Synthesis and antibacterial activity of novel 4-pyrrolidinylthio carbapanems. Part III: Novel 2-alkyl substituents containing cationic heteroaromatics linked via a C-N bond

Bioorganic & Medicinal Chemistry 7 (1999) 1665±1682

Synthesis and Antibacterial Activity of Novel 4-Pyrrolidinylthio

Carbapenems. Part III: Novel 2-Alkyl Substituents Containing

Cationic Heteroaromatics Linked Via a C±N Bond

Hidenori Azami,* David Barrett, Keiji Matsuda, Hideo Tsutsumi, Kenichi Washizuka,

Minoru Sakurai, Satoru Kuroda, Fumiyuki Shirai, Toshiyuki Chiba,

Toshiaki Kamimura and Masayoshi Murata

Medicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532±8514, Japan

Received 8 January 1999; accepted 16 March 1999

AbstractÐThe synthesis and biological activity of a novel series of 2-alkyl-4-pyrrolidinylthio-b-methylcarbapenems containing a

variety of cationic heteroaromatic substituents is described. As a result of these studies, we uncovered a relationship between in

vitro antibacterial activity and the length of the alkyl spacer part, and discovered FR20950 (1c), containing a two methylene spacer

moiety and an imidazolio group, which possesses a balanced spectrum of antibacterial activity, including Pseudomonas aeruginosa

and Methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, FR20950 exhibited excellent urinary recovery, and com-

parable stability against renal dehydropeptidase-I (DHP-I) to Biapenem. DHP-I stability could be improved by introduction of a

Introduction

for novel antibiotics represents an enormous, and

ongoing eort, that has focused predominantly on

established classes.

To control bacterial infections, many researchers and

pharmaceutical companies have researched and devel-

oped many kinds of antibacterial agents, including b-

lactams, tetracyclines, macrolides, quinolones, amino-

glycosides, etc. However, in modern chemotherapy the

acquisition of resistance by pathogens is becoming a

serious problem.^1±3This problem has occurred for

almost all types of antibiotic agent and for most bac-

terial strains, and is often induced by the excessive and/

or inappropriate usage of antibiotics. The resistance

mechanisms in Gram positive and negative bacteria can

be classi®ed in the following way: (1) mutation of the

target protein for the antibiotic (for example, penicillin-

binding proteins, ie PBP-2⁰), (2) poor outer membrane

permeability in Gram negative bacteria (especially Ps.

aeruginosa), (3) production of enzymes that destroy the

antibiotic (for example b-lactamase), and (4) the active

exclusion of antibiotics from bacteria cells (P-glycopro-

tein). To overcome these resistance problems, the search

Certain antibiotics are intrinsically active against resis-

tant bacteria; one successful example is Vancomycin,⁴

however, its activity is restricted to Gram positive bac-

teria, including Methicillin-resistant S. aureus (MRSA).

Furthermore, strains that are resistant to Vancomycin⁵

are now beginning to be isolated. With the aim of solving

this resistance problem in a broad range of bacteria,

including Gram positive (especially MRSA) and negative

bacteria (especially resistant strains of Ps. aeruginosa),

we have been searching for novel antibiotics. We selec-

ted the carbapenem skeleton⁶as the prototype for a new

antibiotic for the following reasons; (1) good selective

toxicity for the target proteins, penicillin binding pro-

teins (PBP's), which do not exist in humans, (2) the

superior ecacy pro®le of the antibacterial activity.

Thus, the bactericidal activity of carbapenems is super-

ior to the bacteriostatic activity of cephalosporins, and

(3) broad antibacterial spectrum. Whilst the weak point

of the carbapenem skeleton is instability to the renal

enzyme dehydropeptidase-I (DHP-I), several approa-

ches exist to improve stability and improve bioavail-

ability and in vivo antibacterial activity.

Key words: Carbapenems; antibacterials; cationic heteroaromatics;

DHP-I stability.

* Corresponding author. Tel.: +81-6-6390-1457; fax: +81-6-6304-

5435; e-mail: hidenori_azami@po.fujisawa.co.jp

PII: S0968-0896(99)00085-1

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H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

From the literature of carbapenem antibiotics, espe-

cially the SAR related to Meropenem^7,8and Panipe-

nem,⁹the importance of a pyrrolidine ring for potent

activity and high PBP anity were noted. It was also

reported that the good activity of Biapenem^10,11against

Ps. aeruginosa was related to its good outer membrane

permeability. The quaternary ammonium cationic cen-

ter present in the side chain of Biapenem was critical to

impart good outer membrane permeability. We postu-

lated that a combination of these two factors in a single

side chain might lead to agents with superior ecacy.

We described in earlier communications a series of azo-

liomethyl substituted pyrrolidines containing hetero-

cycles linked via a carbon±carbon bond to the spacer,^12±14

an eort that culminated in FR21818 (Fig. 1). In this

report, we examine the relationship between the spacer

length and activities in a related series to FR21818 that

contains a carbon±nitrogen bond as the point of

attachment of the heterocycle to the spacer moiety.

We have also reported various alkoxymethyl and

thioalkylmethyl-substituted pyrrolidine derivatives^15,16

however, introduction of cationic centers, in the form of

quaternary heterocyclic salts, did not lead to an

improved spectrum of activity, indicating that the nature

of the spacer group seems to be critical for good activity.

Figure 2. Retrosynthetic analysis of target compounds.

and the resulting products transformed to thiols by a

variety of methods. Finally, construction of the target

compounds (1a±g, j±s) was achieved by coupling of the

thiol with an activated carbapenem, followed by global

deprotection to remove protecting groups.

Results and Discussion

Chemistry

The preparation of pyrrolidine derivatives containing

leaving groups (4, 7, 8, 10) is outlined in Scheme 1. The

starting materials, 2-hydroxymethylpyrrolidine deriva-

tives 2a±c were obtained by standard methods.^15,17,18

Mesyloxymethyl derivative 3b and iodomethyl deriva-

tive 4 were obtained by mesylation of 2b and subsequent

reaction with NaI. The pyrrolidine derivatives required

for preparation of one carbon homologated compounds

(Fig. 2, n=2) were obtained from 2c. The hydro-

xymethyl group of 2c was transformed to a methylene

(5) by Swern oxidation and subsequent Wittig reaction.

The methylene compound 5 was transformed to the

hydroxyethyl derivative 6 by standard hydroboration

The general synthetic route to the target carbapenems

(1a±s) was based on the retrosynthesis shown in Figure

2. The target carbapenems (1a±s) can be retro-

synthetically divided into three parts: the carbapenem

skeleton (A), a pyrrolidine structure which can be pre-

pared from 4-hydroxyproline (B), and various hetero-

aromatics (C). Two dierent approaches were adapted

in this work for preparation of these compounds. The

®rst route involved as the key step, coupling of unit A

and a preformed BC moiety, and the second involved

coupling of an assembled AB unit with the appropriate

heterocycle C. We predominantly employed the ®rst

route, because the carbapenem skeleton A is relatively

unstable and as such it is recommended that the coupling

reaction to aord this moiety occurs towards the ®nal

stage of the synthetic route. Various protected hydroxy-

proline derivatives, suitably activated with a leaving

group, were coupled with appropriate heteroaromatics,

.

(9-BBN, NaBO₃4H₂O) and protecting group inter-

conversion from benzyl to allyloxycarbonyl (Pd-C, H₂,

AocCl). Compound 6 was then activated as the mesylate

7 by the same method used for 3a±b. Dimesylate 8 was

prepared by deprotection of the silyl group (c-HCl) of 6

and subsequent mesylation. The pyrrolidine inter-

mediate (10) for two carbon homologated compounds

(Fig. 2, n=3) was also obtained from 2b. Hydroxy-

methyl compound (2b) was transformed to a,b-unsatu-

rated ester (9) by Swern oxidation and Wittig reaction

(Ph₃P=CHCOOMe) in a similar manner as for 5. The

a,b-unsaturated ester (9) was transformed to alcohol by

one pot 1,4-reduction and ester reduction using sodium

borohydride (NaBH₄) and lithium iodide (LiI) in THF

as a solvent. The obtained alcohol was transformed to

mesylate (10) by the usual method.

The coupling reactions of the pyrrolidine derivatives con-

taining various leaving groups (B) with heteroaromatic

derivatives (C), and subsequent transformation to the

Figure 1. Carbapenem antibiotics.

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1667

Scheme 1. Synthesis of key pyrrolidine intermediates. Reagents and conditions: (i) MsCl, Et₃N, CH₂Cl₂; (ii) NaI, DMF; (iii) DMSO, (ClCO)₂, Et₃N,

CH₂Cl₂; (iv) Ph₃P⁺MeCl , KO Bu, THF; (v) 9-BBN, THF then NaBO₃4H₂O; (vi) 10% Pd±C, H₂, MeOH, then AocCl, THF±H₂O; (vii) c-HCl,

.

t

MeOH; (viii) Ph₃PCHCO₂Me, THF; (ix) NaBH₄±LiI, THF, re¯ux.

thiol derivatives, are summarized in Scheme 2. The het-

eroaromatics used in these reactions were commercially

available or synthesized by known methods.^19±23

reaction of the aromatic nucleophile, since we expected

that a cationic salt of a pyridine ring produced by direct

nucleophilic reaction of pyridine and substrate may

have isolation and puri®cation diculties, and possibly

instability in basic conditions. We adapted an indirect

method via amine intermediate (20b) to obtain cationic

pyridine salt (21). A triphenylmethyl group was selected

as a thiol protecting group rather than acetate, due to

facile deprotection under acidic conditions, as compared

to the basic conditions used for thioester derivatives. To

synthesize amine intermediate 20b, introduction of an

azide group to mesylate 7 was achieved under standard

conditions (NaN₃, NH₄Cl, DMF). The obtained azide

(18a) was then transformed to thioacetate (19) by the

usual three steps. After protecting group transformation

from a thioacetate (19) to a triphenylmethylthio group

(20a), reduction of the azide group gave the amine 20b.

Construction of the cationic pyridine group was

achieved by the coupling of Zincke's salt and amine²⁴to

give 21.

Iodide 4 and mesylates (3b, 7, 10) were coupled with

heteroaromatic nucleophiles under basic conditions

t

(NaH or BuOK and imidazoles, imidazopyrazole, or

1,2,4-triazole) or using the heterocycle as base (coupling

of iodide with imidazole). Subsequent deprotection of

the silyl group aorded alcohols 11 and 13a±d. The

alcohols 11 and 13a,b were transformed to a mesylate

followed by a nucleophilic substitution reaction with

t

thioacetate anion (AcSH, BuOK or NaH) to aord

thioacetates 12 and 14a,b. The 4-hydroxyl group

underwent clean inversion in the transformation from

alcohol to a thioacetate group. One step conversion of

alcohol to thiobenzoate was achieved by Mitsunobu

reaction with thiobenzoic acid, triphenylphosphine and

DEAD, to give 14c,d in high yield. Various substituted

imidazole derivatives were also synthesized. The inter-

mediates 16b and 17b were prepared from the same

mesylate (7) and commercially available 4-formyl-

imidazole. Mesylate (7) was coupled with 4-formyl-

imidazole by the usual basic conditions to aord a

mixture of 5-formylimidazole (15a) and 4-formyl-

imidazole (15b), that were separated by silica gel column

chromatography (isolated yield, 24% for 15a and 36%

for 15b). The 5-formylimidazole (15a) was reduced with

NaBH₄, methylation of the obtained alcohol with

MeI ^tBuOK, and then deprotection of the silyl group

gave methoxymethylimidazole (16a). On the other hand,

4-formylimidazole (15b) was transformed to 17a by

Horner±Wittig reaction and silyl deprotection. The

substituted imidazole derivatives 16a and 17a were simi-

larly transformed to thioacetates 16b and 17b by the

usual methods (mesylation and thioacetate substitution).

Various other substituted imidazole derivatives were

constructed from the reaction of dimesylate 8 with the

appropriate substituted imidazole nucleophile. Imida-

zoles were readily prepared by standard methods. Thus,

dimesylate (8) reacted with the substituted imidazole to

produce imidazole derivatives 22a±e. Reactions occur-

red selectively at the primary mesylate, due to steric

hindrance. Concerning the imidazole regioselectivity,

the 5-substituted imidazole was not isolated in the reac-

tions of 4-nitrile, 4-tert-butyldimethylsiloxymethyl, and

4-amidomethyl substituted imidazoles. After the appro-

priate transformation of 22a±c (deprotection of the silyl

group to give 22f,g, and oxidation from nitrile to amide

22h), the secondary mesylate group of 22c±e, f±h was

transformed by the usual substitution reaction with

thioacetate or thiobenzoate to give thioesters 23a±f.

Using pyrazole as the nucleophile in the reaction with

the dimesylate 8, a selective nucleophilic reaction

Introduction of a pyridine ring to pyrrolidine derivatives

was not achieved by direct nucleophilic substitution

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H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

t

Scheme 2. Preparation of thioester pyrrolidine derivatives. Reagents and conditions: (i) Heteroaromatics, base (NaH or BuOK or no base); (ii) c-

HCl, MeOH; (iii) MsCl, Et₃N, CH₂Cl₂; (iv) AcSH, NaH, DMF, or AcSK, MeCN, or PhCOSH, ^tBuOK, DMF; (v) Ph₃P, DEAD, PhCOSH, THF;

t

(vi) NaBH₄, THF±MeOH; (vii) BuOK, MeI, THF; (viii) (EtO)₂POCH₂CONH₂, BuOK, THF; (ix) NaN₃, NH₄Cl, DMF; (x) NaOMe, THF±

MeOH, then TrCl; (xi) PPh₃, pyridine, NH₃aq.; (xii) ⁿBuOH; (xiii) 30% H₂O₂, K₂CO₃, DMSO.

occurred to give 22i in good yield. Transformation of

mesylate (22i) to thioacetate (23g) was achieved by

nucleophilic substitution with the thioacetate salt.

The coupling of thiol derivatives with an activated car-

bapenem 24^18,25and subsequent cationic salt formation

and deprotection are summarized in Scheme 3. The

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1669

i

Scheme 3. Synthetic route to novel carbapenems. Reagents and conditions: (i) NaOMe, MeOH, (or TFA, Et₃SiH), then 24, Pr₂EtN, DMAC,

MeCN; (ii) MeI, Me₂CO or THF; (iii) ICH₂CONH₂, Me₂CO; (iv) I(CH₂)₃NHAoc, DMF; (v) MeOTf, CH₂Cl₂; (vi) FSO₃Me, CH₂Cl₂; (vii)

Pd(PPh₃)₄, PPh₃, THF±EtOH, ⁿBu₃SnH or morpholine; (viii) Pd(OH)₂-C, H₂, THF±phosphate buer (pH 6.5).

thioesters (12, 14a±d, 16b, 17b, 21, 23a±g) were

smoothly deprotected by NaOMe in MeOH or acetoni-

trile to produce the thiols which were then immediately

coupled with the activated carbapenem (24) in the pre-

sence of Hunig's base in acetonitrile, to give coupling

products 25a±g, l±s. In the case of triphenylmethylthio

derivative 21, the trityl group was deprotected under

acidic conditions using TFA and Et₃SiH to give the thiol

derivative as a crude solution, and this solution was

directly coupled with 24, in a similar manner. The cou-

pling products 25a±h, m±t were transformed to cationic

salts by reaction with the appropriate alkylating

reagent, then deprotected to give target compounds. In

the cationic salt formation step, carbamoylmethyliodide

and allyloxycarbonylaminoethyliodide were used to

produce derivatives 1j and 1k. Other salts usually used

MeI, however, in the case of imidazole derivatives with

poor reactivities, we used stronger methylation reagents

(MeOTf or FSO₃Me for 25d, 25o±p, 25s). After construc-

tion of the cationic center, deprotection was achieved by

two procedures. The allyloxycarbonyl group and allyl

ester moieties were simultaneously deprotected by

Pd(Ph₃P)₄, Ph₃P, and an allyl trapping reagent (morpho-

In contrast to these synthetic routes (Fig. 2), we also

employed another synthetic strategy for several deriva-

tives. After coupling of carbapenem skeleton A and

pyrrolidine B, the coupled compound AB and the het-

eroaromatic moiety C were coupled to give the target

compound. These results are summarized in Scheme 4.

The starting material (2b) was oxidized under Swern

conditions followed by ole®n formation with a Wittig

reagent (Ph₃P=CHCHO), to give a,b-unsaturated

aldehyde (26). This compound was reduced with

NaBH₄in a mixture of THF and EtOH, followed by

deprotection of the silyl group under acidic conditions

to give diol 27. In this reduction step, 1,2-reduction of

the a,b-unsaturated aldehyde occurred selectively to

give allyl alcohol. The primary alcohol group of diol 27

was selectively protected with a tert-butyldimethylsilyl

group, and the remaining secondary alcohol trans-

formed to the thiobenzoate ester 28 under Mitsunobu

conditions (DEAD, Ph₃P, PhCOSH). The thiobenzoate

group of 28 was deprotected (NaOMe in MeOH), and

coupled with carbapenem skeleton 24 to give 29a. The

tert-butyldimethylsilyl group of 29a was deprotected to

give alcohol 29b, which was subsequently transformed

to iodide by a two step transformation (activation of the

alcohol by (PhO)₂P(O)Cl and DMAP, and substitution

reaction with iodide anion) to give iodide 29d. This iodide

n

line or Bu₃SnH),^26,27and the p-nitrobenzyloxycarbonyl

and p-nitrobenzyl groups were removed by hydrogenolysis

(Pd(OH)₂-C, H₂) to give the target carbapenems.

Scheme 4. Synthetic route to novel carbapenems 1h and 1i. Reagents and conditions: (i) DMSO, (ClCO)₂, Et₃N, CH₂Cl₂; (ii) Ph₃P=CHCHO,

toluene; (iii) NaBH₄, THF±EtOH; (iv); c-HCl, MeCN; (v) TBDMSCl, imidazole, DMF; (vi) DEAD, PPh₃, PhCOSH, THF; (vii) NaOMe, MeOH,

then 24, EtNⁱPr₂, MeCN; (viii) TBAF, AcOH, THF; (ix) (PhO)₂P(O)Cl, DMAP, CH₂Cl₂; (x) NaI, Me₂CO; (xi) heteroaromatics (1h, 3-methylimi-

dazole, 1i, pyridine), CH₂Cl₂, then Pd(PPh₃)₄, PPh₃, THF±EtOH, ⁿBu₃SnH.

1670

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

was then coupled with several heteroaromatics (3-

methylimidazole or pyridine) by nucleophilic substitu-

tion. Subsequent deprotection of the allyloxycarbonyl

groups by ordinary methods gave imidazolio derivative

1h and pyridinio derivative 1i, respectively.

Moreover, the three methylene linked compound 1g

(group 3) displayed fourfold higher MRSA activity,

relative to 1a. Compound 1h, containing a double bond

in the spacer part was roughly equal in activity relative

to the saturated compound 1g. As a result, regarding

the activities against S. aureus including MRSA, com-

pounds that possess a three methylene unit (1g, group 3)

were the best compounds, and were two to fourfold

more active than the reference compound Biapenem.

Biological activity

In vitro antibacterial activity, DHP-I stability,^28,29and

urinary recovery of the novel carbapenems prepared in

this work are shown in Tables 1 and 2. We investigated

the structure activity relationships of various lengths of

methylene spacer, which connects the pyrrolidine ring

and the heteroaromatic portion. The results are sum-

marized in Table 1. We can separate compounds in

Table 1 into three dierent groups: (1) compounds that

possess one methylene unit as a spacer (1a,b), (2) com-

pounds that possess a two methylene unit as a spacer

(1c±f), and (3) compounds that possess a three methyl-

ene unit as a spacer, including a trans double bond (1g±

i). In these groups, we compared the N-methylated imi-

dazole rings as the cationic center (1a, 1c, 1g, 1h), and

compared in vitro activities. Regarding antibacterial

activity against S. aureus, including MRSA (S.a.(2, 3)),

as a representative Gram positive bacteria, it was shown

that superior activity was achieved with the longer

spacer groups. Relative to 1a (group 1), compound 1c

(group 2) had fourfold higher activity against MSSA

(Sa.(1)), and twofold higher activity against MRSA.

On the other hand, regarding the activities against Ps.

aeruginosa, the compounds that possess two methylenes

as a spacer (group 2) had the best pro®le. This was

obvious from a comparison of activities against Ps.

aeruginosa of compounds 1a, 1h, 1g, 1c, which increase

in that order. As a result, the two methylene compound

1c (group 2) displayed the best activity against Ps. aer-

uginosa, and this activity was comparable to the refer-

ence compounds. The Ps. aeruginosa activity of

compound 1h, containing a double bond in the spacer,

was marginally improved relative to the saturated com-

pound 1g. Outer membrane permeability of Ps. aerugi-

nosa is one of the important factors to determine

antibacterial activities. Protein D2 of the Ps. aeruginosa

outer membrane is known to facilitate the speci®c per-

meation of imipenem across this membrane barrier.

Competitive inhibition of imipenem ¯ux by basic amino

acids has been reported (there is a similarity of structure

between basic amino acids and the part of imipenem

Table 1. Antibacterial activity (MIC),^aDHP-I stability and urinary recovery of novel carbapenems

R

S.a.(1)

S.a. (2)

S.a.(3)

E.c.

P.v.

P.a.(1)

P.a.(2)

P.a.(3)

P.a.(4)

P.a.(5)

DHP^b

U.R.^c

Meropenen

Biapenem

0.1

0.05

6.25

1.56

25

ꢀ0.025

0.1

3.13

1.56

0.39

0.78

0.2

1.56

0.39

0.2

1

0.19

25%

71%

0.39

1a

1b

0.1

3.13

1.56

25

0.1

0.2

6.25

12.5

3.13

1.56

0.78

0.39

6.25

1.56

0.78

0.46

0.69

76%

NT

0.05

12.5

0.39

1c

1d

<0.025

0.05

1.56

3.13

12.5

0.1

0.2

0.78

1.56

0.78

3.13

0.78

0.39

0.2

1.56

0.39

0.48

0.17

84%

75%

1e

1f

ꢀ0.025

1.56

6.25

0.2

0.1

0.78

1.56

0.78

0.2

1.56

3.13

0.39

0.78

0.25

0.92

69%

74%

1g

1h

1i

ꢀ0.025

0.78

1.56

6.25

12.5

6.25

0.2

3.13

1.56

0.78

3.13

1.56

3.13

1.56

0.78

1.56

0.39

3.13

0.78

0.56

0.64

66%

76%

62%

a

S.a. (1), S. aureus 209P JC-1; S.a. (2), S. aureus 2538; S.a. (3), S. aureus 3004; E.c., E. coli NIHJ JC-2; P.v., P. vulgaris IAM 1025; P.a. (1), Ps.

aeruginosa IAM 1095; P.a. (2), Ps. aeruginosa 2; P.a.(3), Ps. aeruginosa 26; P.a. (4), Ps. aeruginosa 175; P.a.(5), Ps. aeruginosa FP 1457; DHP, DHP-I

stability; U.R., Urinary Recovery.

b

Human DHP-I stability is given relative to meropenem.

Recovery (%) in Mouse after s.c. administration (20 mg/kg).

c

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1671

Table 2. Antibacterial activity (MIC), DHP-I stability and urinary recovery of novel carbapenems^a

R

S.a.(1)

S.a.(2)

1.56

S.a.(3)

12.5

E.c.

0.1

P.v.

0.78

P.a.(1)

0.78

P.a.(2)

0.78

P.a.(3)

0.39

P.a.(4)

1.56

P.a.(5)

0.39

DHP

0.48

U.R.

84%

(1c)

(1j)

ꢀ0.025

0.05

1.56

3.13

12.5

6.25

12.5

0.2

1.56

3.13

1.56

3.13

0.78

0.2

3.13

1.56

3.13

0.78

0.62

0.24

1.28

76%

56%

60%

(1k)

(1l)

ꢀ0.025

0.39

0.2

0.39

(1m)

ꢀ0.025

1.56

6.25

0.2

0.78

1.56

0.78

0.2

3.13

0.78

0.28

64%

(1n)

(1o)

0.05

1.56

12.5

6.25

0.2

1.56

3.13

6.25

1.56

0.78

0.2

6.25

1.56

0.78

0.39

0.45

0.50

67%

61%

ꢀ0.025

(1p)

(1q)

0.05

0.78

3.13

6.25

12.5

0.39

1.56

0.78

0.2

3.13

0.78

0.39

0.10

0.19

72%

73%

0.39

(1r)

(1s)

ꢀ0.025

1.56

6.25

0.1

1.56

6.25

3.13

0.78

0.39

3.13

0.78

0.74

0.12

70%

66%

0.05

0.39

a

S.a. (1), S. aureus 209P JC-1; S.a. (2), S. aureus 2538; S.a. (3), S. aureus 3004; E.c., E. coli NIHJ JC-2; P.v., P. vulgaris IAM 1025; P.a. (1), Ps.

aeruginosa IAM 1095; P.a. (2), Ps. aeruginosa 2; P.a.(3), Ps. aeruginosa 26; P.a. (4), Ps. aeruginosa 175; P.a.(5), Ps. aeruginosa FP 1457; DHP, DHP-I

stability; U.R., Urinary Recovery.

containing the carboxyl group and the substituent on

C2).³⁰As a result, it appears that superior outer mem-

brene permeability on Ps. aeruginosa was observed with

superior binding anities of the carbapenems to D2

protein which was related to the proper distance and

direction of the cationic center from carboxylate ion.

Against other Gram negative bacteria, such as E. coli

and P. vulgaris, there was a small tendency to lower

activities by elongation of the spacer group. Consider-

ing the balance of antibacterial activities against all

strains, 1c was obviously the best compound in Table 1.

It is widely known that carbapenem antibiotics are

relatively easily decomposed by a metabolic enzyme,

DHP-I. The best compound in Table 1 (1c) possessed a

satisfactory pro®le regarding antibacterial activity,

however, its DHP-I stability was not optimum. All

compounds in Table 1, especially 1c, displayed superior

stability against DHP-I compared to Meropenem, how-

ever, except for 1d, most compounds displayed inferior

stability to DHP-I, compared to Biapenem. For good

bioavailabilty and in vivo antibacterial activity,

improvement of DHP-I stability was needed in the imi-

dazole compound 1c. Thus we attempted to introduce

substituents on to the imidazole ring.

Next, to investigate the generality of these SAR obser-

vations on the relationship between the spacer length

and activity, we evaluated other kinds of cationic het-

eroaromatic salts. We evaluated heteroaromatics that

possessed the same spacer parts (1b versus 1a, 1d±f ver-

sus 1c, and 1h versus 1i). As a result, compounds with

dierent heteroaromatic groups (1b, d±f, i) displayed

relatively similar activities compared to the imidazole

compound with the same spacer moiety. Therefore,

for antibacterial activity, we concluded that the length of

the spacer part is a more important factor than the nature

of the heteroaromatic ring in this type of carbapenem.

The antibacterial activities, DHP-I stabilities, and urin-

ary recoveries of substituted imidazole derivatives are

summarized in Table 2. An obvious improvement in

DHP-I stability was achieved by the introduction of an

amido group (1p), amidomethyl group (1q), or nitrile

group (1s). On the other hand, the antibacterial activity

against MRSA (S.a.(3)) was improved by introduction

of an amino group (1k), hydroxy group (1m), amido

group (1o, 1p, 1r), or a nitrile group (1s), compared to

1c. However, against some Ps. aeruginosa strains (P.a.(1)

1672

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

or P.a.(4)) and a strain of Gram negative bacteria (P.v.)

these compounds (1j±1s) showed a reduction in anti-

bacterial activities. A compound possessing a desirable

pro®le of activity comparable to that of 1c was not dis-

covered amongst these analogues. The urinary recov-

eries of all compounds in Tables 1 and 2, were good to

excellent (60±80%). This is superior to that of Mer-

openem and almost equal to that of Biapenem.

study were obtained from commercial sources and used

without further puri®cation. Reaction solvents were

the highest grade available. Selected spectroscopic and

analytical data for intermediates and ®nal compounds

are collected in Tables 4, 5, and 6.

(2S,4R)-4-tert-Butyldimethylsilyloxy-2-iodomethyl-1-(4-

nitrobenzyloxycarbonyl) pyrrolidine (4). To a solution of

2a (20 g) and Et₃N (8.85 mL) in CH₂Cl₂(200 mL) was

added dropwise a solution of methanesulfonylchloride

(MsCl) (4.53 mL) in CH₂Cl₂(20 mL) at 0^ꢁC. After stir-

ring at 0^ꢁC for 30 min, the mixture was quenched with

water and separated. The organic layer was washed with

saturated NaHCO₃aqueous solution and brine, dried

over MgSO₄, and evaporated under reduced pressure

to give (2S,4R)-4-tert-butyldimethylsilyloxy-2-methane-

sulfonyloxymethyl-1-(4-nitrobenzyloxycarbonyl)pyr-

rolidine (3a) as a oil. A mixture of this crude oil and

NaI (10.95 g) in DMF (100 mL) was stirred at 70±75^ꢁC

for 8 h. After pouring into ice-water (500 mL), the

mixture was extracted with AcOEt (300 mLÂ3). The

combined organic extract was dried over MgSO₄, eva-

porated under reduced pressure, and puri®ed by column

chromatography (SiO₂400 mL, CHCl₃elution) to give 4

(15.0 g, 59%) as a solid. Mp 105±106^ꢁC; IR (Nujol)

From these results, we selected 1c as a representative of

this type of carbapenem antibiotic, and investigated

further. Table 3 displays the in vivo protective eect

against systemic infection in mice caused by a strain of

Ps. aeruginosa in comparison to reference compounds.

As a result, a comparable eect to reference compounds

was observed.

Conclusions

We have designed and synthesized novel pyrrolidine

carbapenems connected with cationic heteroaromatics

and investigated their antibacterial activity, DHP-I sta-

bility, and urinary recovery. We found a relationship

between in vitro antibacterial activities and the length of

the spacer connecting the heteroaromatics to the pyrro-

lidine ring, and that a two methylene group was the best

spacer length for a good balance of activity against

Gram-positive (include MRSA) and Gram-negative

bacteria (especially Ps. aeruginosa). The representative

compound 1c possessed relatively poor stability to

DHP-I which was improved by the introduction of

substituents to the imidazole ring, however, the good

antibacterial spectrum of 1c was weakened in these

derivatives. As a result, the representative compound 1c

(FR20950) displayed comparable in vivo activity to

reference compounds, and is undergoing further eva-

luation as a potential new carbapenem antibiotic.

1

cm 1690; H NMR (90 MHz, CDCl₃) d 0.05 (6H, s),

0.87 (9H, s), 1.80±2.28 (2H, m), 3.41±4.00 (5H, m),

4.30±4.50 (1H, m), 5.27 (2H, s), 7.52 (2H, d, J=8.1 Hz),

8.22 (H, d, J=8.1 Hz).

(2S,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-methanesulfonyloxymethylpyrrolidine (3b). 3b was pre-

pared from 2b (63.1 g) by a similar method to that

described for preparation of 3a. An orange oil (43.19 g,

1

ꢂ100%). IR (Neat) cm 1699; H NMR (200 MHz,

CDCl₃) d 0.00 (6H, s), 0.78 (9H, s), 1.50±1.70 (1H, m),

1.95±2.10 (2H, m), 2.93 (3H, s), 3.38ꢂ3.50 (2H, m),

4.10±4.40 (3H, m), 4.50±4.60 (3H, m), 5.13±5.28 (2H,

m), 5.79±6.00 (1H, m).

Experimental

(2S,4R)-1-Benzyl-4-tert-butyldimethylsilyloxy-2-vinyl-

pyrrolidine (5). To a solution of oxalyl chloride (20 mL)

in CH₂Cl₂(700 mL) was added, dropwise, DMSO

(34.0 mL) at 40 to 50^ꢁC. After stirring for 5 min, a

solution of 2c (70.1 g) in CH₂Cl₂(300 mL) was added to

the mixture at 40 to 50^ꢁC. After stirring for 10 min,

Et₃N (151.9 mL) was added, dropwise, to the solution

and the mixture was stirred at room temperature for 1 h.

The reaction mixture was poured into 1N HCl and

extracted with AcOEt. The organic layer was washed

with saturated NaHCO₃, water and brine, dried over

MgSO₄, and evaporated under reduced pressure to give

(2S,4R)-1-benzyl-4-tert-butyldimethylsilyloxy-2-formyl-

pyrrolidine as a crude oil. This oil was used in the next

reaction immediately because of its instability. To a

suspension of methyltriphenylphosphonium chloride

(62.67 g) in THF (300 mL) was added, portionwise,

^tBuOK (24.20 g) at 0 5^ꢁC. After stirring at room tem-

perature for 2 h, the mixture was added, dropwise, to a

solution of the above crude aldehyde in THF (200 mL)

at 0 5^ꢁC, and then stirred at the same temperature for

1 h. The mixture was then poured into water and

extracted with AcOEt. The organic layer was washed

General procedures

IR spectra were recorded on a Horiba Spectradesk FT-

210 (FT-IR) or a Hitachi 260-10 spectrometer. NMR

spectra were measured on a Bruker R-90H spectrometer

(¹H, 90 MHz) or a Bruker AC200P spectrometer (¹H,

200 MHz). Chemical shifts are given in parts per mil-

lion, and TMS was used as the internal standard for

spectra obtained in DMSO-d₆and CDCl₃. DSS was

used for spectra run in D₂O. MS spectra were measured

on a Hitachi Model M-80 mass spectrometer (EI-MS),

a Finnigan MAT TSQ-70 (FAB-MS), and a Hitachi

M-1000 LC/9MS (APCI-MS). Reagents used in this

Table 3. In vivo protective eect against infection in mouse^a

1c

IPM^b

MPM^c

Biapenem

ED₅₀(mg/kg)

MIC (mg/mL)

0.289

1.56

0.289

1.56

0.289

1.56

0.219

1.56

a

Ps. aeruginosa 93, 8.0Â10⁸Cells/head.

IPM/CS, Imipenem+cilastatin.

MPM/CS, Meropenem+cilastatin.

b

c

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1673

Table 4. NMR data for thiols

No.

¹H NMR (200 MHz, CDCl₃) d ppm

Yield

40%

14b

1.45±1.80 (1H, m), 1.85±2.10 (1H, m) 2.26±2.60 (2H, m) 2.34 (3H, s), 2.34 (3H, s), 3.22 (1H, dd, J=7.6 Hz, 3.9 Hz), 3.80±

4.20 (5H, m), 4.59 (2H, d, J=5.7 Hz), 5.21±5.36 (2H, m), 5.84±6.03 (1H, m), 6.96 (1H, brs), 7.06 (1H, s), 7.50 (1H, s).

14c

1.65±1.85 (1H, m), 2.05±2.30 (1H, m), 2.40±2.75 (2H, m), 3.32 (1H, dd, J=11.3Hz, 6.8Hz), 4.00±4.40 (5H, m), 4.61 (2H,

dd, J=4.3 Hz, 1.2 Hz), 5.21±5.36 (2H, m), 5.85±6.08 (1H, m), 7.40±7.75 (4H, m), 7.95 (1H, s), 7.90±7.95 (1H, m), 8.14±

8.35 (1H, m).

94%

14d

16b

1.40±2.20 (5H, m), 2.55±2.80 (1H, m), 3.26 (1H, dd, J=11.1 Hz, 7.3 Hz), 3.95±4.25 (5H, m), 4.57±4.61 (2H, m), 5.20±5.35

(2H, m), 5.84±6.03 (1H, m), 6.93 (1H, s), 7.07 (1H, s), 7.39±7.64 (4H, m), 7.90±7.95 (2H, m).

86%

52%

1.50±1.75 (1H, m), 1.85±2.10 (1H, m), 2.34 (3H, s), 2.30±2.70 (2H, m), 3.21 (1H, dd, J=11.4 Hz, 7.1 Hz), 3.30 (3H, s),

3.50±3.65 (1H, m), 3.90±4.15 (4H, m), 4.40 (2H, s), 4.62 (2H, d, J=5.5 Hz), 5.15±5.36 (2H, m), 5.85±6.04 (1H, m), 7.01

(1H, s), 7.55 (1H, br.s).

23a

23b

23c

23d

23e

23f

23g

1.55±1.75 (1H, m), 1.88±2.14 (1H, m), 2.34 (3H, s), 2.40±2.69 (2H.m), 3.22 (1H, dd, J=10.9 Hz, 7.3 Hz), 3.80±4.20 (5H,

m), 4.60 (2H, d, J=6.2 Hz) 4.65 (2H,s) 5.10±5.40 (2H, m), 5.80±6.00 (1H, m), 6.80±7.10 (2H, m).

40%

96%

49%

98%

70%

76%

60%

1.50±2.60 (4H, m), 2.34 (3H, s), 3.16±3.28 (1H, nm), 3.80±4.15 (5H, m), 4.58±4.65 (4H, m), 5.21±5.35 (2H, m), 5.84-6.00

(1H, m), 6.91±6.98 (1H, m), 7.46±7.50 (1H, m).

1.65±1.85 (1H, m), 1.95±2.15 (1H, m), 2.40±2.80 (2H, m), 3.34 (1H, dd, J=11.1 Hz, 6.2 Hz), 3.95±4.26 (5H, m), 4.60 (2H, d,

J=5.6 Hz), 5.22±5.36 (2H, m), 5.85±6.10 (2H, m, CONH₂), 7.04 (1H, brs,CONH₂),7.30±7.64 (5H, m), 7.90±7.95 (2H, m).

1.50±1.80 (1H, m), 1.90±2.10 (1H, m), 2.30±2.60 (2H, m), 2.35 (3H, s), 3.22 (1H, dd, J=11.6 Hz, 6.9 Hz), 3.80±4.30 (5H,

m), 4.59 (2H, d, J=5.6 Hz), 5.20±5.40 (2H, m), 5.80±6.00 (1H, m), 7.59 (2H, brs).

1.69±1.96 (1H, m), 2.04±2.18 (1H, m), 2.40±2.75 (2H, m), 3.31 (1H, dd, J=11.1 Hz, 7.1 Hz), 3.90±4.26 (3H, m), 4.50±4.61

(4H, m), 5.20±5.35 (2H, m), 5.46 (1H, brs, CONH₂), 5.84±6.03 (1H, m), 7.00±8.00 (8H, m, CONH₂).

1.70±2.20 (2H, m), 2.40±2.70 (2H, m), 3.33 (1H, dd, J=10.9 Hz, 6.3 Hz), 3.51 (2H, s), 3.90±4.30 (5H, m), 4.60 (2H, d,

J=5.4 Hz), 5.20±5.40 (3H, m, CONH₂), 5.90±6.10 (1H, m), 6.90±8.00 (8H, m).

1.40±1.70 (1H, m), 1.98±2.19 (1H, m), 2.32 (3H, s), 2.30±2.57 (2H, m), 3.17 (1H, dd, J=11.4 Hz, 7.5 Hz), 3.77±3.99 (2H,

m), 4.04±4.24 (3H, m), 4.58 (2H, d, J=5.9 Hz), 5.18±5.34 (2H, m), 5.88±6.02 (1H, m), 6.24 (1H, t, J=2.0 Hz), 7.30±7.50

(2H, m).

with brine, dried over MgSO₄, evaporated under

reduced pressure, and puri®ed by column chromato-

(200 mL) was stirred vigorously for 3 h under an atmo-

spheric pressure of hydrogen at room temperature.

After the catalyst was ®ltered o, the ®ltrate was eva-

porated under reduced pressure to give a crude residue.

The obtained crude residue was dissolved in a mixture

of THF (80 mL) and water (80 mL), and treated drop-

wise with a solution of allyl chloroformate (7.27 mL) in

THF (20 mL) at 0±10^ꢁC adjusting pH (8±10) with 6

NNaOH. After stirring for 30 min, the mixture was

extracted with AcOEt (Â3). The combined organic layer

was washed with brine, dried over MgSO₄, evaporated

under reduced pressure, and puri®ed by column chro-

matography (SiO₂, n-hexane:AcOEt (2:1) elution) to

graphy (SiO₂, n-hexane:AcOEt (15:1) elution) to give 5

1

(35.10 g, 50.4%) as a yellow oil. IR (Neat) cm 1467,

1

1371; H NMR (200 MHz, CDCl₃) d 0.00 (6H, s), 0.85

(9H, s), 1.82±1.90 (2H, m), 2.11 (1H, dd, J=9.7 Hz,

5.5 Hz), 3.09±3.22 (2H, m), 3.14 (1H, d, J=13.1 Hz),

4.00 (1H, d, J=13.0 Hz), 4.25±4.35 (1H, m), 5.12 (1H,

dd, J=10.0 Hz, 1.9 Hz), 5.22 (1H, dd, J=17.2 Hz,

1.9 Hz), 5.72 (1H, ddd, J=17.2 Hz, 9.9 Hz, 8.2 Hz),

7.20±7.30 (5H, m); APCI-MS m/z 318 (MH)⁺.

(2R,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-(2-hydroxyethyl)pyrrolidine (6). To a solution of 5

(24.03 g) in THF (120 mL) was added 9-borabicyclo-

[3.3.1]nonane (0.5 M in THF, 318 mL) at 0 5^ꢁC and the

mixture stirred at room temperature for 4 h. The

reaction mixture was quenched with a solution of

1

give 6 (18.52 g, 90.3%) as an oil. IR (Neat) cm 1684;

¹H NMR (200 MHz, CDCl₃) d 0.00 (6H, s), 0.81 (9H, s),

1.40±1.80 (3H, m), 1.95±2.08 (1H, m), 3.35±3.40 (2H,

m), 3.52±3.60 (2H, m), 4.10±4.30 (1H, m), 4.30±4.45

(1H, m), 4.54 (2H, d, J=5.5 Hz), 5.13±5.30 (2H, m),

5.79±5.98 (1H, m); APCI-MS m/z 330 (MH)⁺.

.

NaBO₃4H₂O (87 g) in water (200 mL) and at room

temperature with vigorous stirring and then stirred for

12 h. After ®ltration of the mixture, the ®ltrate was

separated. The organic layer was dried over MgSO₄,

evaporated under reduced pressure, and puri®ed by

column chromatography (SiO₂, n-hexane:AcOEt (1:1)

elution) to give (2R,4R)-1-benzyl-4-tert-butyldimethyl-

silyloxy-2-(2-hydroxyethyl)pyrrolidine (20.89 g, 82.3%)

(2R,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-(2-methanesulfonyloxyethyl)pyrrolidine (7). 7 was pre-

pared from 6 (18.52 g) by a similar method to that

described for the preparation of 3a. Oil (17.31 g,

1

75.6%); H NMR (200 MHz, CDCl₃) d 0.02 (6H, s),

0.80 (9H, s), 1.65±2.35 (4H, m), 2.96 (3H, s), 3.29±3.46

(1H, m), 3.33 (1H, dd, J=11.4 Hz, 4.3 Hz), 4.00±4.15

(1H, m), 4.20±4.35 (3H, m), 4.50±4.60 (2H, m), 5.12±

5.28 (2H, m), 5.78±5.94 (1H, m).

1

as an oil. H NMR (200 MHz, CDCl₃) d 0.00 (6H, s),

0.86 (9H, s), 1.52 (1H, ddt, J=14.8 Hz, 3.1 Hz, 3.1 Hz),

1.84 (1H, ddd, J=13.0 Hz, 8.4 Hz, 4.5 Hz), 1.99±2.22

(2H, m), 2.24 (1H, dd, J=10.4 Hz, 5.4 Hz), 3.03 (1H,

dd, J=10.4 Hz, 5.5 Hz), 3.21±3.34 (1H, m), 3.31 (1H, d,

J=12.6 Hz), 3.73 (1H, dt, J=11.0 Hz, 4.0 Hz), 4.00

(1H, td, J=11.0 Hz, 2.8 Hz), 4.21 (1H, d, J=12.6 Hz),

4.27±4.38 (1H, m), 7.22±7.36 (6H, m). A mixture of this

oil (20.89 g) and 10% Pd/C (50% wet, 8 g) in MeOH

(2R,4R)-1-Allyloxycarbonyl-4-methanesulfonyloxy-2-(2-

methanesulfonyloxyethyl)pyrrolidine (8). To a solution

of 6 (5.0 g) in MeOH (25 mL) was added concentrated

HCl (2.53 mL) at 0±5^ꢁC and the mixture stirred for 1 h.

The mixture was quenched with NaOMe (28% in

1674

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

Table 5. Physical data for carbapenems^a

No.

¹H NMR (200 MHz, D₂O) d ppm

MS

446

IR

1b

1.23 (3H, d, J=7.2 Hz), 1.29 (3H, d, J=6.4 Hz), 1.77±1.92 (1H,m), 2.75±2.91 (1H, m), 3.33±3.50 (3H, m), 3.68

(1H, dd J=12.5 Hz, 6.7 Hz), 4.08 (3H, s), 4.00±4.30 (4H,m), 4.63±4.68 (2H, m), 6.54 (1H, d, J=3.6 Hz), 7.61

(1H, dd, J=2.4 Hz, 1.1 Hz), 7.93 (1H, d, J=2.3 Hz), 7.99 (1H, d, J=3.5 Hz).

1755±1740

1590±1580

1e

1g

1h

1j

1.22 (3H, d, J=7.2 Hz), 1.30 (3H, d J=6.4 Hz), 1.72±1.87 (1H, m), 2.46±2.69 (2H, m), 2.78±2.90 (1H, m), 3.34±

3.50 (3H, m), 3.70 (1H, dd, J=12.4 Hz, 6.7 Hz), 3.79±3.95 (1H, m), 4.00 (3H, s), 4.00±4.15 (1H, m), 4.23±4.29

(2H, m), 4.57±4.66 (2H, m), 8.86 (1H, s).

1.20 (3H, d, J=7.2 Hz), 1.28 (3H, d, J=6.4 Hz), 1.59±2.08 (5H, m), 2.67±2.87 (1H, m), 3.35±3.48 (3H, m),

3.59±3.77 (2H, m), 3.89 (3H, s), 3.94±4.10 (1H, m), 4.19Ð4.30 (4H, m), 7.45 (1H, d, J=1.7 Hz), 7.49 (1H, d,

J=1.8 Hz), 8.75 (1H, s).

1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.3 Hz), 1.70±2.00 (1H, m), 2.60±2.90 (1H, m), 3.30±3.50 (3H, m), 3.63

(1H, dd, J=12.4 Hz, 6.6 Hz), 3.92 (3H, s), 4.00±4.10 (1H, m), 4.10±4.40 (3H, m), 4.89 (2H, d, J=5.7 Hz), 5.90±

6.30 (2H, m), 7.48 (2H, s), 8.75 (1H, s).

1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.3 Hz), 1.69±1.83 (1H, m), 2.40±2.70 (2H, m), 2.75±2.90 (1H, m),

3.35±3.55 (3H, m), 3.65±3.85 (2H, m), 4.00±4.15 (1H, m), 4.20±4.35 (2H, m), 4.43 (2H, t, J=7.6 hz), 5.14 (2H,

s), 7.57 (1H, s), 7.65 (1H, s), 8.98 (1H, s).

422

NT

464

1745, 1580

1724, 1570

1740, 1570

1740±1760

1660±1690

1k

1l

1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.3 Hz), 1.70±1.90 (1H, m), 2.20±2.90 (5H, m), 3.00±3.10 (2H, m),

3.30±3.50 (3H, m), 3.60±3.90 (2H, m), 4.00±4.10 (1H, m), 4.20±4.50 (6H, m), 7.61 (2H, s), 8.97 (1H, s)

1.22 (3H, d, J=7.2 Hz), 1.29 (3H, d, J=6.3 Hz), 1.70±1.85 (1H, m), 2.35±2.60 (2H, m), 2.70±2.90 (1H, m),

3.35±4.55 (10H, m), 3.92 (3H, s), 4.94 (2H, s), 7.49±7.57 (2H, m).

1.22 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.4 Hz), 1.70±1.85 (1H, m), 2.38±2.65 (2H, m), 2.70±2.90 (1H, m),

3.30±3.52 (3H, m), 3.63±3.85 (2H, m), 3.89 (3H, s), 4.00±4.10 (1H, m), 4.20±4.40 (4H, m), 4.73 (2H, br s), 7.48±

7.58 (1H, m), 8.84 (1H, brs).

NT

1750, 1565

1755, 1585

1750, 1580

1m

1n

1o

1q

1r

1s

1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d,J=6.4 Hz), 1.70±1.85 (1H, m), 2.35±2.58 (2H, m), 2.75±2.92 (1H, m), 3.42

(3H, s), 3.35±3.50 (3H, m), 3.64±3.86 (2H, m), 3.90 (3H, s), 4.00±4.15 (1H, m), 4.23±4.38 (4H, m), 4.63 (2H, s),

7.58 (1H, s), 8.90 (1H, s).

1.23 (3H, d, J=7.3 Hz), 1.30 (3H, d, J=6.3 Hz), 1.65±1.83 (1H, m), 2.38±2.60 (2H, m) 2.72±2.90 (1H, m), 3.32±

3.50 (3H, m), 3.63±3.81 (3H, m), 4.02 (3H, s), 4.20±4.30 (2H, m), 4.46 (2H, t, J=8.6 Hz), 7.62 (1H, d, J=2.0

Hz), 7.71 (1H, d, J=2.0 Hz).

1.22 (3H, d, J=7.2 Hz), 1.29 (3H, d, J=6.3 Hz), 1.65±1.85 (1H, m), 2.35±2.60 (2H, m), 2.70±2.85 (1H, m),

3.30±3.50 (3H, m), 3.63±3.80 (2H, m), 3.80 (3H, s), 3.88 (2H, s), 3.95±4.10 (1H, m), 4.22±4.38 (4H, m), 7.53

(1H, s), 8.84 (1H, s).

1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.4 Hz), 1.70±1.85 (1H, m), 2.35±2.65 (2H, m), 2.75±2.90 (1H, m),

3.30±3.55 (3H, m), 3.65±3.90 (2H, m), 3.95 (3H, s), 4.00±4.15 (1H, m), 4.20±4.35 (2H, m), 4.38 (2H, t, J=7.7

Hz), 6.76 (1H, d, J=15.9 Hz), 7.41 (1H, d, J=16.3 Hz), 8.00 (1H, s), 8.92 (1H, s).

1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.4 Hz), 1.70±1.90 (1H, m), 2.40±2.60 (2H, m), 2.80±2.90 (1H, m),

3.33±3.55 (3H, m), 3.64±3.85 (2H, m), 4.00±4.15 (1H, m), 4.06 (3H, s), 4.22±4.30 (2H, m), 4.46 (2H, t, J=8.0

Hz), 8.46 (1H, s).

NT

1753, 1585

1750, 1595

1750, 1675,

1575

1735,

1580±1510

1750, 1580

a

1

MS, FAB-MS (MH⁺) (Free), m/z; IR, IR (Nujol), cm

.

Table 6. Elemental analysis data for selected carbapenems

No.

Found

H(%)

Calcd

H(%)

Molecular formula

C(%)

N(%)

C(%)

N(%)

.

C₂₁H₂₈ClN₅O₄S 3.6H₂O

1b

1c

1e

1g

1j

46.19

45.63

44.34

45.09

45.61

46.13

6.61

6.84

6.78

7.14

6.51

6.87

12.75

10.68

13.57

10.00

12.62

10.05

46.12

45.72

44.26

45.10

45.67

46.16

6.49

7.02

6.72

7.35

6.53

6.93

12.81

10.66

13.58

10.02

12.68

10.25

.

C₂₀H₂₉ClN₄O₄S 3.8H₂O

.

C

C₂₁H₃₁ClN₄O₄S 4.9H₂O

₁₉H₂₈ClN₅O₄S 3.2H₂O

.

C₂₁H₃₀ClN₅O₄S 2.9H₂O

C₂₁H₃₁ClN₄O₅S 3.3H₂O

1m

MeOH, 5.84 mL) at 0^ꢁC and the resulting precipitates

were ®ltered o. The ®ltrate was evaporated under

reduced pressure to give a residue which was dissolved

in CH₂Cl₂(15 mL). The solution was dried over MgSO₄

and evaporated under reduced pressure to give a crude

oily residue. This residue was washed with n-hexane and

dried under reduced pressure to give (2R,4R)-1-allyl-

oxycarbonyl - 4 - hydroxy - 2 - (2 - hydroxyethyl)pyrrolidine

(3.10 g, 94.9%) as an oil. ¹H NMR (200 MHz, CDCl₃) d

1.60±1.80 (2H, m), 1.80±1.95 (1H, m), 2.10±2.25 (1H,

m), 3.45 (1H, dd, J=11.7 Hz, 4.9 Hz), 3.55±3.75 (3H,

m), 4.20±4.40 (1H, m), 4.40±4.55 (1H, m), 4.60 (2H, d,

J=5.4 Hz), 5.19±5.36 (2H, m), 5.84±6.04 (1H, m).

Mesylation of this diol (8.28 g) was achieved by a simi-

lar method to that described for the preparation of 3b,

using MsCl (6.26 mL, 2.1 eq.) and Et₃N (11.26 mL, 2.1

1

eq.). Oil (10.32 g, 72.2%); H NMR (200 MHz, CDCl₃)

d 1.85±2.15 (2H, m), 2.30±2.65 (2H, m), 3.04 (3H, s),

3.05 (3H, s), 3.50±3.65 (1H, m), 3.92±4.45 (4H, m), 4.61

(2H, d, J=4.4Hz), 5.21±5.36 (3H, m), 5.85±6.05 (1H, m).

(2S,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-[(E)-3-methoxy-3-oxo-1-propenyl]pyrrolidine (9). Swern

oxidation of 2b (200 g) was achieved by the same

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1675

method as described for the preparation of 5, to give

(2S,4R)-1-allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-formylpyrrolidine (202.7 g, ꢂ100%) as a crude brown

preparation of 8 to give 11 (0.8 g, 69%) as a solid. Mp.

147±148^ꢁC; IR (CHCl₃) cm

1

1710±1690; ¹H NMR

(90 MHz, CDCl₃) d 1.50±2.20 (2H, m), 3.29 (1H, dd,

J=10.8 Hz, 4.5 Hz), 3.40±3.70 (1H, m), 3.90±4.50 (4H,

m), 5.26 (2H, s), 6.78 (1H, br s), 6.97 (1H, br s), 7.32

(1H, br s), 7.50 (2H, d, J=8.1 Hz), 8.20 (2H, d,

J=8.1 Hz).

1

oil. IR (Neat) cm 1710, 1696; H NMR (200 MHz,

CDCl₃) d 0.07 (6H, s), 0.87 (9H, s), 1.90±2.09 (2H, m),

3.42±3.64 (2H, m), 4.30±4.41 (2H, m), 4.59±4.64 (2H,

m), 5.17±5.37 (2H, m), 5.81±5.97 (1H, m), 9.50 (0.5H,

d, J=3.3 Hz), 9.58 (0.5H, d, J=2.6 Hz), two con-

formational isomers. To a solution of the obtained

crude aldehyde (30 g) in THF (300 mL) was added

methyl (triphenylphosphoranylidene)acetate (Ph₃P=

CHCOOMe) (35.2 g). After stirring at room tempera-

ture overnight, the mixture was evaporated and puri®ed

by column chromatography (SiO₂600 mL) to give 9

(2S,4S)-4-Acetylthio-2-(imidazol-1-yl)methyl-1-(4-nitro-

benzyloxycarbonyl)pyrrolidine (12). Mesylation of 11

(0.79 g) was achieved by a similar method to that

described for preparation of 3a to give (2S,4R)-2-(imi-

dazol-1-yl)methyl-4-methanesulfonyloxy-1-(4-nitrobenzyl-

oxycarbonyl)pyrrolidine (0.78 g, 80.4%) as an oil. IR

(CHCl₃) cm ¹1710, 1525; ¹H NMR (90 MHz, CDCl₃) d

1.75±2.62 (2H, m), 3.00 (3H, s), 3.00±3.40 (1H, m),

3.88±4.60 (4H, m), 4.80±4.98 (1H, m), 5.38 (2H, s), 6.80

(1H, br s), 7.04 (1H, br s), 7.39 (1H, br s), 7.51 (2H, d,

J=9.0 Hz), 8.22 (2H, d, J=9.0 Hz). To a suspension of

NaH (62.8% in oil, 0.08 g) in DMF (5 mL) was added

S-thioacetic acid (0.16 mL) at 0^ꢁC. After stirring at

room temperature for 10 min, a solution of the above

mesylate (0.76 g) in DMF (2 mL) was added to the sus-

pension, and the mixture stirred at 70±75^ꢁC for 5 h. The

mixture was then quenched with water (70 mL) and

extracted with AcOEt (70 mL). The organic extract was

dried over MgSO₄, evaporated under reduced pressure,

and puri®ed by column chromatography (SiO₂40 mL,

MeOH:CHCl₃(2:98) elution) to aord 12 (0.38 g,

1

(29.8 g, 84.2%) as a colorless oil. H NMR (200 MHz,

CDCl₃) d 0.06 (6H, s), 0.87 (9H, s), 1.74±1.95 (1H, m),

2.01±2.22 (1H, m), 3.40±3.60 (2H, m), 3.73 (3H, s),

4.30±4.43 (1H, m), 4.49±4.68 (3H, m), 5.10±5.39 (2H, m),

5.75±6.00 (2H, m), 6.85 (1H, dd, J=15.6 Hz, 6.4 Hz).

(2S,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-(3-methanesulfonyloxypropyl)pyrrolidine (10). To a

solution of 9 (18.7 g) in THF (190 mL) were added suc-

cessively NaBH₄(3.83 g) and LiI (13.5 g) at room tem-

perature and the mixture was stirred under re¯ux for

4 h. After cooling to room temperature, the mixture was

diluted with water (200 mL) and extracted with AcOEt

(200 mLÂ3). The combined extracts were washed with

brine (400 mL), dried over MgSO₄, and evaporated

under reduced pressure to give (2S,4R)-1-allyloxy-

carbonyl-4-tert-butyldimethylsilyloxy-2-(3-hydroxy-

propyl)pyrrolidine (17.2 g, 98.9%) as a pale yellow

1

52.8%) as an oil. IR (Neat) cm 1710, 1685; H NMR

(90 MHz, CDCl₃) d 1.50±1.90 (1H, m), 2.32 (3H, s),

2.07±2.60 (1H, m), 2.93±3.12 (1H, m), 3.68±4.30 (5H, m),

5.21 (2H, s), 6.81 (1H, br s), 7.02 (1H, br s), 7.37 (1H, br

s), 7.47 (2H, d, J=8.1 Hz), 8.19 (2H, d, J=8.1 Hz).

1

paste. H NMR (200 MHz, CDCl₃) d 0.06 (6H, s), 0.86

(9H, s), 1.22±2.34 (6H, m), 3.36±3.37 (2H, m), 3.61 (2H,

br t, J=6.7 Hz), 3.95±4.11 (1H, m), 4.26±4.35 (1H, m),

4.52±4.54 (2H, m), 5.10±5.29 (2H, m), 5.79±5.98 (1H,

m). The mesylation of the alcohol (17.1 g) was achieved

by a similar method to that described for preparation of

(2S,4R)-1-Allyloxycarbonyl-4-hydroxy-2-(imidazo[1,2-b]-

pyrazol-1-ylmethyl)pyrrolidine (13a). To a solution of

imidazo[1,2-b]pyrazole (1.75 g) in DMF (20 mL) was

added NaH (60% in oil, 686 mg) at 0^ꢁC, and the mix-

ture stirred for 30 min at the same temperature. The

mixture was then added to a solution of 3b (5 g) in

DMF (50 mL) and stirred at 80±90^ꢁC for 2 h. The mix-

ture was poured into ice-water (100 mL) and extracted

with AcOEt (150 mLÂ2). The combined extracts were

washed with brine, dried over MgSO₄, evaporated

under reduced pressure, and puri®ed by column chro-

matography (SiO₂200 mL, CH₂Cl₂:Me₂CO (3:1) elu-

tion) to give (2S,4R)-1-allyloxycarbonyl-4-tert-butyl-

dimethylsilyloxy-2-(imidazo[1,2-b]pyrazol-1-ylmethyl)-

pyrrolidine (4.24 g, 82.5%) as an oil. ¹H NMR

(200 MHz, CDCl₃) d 0.00 (6H, s), 0.80 (9H, s), 1.78±2.05

(2H, m), 3.15±3.50 (2H, m), 3.85±4.45 (4H, m), 4.67

(2H, d, J=5.4 Hz), 5.20±5.39 (2H, m), 5.61 (1H, d,

J=1.8 Hz), 5.87±6.07 (1H, m), 6.64 (1H, br s), 7.30±7.31

(1H. m), 7.61 (1H, br s). Desilylation of this oil (4.23 g)

was achieved by a similar method to that described for

preparation of 8 to give 13a (3.18 g, ꢂ100%) as an oil.

¹H NMR (200 MHz, CDCl₃) d 1.70±2.15 (2H, m), 3.24

(1H, dd, J=11.8 Hz, 4.5 Hz), 3.45±3.65 (1H, m), 4.00±

4.20 (2H, m), 4.30±4.50 (2H, m), 4.65 (2H, d,

J=5.6 Hz), 5.24±5.39 (2H, m), 5.61 (1H, d, J=2 Hz),

5.90±6.04 (1H, m), 6.65 (1H, br s), 7.28 (1H. br s), 7.58

(1H, br s).

1

3a to give 10 (21.2 g, ꢂ100%) as a yellow paste. H

NMR (200 MHz, CDCl₃) d 0.06 (6H, s), 0.86 (9H, s),

1.43±2.04 (6H, m), 3.01 (3H, s), 3.39 (1H, dd, J=

11.4 Hz, 4.5 Hz), 3.35±3.67 (1H, m), 3.91±4.11 (1H, m),

4.24 (2H, t, J=6.0 Hz), 4.30±4.40 (1H, m), 4.49±4.69

(2H, m), 5.17±5.35 (2H, m), 5.84±6.03 (1H, m).

(2S,4R)-4-Hydroxy-2-(imidazol-1-yl)methyl-1-(4-nitro-

benzyloxycarbonyl)pyrrolidine (11). A mixture of 4 (3 g)

and imidazole (15 g) was melted at 100±110^ꢁC under

stirring for 5 h. The mixture was poured into water

(150 mL) and extracted with CHCl₃(50 mLÂ3). The

combined organic extracts were washed with water,

dried over MgSO₄, evaporated under reduced pressure,

and puri®ed by column chromatography (SiO₂60 mL,

MeOH:CHCl₃(1:99) elution) to give (2S,4R)-4-tert-

butyldimethylsilyloxy-2-(imidazol-1-yl)methyl-1-(4-nitro-

benzyloxycarbonyl)pyrrolidine (1.57 g, 59.2%) as a

1

syrup. IR (Neat) cm 1705, 1525; H NMR (90 MHz,

CDCl₃) d 0.01 (6H, s), 0.82 (9H, s), 1.50±2.20 (2H, m),

3.21 (1H, dd, J=10.8 Hz, 4.5 Hz), 3.20±3.50 (1H, m),

3.75±4.60 (4H, m), 5.24±5.28 (2H, m), 6.78 (1H, s),

7.01 (1H, s), 7.33 (1H, s), 7.48 (2H, d, J=8.1 Hz), 8.20

(2H, d, J=8.1 Hz). Desilylation of the syrup (1.55 g)

was achieved by a similar method to that described for

1676

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

(2R,4R)-1-Allyloxycarbonyl-4-hydroxy-2-[2-(imidazol-1-

yl)ethyl]pyrrolidine (13b). To a solution of imidazole

(1.52 g) in DMF (83 mL) was added successively ^tBuOK

(2.51 g) and 7 (8.3 g) at room temperature. After stirring

for 1 h at ꢂ60^ꢁC, the mixture was quenched with water

and extracted with AcOEt. The organic extract was

washed with brine, dried over MgSO₄, evaporated

under reduced pressure, and puri®ed by column chro-

matography (SiO₂, CHCl₃:Me₂CO (100:0±97:3) elution)

to give (2R,4R)-1-allyloxycarbonyl-4-tert-butyldimethyl-

silyloxy-2-[2-(imidazol-1-yl)ethyl]pyrrolidine (11.11 g,

and extracted with AcOEt (100 mL). The extract was

washed with brine, dried over MgSO₄, evaporated

under reduced pressure, and puri®ed by column chro-

matography (SiO₂200 mL, CH₂Cl₂:Me₂CO (5:1) elu-

1

tion) to give 14a (2.33 g, 81%). H NMR (200 MHz,

CDCl₃) d 1.71±1.85 (1H, m), 2.33 (3H, s), 2.30±2.60

(1H, m), 3.00±3.30 (1H, m), 3.80±4.30 (5H, m), 4.63

(2H, d, J=5.5 Hz), 5.24±5.38 (2H, m), 5.66 (1H, br s),

5.85±6.10 (1H, m), 6.67 (1H, br s), 7.30±7.31 (1H. m),

7.60±7.62 (1H, m).

1

ꢂ100%) as an oil. H NMR (200 MHz, CDCl₃) d 0.00

The preparation of 14b (40%) was achieved from 13b by

a similar method to that described for preparation of

14a.

(6H, s), 0.81 (9H, s), 1.45±1.70 (1H, m), 1.77±2.10 (2H,

m), 2.15±2.40 (1H, m), 3.34 (1H, dd, J=11.4 Hz,

4.4 Hz), 3.30±3.47 (1H, m), 3.85±4.05 (1H, m), 4.25±4.40

(1H, m), 4.50±4.60 (2H, m), 5.10±5.30 (2H, m), 5.80±

6.00 (1H, m), 6.86±6.92 (1H, m), 7.01 (1H, m), 7.45±7.50

(1H, m): FAB-MS m/z 380.2 (MH)⁺. Desilylation of

the oil (11.11 g) was achieved by a similar method to

that described for preparation of 8 to give 13b (9.19 g,

Mitsunobu reaction of 13c and 13d was respectively

achieved by a similar method to that described for pre-

paration of 28 to give 14c (94%) and 14d (86%).

(2R,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-[2-(5-formylimidazol-1-yl)ethyl]pyrrolidine (15a) and

(2R,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-[2-(4-formylimidazol-1-yl)ethyl]pyrrolidine (15b). To a

solution of 7 (63 g) and 4-formylimidazole (17.8 g) in

1

ꢂ100%) as an oil. H NMR (200 MHz, CDCl₃) d 1.68±

2.00 (2H, m), 2.15±2.23 (1H, m), 2.35±2.60 (1H, m), 3.43

(1H, dd, J=11.9 Hz, 4.1 Hz), 3.60±3.75 (1H, m), 3.95±

4.30 (3H, m), 4.40±4.45 (1H, m), 4.56 (2H, d, J=

5.4 Hz), 5.19±5.34 (2H, m), 5.83±6.02 (1H, m), 7.17 (2H,

br s), 8.01 (1H, br s).

t

DMF (300 mL) was added BuOK (20.8 g) and the mix-

ture stirred at 45^ꢁC for 2 h. After evaporation of the

solvent, the residue was dissolved in a mixture of AcOEt

(1.5 L) and water (200 mL). The organic layer was

separated, washed in turn with 1N HCl (100 mL) and

brine (300 mLÂ3), dried over MgSO₄, evaporated under

reduced pressure, and puri®ed by column chromato-

graphy (SiO₂2000 L, hexane:AcOEt (1:1±1:2±0:1)

elution). The former fractions were collected and evapo-

rated to give 15a (14.9 g, 24%) as an oil. ¹H NMR

(200 MHz, CDCl₃) d 0.02 (6H, s), 0.80 (9H, s), 1.70±2.30

(4H, m), 3.36 (1H, dd, J=11.4 Hz, 4.5 Hz), 3.30±3.60

(1H, m), 3.95±4.10 (1H, m), 4.20±4.40 (3H, m), 4.50±4.55

(2H, m), 5.10±5.30 (2H, m), 5.78±6.27 (1H, m), 7.61±7.78

(2H, m), 9.68 (1H, s). The latter fractions were collected

(2R,4R)-1-Allyloxycarbonyl-4-hydroxy-2-[2-(1,2,4-tri-

azol-1-yl)ethyl]pyrrolidine (13c). The preparation of 13c

was achieved from 7 by a similar method to that

described for preparation of 13a using 1,2,4-triazole

1

sodium salt. A yellow paste, 91%; H NMR (200 MHz,

CDCl₃) d 1.65±1.82 (1H, m), 2.00±2.20 (2H, m), 2.32±

2.50 (1H, m), 3.35±3.50 (1H, m), 3.60±3.80 (1H, m),

4.00±4.13 (1H, m), 4.20±4.50 (3H, m), 4.58 (2H, d,

J=5.5 Hz), 5.20±5.35 (2H, m), 5.84±6.03 (1H, m), 7.93

(1H, s), 8.05±8.23 (1H, m).

(2R,4R)-1-Allyloxycarbonyl-4-hydroxy-2-[3-(imidazol-1-

yl)propyl)pyrrolidine (13d). The preparation of 13d was

achieved from 10 by a similar method to that described

1

and evaporated to give 15b (22.4 g, 36%) as an oil. H

NMR (200 MHz, CDCl₃) d 0.02 (6H, s), 0.80 (9H, s),

1.50±1.70 (1H, m), 1.79±2.00 (2H, m), 2.10±2.30 (1H, m),

3.33 (1H, dd, J=11.5 Hz, 4.4 Hz), 3.28±3.55 (1H, m),

3.90±4.10 (3H, m), 4.25±4.36 (1H, m), 4.55 (2H, d,

J=5.3 Hz), 5.15±5.30 (2H, m), 5.79±6.00 (1H, m), 7.50±

7.75 (2H, m), 9.81 (1H, s).

1

for preparation of 13b. A yellow paste, 94%; H NMR

(200 MHz, CDCl₃) d 1.29±1.50 (1H, m), 1.65±2.20 (5H,

m), 3.27±3.74 (2H, m), 3.85±4.12 (3H, m), 4.33±4.46

(1H, m), 4.58 (2H, d, J=5.6 Hz), 5.18±5.33 (2H, m),

5.78±6.06 (1H, m), 6.90 (1H, s), 7.04 (1H, s), 7.46 (1H,

s).

(2R,4R)-1-Allyloxycarbonyl-4-hydroxy-2-[2-(5-methoxy-

methylimidazol-1-yl)ethyl]pyrrolidine (16a). To a solu-

tion of 15a (12.65 g) in a mixture of THF (60 mL) and

MeOH (60 mL) was added NaBH₄(1.17 g) at 0^ꢁC. After

stirring for 1 h, the mixture was adjusted to pH 8 with

6N HCl. The resulting precipitates were ®ltered o and

the ®ltrate was evaporated under reduced pressure to

give a residue which was puri®ed by column chromato-

graphy (SiO₂500 mL, CHCl₃:MeOH (9:1) elution) to

give (2R,4R)-1-allyloxycarbonyl-4-tert-butyldimethyl-

silyloxy-2-[2-(5-hydroxymethylimidazol-1-yl)ethyl]pyrro-

(2R,4S)-4-Acetylthio-1-allyloxycarbonyl-2-(imidazo-[1,2-

b]pyrazol-1-yl)methylpyrrolidine (14a). Mesylation of

13a (3.16 g) was achieved by a similar method to that

described for the preparation of 3a to give (2R,4R)-1-

allyloxycarbonyl-2-(imidazo[1,2-b]pyrazol-1-yl)methyl-

4-methanesulfonyloxypyrrolidine (3.06 g, 76.3%) as an

1

oil. H NMR (200 MHz, CDCl₃) d 1.99±2.13 (1H, m),

2.35±2.50 (1H, m), 2.98 (3H, s), 3.24±3.36 (1H, m),

3.83±4.52 (4H, m), 4.66±4.70 (2H, m), 4.85±5.00 (1H,

m), 5.20±5.40 (2H, m), 5.61 (1H, d, J=1.9 Hz), 5.88±

6.07 (1H, m), 6.64 (1H, br s), 7.31±7.32 (1H. m), 7.60±

7.62 (1H, m). To a solution of this oil (3.04 g) in MeCN

(60 mL) was added potassium S-thioacetate (1.41 g) and

the solution stirred at 80±90^ꢁC for 4 h. The mixture was

quenched with a mixture of water and brine (1:1, 50 mL)

1

lidine (10.23 g, 81%) as an amorphous solid. H NMR

(200 MHz, CDCl₃) d 0.05 (6H, s), 0.86 (9H, s), 1.65±

2.50 (4H, m), 3.30±3.50 (2H, m), 3.95±4.15 (3H, m),

4.30±4.45 (1H, m), 4.55±4.65 (2H, m), 4.60 (2H, s),

5.18±5.35 (2H, m), 5.84±6.03 (1H, m), 6.87 (1H, s), 7.45

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1677

(1H, br s). To a solution of the above alcohol (9.22 g) in

THF (100 mL) was added BuOK (3.54 g) at 0^ꢁC. After

d, J=6.8 Hz), 5.22±5.36 (3H, m), 5.83±6.03 (1H, m),

6.65 (1H, d, J=15.3 Hz), 7.14 (1H, br s), 7.47 (1H, d,

J=15.3 Hz), 7.54 (1H, br s). To a mixture of BuOK

t

stirring for 10 min, MeI (2.80 mL) was added to the

mixture, which was then stirred for 1 h at 0^ꢁC. The

mixture was dissolved in AcOEt (100 mL), washed with

water, saturated NaHCO₃and brine, dried over

MgSO₄, evaporated under reduced pressure and pur-

i®ed by column chromatography (SiO₂400 mL,

CHCl₃:MeOH (19:1) elution). Fractions containing the

desired compound were collected and evaporated to

give (2R,4R)-1-allyloxycarbonyl-4-tert-butyldimethylsi-

lyloxy-2-[2-(5-methoxymethylimidazol-1-yl)ethyl]pyrro-

lidine as a residue (7.79 g). Desilylation of this residue

(7.79 g) was achieved by a similar method to that

described for preparation of 8 to give 16a (3.63 g, 52%)

as an oil. ¹H NMR (200 MHz, CDCl₃) d 1.50±2.50 (4H,

m), 3.29 (3H, s), 3.40±3.50 (1H, m), 3.55±3.80 (1H, m),

3.95±4.15 (3H, m), 4.35±4.50 (1H, m), 4.38 (2H, s), 4.59

(2H, d, J=5.4 Hz), 5.19±5.35 (2H, m), 5.84±6.01 (1H,

m), 6.98 (1H, s), 7.55 (1H, br s). 16b (52%) was pre-

pared from 16a by a similar method to that described

for preparation of 14a.

(1.8 g) in DMF (50 mL) was added dropwise PhCOSH

(1.9 mL) at 0^ꢁC. After stirring for 30 min, a solution of

the obtained mesylate (5.5 g) in DMF (20 mL) was

added to the mixture and stirred at 80^ꢁC for 4 h. The

mixture was evaporated under reduced pressure and

puri®ed by column chromatography (SiO₂300 mL,

CH₂Cl₂:MeOH (9:1)) to give 17b (5.33 g, 88%) as an oil.

¹H NMR (200 MHz, CDCl₃) d 1.65±2.70 (4H, m), 3.34

(1H, dd, J=11.1 Hz, 6.3 Hz), 3.90±4.30 (5H, m), 4.60

(2H, d, J=5.6 Hz), 5.22±5.37 (2H, m), 5.65 (2H, br s,

CONH₂), 5.85±6.04 (1H, m), 6.63 (1H, d, J=6.6 Hz),

7.14 (1H, br s), 7.40±7.65 (5H, m), 7.90±7.95 (2H, m).

(2R,4R)-1-Allyloxycarbonyl-2-(2-azidoethyl)-4-tert-butyl-

dimethylsilyloxy pyrrolidine (18a). To a solution 7 (37 g)

in DMF (185 mL) was added NH₄Cl (5.84 g) and NaN₃

(7.1 g). After stirring at 70^ꢁC for 3 h, the mixture was

quenched with water and extracted with AcOEt (Â3).

The organic layer was washed with water (Â2) and

brine, dried over MgSO₄, and evaporated under reduced

(2R,4R)-1-Allyloxycarbonyl-2-[2-[4-[(E)-2-carbamoyl-

ethenyl]imidazol-1-yl]ethyl]-4-hydroxypyrrolidine (17a).

To a solution of diethyl carbamoylmethylphosphonate

(12.7 g) and ^tBuOK (13.9 g) in THF (400 mL) was added

a solution of 15b (24 g) in THF (50 mL) at 45^ꢁC. After

stirring for 1 h, the mixture was quenched with water

(3 mL) and evaporated under reduced pressure. The

residue was dissolved in a mixture of AcOEt (500 mL)

and water (50 mL). The organic layer was separated,

washed with water (50 mLÂ2) and brine (50 mLÂ2),

dried over MgSO₄, evaporated under reduced pressure,

and puri®ed by column chromatography (SiO₂500 mL,

CH₂Cl₂:MeOH (10:1)) to give (2R,4R)-1-allyloxy-

carbonyl-4-tert-butyldimethylsilyloxy-2-[2-[4-[(E)-2-carb-

amoylethenyl]imidazol-1-yl]ethyl]pyrrolidine (13.87 g,

53%) as an oil. ¹H NMR (200 MHz, CDCl₃) d 0.04

(6H, s), 0.84 (9H, s), 1.50±1.75 (1H, m), 1.75±2.10

(2H, m), 2.16±2.40 (1H, m), 3.36 (1H, dd, J=11.5 Hz,

4.4 Hz), 3.33±3.65 (1H, m), 3.90±4.15 (3H, m), 4.30±4.45

(1H, m), 4.59 (2H, d, J=5.3 Hz), 5.19±5.34 (2H, m),

5.57 (2H, br s, CONH₂), 5.83±6.02 (1H, m), 6.61 (1H, d,

J=15.0 Hz), 7.11 (1H, m), 7.50 (1H, d, J=15.0 Hz),

7.51 (1H, br s). Desilylation of the oil (13.87 g) was

achieved as described for 8 to give 17a (9.92 g, 96%) as

a pale yellow foam. ¹H NMR (200 MHz, CDCl₃) d

1.63±2.18 (3H, m), 2.30±2.50 (1H, m), 3.35±3.70 (2H,

m), 3.90±4.15 (3H, m), 4.35±4.43 (1H, m), 4.58 (2H, d,

J=5.6 Hz), 5.20±5.35 (2H, m), 5.83±6.03 (1H, m), 6.57

(1H, d, J=15.4 Hz), 7.16 (1H, br s), 7.34 (1H, d,

J=15.9 Hz), 7.57 (1H, br s).

pressure to give 18a (29.65 g, 92%) as an oil. IR (Neat)

1

cm

2925, 2080, 1665, 1400; ¹H NMR (200 MHz,

CDCl₃) d 0.06 (6H, s), 0.86 (9H, s), 1.50±2.35 (4H, m),

3.30±3.50 (3H, m), 3.75±4.45 (3H, m), 4.60±4.70 (2H,

m), 5.18±5.85 (2H, m), 5.85±6.01 (1H, m).

(2R,4S)-4-Acetylthio-1-allyloxycarbonyl-2-(2-azidoethyl)-

pyrrolidine (19). Desilylation of 18a (29.65 g) was

achieved as described for the preparation of 8 to give

18b (20 g, 100%) as an oil. Mesylation and subsequent

thioacetylation of 18b (20 g) was achieved as described

for the preparation of 14a, to give 19 (14.68 g, 58%) as

an oil. IR (Neat) cm ¹2925, 2080, 1665, 1395; ¹H NMR

(200 MHz, CDCl₃) d 1.65±1.90 (2H, m), 2.15±2.35 (1H,

m), 2.35 (3H, s), 2.50±2.70 (1H, m), 3.18 (1H, dd,

J=11.4 Hz, 7.4 Hz), 3.30±3.45 (2H, m), 3.81±4.14 (3H,

m), 4.59 (2H, d, J=4.9 Hz), 5.21±5.36 (2H, m), 5.84±

6.04 (1H, m).

(2R,4S)-1-Allyloxycarbonyl-2-(2-azidoethyl)-4-triphenyl-

methylthiopyrrolidine (20a). A solution of 19 (14.68 g)

in a mixture of THF (74 mL) and MeOH (74 mL) was

treated with NaOMe (28% in MeOH, 11.3 mL) at 0^ꢁC

for 1 h. To the mixture was added triphenylmethyl

chloride (14.4 g), followed by stirring for 5 h at 0^ꢁC. The

mixture was quenched with water and extracted with

AcOEt. The combined organic extracts were washed

with water and brine, dried over MgSO₄, evaporated

under reduced pressure, and puri®ed by column chro-

matography (SiO₂, hexane: AcOEt (10:1±5:1) elution) to

1

give 20a (16.48 g, 67%) as an oil. IR (Neat) cm 2925,

1

(2R,4S)-1-Allyloxycarbonyl-4-benzoylthio-2-[2-[4-[(E)-2-

carbamoylethenyl]imidazol-1-yl]ethyl]pyrrolidine (17b).

Mesylation of 17a (9.9 g) was achieved by a similar

method to that described for preparation of 3a to give

(2R,4R)-1-allyloxycarbonyl-2-[2-[4-[(E)-2-carbamoyl-

ethenyl]imidazol-1-yl]ethyl]-4-methanesulfonyloxypyr-

2080, 1665, 1395; H NMR (200 MHz, CDCl₃) d 1.35±

1.71 (2H, m), 1.90±2.25 (2H, m), 2.70±3.40 (5H, m),

3.62±3.75 (1H, m), 4.40±4.60 (2H, m), 5.19±5.29 (2H,

m), 5.78±5.97 (1H, m), 7.18±7.49 (15H, m).

(2R,4S)-1-Allyloxycarbonyl-2-(2-aminoethyl)-4-triphenyl-

methylthiopyrrolidine (20b). To a solution of 20a (10.1 g)

in pyridine (30 mL) was added Ph₃P (8.5 g) at room

temperature. After stirring for 1 h, aqueous NH₃(28%,

1

rolidine (8.84 g, 72%) as an oil. H NMR (200 MHz,

CDCl₃) d 1.80±2.05 (2H, m), 2.30±2.70 (2H, m), 3.04

(3H, s), 3.49±3.65 (1H, m), 3.95±4.20 (4H, m), 4.61 (2H,

1678

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

2.7 mL) was added to the mixture, followed by stirring

at room temperature overnight. The mixture was eva-

porated under reduced pressure and puri®ed by column

chromatography (SiO₂) to give 20b (9.03 g, 94%) as an

oil. IR (Neat) cm 1660, 1390; H NMR (200 MHz,

CDCl₃) d 1.35±2.15 (4H, m), 2.60±3.00 (5H, m), 3.60±

3.80 (1H, m), 4.40±4.60 (2H, m), 5.20±5.30 (2H, m),

5.77±5.96 (1H, m), 7.15±7.50 (15H, m).

m), 3.90±4.20 (4H, m), 4.62 (2H, d, J=5.7 Hz), 5.20±

5.40 (3H, m), 5.80±6.00 (1H, m), 7.50±7.70 (2H, m).

(2R,4R)-1-Allyloxycarbonyl-2-[2-(2-carbamoylimidazol-

1-yl)ethyl]-4-methanesulfonyloxypyrrolidine (22d). A yel-

1

low solid (80%); H NMR (200 MHz, CDCl₃) d 1.92±

2.21 (2H, m), 2.40±2.60 (2H, m), 3.04 (3H, s), 3.50±3.65

(1H, m), 3.94±4.20 (2H, m), 4.49 (2H, t, J=7.7 Hz), 4.60

(2H, d, J=5.5 Hz), 5.15±5.34 (3H, m), 5.63 (1H, br s,

CONH₂), 5.84±6.03 (1H, m), 7.03±7.28 (2H, m).

(2R,4S)-1-Allyloxycarbonyl-2-[2-(1-pyridinio)ethyl]-4-

(triphenylmethylthio)pyrrolidine chloride. (21) A solution

of 20b (5.78 g) and 1-(2,4-dinitrophenyl)pyridinium

chloride (4.19 g) in n-butanol (60 mL) was stirred under

re¯ux for 4 h, then evaporated under reduced pressure.

The residue was puri®ed by column chromatography

(SiO₂, CHCl₃:MeOH (5:1±4:1) elution) to give 21 (5.88 g,

84%) as an oil. ¹H NMR (200 MHz, CDCl₃) d 1.55±1.75

(1H, m), 2.30±2.90 (6H, m), 3.55±3.75 (1H, m), 4.43 (2H,

d, J=5.4 Hz), 4.85±5.05 (1H, m), 5.20±5.30 (3H, m),

5.75±5.94 (1H, m), 7.17±7.45 (15H, m), 8.07 (2H, t, J=

7.0 Hz), 8.44 (1H, t, J=7.8 Hz), 9.66 (2H, d, J=5.5 Hz).

(2R,4R)-1-Allyloxycarbonyl-2-[2-(4-carbamoylmethylimi-

dazol-1-yl)ethyl]-4-methanesulfonyloxypyrrolidine (22e).

1

A yellow paste (13%); H NMR (200 MHz, CDCl₃) d

1.75±2.00 (2H, m), 2.30±2.55 (2H, m), 3.04 (3H, s), 3.51

(2H, s), 3.51±3.65 (1H, m), 3.85±4.15 (4H, m), 4.61 (2H,

d, J=5.5 Hz), 5.20±5.36 (3H, m), 5.84±6.04 (1H, m),

6.85 (1H, br s), 7.06 (1H, br s, CONH₂), 7.49 (1H, br s).

22f and 22g were prepared from 22a, and 22b, respec-

tively, by a similar deprotection method as described for

preparation of 8.

(2R,4R)-1-Allyloxycarbonyl-4-methanesulfonyloxy-2-[2-

(pyrazol-1-yl)ethyl]pyrrolidine (22i). To a solution of 8

(27.14 g) in DMF (270 mL) was added pyrazole (5.47 g)

and ^tBuOK (9.02 g). The mixture was stirred at 50±60^ꢁC

for 2 h, poured into water, and extracted with AcOEt

(Â3). The combined organic extracts were washed with

brine, dried over MgSO₄, evaporated under reduced

pressure, and puri®ed by column chromatography

(SiO₂, hexane: AcOEt (1:1±1:3) elution) to give 22i

(2R,4R)-1-Allyloxycarbonyl-2-[2-(2-hydroxymethyl)imi-

dazol-1-yl]ethyl]-4-methanesulfonyloxypyrrolidine (22f).

A light brown paste (60%); ¹H NMR (200 MHz,

CDCl₃) d 1.80±2.10 (2H, m), 2.30±2.60 (2H, m), 3.02

(3H, s), 3.50±3.70 (1H, m), 3.90±4.20 (4H, m), 4.50±4.70

(4H, m), 5.10±5.40 (3H, m), 5.80±6.00 (1H, m), 6.80±

7.00 (2H, m).

1

(14.85 g, 59%) as an oil. H NMR (200 MHz, CDCl₃) d

(2R,4R)-1-Allyloxycarbonyl-2-[2-(4-hydroxymethyl)imi-

dazol-1-yl]ethyl]-4-methanesulfonyloxypyrrolidine. (22g)

1.70±1.90 (1H, m), 1.95±2.15 (1H, m), 2.20±2.60 (2H,

m), 3.04 (3H, s), 3.45±3.60 (1H, m), 3.90±4.35 (4H, m),

4.60 (2H, d, J=5.5 Hz), 5.14±5.35 (3H, m), 5.84±6.03

(1H, m), 6.25 (1H, t, J=1.9 Hz), 7.41±7.53 (2H, m).

1

A yellow paste (33%); H NMR (200 MHz, CDCl₃) d

1.75±2.15 (2H, m), 2.20±2.60 (2H, m), 3.03 (3H, s),

3.50±3.70 (1H, m), 3.90±4.20 (4H, m), 4.50±4.70 (4H,

m), 5.20±5.40 (3H, m), 5.85±6.04 (1H, m), 6.90±7.00

(1H, m), 7.40±7.60 (1H, m).

Using a similar procedure 22a±e were also prepared by

the same method as described for 22i from the dimesy-

late 8 and the appropriate substituted imidazole.

(2R,4R)-1-Allyloxycarbonyl-2-[2-(4-carbamoylimidazol-

1-yl)ethyl]-4-methanesulfonyloxypyrrolidine (22h). To a

solution of 22c (24.8 g) in DMSO (100 mL) was added

K₂CO₃(1.86 g) and H₂O₂(30% aqueous solution,

9.92 mL) at room temperature. After stirring at 60^ꢁC for

5 h, the mixture was poured into brine (400 mL) and

extracted with a mixture of AcOEt and THF (1:1,

200 mLÂ4). The combined extracts were dried over

MgSO₄, evaporated under reduced pressure, and pur-

i®ed by column chromatography (SiO₂600 mL, CHCl₃:

MeOH (14:1) elution) to give 22h (11.54 g, 44%) as an

(2R,4R)-1-Allyloxycarbonyl-2-[2-[2-(tert-butyldimethyl-

silyloxymethyl)imidazol-1-yl]ethyl]-4-methanesulfonyloxy-

1

pyrrolidine (22a). A yellow paste (91%); H NMR (200

MHz, CDCl₃) d 0.08 (6H, s), 0.90 (9H, s), 1.75±2.10

(2H, m), 2.31±2.57 (2H, m), 3.04 (3H, s), 3.43±3.62 (1H,

m), 3.92±4.22 (4H, m), 4.51 (2H, d, J=8.0 Hz), 4.77

(2H, s), 5.13±5.39 (3H, m), 5.74±5.93 (1H, m), 6.82±7.07

(2H, m).

1

(2R,4R)-1-Allyloxycarbonyl-2-[2-[4-(tert-butyldimethyl-

silyloxymethyl)imidazol-1-yl]ethyl]-4-methanesulfonyloxy-

oil. H NMR (200 MHz, CDCl₃) d 1.80±2.10 (2H, m),

2.30±2.50 (2H, m), 3.05 (3H, s), 3.50±3.62 (1H, m),

3.95±4.20 (4H, m), 4.61 (2H, d, J=5.6 Hz), 5.22±5.36

(3H, m), 5.84±6.04 (2H, m, CONH₂), 7.05±7.60 (2H, m,

CONH₂), 7.65 (1H, br s); APCI-MS m/z 387 (MH)⁺.

1

pyrrolidine. (22b) A brown paste (ꢂ100%); H NMR

(200 MHz, CDCl₃) d 0.10 (6H, s), 0.93 (9H, s), 1.65±1.96

(2H, m), 2.20±2.40 (2H, m), 2.94 (3H, s), 3.35±3.55 (1H,

m), 3.80±4.09 (4H, m), 4.50±4.60 (2H, m), 4.64 (2H, s),

5.13±5.27 (3H, m), 5.76±6.00 (1H, m), 6.85 (1H, s), 7.50

(1H, s).

23a±g were prepared from 22c±i, respectively, by a

similar method as described for the preparation of 14a

(as a thioacetate) or 17b (as a thiobenzoate).

(2R,4R)-1-Allyloxycarbonyl-2-[2-(4-cyanoimidazol-1-yl)-

ethyl]-4-methanesulfonyloxypyrrolidine (22c). A yellow

paste (39%); H NMR (200 MHz, CDCl₃) d 1.80±2.00

(2H, m), 2.20±2.60 (2H, m), 3.05 (3H, s), 3.50±3.60 (1H,

(4R,5S,6S)-6-[(1R)-1-Hydroxyethyl]-4-methyl-3-[(2R,4S)-

2 - [ 2 - ( 3 - methyl - 1- imidazolio ) ethyl ] pyrrolidine - 4- yl]

thio-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate

1

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1679

hydrochloride (1c). To a solution of 14b (4.54 g) in a

mixture of THF (45 mL) and MeOH (45 mL) was added

dropwise a solution of NaOMe in MeOH (28%,

2.97 mL) at 0^ꢁC. After stirring for 30 min at 0^ꢁC, the

mixture was quenched with AcOH (0.8 mL) at 0^ꢁC and

evaporated under reduced pressure. The obtained resi-

due was diluted with AcOEt (200 mL), washed water

(50 mL) and brine (50 mL), dried over MgSO₄, and ®l-

tered. To the obtained ®ltrate dimethylaminopyridine

(DMAC) was added and the solution was evaporated

under reduced pressure to give a solution of (2S,4S)-1-

allyloxycarbonyl-2-(2-imidazoylethyl)-4-mercaptopyrro-

lidine in DMAC. This solution was used immediately in

the next reaction because of instability of the thiol

function. To a solution of activated carbapenem (24)

(12.8 mmol) in MeCN (38 mL) was added the above

Compounds 1b, 1e, 1g, 1l±1n, 1q, 1r were all prepared

using the same methodology as described for prepara-

tion of 1c from the appropriate thioacetate or thio-

benzoate. Compound 1j and 1k were prepared similarly

from 25c using iodoacetamide or N-allyloxycarbonyl-3-

iodopropylamine as the alkylating agent.

(4R,5S,6S)-6-[(1R)-1-Hydroxyethyl]-4-methyl-3-[(2S,4S)-

2-(3-methylimidazoliomethyl)pyrrolidine-4-yl]thio-7-oxo-

1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate hydrochloride

(1a). The coupling reaction of activated carbapenem

(24) and thioacetate (12) was achieved by a similar

method as described for the preparation of 25c to give

25a (37%) as an oil. A solution of 25a (610 mg) and MeI

(2 mL) in THF (3 mL) was stirred at room temperature

for 15 h and the solution was evaporated under reduced

pressure to give a crude oil (0.8 g, ꢂ100%). The

obtained crude oil was dissolved in a mixture of THF

(50 mL) and phosphate buer (0.1 M, pH 6.5), and to

the mixture was added Pd(OH)₂C (20%, 0.3 g). After

stirring at room temperature under hydrogen for 5 h,

the mixture was ®ltered and THF was evaporated under

reduced pressure. The residual solution was washed

with AcOEt (50 mLÂ2), organic solvent removed from

the aqueous layer by evaporation under reduced pres-

sure, loaded onto a HP-20 column (50 mL), washed with

water, and then eluted with a mixture of Me₂CO and

water (1:99). The eluate was evaporated and lyophilized

solution and Pr₂EtN (2.7 mL). After stirring at 0^ꢁC

i

overnight, the mixture was poured into water (200 mL)

and extracted with a mixture of AcOEt and THF (1:1)

(200 mL). The organic layer was washed with brine,

dried over MgSO₄, ®ltered, evaporated under reduced

pressure, and puri®ed by column chromatography (SiO₂

200 mL, CHCl₃:MeOH (20:1) elution) to give 25c

1

(4.74 g, 70%) as an oil. H NMR (200 MHz, CDCl₃) d

1.25 (3H, d, J=7.2 Hz), 1.36 (3H, d, J=6.2 Hz), 1.50±

1.75 (1H, m), 1.90±2.10 (1H, m), 2.30±2.60 (3H, m),

3.20±3.40 (3H, m), 3.50±3.65 (1H, m), 3.95±4.15 (4H,

m), 4.18±4.30 (2H, m), 4.59 (2H, d, J=4.9 Hz), 4.68

(1H, dd, J= 12.8 Hz, 5.5 Hz), 4.83 (1H, dd, J=12.8 Hz,

5.5 Hz), 5.23±5.49 (4H, m), 5.88±6.04 (2H, m), 6.98 (1H,

br s), 7.07 (1H, s), 7.52 (1H, br s). The oil was used

immediately in the next reaction because of instability

of the b-lactam function. To a solution of the above oil

(6.94 g) in Me₂CO (35 mL) was added MeI (8.14 mL) at

room temperature. After stirring at room temperature

overnight, the mixture was evaporated to give a crude

salt. To a solution of this salt in a mixture of THF

(88 mL) and EtOH (88 mL) was added Ph₃P (0.69 g),

Pd(Ph₃P)₄(0.6 g), and n-Bu₃SnH (14.1 mL) at room

temperature, and then the mixture was stirred for

30 min at room temperature. The resulting precipitate

was ®ltered, washed with THF and EtOH, and dried

under reduced pressure to give a crude powder. A solu-

tion of the powder in water (45 mL) was loaded onto a

HP-20 column (440 mL), washed with water, then eluted

with a mixture of Me₂CO and water (5:95). The com-

bined product-containing fractions were evaporated and

lyophilized to give a powder (4 g). The powder was

puri®ed by column chromatography (ODS 400 mL,

phosphate buer (pH=6.86):MeCN (5:1) elution). The

combined product-containing fraction was evaporated

to remove MeCN, adjusted to pH (6.0), then re-loaded

onto a HP-20 column (200 mL), washed with water,

then eluted with a mixture of Me₂CO and water (5:95).

The eluate was evaporated, passed through a Amberlyst

A-26 column (200 mL) and then treated with charcoal,

1

to give 1a (0.25 g, 65%) as a white powder. H NMR

(90 MHz, D₂O) d 1.21 (3H, d, J=6.3 Hz), 1.28 (3H, d,

J=6.3 Hz), 1.60±2.05 (1H, m), 2.65±3.00 (1H, m), 3.24±

4.80 (10H, m), 3.93 (3H, s), 7.52 (1H, br s), 7.60 (1H, br

s), 8.88 (1H, br s).

(4R,5S,6S)-6-[(1R)-1-Hydroxyethyl]-4-methyl-3-[(2R,4S)-

2-[2-(1-pyridinio)ethyl]pyrrolidine-4-yl]thio-7-oxo-1-aza-

bicyclo[3.2.0]hept-2-ene-2-carboxylate hydrochloride (1f).

To a solution of 21 (2.5 g) in CH₂Cl₂(8.8 mL) was

added dropwise Et₃SiH (0.84 mL) at 5^ꢁC, and then TFA

(8.75 mL). After stirring for 1 h at room temperature,

the mixture was evaporated under reduced pressure.

The residue was washed with hexane, and treated with

DMAC (10 mL) and MeCN (10 mL) to give a solution

of 25f. This solution was used immediately in the next

reaction because of instability of thiol function. Cou-

pling this solution and activated carbapenem 24 by a

similar method as described for the preparation of 1c

gave 1f (0.42 g, 21%) as a white powder. IR (Nujol)

1

cm 1730, 1540±1580; ¹H NMR (200 MHz, D₂O) d

1.22 (3H, d, J=7.2 Hz), 1.30 (3H, d, J=6.6 Hz), 1.72±

1.88 (1H, m), 2.55±2.91 (3H, m), 3.34±3.50 (3H, m),

3.66±3.95 (4H, m), 4.00±4.15 (1H, m), 4.20±4.35 (2H,

m), 8.13 (2H, t, J=7.3 Hz), 8.61 (1H, t, J=7.9 Hz), 8.92

(1H, d, J=6.9 Hz). MS(FAB+) 418(MH+) (Free).

(4R,5S,6S)-3-[(2R,4S)-2-[2-(4-Carbamoyl-3-methyl-1-

imidazolio)ethyl]pyrrolidine-4-yl]thio-6-[(1R)-1-hydroxy-

ethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-

carboxylate hydrochloride (1p). The coupling reaction

of 23c (6.37 g) and activated carbapenem 24 was

achieved by a similar method to that of the preparation

of 25c to give 25p (2.23 g, 38%) as an oil. To a solution of

25p (2.07 g) in CH₂Cl₂(20 mL) was added CF₃SO₃Me

®ltered, and lyophilized to give 1c (3.49 g, 58%) as a

1

white powder. IR (Nujol) cm

1750; ¹H NMR

(200 MHz, D₂O) d 1.23 (3H, d, J=7.2 Hz), 1.30 (3H, d,

J=6.6 Hz), 1.65±1.80 (1H, m), 2.35±2.60 (2H, m), 2.70±

2.85 (1H, m), 3.34±3.50 (3H, m), 3.57±3.74 (2H, m), 3.91

(3H, s), 3.90±4.10 (1H, m), 4.21±4.40 (4H, m), 7.49 (1H,

d, J=1.7 Hz), 7.55 (1H, d, J=1.8 Hz), 8.81 (1H, br s).

1680

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

(1.16 mL) at 0^ꢁC, and the solution was stirred for

30 min. The obtained mixture was treated with a sus-

pension of ion exchange resin (Amberlyst A-26, Cl-

type) (10 mL) in CH₂Cl₂(10 mL) for 5 min. After ®ltra-

tion to remove the resin, the resin was washed with a

mixture of CH₂Cl₂and MeOH (4:1). The ®ltrate and the

washings were collected and concentrated under

reduced pressure to give imidzolio salt as a pale yellow

solid (2.75 g, ꢂ100% ). Deprotection of this carbape-

nem compound was achieved by a similar method as

shown in the preparation of 1d to give 1p (421 mg,

(2S,4R)-1-allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-[(E)-3-hydroxy-1-propen-1-yl]pyrrolidine (59.4 g, 98.8%)

as a light brown oil. ¹H NMR (200 MHz, CDCl₃) d 0.06

(6H, s), 0.88 (9H, s), 1.80 (1H, m), 1.90±2.20 (1H, m),

3.30±3.60 (2H, m), 4.16 (2H, m), 4.30±4.70 (4H, m),

5.20±5.29 (2H, m), 5.50±6.10 (3H, m). Desilylation of

this oil (9.59 g) was achieved by a similar method to that

described for preparation of 8 to give crude 27 (7.04 g,

ꢂ100%) as a pale yellow oil that was used directly in

1

the next step. IR (Neat) cm 1670, 1405; H NMR

(200 MHz, CDCl₃) d 2.00±2.30 (2H, m), 3.63 (2H, m),

4.12 (2H, d, J=5.1 Hz), 4.40±4.70 (4H, m), 5.10±5.50

(2H, m), 5.50±6.10 (3H, m).

1

19.2%) as a white power. H NMR (200 MHz, D₂O) d

1.22 (3H, d, J=7.7 Hz), 1.29 (3H, d, J=6.2 Hz), 1.70±

1.90 (1H, m), 2.40±2.60 (2H, m), 2.70±2.90 (1H, m),

3.30±3.80 (6H, m), 4.04 (3H, s), 4.20±4.50 (4H, m), 8.15

(1H, s), 9.01 (1H, s).

(2S,4S)-1-Allyloxycarbonyl-4-benzoylthio-2-[(E)-3-tert-

butyldimethylsilyloxy-1-propen-1-yl]pyrrolidine (28). To

a solution of 27 (7.04 g) in DMF (30 mL) was added t-

BuMe₂SiCl (5.0 g) and imidazole (2.5 g). After stirring

for 2 h, the mixture was quenched with water and

extracted with AcOEt (100 mLÂ2). The combined

organic layer was washed with brine (50 mLÂ2), dried

over MgSO₄, evaporated under reduced pressure, and

puri®ed by column chromatography (SiO₂) to give

(2S,4R)-1-allyloxycarbonyl-2-[(E)-3-tert-butyldimethyl-

silyloxy-1-propen-1-yl]-4-hydroxypyrrolidine (4.53 g,

44.3%) as a pale yellow oil. ¹H NMR (200 MHz,

CDCl₃) d 0.09 (6H, s), 0.91 (9H, s), 1.80±2.00 (1H, m),

2.00±2.20 (1H, m), 3.57 (2H, m), 4.16 (2H, d, J=

2.9 Hz), 4.30±4.70 (4H, m), 5.10±5.40 (2H, m), 5.50±5.70

(2H, m), 5.89 (1H, m). To a solution of the above

mono-silyl derivative (4.53 g) in THF (40 mL) was

added Ph₃P (5.25 g), followed by diethyl azodicarboxy-

late (3.1 mL) dropwise at 0^ꢁC. After stirring for 30 min,

PhCOSH (2.8 mL) was then added dropwise at 0^ꢁC, and

the mixture stirred for 30 min at 0^ꢁC. The mixture was

poured into saturated NaHCO₃(40 mL) and extracted

with AcOEt (100 mLÂ2). The combined extracts were

washed with brine (50 mLÂ2), dried over MgSO₄, eva-

porated under reduced pressure, and puri®ed by column

chromatography (SiO₂) to give 28 (5.92 g, 93.2%) as a

pale yellow oil. ¹H NMR (200 MHz, CDCl₃) d 0.07 (3H,

s), 0.10 (3H, s), 0.91 (9H, s), 1.90±2.70 (2H, m), 3.41±

4.40 (5H, m), 4.50±4.70 (3H, m), 5.10±5.40 (2H, m),

5.60±6.20 (3H, m), 7.30±7.80 (3H, m), 8.10 (2H, m).

1o, 1s were prepared from 23d and 23c, respectively, by

a similar method as described for the preparation of 1p.

(4R,5S,6S)-6-[(1R)-1-Hydroxyethyl]-4-methyl-3-[(2R,4S)-

2-[2-(2-methyl-1-pyrazolio)ethyl]pyrrolidine-4-yl]thio-7-

oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate hydro-

chloride (1d). The coupling reaction of 23g and acti-

vated carbapenem (24) was achieved by a similar

method to that of the preparation of 25c to give 25d

(10.49 g, 98%) as an oil. To a solution of 25d in CH₂Cl₂

(150 mL) was added FSO₃Me (2.33 mL) at 5^ꢁC. After

stirring at room temperature for 1.5 h, the mixture was

evaporated under reduced pressure to give pyrazolio

salt (12.75 g, ꢂ100%) as an oil. Deprotection of this

carbapenem compound was achieved by similar method

as shown in the preparation of 1c to give 1d (2.34 g,

25.9%) as a white powder. IR (Nujol) cm ¹1765--1740,

1

1580- -1570; H NMR (200 MHz, D₂O) d 1.22 (3H, d,

J=7.2 Hz), 1.29 (3H, d, J=6.4 Hz), 1.71--1.86 (1H, m),

2.42--2.64 (2H, m), 2.76--2.91 (1H, m), 3.30--3.49 (3H,

m), 3.67--3.95 (2H, m), 4.03--4.17 (1H, m) , 4.17 (3H, s),

4.17--4.28 (2H, m), 4.62 (2H, t, J=8.0 Hz), 6.80 (1H, t,

J=3.0 Hz), 8.22 (1H, d, J=2.8 Hz), 8.29 (1H, d,

J=2.3 Hz); MS (FAB+) 421.2 (MH+).

(2S,4R)-1-Allyloxycarbonyl-4-tert-butyldimethylsilyloxy-

2-[(E)-2-formylethenyl) pyrrolidine (26). 26 was obtained

from 2b (79.5 g) using (triphenylphosphoranylidene)-

acetaldehyde (107 g) instead of Ph₃P=CHCOOMe by a

similar method as for 9. Brown oil (59.7 g, 69.3%); IR

Allyl (4R,5S,6S)-3-[(2S,4S)-1-allyloxycarbonyl-2-[(E)-3-

tert-butyldimethylsilyloxy-1-propen-1-yl]pyrrolidine-4-

yl]thio-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-aza-

bicyclo[3.2.0]hept-2-ene-2-carboxylate (29a). The pre-

paration of 29a (1.65 g, 22%) was achieved from 28

(5.9 g) by a similar method as described for preparation

of 25c. A pale yellow oil. ¹H NMR (200 MHz, CDCl₃) d

0.06 (6H, s), 0.90 (9H, s), 1.26 (3H, d, J=7.2 Hz), 1.35

(3H, d, J=6.2 Hz), 2.00±2.70 (2H, m), 3.10±3.57 (4H,

m), 4.00±4.30 (5H, m), 4.49 (1H, m), 4.68 (2H, d, J=

12.0 Hz), 4.68±4.84 (2H, m), 5.10±5.44 (4H, m), 5.50±

6.10 (4H, m).

1

(Neat) cm 1682, 1400; H NMR (200 MHz, CDCl₃) d

0.07 (6H, s), 0.87 (9H, s), 1.80±2.00 (1H, m), 2.05±2.30

(1H, m), 3.40±3.60 (2H, m), 4.34±4.42 (1H, m), 4.50±

4.80 (3H, m), 5.20±5.30 (2H, m), 5.70±6.10 (1H, m), 6.16

(1H, dd, J=15.7 Hz, 7.7 Hz), 6.60±6.90 (1H, m), 9.56

(1H, d, J=7.7 Hz).

(2S,4R)-1-Allyloxycarbonyl-2-[(E)-3-hydroxy-1-propen-

1-yl]-4-hydroxypyrrolidine (27). To a solution of 26

(59.8 g) in a mixture of EtOH (300 mL) and THF

(300 mL) was added NaBH₄(6.66 g) at room tempera-

ture. After stirring for 1 h, brine (600 mL) was added to

the mixture which was then extracted with AcOEt

(300 mLÂ3). The combined extracts were washed with

water (500 mLÂ5) and brine (500 mLÂ2), dried over

MgSO₄, and evaporated under reduced pressure to give

Allyl (4R,5S,6S)-3-[(2S,4S)-1-allyloxycarbonyl-2-[(E)-3-

hydroxy-1-propen-1-yl]pyrrolidine-4-yl]thio-6-[(1R)-1-

hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-

ene-2-carboxylate (29b). To a solution of 29a (1.65 g) in

THF (10 mL) was added AcOH (0.54 mL) at room

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

1681

temperature. After a short stirring, a solution of TBAF

(70% aqueous solution, 3.0 g) in THF (5 mL) was added

to the mixture. After stirring for 4 h, the mixture was

poured into a mixture of AcOEt (100 mL) and water

(50 mL). The organic layer was separated, and the aqu-

eous layer was extracted with AcOEt (100 mL). The

combined organic layer was washed with water (50 mL),

saturated NaHCO₃(50 mL), and brine (50 mLÂ2), dried

over MgSO₄, evaporated under reduced pressure, and

puri®ed by column chromatography to give 29b (0.77 g,

57%) as a pale yellow oil. IR (CH₂Cl₂) cm ¹1765, 1695;

¹H NMR (200 MHz, CDCl₃) d 1.27 (3H, d, J=7.2 Hz),

1.36 (3H, d, J=6.2 Hz), 1.60±2.00 (2H, m), 2.59 (1H, dt,

J=6.4 Hz, 13.3 Hz), 3.20±3.50 (3H, m) 3.64 (1H, m),

4.00±4.40 (5H, m), 4.40±4.90 (4H, m), 5.10±5.60 (4H,

m), 5.60±6.10 (4H, m).

was added dropwise nBu₃SnH (0.43 mL) at room tem-

perature. After stirring for 30 min, the mixture was

diluted with THF (4.5mL). The resultant precipitate was

collected by decantation, washed with THF (4.5mLÂ4),

and then dissolved in water (9 mL). The solution was

washed with AcOEt (9.0 mLÂ2), concentrated to ca.

5 mL, and the obtained solution loaded onto nonionic

adsorption resin (Dianion HP-20=45mL, water:Me₂CO

(100:0±96:4) elution). The combined product-containing

fractions were concentrated to ca. 10 mL, then passed

through ion exchange resin (Amberlyst A-26=1.4 mL,

water (10 mL) elution), and lyophilized to give 1i (51 mg,

15%) as a white solid. IR (Nujol) cm 1730; H NMR

(200 MHz, CDCl₃) d 1.21 (3H, d, J=7.2 Hz), 1.28 (3H,

d, J=6.4 Hz), 1.83±1.96 (1H, m), 2.74±2.95 (1H, m),

3.30±3.50 (3H, m), 3.71 (1H, dd, J=12.5 Hz, 6.8 Hz),

4.00±4.15 (1H, m), 4.20±4.46 (3H, m), 5.32 (2H, d,

J=5.8 Hz), 6.07±6.38 (2H, m), 8.11 (2H, t, J=7.2 Hz),

8.60 (1H, t, J=7.9 Hz), 8.85 (2H, d, J=5.5 Hz).

1

Allyl (4R,5S,6S)-3-[(2S,4S)-1-allyloxycarbonyl-2-[(E)-3-

diphenoxyphosphoryloxy-1-propen-1-yl]pyrrolidine-4-yl]-

thio-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo-

[3.2.0]hept-2-ene-2-carboxylate (29c). To a solution of

29b (370 mg) in CH₂Cl₂(1.9 mL) was added succes-

sively DMAP (106 mg) and diphenylchlorophosphate

(0.159mL) at 50^ꢁC. After stirring at 50^ꢁC for 40min,

the mixture was quenched with saturated NaHCO₃

(2 mL) and extracted with AcOEt (12 mL). The organic

extract was washed with aqueous HCl (0.1N, 5 mL),

water (10 mL), and brine (10 mL), then dried over

MgSO₄, and evaporated under reduced pressure to give

29c (536 mg, 99%) as a light brown paste. IR (CH₂Cl₂)

The preparation of 1h (1.18 g, 30%) was achieved from

29d (5.0 g) and 1-methylimidazole (1.98 mL) by a similar

method as described for preparation of 1i.

Measurement of in vitro antibacterial activity

According to the method of the Japan Society of Che-

motherapy, the MICs of compound were determined by

the twofold agar dilution method using heart infusion

agar (Eiken). The inoculum size was adjusted to 10⁶cfu/

mL, and incubation was carried out at 37^ꢁC for 20 h.

1

cm 1764, 1692, 1592; H NMR (200 MHz, CDCl₃) d

1.25 (3H, d, J=7.2 Hz), 1.36 (3H, d, J=6.3Hz), 1.61±1.93

(1H, m), 2.43±2.66 (1H, m), 3.14±3.48 (3H, m), 3.48±

3.76 (1H, m), 3.88±4.50 (4H, m), 4.50±4.95 (6H, m), 5.09±

5.58 (4H, m), 5.60±6.14 (4H, m), 7.20±7.40 (10H, m).

Stability to DHP-I

The stability of carbapenems against recombinant

human renal DHP-I was determined spectrophoto-

merically and expressed as the ratio of hydrolysis to that

of meropenem at 50 mg/mL.

Allyl (4R,5S,6S)-3-[(2S,4S)-1-allyloxycarbonyl-2-[(E)-3-

iodopropen-1-yl]pyrrolidine-4-yl]thio-6-[(1R)-1-hydroxy-

ethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-

carboxylate (29d). To a solution of 29c (431 mg) in

Me₂CO (3 mL) was added NaI (178 mg), and the mix-

ture was stirred at 50^ꢁC for 30 min. The mixture was

quenched with water (5 mL) and extracted with AcOEt

(15 mL). The organic layer was washed with water

(10 mL), saturated sodium thiosulfate (10 mL) and brine

(10 mLÂ2), dried over MgSO₄, and evaporated under

reduced pressure to give 29d (451 mg, 100%) as a yellow

solid. IR (CHCl₃) cm ¹1762, 1682; ¹H NMR (200 MHz,

CDCl₃) d 1.26 (3H, d, J=7.3 Hz), 1.36 (3H, d, J=

6.3 Hz), 1.64±2.70 (2H, m), 3.17±3.45 (3H, m), 3.45±3.71

(1H, m), 3.70±4.50 (6H, m), 4.50±4.92 (4H, m), 5.10±

5.60 (4H, m), 5.60±6.10 (4H, m).

Ecacy in lethal systemic infection

A strain was intraperitoneally inoculated in groups of

eight male ICR mice aged 4 weeks with 0.5 mL of bac-

terial suspension in 5% gastric mucin, given at one to

®ve times minimum lethal dose (MLD). The infected

mice were treated subcutaneously with serially diluted

drugs 1 h after infection. The survival of the infected

mice was observed for 3±5 days, and the 50% eective

dose (ED₅₀) was determined from the ®nal survival rates

by the Probit method.

Urinary recovery

(4R,5S,6S)-6-[(1R)-1-Hydroxyethyl]-4-methyl-7-oxo-3-

[(2S,4S)-2-[(E)-3-(1-pyridinio)-1-propen-1-yl]pyrrolidine-4-

yl]thio-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate hydro-

chloride (1i). To a solution of 29d (441 mg) in CH₂Cl₂

(4.4 mL) was added pyridine (0.594 mL) at room tem-

perature. After stirring for 1.5 h, the mixture was eva-

porated and then triturated with ether to give a crude

yellow powder (468 mg). To the solution of this powder

(453 mg), Ph₃P (17 mg), AcOH (0.1 mL), and Pd(Ph₃P)₄

(23 mg) in a mixture of EtOH (4.5mL) and THF (4.5 mL)

Rats were used in groups of nine to ten. The animals

were housed individually in a metabolism cage and urine

was collected 0±24 h after dosing from each animal.

Acknowledgements

The authors are grateful to Dr. Kazuo Sakane for useful

suggestions and encouragement during the preparation

of this paper.

1682

H. Azami et al. / Bioorg. Med. Chem. 7 (1999) 1665±1682

K.; Sakurai, M.; Matsumoto, Y.; Tawara, S.; Chiba, T.;

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Synthesis and antibacterial activity of novel 4-pyrrolidinylthio carbapanems. Part III: Novel 2-alkyl substituents containing cationic heteroaromatics linked via a C-N bond

DOI: 10.1016/S0968-0896(99)00085-1

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Full text of DOI:10.1016/S0968-0896(99)00085-1

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