5S,6S)-2-[(3S,5S)-5-[5-(N-allylox-
ycarbonyl)isoindolinyl]-1-allyloxycarbonylpyrrolidin-3-
ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-em-
3-carboxylate (diastereomer II, 219.8 mg, 35.0%) as
foams. diastereomer I: IR nmax (KBr) 3378, 2988, 1759,
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-[4-[N-
(methoxy)aminomethyl]phenyl]pyrrolidin - 3 - ylthio] - 1 -
methyl-1-carbapen-2-em-3-carboxylic acid hydrochloride
1i. IR nmax (KBr) 1747, 1650, 1558, 1540 cmꢁ1 1H
;
NMR (300 MHz, D2O) d 1.23 (3H, d, J=7.1 Hz), 1.27
(3H, d, J=6.3 Hz), 2.52 (1H, m), 2.78 (1H, m), 3.44
(6H, m), 3.88 (1H, m), 4.05 (2H, s), 4.25 (3H, m), 5.08
(1H, m), 7.49 (4H, m); FAB-MS m/z 448 (M+H)+; UV
lmax 297 (e 10,100).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-(1,2,3,4
-tetrahydrophthalazin-6-yl)pyrrolidin-3-ylthio]-1-methyl-
1-carbapen-2-em-3-carboxylic acid hydrochloride 1j. IR
;
nmax (KBr) 3390, 1749, 1591, 1392 cmꢁ1 1H NMR
(300 MHz, D2O) d 1.24 (3H, d, J=7.3 Hz), 1.29 (3H, d,
J=6.4 Hz), 2.51 (1H, m), 2.77 (1H, m), 3.41 (3H, m),
3.88 (1H, dd, J=12.5, 6.9 Hz), 4.06 (4H, s), 4.24 (3H,
m), 5.03 (1H, dd, J=11.2, 6.9 Hz), 7.27 (3H, m); FAB-
HRMS m/z calcd for C22H29N4O4S (M+H)+:
445.1910, found: 445.1902; UV lmax 298 (e 11,100).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-[4-(iso-
thioureidomethyl)phenyl]pyrrolidin-3-ylthio]-1-methyl-1-
carbapen-2-em-3-carboxylic acid hydrochloride 1k. To
an ice-cooled solution of allyl (1R,5S,6S)-2-[(3S,5R)-1-
allyloxycarbonyl-5-[4-(iodomethyl)phenyl]pyrrolidin - 3 -
ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-em-
3-carboxylate (3.8 g, 5.82 mmol) in CH3CN (100 mL)
was added thiourea (443 mg, 5.82 mmol). After being
stirred overnight at 4 ꢀC, the mixture was poured into
H2O and the whole was 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
(CHCl3/acetone=4/1ꢃ1/1) to give allyl (1R,5S,6S)-2-
[(3S,5R)-1-allyloxycarbonyl-5-[4-(isothioureidomethyl)-
phenyl]pyrrolidin-3-ylthio]-6-[( R)-1-hydroxyethyl]-1-
methyl-1-carbapen-2-em-3-carboxylate hydroiodide salt
as a foam (2.35 g, 55.4%). IR nmax (KBr) 1754, 1682,
1
1086, 776 cmꢁ1; H NMR (300 MHz, CDCl3) d 1.26
(3H, d, J=6.9 Hz), 1.36 (3H, d, J=6.2 Hz), 2.23 (1H,
m), 2.42 (1H, m), 3.24 (1H, dd, J=7.1, 2.6 Hz), 3.30
(1H, m), 3.69 (1H, m), 3.81 (1H, m), 4.04 (1H, m), 4.26
(2H, m), 4.50 (2H, m), 4.74 (6H, m), 4.77 (4H, m), 5.30
(6H, m), 5.96 (3H, m), 7.20 (3H, m); FAB-HRMS m/z
calcd for C39H51N4O10S (M+H)+: 767.3326, found:
767.3326.

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1605
1
808, 777 cmꢁ1; H NMR (300 MHz, CD3OD) d 1.22
carboxylate (2.896 g, 57.2%) as a yellow foam. IR nmax
(KBr) 3338, 1760, 1680, 1560, 1390, 1266, 1080 cmꢁ1
(3H. d, J=7.3 Hz), 1.28 (3H, d, J=6.4 Hz), 2.28 (1H,
m), 2.46 (1H, m), 3.52 (1H, m), 3.76 (1H, m), 4.06 (2H,
m), 4.24 (1H, d, J=10.8, 2.8 Hz), 4.42 (4H, br s), 4.51
(1H, m), 4.70 (1H, m), 5.18 (4H, m), 5.40 (1H, m), 5.93
(2H, m), 7.29 (2H, d, J=8.4 Hz), 7.40 (2H, d, J=8.4
Hz); FAB-MS m/z 601 (M+H)+.
;
1H NMR (300 MHz, CDCl3) d 1.27 (3H, m), 1.38 (3H,
d, J=6.2 Hz), 2.21 (2H, m), 2.27 (2H, m), 2.44 (1H, m),
3.31 (2H, m), 3.80 (2H, m), 4.04 (1H, m), 4.28 (2H, m),
4.53 (2H, m), 5.15 (5H, m), 5.52 (1H, m), 6.98 (1H, m),
7.21 (5H, m), 7.55 (2H, d, J=8.5 Hz), 7.63 (2H, d,
J=8.5 Hz), 8.02 (1H, m), 8.23 (4H, m); FAB-MS m/z
974 (M+Na)+; FAB-HRMS m/z calcd for
C46H46N7O14S (M+H)+: 952.2823, found: 952.2803.
To the ice-cooled solution of obtained above (1.65 g,
2.27 mmol) in CH2Cl2 (100 mL) were successively added
H2O (203 mL), bis(triphenylphosphine)palladium(II)
dichloride (80.0 mg, 0.11 mmol), and tributylthin
hydride (2.01 g, 7.49 mmol). After being stirred for 20
min at the same temperature, the reaction mixture was
concentrated under reduced pressure and poured into
H2O (20 mL). The aqueous layer was washed with
CHCl3. After removal of the insoluble matter by filtra-
tion, the concentrated aqueous layer (100 mL) was sub-
jected to reversed-phase column chromatography. The
eluent was monitored by HPLC, and the fractions
eluted with 30% MeOH/H2O were combined and
adjusted to pH 6.0 with 1 M aqueous HCl. The com-
bined fractions were concentrated under reduced pres-
sure and lyophilized to give 1k as an amorphous powder
(71.3 mg, 4.7%). IR nmax (KBr) 3415, 2971, 1751, 1650,
1583, 1396, 773, 707 cmꢁ1; 1H NMR (300 MHz, D2O) d
1.22 (3H, d, J=7.7 Hz), 1.27 (3H, d, J=6.6 Hz), 2.51
(1H, m), 2.76 (1H, m), 3.41 (4H, m), 3.88 (1H, m), 4.23
(2H, m), 4.42 (2H, s), 5.06 (1H, m), 7.48 (2H, d, J=8.0
Hz), 7.53 (2H, d, J=8.0 Hz); FAB-MS m/z 499
(M+H)+; FAB-HRMS m/z 499 (M+Na)+; FAB-
HRMS m/z calcd for C22H29N4O4S2 (M+H)+:
477.1630, found: 477.1642; UV lmax 298 (e 5490).
The mixture obtained above (1.22 g, 1.28 mmol) and
10% Pd/C (1.22 g) in THF (86 mL), EtOH (11 mL), and
0.2 M sodium 3-morpholinopropanesulfonate buffer
(MOPS buffer, pH 6.5, 64 mL) was stirred for 15 h at
room temperature under a hydrogen atmosphere. The
catalyst was removed by filtration and washed with
H2O. The combined filtrate and washings were con-
centrated under reduced pressure to ca. 20 mL. After
the insoluble portion of the aqueous layer was removed
by filtration, the filtrate was subjected to reversed-phase
column chromatography. The eluent was monitored by
HPLC, and the fractions eluted with 30% MeOH/H2O
were combined and adjusted to pH 6.0 with 0.1 M aqu-
eous HCl. The combined fractions were concentrated
under reduced pressure and lyophilized to give 1l as an
amorphous powder (282.6 mg, 44.6%). IR nmax (KBr)
3338, 1749, 1681, 1648, 1560, 1394, 1263, 1180, 1147,
1
1072, 1054, 808, 773, 723 cmꢁ1; H NMR (300 MHz,
D2O) d 1.21 (3H, d, J=7.1 Hz), 1.26 (3H, d, J=6.3 Hz),
2.26 (3H, s), 2.48 (1H, m), 2.78 (1H, m), 3.36 (1H, m),
3.48 (2H, m), 3.87 (1H, dd, J=12.5, 5.9 Hz), 4.21 (3H,
m), 4.51 (2H, s), 5.05 (1H, m), 7.42 (2H, d, J=8.3 Hz),
7.49 (2H, d, J=8.3 Hz); FAB-HRMS m/z calcd for
C23H31N4O4S (M+H)+: 459.2066, found: 459.2094;
UV lmax 298 (e 7590).
(1R,5S,6S)-2-[(3S,5R) - 5 - [4 - [N - (Acetimidoyl)aminome-
thyl]phenyl]pyrrolidin-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-
methyl-1-carbapen-2-em-3-carboxylic acid hydrochloride
1l. To an ice-cooled solution of (2R,4S)-4-acetylthio-2-
[4 -[ N - (p -nitrobenzyloxycarbonylacetimidoyl)amino-
methyl]phenyl]-1-p-nitrobenzyloxycarbonylpyrrolidine 9l
(3.45 g, 5.32 mmol) in MeOH (70 mL) and THF (30
mL) was added 1 M aqueous NaOH (5.3 mL). After
being stirred for 10 min at the same temperature, the
reaction mixture was adjusted to pH 7.0 with 1 M aqu-
eous HCl and concentrated under reduced pressure. The
mixture was poured into H2O, and the whole was
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated under
reduced pressure. To a stirred solution of the residue
and p-nitrobenzyl (1R,5S,6S)-2-diphenylphosphoryloxy-
6-[(R)-1-hydroxyethyl] - 1 - methyl - 1 - carbapen-2-em-3-
carboxylate 11 (3.16 g, 5.32 mmol) in CH3CN (140 mL)
was added N,N-diisopropylethylamine (1.29 mL, 7.44
mmol) in a dropwise fashion at 0 C. After being stirred
for 17 h at 6 ꢀC, the mixture was poured into H2O and
the whole was extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and evapo-
rated under reduced pressure. The residue was purified
by silica gel column chromatography (EtOAc/ace-
tone=4/1) to give p-nitrobenzyl (1R,5S,6S)-2-[5-[4-[N-
(p -nitrobenzyloxycarbonylacetimidoyl)aminomethyl]-
phenyl]-1-p-nitrobenzyloxycarbonylpyrrolidine-3-ylthio]-
6-[(R)-1-hydroxyethyl] - 1 - methyl - 1 - carbapen-2-em-3-
(1R,5S,6S)-2-[(3S,5R)-5-[3,4-Bis(aminomethyl)phenyl]-
pyrrolidin-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-
carbapen-2-em-3-carboxylic acid dihydrochloride 1m. IR
;
nmax (KBr) 1737, 1650, 1558, 1540, 1070 cmꢁ1 1H
NMR (300 MHz, D2O) d 1.23 (3H, d, J=7.3 Hz), 1.28
(3H, d, J=6.3 Hz), 2.48 (1H, m), 2.63 (1H, m), 3.39
(3H, m), 3.81 (1H, m), 4.20 (7H, m), 7.54 (3H, m); FAB-
HRMS m/z calcd for C22H31N4O4S (M+H)+:
447.2066, found: 447.2037; UV lmax 298 (e 9640).
(2R,4R)-1-tert - Butoxycarbonyl - 4 - tert - butyldimethylsi-
loxy-2-[(E)-2-(ethoxycarbonyl)vinyl] pyrrolidine 13. To a
mixture of 60% NaH (6.52 g, 163 mmol) in THF (500
mL) was added triethyl phosphonoacetate (36.6 g, 163
mmol) in a dropwise manner at 0 ꢀC and the mixture
was stirred for 1 h at the same temperature. A solution
of 12 (53.5 g, 163 mmol) in THF (100 mL) was added in
a dropwise manner at a temperature below 5 ꢀC, and the
mixture was stirred for 10 min at the same temperature.
The mixture was poured into saturated aqueous NH4Cl,
and the whole was 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 (n-hex-
ane/EtOAc=9/1ꢃ6/1) to give 13 (61.5 g, 94.8%) as a
colorless oil. IR (Nujol) nmax 1698 cmꢁ1 1H NMR
;

1606
H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
(300 MHz, CDCl3) d 0.08 (6H, s), 0.89 (9H, s), 1.24 (9H,
s), 1.24 (3H, t, J=7.0 Hz), 1.39 (6H, br s), 1.43 (3H, br s),
1.79 (1H, m), 2.18 (1H, m), 3.30 (1H, m), 3.54 (1H, m),
4.14 (2H, q, J=7.0 Hz), 4.38 (2H, m), 5.78 (1H, m), 6.96
(1H, dd, J=15.4, 7.5 Hz); FAB-MS m/z 422 (M+Na)+.
4.24 (1H, m), 4.43 (1H, m), 5.78 (1H, m), 6.74 (1H, m);
FAB-MS m/z 422 (M+Na)+.
(2S,4S)-1-tert-butoxycarbonyl-4-tert-butyldimethylsiloxy-
2-(1,5-pentanediol-3-yl)pyrrolidine 17. To an ice-cooling
solution of 60% NaH (4.12 g, 103 mmol) in THF
(600 mL) was added diethyl malonate (15.6 mL, 103
mmol), and the mixture was stirred for 30 min at the
same temperature. To the mixture were added tetra-
n-butylammonium bromide (3.3 g, 10.3 mmol) and a
solution of 14 (34.1 g, 85.5 mol) in THF (80 mL). The
reaction mixture was stirred for 24 h at 50 ꢀC. The mix-
ture was poured into H2O, and the whole was extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and evaporated under reduced pres-
sure. The residue was passed through silica gel (500 g,
n-hexane/acetone=20/1ꢃ10/1) to give crude 15 as a
yellow oil. The residue was used for the next reaction
without further purification. FAB-MS m/z 560
(M+H)+. To a solution of crude 15 in DMSO (300
mL) were added NaCl (5.0 g, 85.4 mmol) and H2O (3.1
mL), and the mixture was stirred for 3 h at 160 ꢀC. The
mixture was poured into H2O, and the whole was
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated under
reduced pressure to give crude 16 (27.94 g) as a brown
oil. The residue was used for next reaction without fur-
ther purification. FAB-MS m/z 510 (M+Na)+. To a
solution of crude 16 (27.9 g) in THF (500 mL) was
added LiAlH4 (5.4 g, 143 mmol) in a dropwise fashion
at ꢁ20 ꢀC, and the mixture was stirred for 30 min at the
same temperature. The reaction mixture was quenched
with saturated aqueous NH4Cl. Subsequently, the mix-
ture was poured into H2O, and the whole was extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and evaporated under reduced pres-
sure. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc=1/3ꢃEtOAc) to
give 17 (9.09 g, 26.4%, three steps) as a yellow oil. IR
(2R,4S)-1-tert-Butoxycarbonyl-4-tert-butyldimethylsiloxy-
2 - [(E) - 2 - (ethoxycarbonyl)vinyl] pyrrolidine 14. To a
solution of 13 (55.8 g, 140 mmol) in THF (500 mL) was
added tetra-n-butylammonium fluoride (1 M in THF,
140 mL, 140 mmol) in a dropwise manner at 0 ꢀC, and
the mixture was stirred for 30 min at the same tem-
perature. The mixture was poured into 0. 4 M sodium
phosphate buffer (pH 6.5), and the whole was extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and evaporated under reduced pres-
sure. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc=1/2) to give (2R,4R)-
1-tert-butoxycarbonyl-2-[(E)-2-(ethoxycarbonyl)vinyl]-4-
hydroxypyrrolidine (39.1 g, 98.1%) as a yellow oil. IR
1
(Nujol) nmax 1698 cmꢁ1; H NMR (300 MHz, CDCl3) d
1.24 (3H, t, J=7.2 Hz), 1.43 (9H, s), 1.90 (1H, m), 2.32
(1H, m), 3.44 (1H, m), 3.62 (1H, m), 4.18 (2H, q, J=7.0
Hz), 4.43 (2H, m), 5.87 (1H, m), 7.01 (1H, dd, J=15.3,
8.3 Hz); FAB-MS m/z 308 (M+Na)+. To the ice-cool-
ing solution obtained above (37.9 g, 133 mmol) in THF
(1000 mL) were successively added triphenylphosphine
(105 g, 399 mmol), diisopropyl azodicarboxylate (80.7 g,
399 mmol), and acetic acid (24 g, 399 mmol) in a drop-
wise fashion and the mixture was stirred for 30 min at
the same temperature. The mixture was then poured
into H2O, and the whole was extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4,
and evaporated under reduced pressure. To a solution
of the residue in EtOH (600 mL) was added 5 M aqu-
eous NaOH (28 mL) at room temperature, and the
mixture was stirred for 10 min at same temperature.
After neutralization with 5 M aqueous HCl, the mixture
was poured into H2O, and the whole was 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 chroma-
tography (n-hexane/EtOAc=1/2) to give (2R,4S)-1-tert-
butoxycarbonyl -2 -[( E) -2 -(ethoxycarbonyl)vinyl]-4-
hydroxypyrrolidine (31.1 g, 82.2%) as a yellow oil. IR
(Nujol) nmax 3402, 1690 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 0.03 (6H, s), 0.83 (9H, s), 1.42 (9H, s), 1.55
(5H, m), 1.83 (1H, m), 2.39 (1H, m), 3.23 (1H, dd,
J=11.6, 3.7 Hz), 3.63 (2H, t, J=6.7 Hz), 3.70 (2H, m),
4.06 (1H, m), 4.27 (1H, m); FAB-HRMS m/z calcd for
C20H41NO5SiNa
426.2645.
(M+Na)+:
426.2652,
found:
(Nujol) nmax 3400, 1698 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 1.24 (3H, t, J=7.2 Hz), 1.42 (9H, s), 1.83 (1H,
m), 2.16 (1H, m), 3.54 (2H, m), 4.18 (2H, q, J=7.0 Hz),
4.46 (2H, m), 5.85 (1H, d, J=15. 4 Hz), 6.83 (1H, m);
FAB-MS m/z 308 (M+Na)+. To the solution obtained
above (31.1 g, 109 mmol) in DMF (500 mL) were added
imidazole (14.8 g, 218 mmol) and tert-butyldimethyl-
chlorosilane (18.1 g, 120 mmol) at room temperature
and the mixture was stirred for 3 h at same temperature.
The mixture was poured into H2O and the whole was
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 (n-hexane/EtOAc=10/1) to
give 14 (34.42 g, 78.9%) as a yellow oil. IR (Nujol) nmax
(2S,4S)-1-tert-Butoxycarbonyl-4-tert-butyldimethylsiloxy-
2 - (4 - cyano - 4 - phenylsulfonylcyclohexyl)pyrrolidine 18.
To a solution of 17 (7.09 g, 17.6 mmol) in THF (300
mL) were successively added 1,10-(azodicarbonyl)dipi-
peridine (20 g, 79.3 mmol), phenylsulfonylacetonitrile
(4.08 g, 22.5 mmol), and trimethylphosphine (1 M in
THF, 70.4 mL, 70.4 mmol), and the mixture was stirred
for 13 h at 50 ꢀC. The resulting reaction mixture was
evaporated under reduced pressure. The reaction mix-
ture was quenched with saturated aqueous NH4Cl.
Subsequently, the mixture was poured into H2O, and
the whole was extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and evapo-
rated under reduced pressure. The residue was purified
by silica gel column chromatography (n-hexane/
EtOAc=5/1ꢃ4/1) to give 18 (7.22 g, 74.9%) as a solid.
1
1698 cmꢁ1; H NMR (300 MHz, CDCl3) d 0.04 (6H, s),
0.82 (9H, s), 1.22 (3H, t, J=7.1 Hz), 1.38 (9H, br s),
1.73 (1H, m), 2.01 (1H, m), 3.38 (2H, m), 4.12 (2H, m),

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1607
1
IR (Nujol) nmax 2235, 1692 cmꢁ1; H NMR (300 MHz,
washed with brine and dried over MgSO4, and the mix-
ture was evaporated under reduced pressure. The resi-
due was purified by silica gel column chromatography
(n-hexane/EtOAc=1/2) to give 21 (3.41 g, 80.7%) as a
CDCl3) d 0.03 (6H, s), 0.83 (9H, s), 1.43 (9H, s), 1.73
(7H, m), 1.96 (2H, m), 2.11 (2H, m), 3.11 (1H, dd,
J=11.3, 3.8 Hz), 3.50 (1H, m), 3.88 (1H, m), 4.22 (1H,
m), 7.60 (2H, m), 7.72 (1H, m), 7.94 (2H, d, J=8.3 Hz);
FAB-HRMS m/z calcd for C28H45N2O5SSi (M+H)+:
549.2818, found: 549.2798.
1
colorless oil. IR nmax (KBr) 3411, 1732, 1695 cmꢁ1; H
NMR (300 MHz, CDCl3) d 0.92 (2H, m), 1.21 (1H, m),
1.53 (4H, m), 1.85 (5H, m), 2.36 (1H, m), 3.01 (1H, m),
3.19 (1H, m), 3.32 (1H, m), 3.68 (1H, m), 4.08 (1H, m),
4.39 (1H, br s), 4.57 (4H, m), 4.74 (1H, m), 5.24 (4H,
m), 5.93 (2H, m); FAB-HRMS m/z calcd for
C19H31N2O5 (M+H)+: 367.2233, found: 367.2269.
(2S,4S)-1-tert-Butoxycarbonyl-4-tert-butyldimethylsiloxy-
2-(4-cyanocyclohexyl)pyrrolidine 19. To an ice-cooling
solution of 18 (6.72 g, 12.3 mmol) in THF (40 mL) and
MeOH (40 mL) were successively added HgCl2 (33 mg,
0.12 mmol) and Mg (1.5 g, 61.3 mmol), and the mixture
was stirred for 1 h at 18 ꢀC. The reaction mixture was
quenched with saturated aqueous NH4Cl. The mixture
was poured into H2O, and the whole was 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 chroma-
tography (n-hexane/EtOAc=4/1) to give 19 (4.88 g,
97.5%) as a colorless oil. IR (Nujol) nmax 2230, 1694
(2S,4R)-2 - [4 - (N - Allyloxycarbonylaminomethyl)cyclo-
hexyl]-1-allyloxycarbonyl-4-hydroxypyrrolidine 22. To
an ice-cooled solution of 21 (3.41 g, 9.34 mmol) in THF
(100 mL) were successively added triphenylphosphine
(7.34 g, 28.0 mmol), diisopropyl azodicarboxylate (5.51
mL, 28.0 mmol), and acetic acid (1.6 mL, 28.0 mmol),
and the mixture was stirred for 30 min at same tem-
perature. The resulting mixture was evaporated under
reduced pressure. The residue was purified by silica gel
column chromatography (n-hexane/EtOAc=2/1) to
give (2R,4R)-4-acetoxy-2-[4-(N-allyloxycarbonylamino-
methyl)cyclohexyl]-1-allyloxycarbonylpyrrolidine (3.56 g,
93.7%) as a colorless oil. IR nmax (KBr) 1730, 1693
cmꢁ1; 1H NMR (300 MHz, CDCl3) d 0.96 (2H, m), 1.33
(1H, m), 1.42 (3H, m), 1.75 (5H, m), 2.05 (3H, s), 2.18
(1H, m), 3.02 (1H, m), 3.19 (2H, m), 3.81 (1H, m), 4.03
(1H, m), 4.57 (4H, m), 4.74 (1H, m), 5.24 (4H, m), 5.90
(2H, m); FAB-MS m/z 409 (M+H)+. To the solution
obtained above (3.57 g, 8.75 mmol) in MeOH (50 mL)
was added 1 M aqueous NaOH (9 mL) at room tem-
perature, and the mixture was stirred for 30 min at same
temperature. After neutralization by 1 M aqueous HCl,
the mixture was poured into H2O and the whole was
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 (n-hexane/EtOAc=1/1) to
give 22 (3.2 g, 100%) as a colorless oil. IR (thin film)
cmꢁ1; H NMR (300 MHz, CDCl3) d 0.03 (3H, s), 0.05
1
(3H, s), 0.84 (9H, s), 1.44 (9H, s), 1.68 (8H, m), 2.01
(1H, m), 2.12 (1H, m), 2.31 (1H, m), 2.94 (1H, br s),
3.18 (1H, m), 3.52 (1H, m), 3.89 (1H, m), 4.27 (1H, m);
FAB-HRMS m/z calcd for C22H40N2O3SiNa
(M+Na)+: 431.2706, found: 431.2697.
(2S,4S)-2 - [4 - (N - Allyloxycarbonylaminomethyl)cyclo-
hexyl]-1-tert-butoxycarbonyl-4-tert-butyldimethylsiloxy-
pyrrolidine 20. To a solution of 19 (4.88 g, 12.0 mmol)
in MeOH (150 mL) was added Raney Ni (NDT-65,
kawaken, 4.88 g), and the mixture was stirred for 22 h
under 3.5 hydrogen atmosphere at room temperature.
The catalyst was filtered off and washed with MeOH.
The filtrate and washings were combined and evapo-
rated under reduced pressure. To an ice-cooling solution
of the residue in THF (90 mL) and H2O (10 mL) were
added N,N-diisopropylethylamine (10.5 mL, 60.6 mmol)
and allyl chloroformate (1.9 mL, 18.0 mmol). The mix-
ture was poured into H2O, and the whole was extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and evaporated under reduced pres-
sure. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc=4/1) to give 20
(5.74 g, 96.8%) as a colorless oil. IR (thin film) nmax
nmax 3433, 1730, 1696 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 1.00 (2H, m), 1.41 (4H, m), 1.76 (4H, m), 1.91
(1H, m), 2.11 (1H, m), 3.11 (3H, m), 3.88 (2H, m), 4.38
(1H, m), 4.58 (4H, m), 4.77 (1H, m), 5.26 (4H, m), 5.91
(2H, m); FAB-MS m/z 367 (M+H)+.
1688 cmꢁ1; H NMR (300 MHz, CDCl3) d 0.03 (6H, s),
(2S,4S)-4-Acetylthio-2-[4-(N-allyloxycarbonylaminome-
thyl)cyclohexyl]-1-allyloxycarbonylpyrrolidine 23. To an
ice-cooled solution of 22 (3.16 g, 8.75 mmol) in THF (80
mL) were successively added triphenylphosphine (5.74 g,
21.9 mmol), diisopropyl azodicarboxylate (4.30 mL,
21.9 mmol), and thioacetic acid (1.57 mL, 21.9 mmol),
and the mixture was stirred for 30 min at the same
temperature. The resulting mixture was evaporated
under reduced pressure. The residue was purified by
silica gel column chromatography (n-hexane/EtOAc=3/
1) to give a mixture of 23 and triphenylphosphine oxide
(5.15 g) as a colorless solid. 1H NMR (300 MHz,
CDCl3) d 0.96 (2H, m), 1.50 (7H, m), 1.80 (2H, m), 2.18
(1H, s), 2.32 (3H, s), 3.03 (1H, m), 3.17 (1H, m), 3.49
(1H, m), 3.69 (1H, m), 3.91 (2H, m), 4.58 (4H, m), 5.26
(4H, m), 5.92 (2H, m), 6.29 (1H, br s); FAB-MS m/z 425
(M+H)+.
1
0.80 (9H, s), 1.40 (9H, s), 1.48 (6H, m), 1.72 (4H, m),
2.98 (1H, m), 3.14 (1H, m), 3.42 (1H, m), 3.84 (1H, m),
4.23 (1H, m), 4.67 (1H, m), 5.20 (2H, m), 5.86 (1H, m);
FAB-MS m/z 497 (M+H)+.
(2S,4S) - 2 - [4 - (N - Allyloxycarbonylaminomethyl)cyclo-
hexyl]-1-allyloxycarbonyl-4- hydroxypyrrolidine 21. To
a solution of 20 (5.74 g, 11.6 mmol) in MeOH (10 mL)
was added 10% HCl/MeOH (100 mL), and the mixture
was stirred for 20 h at room temperature. The resulting
mixture was evaporated under reduced pressure. To a
solution of the residue in 1,4-dioxane (90 mL) and H2O
(9 mL) was added allyl chloroformate (1.84 mL); the pH
was maintained at 8.5 with 1 M aqueous NaOH at room
temperature. The mixture was poured into H2O, and the
whole was extracted with EtOAc. The organic layer was

1608
H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
(1R,5S,6S)-2-[(3S,5S)-5-[4-(Aminomethyl)cyclohexyl]-
pyrrolidin-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-
carbapen-2-em-3-carboxylic acid hydrochloride 2. To an
ice-cooled solution of crude 23 (5.15 g) in MeOH (60
mL) and THF (30 mL) was added 1 M aqueous NaOH
(1.0 mL). After being stirred for 20 min at the same
temperature, the reaction mixture was adjusted to pH
7.0 with 1 M aqueous HCl and concentrated under
reduced pressure. The mixture was poured into H2O,
and the whole was extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and
evaporated under reduced pressure. To an ice-cooled
solution of the residue and 10 (4.99 g, 10.0 mmol) in
CH3CN (90 mL) was added N,N-diisopropylethylamine
(1.98 mL, 11.4 mmol) in a dropwise fashion at 0 ꢀC.
After being stirred overnight at 4 ꢀC, the mixture was
poured into H2O and the whole was 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 chroma-
tography (n-hexane/EtOAc=1/4) to give allyl
(1R,5S,6S) -2 -[(3 S,5R) - 5 - [4 - (N -allyloxycarbonyl-
aminomethyl)cyclohexyl]-1-allyloxycarbonylpyrrolidin-3-
ylthio] -6 -[( R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-
em-3-carboxylate (2.37 g, 42.9%) as a yellow foam. IR
mmol), and the mixture was stirred for 20 h at room
temperature. The reaction mixture was quenched with
saturated aqueous NH4Cl. Subsequently, the mixture
was poured into H2O, and the whole was extracted with
EtOAc. The organic layer was washed with brine, dried
over MgSO4, and evaporated under reduced pressure.
To an ice-cooled solution of the residue in 1,4-dioxane
(90 mL) and H2O (10 mL) was added p-nitrobenzyl
chloroformate (2.52 g, 11.7 mmol); the pH was main-
tained at 8ꢃ10 with 1 M aqueous NaOH. The mixture
was poured into H2O, and the whole was extracted with
EtOAc. The organic layer was washed with brine and
dried over MgSO4, and the mixture was evaporated
under reduced pressure. The residue was purified by
silica gel column chromatography (n-hexane/EtOAc=
3/2) to give 26 (2.08 g, 32.6%) as a brown oil. IR nmax
1
(Nujol) 1745 cmꢁ1; H NMR (300 MHz, CDCl3) d 0.96
(9H, s), 3.42 (2H, t, J=6.7 Hz), 4.42 (2H, t, J=6.7 Hz),
4.44 (4H, m), 5.26 (4H, m), 5.29 (1H, br s), 7.63 (18H,
m), 8.22 (4H, m); FAB-HRMS m/z calcd for
C34H39N2O6Si (M+H)+: 599.2577, found: 599.2589.
2-Acetoxy-[N-(4-p-nitrobenzyloxycarbonyl)aminomethyl-
benzyl-N-p-nitrobenzyloxycarbonyl]ethylamine 27. To a
solution of the residue in pyridine (25 mL) were added
acetic anhydride (10 mL) and catalytic amount of
4-dimethylaminopyridine, and the mixture was stirred
for 18 h at room temperature. The mixture was poured
into H2O, and the whole was extracted with EtOAc. The
organic layer was washed with brine and dried over
MgSO4, and the mixture was evaporated under reduced
pressure. To a solution of the residue in THF (30 mL)
was added tetra-n-butylammonium fluoride (1 M in
THF, 5.24 mL, 5.24 mmol), and the mixture was stirred
for 1 h at room temperature. The mixture was poured
into H2O, and the whole was extracted with EtOAc. The
organic layer was washed with brine and dried over
MgSO4, and the mixture was evaporated under reduced
pressure. To an ice-cooled solution of the residue in
THF (30 mL) were added N,N-diisopropylethylamine
(1.22 mL, 7.0 mmol) and methanesulfonyl chloride (351
mL, 4.54 mL), and the mixture was stirred for 30 min at
the same temperature. The mixture was poured into
H2O, and the whole was extracted with EtOAc. The
organic layer was washed with brine and dried over
MgSO4, and the mixture was evaporated under reduced
pressure. To a solution of the residue in DMF (80 mL)
was added sodium azide (340 mg, 5.24 mmol), and the
mixture was stirred for 1 h at room temperature. The
organic layer was washed with brine and dried over
MgSO4, and the mixture was evaporated under reduced
pressure. To a solution of the residue in THF (60 mL)
were added triphenylphosphine (1.37 g, 5.24 mmol) and
H2O (10 mL). After being stirred for 14 h at room
temperature, the reaction mixture was concentrated
under reduced pressure. To an ice-cooled solution of the
residue in 1,4-dioxane (80 mL) and H2O (20 mL) was
added p-nitrobenzyl chloroformate (980 mg, 4.54
mmol); the pH was maintained at 8.5 with 1 M aqueous
NaOH. The mixture was poured into H2O, and the
whole was extracted with EtOAc. The organic layer was
washed with brine and dried over MgSO4, and the mix-
ture was evaporated under reduced pressure. The resi-
nmax (KBr) 1749, 1688, 1554, 1396 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 0.91 (2H, m), 1.26 (3H, m), 1.37
(3H, d, J=6.7 Hz), 1.66 (8H, m), 1.90 (1H, m), 2.18
(1H, m), 3.08 (2H, m), 3.27 (2H, m), 3.66 (3H, m), 3.99
(1H, m), 4.24 (2H, m), 4.57 (4H, m), 4.73 (3H, m), 5.30
(6H, m), 5.92 (3H. m); FAB-HRMS m/z calcd for
C32H46N3O8S (M+H)+: 632.3006, found: 632.2987.
To the ice-cooled solution obtained above (631 mg, 1.00
mmol) in CH2Cl2 (20 mL) were successively added H2O
(90 mL), bis(triphenylphosphine)palladium(II) dichlor-
ide (35 mg, 0.05 mmol) and tributyltin hydride (968 mL,
3.6 mmol). After being stirred for 30 min at the same
temperature, the reaction mixture was concentrated
under reduced pressure and poured into H2O. The
separated aqueous layer was concentrated under
reduced pressure to ca. 10 mL. After removal of the
insoluble matter by filtration, the concentrated aqueous
layer was subjected to reversed (phase column chroma-
tography. The eluent was monitored by HPLC, and the
fractions eluted with 30% MeOH/H2O were combined
and adjusted to pH 6.4 with 1 M aqueous HCl. The
combined fractions were concentrated under reduced
pressure and lyophilized to give 2 (269 mg, 58.6%) as an
amorphous powder. IR nmax (Nujol) 1749, 1581 cmꢁ1
;
1H NMR (300 MHz, D2O) d 1.04 (1H, m), 1.20 (3H, d,
J=7.2 Hz), 1.25 (3H, d, J=7.2 Hz), 1.46 (2H, m), 1.61
(3H, m), 1.82 (3H, m), 2.22 (2H, m), 2.83 (1H, d, J=7.1
Hz), 2.95 (1H, d, J=7.1 Hz), 3.32 (2H, m), 3.43 (1H,
m), 3.68 (2H, m), 3.89 (1H, m), 4.03 (1H, m), 4.21 (2H,
m); FAB-HRMS m/z calcd for C22H36N3O4S (M+H)+:
424.2270, found: 424.2312; UV lmax 299 (e 9760).
2-[N-(4-tert-Butyldiphenylsiloxymethylbenzyl)-N-p-nitro-
benzyloxycarbonylamino]ethanol 26. To a solution of 24
(716 mg, 11.7 mmol) in THF (100 mL) were successively
added 25 (4.0 g, 10.7 mmol), sodium triacetoxyborohy-
dride (3.41 g, 16.1 mmol), and acetic acid (612 mL, 10.7

H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1609
due was purified by silica gel column chromatography
(n-hexane/EtOAc=3/2) to give 27 (990 mg, 48.9%) as a
J=7.3 Hz), 1.12 (1.8H, d, J=7.3 Hz), 1.37 (3H, d,
J=6.3 Hz), 2.74 (3H, m), 3.20 (2H, m), 3.42 (1H, m),
4.22 (2H, m), 4.55 (4H, m), 5.37 (7H, m), 7.19 (4H, m),
7.50 (6H, m), 8.20 (6H, m); FAB-HRMS m/z calcd for
C43H43N6O14S (M+H)+: 899.2558, found: 899.2529.
1
brown oil. IR nmax (Nujol) 1745, 1735, 757 cmꢁ1; H
NMR (300 MHz, CDCl3) d 1.99 (3H, s), 3.54 (2H, m),
4.18 (2H,m), 4.39 (2H, d, J=6.4 Hz), 4.57 (2H, s), 5.26
(4H, m), 5.29 (1H, br s), 7.54 (8H, m), 8.22 (4H, m);
FAB-HRMS m/z calcd for C28H29N4O10 (M+H)+:
581.1884, found: 581.1868.
The mixture obtained above (267 mg, 0.297 mmol) and
10% Pd/C (100 mg) in THF (16 mL), EtOH (8 mL),
and 0.2 M sodium 3-morpholinopropanesulfonate buf-
fer (MOPS buffer, pH 6.5, 16 mL) was stirred for 15 h at
room temperature under a hydrogen atmosphere. The
catalyst was removed by filtration and washed with
H2O. The combined filtrate and washings were con-
centrated under reduced pressure to ca. 40 mL. After
the insoluble portion of the aqueous layer was removed
by filtration, the filtrate was subjected to reversed-phase
column chromatography. The eluent was monitored by
HPLC, and the fractions eluted with 10ꢃ20% MeOH/
H2O were combined and adjusted to pH 6.0 with 0.05 M
aqueous HCl. The combined fractions were con-
centrated under reduced pressure and lyophilized to give
3 as an amorphous powder (28.1 mg, 24.4%). IR nmax
2-Acetylthio-[N-(4-p-nitrobenzyloxycarbonyl)aminomethyl-
benzyl-N-p-nitrobenzyloxycarbonyl]ethylamine 28. To a
solution of 27 (990 mg, 1.71 mmol) in MeOH (20 mL)
was added 1 M aqueous NaOH (2 mL) at room tem-
perature, and the mixture was stirred for 30 min at same
temperature. After neutralization by 1 M aqueous HCl,
the mixture was poured into H2O and the whole was
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 (n-hexane/EtOAc=1/2) to
give an alcohol (675 mg, 73.5%) as a colorless oil. To an
ice-cooled solution of the alcohol (675 mg, 1.25 mmol)
in THF (15 mL) were successively added triphenylphos-
phine (658 mg, 2.51 mmol), diisopropyl azodicarboxy-
late (494 mL, 2.51 mmol), and thioacetic acid (180 mL,
2.51 mmol), and the mixture was stirred for 30 min at
same temperature. The resulting mixture was evapo-
rated under reduced pressure. The residue was purified
by silica gel column chromatography (n-hexane/
EtOAc=1/1) to give 28 (252 mg, 33.7%) as a colorless
1
(Nujol) 1754, 1575 cmꢁ1; H NMR (300 MHz, D2O) d
1.20 (3H, d, J=7.6 Hz), 1.21 (3H, d, J=5.7 Hz), 2.85
(2H, m), 3.19 (2H, m), 3.34 (2H, m), 3.91 (1H, m), 4.22
(5H, m), 7.46 (2H, d, J=8.5 Hz), 7.51 (2H, d, J=8.5 Hz);
FAB-HRMS m/z calcd for C20H28N3O4S (M+H)+:
406.1801, found: 406.1834; UV lmax 299 (e 7610).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-phenyl-
pyrrolidin-3-ylthio]-1-methyl-1-carbapen-2-em-3-car-
boxylic acid 4a. IR nmax (KBr) 1753, 1589, 1390, 1082
oil. IR nmax (KBr) 1730, 1693, 747 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 2.31 (3H, s), 3.00 (2H, m), 3.42
(2H, m), 4.39 (2H, d, J=6.4 Hz), 4.57 (2H, m), 5.28
(4H, m), 5.29 (1H, m), 7.22 (4H, m), 7.49 (4H, m), 8.22
(4H, m); FAB-HRMS m/z calcd for C28H29N4O9S
(M+H)+: 597.1655, found: 597.1698.
1
cmꢁ1; H NMR (300 MHz, D2O) d 1.22 (3H, d, J=7.3
Hz), 1.26 (3H, d, J=6.3 Hz), 2.54 (1H, m), 2.79 (1H,
m), 3.42 (3H, m), 3.87 (1H, m), 4.22 (3H, m), 5.07 (1H,
m), 7.47 (4H, s); FAB-HRMS m/z calcd for
C20H25N2O4S (M+H)+: 389.1535, found: 389.1539;
UV lmax 298 (e 9120).
(1R,5S,6S)-2-[4-(Aminomethyl)benzyl]aminoethylthio]-6-
[(R)-1-hydroxyethyl]-1-methyl-1-carbapen-2-em-3-car-
boxylic acid hydrochloride 3. To an ice-cooled solution
of 28 (252 mg, 0.423 mmol) in MeOH (20 mL) and THF
(10 mL) was added 1 M aqueous NaOH (486 mL). After
being stirred for 20 min at the same temperature, the
reaction mixture was adjusted to pH 7.0 with 1 M aqu-
eous HCl and concentrated under reduced pressure. The
mixture was poured into H2O, and the whole was
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated under
reduced pressure. To a stirred solution of the residue
and 11 (289 mg, 0.468 mmol) in CH3CN (10 mL) was
added N,N-diisopropylethylamine (96 mL, 0.55 mmol)
in a dropwise manner at 0 ꢀC. After being stirred over-
night at 6 ꢀC, the mixture was poured into H2O and the
whole was 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 (EtOAc) to give
p-nitrobenzyl (1R,5S,6S)-2-[N-[4-( N-p-nitrobenzyloxy-
carbonylaminomethyl)benzyl] - N - p -nitrobenzyloxy-
carbonylaminoethylthio] -6 -[( R) -1 -hydroxyethyl]-1-
methyl-1-carbapen-2-em-3-carboxylate (267 mg, 70.3%)
as a yellow foam. IR nmax (KBr) 3373, 1751, 1587, 1086
Sodium (1R,5S,6S)-2-[(3S,5R)-5-(4-carboxyphenyl)pyr-
rolidin-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carba-
pen-2-em-3-carboxylate 4b. IR nmax (KBr) 1755, 1641,
1
1552, 1402, 1107 cmꢁ1; H NMR (300 MHz, D2O) d
1.23 (3H, d, J=7.4 Hz), 1.28 (3H, d, J=6.5 Hz), 2.47
(1H, m), 2.68 (1H, m), 3.37 (2H, m), 3.46 (1H, dd,
J=6.2, 2.4 Hz), 3.80 (1H, dd, J=12.7, 5.8 Hz), 4.15
(1H, m), 4.23 (2H, m), 4.93 (1H, m), 7.49 (2H, d, J=7.9
Hz), 7.88 (2H, d, J=7.9 Hz); FAB-MS m/z 454
(M+Na)+; UV lmax 297 (e 8790).
Sodium (1R,5S,6S)-2-[(3S,5S)-5-(4-carboxyphenyl)pyrro-
lidin-3-ylthio]-6-[(R)-1-hydroxyethyl]-1-methyl-1-carba-
pen-2-em-3-carboxylate 4c. IR nmax (KBr) 3388, 1749,
1653, 1558, 1394, 1090 cmꢁ1; 1H NMR (300 MHz, D2O)
d 1.23 (3H, d, J=7.3 Hz), 1.28 (3H, d, J=6.7 Hz), 2.08
(1H, m), 2.94 (1H, m), 3.37 (2H, m), 3.43 (1H, m), 3.69
(1H, dd, J=12.2, 7.6 Hz), 4.06 (1H, m), 4.24 (2H, m),
7.52 (2H, d, J=7.9 Hz), 7.88 (2H, d, J=7.9 Hz); FAB-
MS m/z 454 (M+Na)+; UV lmax 298 (e 6807).
(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5R)-5-(4-hy-
droxymethylphenyl)pyrrolidin-3-ylthio]-1-methyl-1-carba-
pen-2-em-3-carboxylic acid 4d. IR nmax (KBr) 3404,
cmꢁ1 1H NMR (300 MHz, CDCl3) d 0.96 (1.2H, d,
;

1610
H. Sato et al. / Bioorg. Med. Chem. 10 (2002) 1595–1610
1
1755, 1583, 1385, 1082, 609 cmꢁ1; H NMR (300 MHz,
D2O) d 1.26 (3H, d, J=7.3 Hz), 1.30 (3H, d, J=6.2 Hz),
2.55 (1H, dd, J=10.9, 7.0 Hz), 2.81 (1H, m), 3.40 (1H, m),
3.49 (2H, m), 3.90 (1H, dd, J=12.5, 6.0 Hz), 4.27 (3H, m),
4.66 (2H, m), 5.11 (1H, dd, J=10.6, 6.6 Hz), 7.49 (4H, s);
FAB-MS m/z 419 (M+H)+; UV lmax 298 (e 9962).
3. Sunagawa, M.; Mastumura, H.; Inoue, T.; Fukasawa, M.;
Kato, M. J. Antibiot. 1990, 43, 519.
4. (a) Nagano, R.; Adachi, Y.; Imamura, H.; Yamada, K.;
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Sakuraba, S.; Nakajima, S.; Abe, S.; Miura, K.; Nishimura, I.;
Yamada, K.; Morishima, H. Tetrahedron 2000, 56, 7705. (b)
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(1R,5S,6S)-6-[(R)-1-Hydroxyethyl]-2-[(3S,5S)-5-(4-hy-
droxymethylphenyl)pyrrolidin-3-ylthio]-1-methyl-1-carba-
pen-2-em-3-carboxylic acid 4e. IR nmax (KBr) 3394 1755,
1402, 1082, 609cmꢁ1; H NMR (300 MHz, D2O) d 1.24
1
(3H, d, J=7.2 Hz), 1.29 (3H, d, J=6.6 Hz), 2.29 (1H,
m), 2.98 (1H, m), 3.42 (3H, m), 3.80 (1H, dd, J=10.8,
8.2 Hz), 4.13 (1H, m), 4.23 (3H, m), 4.66 (2H, m), 7.45
(2H, d, J=8.4 Hz), 7.49 (2H, d, J=8.4 Hz); FAB-MS
m/z 419 (M+H)+; UV lmax 299 (e 9627).
Acknowledgement
6. Dangles, O.; Guibe, F.; Balavoine, G.; Lavielle, S.; Mar-
quet, A. J. Org. Chem. 1987, 52, 4984.
We are grateful to Ms. J. Winward, Merck & Co., for
her critical reading of this manuscript.
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References and Notes
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