Background of the Invention

Field of the Invention

The present invention relates to novel carbapenem

(3-pyrrolidinylthio-1-azabicyclo[3,2,0]hept-2-ene-7-one- 2-carboxylic acid compounds) derivatives, processes for preparing the derivatives and antibiotics containing such compounds as active ingredients.

Description of the Related Art

Thienamycin, which is structurlly related to carbapenem, has strong antibacterial activities, however, thienamycin itself is chemically unstable and has been reported to be decomposed by enzymes such as mammalian renal dehydropeptidase I (hereinafter referred to DHP-I) , whereby the antibacterial activities decrease. Also the decomposed products are toxic to the kidneys. Thienamycin must be used in combination with a DHP-I inhibitor.

Merck & Co. Inc. have synthesized many thienamycin analogues to maintain the excellent antibacterial activities of thienamycin and to secure chemical stability. As a result, imipenem obtained by formimidation of the amino group of thienamycin, has been developed as a pharmaceutical product (U.S. Pat.

4,194,047). However, like thienamycin, imipenem is likely to be decomposed by DHP-I in the human kidneys.

Therefore, it cannot be used for treatment of infection in the genito-urinary tract. Furthermore the decomposed products are toxic to the kidneys. Therefore, imipenem

cannot be administered alone and is required to be used in combination with a DHP-I inhibitor such as cilastatin (Antimicrob. Agents Chemother. , vol 12 (Suppl. D), 1(1983) ) .

However, in the 1980 's, there was a growing demand for stable carbapenem compounds, and various carbapenem compounds having a 1/3-methyl carbapenem basic skeleton were developed. Sumitomo Co. has synthesized meropenem which is much more stable against DHP-I than imipenem, and has a broad antibacterial spectrum. This product is expected to be marketed in the near future (EP 0126587; Antimicrobial Agents and Chemotherapy vol. 33, No. 7, 1009(1989). Further Lederle has synthesized biapenem which shows improved antibacterial activities and stability against DHP-I [EP 028801, Antimicrobial agents and chemotherapy vol 35, No. 1, 203 (1991)].

0-Lactam antibiotics are specific for bacteria and show little or no toxic effects against animal cells. Therefore, they are widely used in treatment of infectious disease caused by bacteria. Carbapenem compounds have a broad antibacterial spectrum against gram positive and gram negative, and lower toxicity than other antibacterial agents. However, imipenem resistant bacteria have been clinically isolated and they pose serious problem for the treatment of the disease.

Carbapenem derivatives having an aminocarbonylvinyl group or subsituted or nonsubstituted amino loweralkyl group at the 2 '-position of pyrrolidine whose 4' position is linked as S(sulfur) to the carbapenem skeleton are known in the art. Representative compounds of these classes are (1R, 5S, 6S)-2-[ (2S, 4S)-2-[ (E)-2- aminocar onyl )vinyl ]pyrrolidin-4-y1thio]-6-[ (R)-l- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylic acid

[Compound of Example 3 in EP 0411664; hereinafter referred to simply as BO-2171] and (1R,5S, 6S)-2- [ (2S, 4S)-2-[ (Z)-3-amino-l-propenyl ]-pyrrolidin-4- ylthio]-6-[ (R)-l-hydroxyethyl]-l-methyl-l-carbapen-2-em- 3-carboxylic acid [Compound of Example 2 in WO 93/01192 ; hereinafter referred to simply as BO-A]

According to the above references, BO-A is reported to have strong antibacterial activity and to be more stable than BO-2171. Therfore we have selected the BO-A as a reference compound, in addition to imipenem and meropenem, and compared our newly synthesized compounds with these comparative references.

The present invention describes structures which contain halo-loweralkenyl, loweralkylsulfonylamino, loweralkysulfamoylamino, side chains attached to 2 position of the pyrrolidine ring of the lactam nucleus, and processes for preparing these compounds. All compounds of these categories have not been introduced or reported in any periodicals or patents.

Extensive research has been directed at identifying compounds with strong antibacterial actvities against various gram positive and gram negative bacteria, including imipenem-resistant strains and clinical isolates of Pseudomonas aeruginosa. which exhibit stability against DHP-1. The present invention describes derivatives with loweralkanesulfonylamino, loweralkylsulfamoylamino side chain attached to the 2 position of pyrrolidine ring of the lactam nucleus exhibit the following properties:

1) strong antibacterial activity against various gram positive and negative strains including beta-lactamase producing strains ;

2) 2-4 times the active against imipenem resistant strains as oll as clinical isolates of PsGUUomon G aeruginosa than meropenem and BO-A ;

3) increased stable against DHP-1 than meropenem ;

4) more favorable pharmacokinetic properties such as longer half-life, higher under the curve (AUC), higher urinary excretion than imipenem, meropenem, and BO-A ;

5) much lower mortality and toxic effects on the central nervous system than imipenem.

Summary of the Invention

The present invention provides a compound of formula (I)

wherein R. is a hydrogen atom or a methyl group, R ₂ is a hydrogen atom, or a metal or a nonmetal salt group, or a carboxy protecting group or a negative charge.

The metal or nonmetal salt group of the general formula (I) represents an alkali metal salt such as sodium salt or potassium salt; an alkaline earth metal salt such as magnesium salt or calcium salt; an ammonium salt; an salt composed of an organic base such as triethylamine salt, pyridine salt, ethanolamine salt; or intermolecular quaternary salt.

The carboxy protecting group may, for example, be a lower alkyl ester group or an esterified carboxyl group which is mentioned below.

The above-mentioned ester group includes at least one appropriate substituent, for example, a lower alkanoyloxy(lower)alkyl ester group such as an acetoxymethyl ester group, a butyryloxymethyl ester group, a pivaloyioxymethyl ester group; a lower alkanesulfonyl(lower)alkyl ester group such as a 2- methylethyl ester group; a lower alkoxycarbonyloxy (lower)alkyl group such as a methoxycarbonyloxymethyl ester group, an ethoxycarbonyloxymethyl ester group, a propoxycarbonyloxymethyl group, a t-butoxycarbonyloxy methyl group; a lower alkenyl group such as a vinyl ester group, an allyl ester group, an isopropenyl ester group; a lower alkynyl group such as an ethynyl ester group, propynyl ester group; an ar(lower) alkyl group capable of having at least more than one substituent such as a benzyl ester group, a 4-methoxybenzyl ester group, a 4-nitrobenzyl ester group, a 2-nitrobenzyl ester group, a trityl ester group, a benzhydryl ester group; an aryl ester group capable of having at least more than one substituent such as a phenyl ester group, a 4-chlorophenyl ester group, a tolyl ester group, and a t-butylphenyl ester group.

"Lower" means that the number of carbon is l to 6. The "Lower alkyl" includes a linear or branched alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, and hexyl. "Lower alkoxy" also includes a linear alkoxy or branched alkoxy such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec- butoxy, t-butoxy.

R-. represents a hydrogen atom, an imino protecting

group or a pharmaceutically acceptable salt. An appropriate "imino protecting group" may be a carbamoyl, an aliphaticacyl, an aromaticacyl, heterocyclicacyl, an aliphaticacyl substituted with an aromatic group, an aliphaticacyl substituted with a heterocyclic group, all of which are derived from a carboxylic acid, a carbonic acid, and a carbamic acid.

And "a pharmaceutically acceptable salt" may be a salt which has attached hydrobromic acid, hydrochloric acid and sulfuric acid etc.

An appropriate "imino protecting group" may be, for example, (C_-C ₄ ) alkenyloxycarbonyl, phenyl(0,-0^)alkoxy carbonyl, o-niro (or m-nitro or p-nitro) benzyl oxycarbonyl and o-methoxy (or m-methoy or p-methoxy) benzyloxycarbonyl group.

R ₄ and R _s independently represent a hydrogen atom, hydroxy group, cyano group, halogen atom, such as chlorine, bromine, iodine, and fluorine; methanesulfonyl group, N-methylsulfamoyl group, N,N-dimethylsulfamoyl group, lower alkyl group which has a substituent, or no substituent and carbamoyl group which has a substituent or no substituent.

A represents a halogen atom such as chlorine, bromine, iodine and fluorine, or one of the following general formula (l)-(2):

R,

(1) N

R,

wherein R ₆ and R ₇ respectively are selected from the

group consisting of a hydrogen atom or a lower alkanesulfonyl group, a N-(lower)alkylsulfamoyl group, or a N,N-(lower)dialkyl sulfamoyl group, wherein either of R ₆ or R ₇ is hydrogen, the other group is a nonhydrogen group.

/

(2) — N

\ ^S0 2 ^R 8

wherein R ₆ is the same as defined above and R ₈ is selected from the group consisting of a carbamoyl group which may be optionally substituted with appropriate substituent, or heterocyclic group of 5 or 6 membered ring consisting of 1 to 4 heteroatoms such as a nitrogen atom or an oxygen atom or a sulfur atom which may be optionally substituted with an appropriate substituent;

5- to 6- membered heterocyclic group containing 1 to 4 hetero atoms such as a nitrogen atom, an oxygen atom or a sulfur atom which may be optionally substituted by appropriate substituent, or may be an unsaturated 5- to

6- membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolidinyl, imidazolyl, imidazolidinyl, pyrazolyl, pyrazolidinyl, pyridyl , pyridinio, d i h d r o pyr i dy 1 , tetrahydropyridyl(such as 1,2,3,6-tetrahydropyridyl), pyrimidinyl, pyrimidinio, pyrazinyl, pyrazinio, pyridazinyl(such as 1, 3,5-triazinyl, 1, 2, 4-triazinyl and l,2,3-triazinyl) , tetrahydrotriazinyl(such as 1,2,5,6- tetrahydro-1,2,4-triazinyl, 1,4,5,6-tetrahydro-l,2,4- triazinyl), triazinio, triazoiyl(such as 1H-1,2,4- triazolyl, 1H-1, 2, 3-triazolyl, and 2H-1, 2, 3-triazolyl), triazolio, tetrazinyl, tetrazinio, tetrazoiyl(εuch as lH-tetrazolyl and 2H-tetrazolyl), tetrazolio; an unsaturated 5- to 6- membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen

atoms, for example, thiazolyl, thiazolio, isothiazoiyl, thiadiazolyl (such as l, 2, 3-thiadiazoyl, 1,2,4- thiαdiαzoiyl, 1, 3, -thiαdiazolyl, 1, 2, 5-thiadiazolyi) , thiadiazolio, thiazolydinyl, dihydrothiazynyl; an unsaturated 5- to 6- membered heteromonocyclic group containing l to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxadiazolyl (such as 1,2,4- oxadiazoiyl, 1, 3, 4-oxadiazoiyl, and 1, 2,5-oxadiazoiyl) . The above-mentioned heterocyclic group can be substituted with 1 to 3 substituents selected from the group consisting of an amino group, an amino protecting group which is the same as the imino protecting group defined above, a lower alkylamino, carbamoyl and a halo lower alkyl, wherein n is 0 or an integer of 1 to 3.

Also the present invention provides a process for preparing the compound of the formula (I), which comprises reacting a compound of the formula (II) ;

wherein R, and R ₂ are the same as defined above, R ₉ is a hydrogen or a hydroxyl protecting group, with a compound of the formula (III),

wherein R "3.,,' RA,,' R , A and n are the same as defined above,

to obtain a compound of the formula (IV)

wherein R,, R ₂ , R ₃ , R ₄ , R _s , R ₉ , A and n are the sames as defined above. The compound of the formula (I) also can be produced by removing any protecting group of the compound of formula (IV), if necessary.

Further, the present invention provides antibacterial compositions comprising an antibacterially effective amount of the compound of formula (I) (wherein R,, R ₂ , R ₃ , R ₄ , R _s , A and n are the same as defined above) or a pharmaceutical acceptable salts, or ester thereof, and a pharmaceutically acceptable carrier or diluent.

The compound of the present invention has the basic structure as follows:

ϋ ^' 1 |~ 0' OH

which s systematically referred to as 7-oxo-l- azabιcyclo[3, 2, 0]hept-2-ene-2-carboxylιc acid or 1- azabιcyclo[3, 2, 0]hept-2-ene-7-one-2-carboxylιc acid. In this specification, this basic structure will be

referred to as a i-carbapen-2-em-3-carboxylic acid, by μuLLlncj o nυiubπrr. bnr_.ocl on n commonly used cnrbnμonoui of the following formula;

The present invention includes optical isomers based on the asymmetrical carbon atoms at the l- position, 5-position, 6-position and 8-position of the carbapenem structure. Among these isomers, preferred is a compound of a (5R,6R,8R) configuration, i.e., a compound having the same steric-configuration (5R,6S) (5,6-trans) as thienamycin in which the carbon atom at the 8-position takes an R-configuration, or a compound of a (IR,5S,6S, 8R) configuration in a case where a methyl group is present at the 1-position.

Accordingly, among compounds of the formula (I), a group of compounds having preferred steric configurations are represented by the formula (I-a):

wherein R., R ₂ , R_, R ₄ , R ₅ , A and n are as defined above.

The 2 '-substituted pyrrolidin-4 '-yl-thio group in the side chain at the 2-position also includes all prossible isomers based on the asymmetrical carbon atoms

92

11 at the 2- and 4-positions of the pyrrolidine structure. Among these isomers, preferred are compounds of a (2'S,4'S) configuration and a (2'R,4'S) configuration.

Among the compounds of formula (I-a) are a group of compounds wherein R, is a methyl group, R ₄ is hydrogen atom, R ₅ is hydrogen atom, methyl group or halogen atom and A is loweralkaneεulfonylamino group or N-(lower) alkylsulfamoylamino group which have excellent antibacterial activities.

Further, with respect to the double bond of the 2- subεtituted pyrrolidine group, cis(Z) and trans(E) geometric isomers are preferred. These isomers are also included in the present invention. Of these isomers, the

(E)-isomer has excellent antibacterial activities.

It is desired that the compound of formula (II) is converted to its reactive derivatives before reacting with the compound of formula (III). That is, the compound of formula (II) is added to the inert organic solvent and reacted with activating agents under alkali conditions to obtain the activated derivatives of the formula (Il-a),

wherein R., R ₂ , R ₉ is the same as defined above and B is a leaving group.

The activating reagent to be used for the reaction may, for example, be an acid anhydride such as

methaneεulfonic anhydride, trifluromethaneεulfonic anhydride, p-toluenesulfonic anhydride, and trifluoroacetic anhydride; or an acid chloride such aε methaneεulfonyl chloride, p-toluenesulfonyl chloride or diphenyl chlorophosphate. Particulary preferred iε diphenyl chlorophoεphate.

In the formula (II-a), B iε a leaving group εuch as a trifluoroacetoxy group, a methanesulfonyloxy group, a trifluoromethane.sulfonyloxy group, a p- tolueneεulfonyloxy group or a diphenoxyphosphoryloxy group. Particularly preferred is a diphenoxyphos phoryloxy group.

The inert organic εolvent to be used for the reaction may, for example, be methylene chloride, chloroform, carbon tetrachloride, dichloroethane, trichloroethylene, diethyl ether, tetrahydrofuran, dioxane, benzene, toluene, chlorobenzene, acetone, ethyl acetate, acetonitrile, N,N-dimethylformamide, hexamethylphosphoric triamide or a mixture of εuch solvents. Particulary preferred is acetonitrile, benzene, toluene, a mixture of toluene and benzene, or a mixture of toluene and ethyl acetate.

The base to be used for the reaction may, for example, be trimethylamine, triethylamine, N,N- diisopropylethylamine, N-methylmorpholine, N- methylpyrrolidine, N-methylpiperidine, N,N- dimethylaniline, 1, 8-diazabicyclo[5, 4, 0]endec-7- ene(DBU), or 1, 5-diazabicyclo[4, 3, 0]-non-5-ene(DBN) , or pyridine 4-dimethylaminopyridine, picoline, lutidine, quinoline or isoquinoline. Particularly preferred is N,N-diisopropylethylamine and triethylamine.

For the reaction, from 1 to 3 mol, preferably from

1 to 1.5 mol, of the base and from 1 to 1.3 mol of the activating reagent are used per mol of the compound of formula (II) .

Though the reaction temperature is not critical, the reaction is conducted uεually within a temperature range of from -40° to 50°C, preferably -20° to 20°C, and uεually completed quantitatively in from 0.5 to 3 hourε.

After completion of the reaction, the reaction product iε treated in accordance with a conventional method to obtain the reactive derivative (Il-a) quantitatively.

The compound of formula (II-a) may be reacted with the compound of formula (III) with or without iεoiation. The reaction is conducted using the above-mentioned inert organic solvent and the base and from l to 2 mol, preferably from 1 to 1.5 mol, of the base and from 0.8 to 1.2 mol of the compound of formula (III) are used per mol of the compound of formula (Il-a).

The reaction iε conducted uεually within a temperature range of -40° to 50°C, preferably from -20° to 20°C, and usually completed quantitatively in from 0.5 to 10 hours.

Further, the compound of the formula (IV) can be prepared in one pot reaction from the compound of formula (II), namely, without isolating the reactive derivative of formula (Il-a). In the same reaction system, the compound of formula (IV) may be produced with high yield by reacting the compound of formula (II- a) with the compound of formula (III). Thus, from 2 to 5 mol, preferably from 2.5 to 3.8 mol of the baεe iε employed per mol of the compound of formula (II).

After completion of the reaction, method well known in the art can be used to obtain a crude product of formula (IV), which may be subjected to a reaction for removing a protecting group without purification. However, it iε preferred to purify the crude product (IV) by crystallization or by column chromatography on silica gel.

From the compound of formula (IV) thus obtained, a compound of formula (I) can be obtained, if necessary, by removing a protecting group for a hydroxyi group, an amino group or imino group, and a carboxyl group.

For the removal of the protecting groups, the method varies depending upon the type of the protecting groups. However, the removal can be conducted in accordance with methods known in the art, for example, by addition of a solvent for decomposition; by chemical reduction using a salt of an amine, a metal such as zinc amalgam, a chromic compound such as chromium (II) chloride, chromium (II) acetate together with an organic or inorganic acid such as acetic acid, propionic acid, hydrochloric acid, sulfuric acid; or by catalytic hydrogenation using a platinum or palladium compound. For example, in the formula (IV), the protecting group of the hydroxyi group, amino group or the imino group is an aralkyloxycarbonyl group such as a benzyloxycarbonyl group or a p-nitrobenzyloxycarbonyl group, and the protecting group for the carboxyl group is an aralkyl group such as a benzyl group, a p-nitroben∑yl group or a benzhydryl group. Such protecting groups can be removed by catalytic hydrogenation by means of a platinum catalyst εuch as platinum oxide, platinum wire or platinum black, or a palladium catalyst such as palladium black, palladium oxide, palladium carbon or palladium hydroxide-carbon (Pearlman's catalyst). When

the protecting group of the carboxyl group is an allyl group, isopropenyl, such a protecting group can be removed by catalytic hydrogenation uεing a palladium ligand complex catalyst εuch aε palladium-carbon, palladium black, palladium hydroxide-carbon, palladium

(II) chloride, tetrakiε(triphenylphoεphine)palladium

(0), biε(dibenzγlidenylacetone)-palladium (0), di(l,2- bis(diphenylphospino)ethane)palladium (0), tetrakis

(triphenylphophine)palladium (II) acetate, di (triphenyl phophine)platinum (II) chloride and the like.

The solvent useful for the catalytic hydrogenation includes, for example, water, methyl alcohol, ethyl alcohol, propyl alcohol, tetrahydrofuran, dioxane, acetonitrile, acetic acid and the above described organic solvent with water, a phosphate buffer(pH=6.5 to 7.0) or 3-(N-morpholino)propaneεulfonic acid(MOPS) solution (pH=6.5 to 7.2). The reaction can be completed in from 0.5 to 8 hours at a temperature within a range of from 0° to 40°C under a hydrogen gas stream of from 1 to 3 atm.

In formula (IV), when the hydroxy group, the amino group, the imino protecting group is an allyloxycarbonyl group, and the protecting group for the carboxyl group is an allyl group, such protecting group can be removed by reacting an organo-soluble palladium ligand complex catalyst in an inert organic solvent containing an allyl group capturing agent (J. Org. Chem. , vol 47, 587, 1982).

A solvent useful for the reaction includes, for example, acetone, diethyl ether, tetrahydrofuran, dioxane, ethyl acetate, acetonitrile, methylene chloride, chloroform and a solvent mixture thereof. The allyl group-capturing agent may be, for example, sodium

2-ethylhexanoate, potasεiu 2-ethylhexanoate, pyridine and piperidine.

The reaction iε conducted usually within a temperature range of from -10° to 50°C, preferably from 0° to 30 C° using from 0.01 to 0.5 mol of the palladium ligand complex catalyst and from 0.5 to 5 mol of the nucleophilic agent relative to 1 mol of the compound of the formula (IV), and the reaction is completed usually in from 0.5 to 5 hours.

After completion of the reactionε for removing the protecting group, the compound of formula (I) can be isolated by column chromatography on silica gel or adεorptive resin, such as Diaion HP-20, freeze drying or crystallization.

The compound of formula (II) as the starting material, can be obtained by the Salzmann method (J. Am. Chem. Soc. Vol 102, 6161-6163, 1980) in the case where R, is a hydrogen atom, and by the Shih method (Heterocycles, Vol. 21, 29-40, 1984 or EP 0272,455) in the case where R. is a methyl group.

The compound of formula (III) as the starting material, can be obtained by the following scheme I or Scheme II. In the case of using εcheme II, the εtarting material can be obtained by methods well known to these skilled in the art.

Compound (8) obtained by activation of the hydroxy group of the compound (3) in accordance with methods well known in the art iε reacted with potasεium thioacetate to convert to an acetylthio derivative (9), followed by alkali or acid hydrolysis to obtain a thiol derivative of formula (III) by Scheme I.

SCHEME I

692

18 wherein R ₃ , R ₄ , R ₅ , A and n are the same as defined abovo and u,. i :. α hydro.inn atom or n hydroxy protect I DM group and R ₎₀ is lower alkyl group and L is a methaneεulfonyloxy group, trifluoro acetoxy group, trifluoro methanesulfonyloxy group or p-toluene- εulfonyloxy group, and Ac is an acetyl group.

Compound (3) can be prepared in accordance with methods known in the art. Compound (6) can be prepared in accordance with the methods described in the reference examples described below.

SCHEME II

692

19

The alkene derivative (6) can be obtained by rnnct.1nu t tin compound (:.) or πohomn TI witli tlin compound (10), ylide, and the resulting compound (6) may be subjected to the same method as Scheme I to obtain the thiol derivatives of formula (III).

The compound (10) can be prepared by the following method.

HO(CH2) _n A -** ^• (CH ₂ ) _n A -^ Br(CH2) _n A (1 1 ) (12) (13)

BuLi Br ^"

PhX (CH2)„A -r- Pt _l3 ⁺ P(CH ₂ ) _n Λ (10) (14)

wherein R ₃ , R ₄ , R _s , R ₉ , A, L and n are the same as defined above. Compound (11) is a commercially available or prepared by methods known in the art. Abbreviation used are as follows:

CIS

I. Antibacterial Tests

The antibacterial activities of the compounds of

present invention were tested by an agar dilution method in accordance with the εtandard method of the Japan Chemotherapy Society [Chemotherapy, Vol. 29,76-79 (1981)] which is incorporated herein by reference. After 10 ml of Mueller Hinton Broth was poured into sterilized test tubes, one platinum loopful of each test micro organism was inoculated and incubated overnight at 37°C. Staphylococcus aureuε was cultured in Trypticase Soy Broth instead of Mueller Hinton Broth. The antibacterial agent solutions were prepared by disεolving 5 to 10 mg of each compound in εterilized diεtilled water to a final the concentration of l mg/ml, and by preparing a two-fold εerial dilution to concentration of 0.25 μg/ml. After one ml of each of the antibacterial εolutionε was poured into separate petri dish and sterilized, 9 ml of Mueller Hinton agar media which had been εterilized and cooled waε added, mixed well, and solidified to prepare the agar plate media the for Minimal Inhibitory Concentration (MIC;μg/ml) test. 0.11 ml of bacterial culture was poured into a sterilized test tube containing 10 ml of buffered εaline gelatin (BSG) εoiution and thoroughly mixed. The agar plate containing the antibacterial agent waε then inoculated with a bacterial εuεpension using a stamp, and cultured at 37°C for 18 hours. After obεerving the growth of bacteria, MIC waε determined to be the lowest drug concentration at which there is no growth. The results are shown in Table 1.

21

Table 1. Minimum Inhibitory Concentration (MIC, /_.g/ml

Test

Microorβani BΓΠ Ceftπzi i p I ipnnpm Mp.-r.jwr.pm nθ-Λ <l-2> <4 !_> <5 _. ^N <.(> .

S.aureus Smith 12.5 ≤0.025 0.1 ≤0.025 ≤.0.025 ≤0.025 ≤0.025 ≤0.0

S.aureus 503 25 ≤O.025 0.1 ≤0.025 ≤0.025 ≤0.025 ≤0.025 ≤0.0

S.pyogenes 308 A 0.39 ≤0.025 ≤0.025 ≤0.025 ≤0.025 ≤0.025 ≤0.025 ≤0.0

E. col i ATCC 9637 0.39 0.2 ≤0.025 0.1 ≤0.025 ≤0.025 0.05 ≤0.0

K.pneumoniae ATCC10031 0.1 0.1 ≤0.025 0.1 ≤O.025 ___0.025 0.05 0.0

E. cloacae P99 >100 0.78 0.1 0.78 0.2 0.2 0.2 0.3

E.aerogenes Bl >100 100 3.13 12.5 3.13 3.13 6.25 6.2

S.marcescens US12 MOO 50 6.25 25 3.13 3.13 6.25 12.5

P.mirabilis ATCC 14273 0.1 3.13 0.1 1.56 0.1 0.1 0.1 0.1

The compounds of the present invention have exelient antibacterial activity against various gram positive and gram negative bacteria, including beta- lactamase producing strains and imipenem reεiεtant εtrains. Example <l-2> was the most active of the compounds tested against Psudomonaε aeruginoεa εtrainε.

II. Porcine Renal dehydropeptidaεe-I εuceptiblity

300g of the swine kidney was homogenized and suspended in a Tris buffer (lOmM Tris, 50mM NaCl, O.OlmM ZnCl ₂ , pH 8.0), and 10% Triton X-100 was added and stirred at 4°C for 24 hours to extract DHP-I. A supernatant, prepared by ultracentrifugation of the above extract at 186,000 x g for 1 hour, was diafiltered to remove Triton X-100. The diafiltered solution was brought to 75% saturation of ammonium sulfate, the resultant precipitate was collected by centrifugation.

The above precipitate was dissolved in Tris buffer, and loaded on DEAE-Sepharoεe faεt flow, and anion exchange chromatography was carried out to give the swine DHP-I. The above DHP-I waε divided into lml portion at a concentration of l unit/ml and εtored at -70°C.

Glycyldehydrophenylalanine (GDP), imipenem, merope¬ nem and BO-A were employed as control compounds.

As shown in Table 2, the compounds of the present invention have higher stability to DHP-l than imipenem and meroDenem.

Table 2. Stability to renal dehydropeptidase-I from porcine

III. Pharmacokinetics in mice

A test compound waε disεolved in sterillized distilled water, and a dose of 20mg/kg of body weight waε inoculated under the εkin of ICR mice weighing 20 ± 2g.

10 μl of blood samples waε collected from the tail vein at preset sampling interval. Urine was collected for 18 hours from another group of mice (n=10) which were housed in metbolism cages. Bioassays were carried out to measure the concentration of a blood and urine by using Bacillus subtilis ATCC 6633 as a test bacterial strain.

Table 3. Pharmacokinetic parameter in mice following subcutaneouε injection of 20 mg/kg

Teεt Pharmacokinetic parameter Urinary Compound recovery

T 1/2 (hr) AUC (μg hr/ml) (0-18 hr,%)

Aε shown in Table 3, compound of the present invention, example 1-2, has longer half-life and higher area under the curve (AUC) than imipenem, meropenem and BO-A and has 1.5 to 3 times higher uninary recovery than the control compounds.

IV. Toxicity in mice

2000, 1500, and 1000 mg/kg of the compounds of present invention were injected into 20±2 g weight ICR mice via the tail vein. After 2 weeks, the mice were observed (n=3). In the case of compounds 1-2 and 5-2, central nervous system (CNS) side effects such aε death and jerkε were not obεerved. But in case of imipenem injection at a dosage of 1,500 mg/Kg, one of three of the mice died and all three of the mice showed jerks as soon as they were injected.

The compounds of the preεent invention have excellent antibacterial activitieε againεt variouε gram poεitive bacteria and gram negative bacteria including beta lactamaεe producing εtrains and imipenem resistant strains. Also, the compounds of the present invention

showed favorable pharmacokinetic properties, good physicochemical εtability and high water slubility. Therfore, the compounds of the preεent invention are uεeful aε antibacterial agents for treatment and prevention of human infectious diseaεeε caused such bacteria. Because of their broad antibacterial spectrum, the compounds of the present invention may be uεed in the form of additives for animal food, preserving agents, and other sterilization and disinfection agents for industrial use aε well as medical use.

The compound of the present invention may be used in the form of a drug formulation suitable for non-oral adminiεtration, oral adminiεtration, external administration; a liquid formulation such aε an injection solutions, syrups or emulsions; solid formulation such aε tabletε, capεuleε or granuleε; and external application formulations such as ointments or suppositories.

Dosages vary depending upon the condition of the patient, the weight, age, sex, type of formulation, and how the dose is to be administered. Usually, however, a preferred daily doεe of the active ingredient to an adult is from about 5 to 50 mg/kg, and a preferred daily dose to a child iε within a range of from about 5 to 25 mg/kg, which is preferably administered once a day or several times a day.

The compound of present invention has high stability to renal DHP-I, therefore, they may be administered by themselveε, and if necessary, may be administered in combination with a DHP-I inhibitory agent such as cilastatin.

The present invention will now be described in further detail with reference to Examples and Reference

Examples. However, it εhould be underεtood that the present invention is by no means restricted by such specific exampleε.

In the Examples and Reference Examples, for the thin layer chromatography, silica gel 60 F ₂₅₄ (Merck) was used as the thinlayer plate material, and an ultraviolet detector or ninhydrin or KMn0 ₄ color development method waε uεed as a detection means. For columns, silicagel 60 (Merck) waε uεed, and UV spectrophotometer DMS 100S (Varian) was used for detecting the UV absorbency. For high εpeed liquid chromatography measurement a M-352 (ACS) model was used. When the NMR spectrum was meaεured uεing dimethyl sulfoxide-d ₆ or chloroform-d, solution, tetramethylεilane (TMS) waε used as the internal standard material, and when measured uεing a deuterium oxide εolution, 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as the internal standad. The measurement was conducted by meanε of AC 200P (200 MHzrBruker) model εpectrometor. All δ valueε are represented by ppm.

The meaning of the abbreviation used in NMR measurement are as follows :

broad doublet

: deuterium oxide coupling constant hertz

REFERENCE EXAMPLE 1

(2S, 4S)-l-allyloxycarbonyl-2f (E)-3-methanesulfonyl amino-1-propenyl .-4-mercaptopyrroiidine

REFERENCE EXAMPLE 1-1

To a solution of 33 g (94.7 mmol) of ethyl tri(phenyiphosphoranylidene)acetate in 100 ml of methylene chloride waε added dropwise a εolution 15 g (47.7 mmol) of (2S, 4R)-l-allyloxycarbonyl-2-formγl-4-t- butyldimethylεilyloxypyrrolidine obtained by known processes in 100 ml of methylene chloride at room temperature. The mixture waε εtirred for 3 hourε, and concentrated under reduced preεεure. 500 ml of a mixture of ethyl acetate and hexane (1:4 v/v) waε added, stirred for 10 minutes, filtered and concentrated under reduced preεsure. The residue waε purified by column chromato graphy on εilica gel (eluted with ethyl acetate:hexane = 1:8) to give 17.15 g (95% yield) of ethyl 3- [ (2S, 4R)-l-allyloxycarbonyl-4-t- butyldimethylεilyloxypyrrolidin-2-yl ]acrylate.

NMR (CDC1 ₃ ) δ

0.01(ε,6H), 0.82(S,9H), 1.20-1.27 (t, 3H) , 1.80-

1.86(m,lH), 2.03-2.07(m,lH), 3.32-3.48(m, 2H) , 4.08-

4.19(q,2H), 4.29-4.36(m, 1H) , 4.51-4.54(m, 3H) , 5.14- 5.29(m,2H), 5.82-5.89(m,2H), 6.73-6.84 (dd, 1H)

REFERENCE EXAMPLE 1-2

To a solution of 11 g (29.06 mmol) of ethyl 3-

[ (2S, 4R)-l-allγloxγcarbonγl-4-t-butyldimethylsilγloxγ pyrrolidin-2-yl]acrylate obtained in REFERENCE EXAMPLE

1-1 in 100 ml of tetrahydrofuran, 84.28 ml (84.27 mmol)

of 1.0 M diisobutylaluminum hydride solution in hexane waε added dropwise at -70°C. The mixture was stirred for 30 minuites at -70°C. Then, the temperature was raised to room temperature. To the reaction solution, 200 ml of ethyl acetate and 20 ml of methanol was added, and the mixture was stirred for another 1 hour. The precipitate which formed waε collected by filtration and the filtrate waε concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate:hexane = 4:1) to give 6.64 g (69 % yield) of (2S,4R)-l-allyloxycarbonyl- 2-[ (E)-3-hydroxy-l-propenyl]-4-t-butyldimethylsilyloxy pyrrolidine.

NMR (CDC1 ₃ ) δ:

0.03(ε,6H), 0.84(s,9H), 1.67-1.71(m, 2H), 3.41- 3.71(m,3H), 4.10-4.12(d,lH), 4.32-4.36(m, 2H), 4.52- 4.58(m,3H), 5.21-5.30(m,2H), 5.62-6.61(m, 2H)

REFERENCE EXAMPLE 1-3

After 6.64 g (19.74 mmol) of (2S, 4R)-l-allyl oxycarbonyl-2-[ (E)-hydroxyl-l-propenyl ]-4-t-butyl dimethylεilyloxypyrrolidine obtained in REFERENCE EXAMPLE 1-2 was dissolved in 65 ml of tetrahydrofuran, 6.21 g (23.69 mmol) of triphenylphosphine and 3.48 g (23.69 mmol) of phthalimide were added to the reaction mixture and cooled in an ice bath. Then, to the mixture, 3.73 ml (23.69 mmol) of diethyi azodicarboxy late was added dropwise. The mixture was stirred for 30 minuteε, and then concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate. The mixture was washed with distilled water and then a saturated sodium chloride agueous εolution. The organic layer waε dried over anhydrouε εodium εulfate, and

concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate.-hexane = 1:8) to give 8.8 g (95 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3- phthalimido-1-propenyl ]-4-t-butyldimethylsilyloxy pyrrolidine.

NMR (CDC1 ₃ ) <5:

0.07(ε,6H), 0.81(s,9H), 1.70-1.82(m, 1H) , 1.96-

2.01(m,lH), 3.41-3.47(m,2H), 4.22-4.33(m,3H) , 4.46-

4.53(m,4H), 4.81-5.28(m, 2H) , 5.62-5.80(m,2H) , 7.68- 7.73(m,2H), 7.77-7.84(m,2H)

REFERENCE EXAMPLE 1-4

To a solution of 8.8 g (18.70 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[(E)-3-phthalimido-l-propenyl]-4-t- butyldimethylεilyloxypyrroiidine prepared in REFERENCE EXAMPLE 1-3 in 90 ml of ethanol, 2.09 ml (43.08 mmol) of hydrazine hydrate waε added. The mixture was stirred for 12 hours, filtered and the filtrate waε concentrated under reduced pressure. The residue waε diluted with ethyl acetate. The mixture was washed in sequence with a 3N-ammonia solution, distilled water and saturated sodium chloride aqueous solution, dried over anhydrous sodium εulfate, and concentrated under reduced preεεure. The residue was diluted with 100ml of dichloromethane. To the mixture, 3.13 ml (22.44 mmol) of triethylamine and 1.8 ml (22.31 mmol) of methanesulfonyl chloride were added and cooled on an ice. The reaction mixture waε εtirred for 30 minuteε, waεhed in εequence with 1N-HC1, εaturated sodium carbonate solution, distilled water, and saturated sodium chloride aqueouε solution, dried over anhydrous sodium sulfate, and then concentrated under reduced presεure. The reεidue was purified by

column chromatography on silica gel (eluted with ethyl acetate:hexane = 1:2) to give 6.26g (80 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3-methanesulfonyl-l- propenyl]-4-t-butyldimethylεilyloxypyrrolidine.

NMR (CDC1 ₃ ) δ:

0.03(ε,6H), 0.84(S,9H), 1.66-1.78(m, 1H), 1.98- 2.07(m,lH), 2.93(S,3H), 3.44~3.46(d, 2H) , 3.70-3.74 (m,2H), 4.30-4.44(m,2H), 4.52-4.56(m, 3H) , 5.14-5.30 (m,2H), 5.62(bε,2H), 5.81-5.98(m, 1H)

REFERENCE EXAMPLE 1-5

To a εolution of 6.26 g (14.95 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[(E)-3-methanesulfonyl-1-propenyl]-4- t-butyldimethylεilyloxypyrrolidine prepared in REFERENCE EXAMPLE 1-4 in 80 ml of tetrahydrofuran, 5.8 g (22.18 mmol) of tetrabutylammonium fluoride hydrate waε added. The mixture waε εtirred for 1.5 hours. After concentration under reduced presεure, the residue was dissolved in chloroform, washed with distilled water and then saturated εodium chloride aqueous εolution and dried over anhydrouε magneεium sulfate, and concentrated under reduced pressure. The residue was diεεolved in 80 ml of dichloromethane. To the mixture, 2.5 ml (17.92 mmol) of triethylamine and 1.45 ml (17.92 mmol) of methaneεulfonyl chloride were added and cooled on an ice. The reaction mixture waε εtirred for 30 minuteε, waεhed with 1N-HC1, distilled water, saturated sodium carbonate solution, and distilled water, dried over anhydrous magneεium εulfate and concentrated under reduced pressure to give 4.3 g (75 % yield) of (2S,4R)- l-allyloxycarbonyl-2-[(E)-3-methaneεulfonylamino-l- propenyl]-4-methaneεulfonyloxypyrrolidine.

NMR (CDC1 δ

1.75(m,lH), 2.01(m,lH), 2.93(s,3H), 3.02(s,3H), 3.62-3.74(m, 3H) , 3.82-3.94 (m, 1H) , 4.48-4.56 (m, 3H) , 5.16- 5.31(m,3H), 5.62-5.64(m, 2H) , 5.81-5.97 (m, 1H)

REFERENCE EXAMPLE 1-6

To a solution of 4.3 g (11.24 mmol) of (2S,4R)-l- allyloxycarbonyl-2- [ (E) -3-methaneεulfonylamino-1- propenyl ]-4-methaneεulfonyloxypyrrolidine prepared in REFERENCE EXAMPLE 1-5 in 150 ml of anhydrous acetonitrile, 1.9 g (16.6 mmol) of potassium thioacetate was added. The mixture was heated to reflux for 4 hours. After cooling in an ice bath, insoluble material waε removed by filtration. The filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate. The residue was waεhed with diεtilled water, dried over anhydrous sodium εulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate: hexane = 1:1) to give 3.26 g (80 % yield) of ( 2S, 4S )-l-allyloxycarbonyl-2-[ (E)-3- methanesul fonyl amino-1-propenyl ] - 4 - thioacetylpyrroiidine.

NMR (CDC1-) δ:

1.62 -1.82(m,lH), 2.31(m,lH), 2.52-2.61(m, 1H) , 2.94(ε,3H), 3.23-3.31(m, 1H), 3.71-3.74 (m, 2H) , 3.88-

4.04(m,2H), 4.36-4.40(m, 1H), 4.51-4.55(m, 2H) , 5.16-

5.31(m,2H), 5.64-5.67(bd,2H), 5.78-5.98 (m, 1H)

REFERENCE EXAMPLE 1-7

To a εolution of 3.26 g (8.99 mmol) of (2S,4S)-1-

allyloxycarbonyl-2-[ (E)-3-methaneεulfonylamino-1- propenyl]-4-thioacetylpyrrolidine prepared in REFERENCE EXAMPLE 1-6 in 50 ml of methanoi, 14.25 ml (28.5 mmol) of 2 N-NaOH waε added under ice cooling. The mixture waε εtirred for 3 minutes. It was neutralized with aqueous citric acid and concentrated under reduced pressure. The reεidue waε purified by column chromatography on silica gel (eluted with ethyl acetate: hexane = 1:1) to give 2.45 g (85 % yield) of (2S,4S)-1- allyloxycarbonyl-2-[ (E)-3-methaneεulfonylamino-1- propenyl]-4-mercaptopyrrolidine.

NMR (CDC1-) δ:

1.60-1.78(m, 1H), 1.69-1.73(d, 1H), 2.54-2.61(m, 1H),

2.94(S,3H), 3.13-3.31(m, 1H), 3.70-3.76(m,2H), 4.01-4.09 (m,lH), 4.28-4.36(m,lH), 4.50-4.55(m, 2H), 5.16-5.22 (m,2H), 5.61-5.71(m,2H), 5.81-6.00(m,1H)

REFERENCE EXAMPLE 2

(2S, 4S)-l-allyloxycarbonyl-2-T (E)-3-methaneεulfonyl amino-l-methylpropenyll-4-mercaptopyrrolidine

REFERENCE EXAMPLE 2-1

To a εolution of 1.0 g (25.7 mmol) of sodium hydride (60 %) in 106 ml of tetrahydrofuran, 1.13 g (25.7 mmol) of triethyl phosphonoacetate waε added dropwiεe at 0 °C. 7.0 g (21.4 mmol) of (2S,4R)-1- allyloxycarbonyl-2-methylcarbonyl-4-t-butyldimethylεilyl oxypyrrolidine obtained by known proceεεes, was added dropwise at the same temperature. The mixture was warmed to room temperature and stirred for 15 hours at the same temperature. Ethyl acetate was added in an excess amount. The mixture waε waεhed with aqueous

εodium chloride, dried over anhydrous magnesium εulfate, and then concentrated under reduced preεεure. The reεidue waε purified by column chromatography on εilica gel (eluted with ethyl acetate:hexane = 1:10) to obtain 3.3 g (95 % yield) of (2S, 4R)-l-allyloxycarbonyl- 2-[ (E)-3-ethoxycarbonyl-l-methylpropenyl ]-4-t-butyl dimethylεilyloxypyrrolidine, together with the recovery of 4.13g of starting material.

NMR (CDCl _j ) δ:

0.03(s,6H), 0.83(s,9H), 1.24(t,3H), 1.80(m,lH), 2.05(ε,3H), 3.40-3.70(m, 2H) , 4.15(q,2H), 4.31(m,lH), 4.58(m,2H), 5.20(m,2H), 5.73(ε,lH), 5.85(m,lH)

REFERENCE EXAMPLE 2-2

To a solution of 3.3 g (8.3 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[ (E ) -3-ethoxycarbonyl-l-methyl propenyl ]-4-t-butyldimethylsilyloxypyrrolidine obtained in reference example 2-1 in 42 ml of tetrahydrofuran, 16 ml (24.1 mmol) of diiεobutylaluminum hydride (1.5 M hexane εolution) waε added dropwise at -78 °C. The mixture waε εtirred for 1.5 hourε at the εame temperature. The reaction mixture waε warmed to 0 °C.

10 ml of methanol was added dropwise to the mixture.

The mixture was εtirred for another about 30 minuteε.

Ethyl acetate was added and insoluble material was removed by filtration. Then the mixture waε concentrated under reduced pressure. The reεidue was refined by column chromatography on εilica gel (eluted with ethyl acetate:hexane = 1:2) to obtain 1.76 g (60 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3-hydroxy-l- methyl propenyl ]-4-t-butyldimethylεilyloxypyrrolidine.

NMR (CDCI ₃ )

0.03(ε,6H), 0.84(Ξ,9H), 1.60(s,3H), 1.79(m,lH), 2.00(m,lH), 3.50(m,2H), 4.15(d,2H), 4.30(m,2H),

4.55(m,2H), 5.20(m,2H), 5.22(t,lH), 5.90(m,lH)

REFERENCE EXAMPLE 2-3

To a εolution of 1.76 g (5.0 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[(E)-3-hydroxy-l-methylpropenyl]-4-t- butyldimethylsilyloxypyrrolidine obtained in REFERENCE EXAMPLE 2-2 in 25 ml of tetrahydrofuran, 1.9 g (7.4 mmol) of triphenylphosphine and 1.3 g (8.7 mmol) of phthalimide were added at 0 °C and 1.2 ml (7.4 mmol) diethyi azodicarboxylate was added dropwise. The mixture was εtirred at the εame temperature and then concentrated under reduced preεεure. The reεidue obtained therein was refined by column chromatography on silica gel (eluted with ethyl acetate:hexane = 1:5) to obtain 2.37 g (98 % yield) of (2S,4R)-1- allyloxycarbonyl-2-[(E)-3-phthalimido-l-methylpropenyl]- 4-t-butyldimethylsilyloxy pyrrolidine.

NMR (CDC1 ₃ ) δ :

0.01(s,6H), 0.81(s,9H), 1.73(ε,3H), 1.97(m, 1H), 3.48(m, 2H) , 4.28(m, 4H) , 4.50(m,2H), 5.80(m,lH), 7.67(m,2H), 7.80(m,2H)

REFERENCE EXAMPLE 2-4

To a εolution of 2.37 g (4.9 mmol) of (2S,4R)-l- allyloxycarbonyl-2-[(E)-3-phthalimido-l-methylpropenγl]- 4-t-butyldimethylsilyloxypyrrolidine obtained in REFERENCE EXAMPLE 2-3 in 40 ml of ethanol, 0.06 ml of hydrazine hydrate waε added. The mixture waε stirred for 13 hours at room temperature. The insoluble material was removed by filtration and the filtrate waε

concentrated under reduced presεure. The reεidue waε dissolved in 20 ml of dichloromethane. To the mixture were added 0.4 ml (5.2 mmol) of methanesulfonyl chloride and 0.72 ml (5.2 mmol) of triethylamine at 0 °C. The mixture waε then stirred for 30 minites at the εame temperature, and waεhed with water and then aqueouε sodium chloride, and dried over anhydrous magnesium sulfate. The organic layer waε then concentrated under reduced preεsure. The residue was refined by column chromatography on silica gel (eluted with ethyl acetate:hexane = 1:1.5) to obtain 1.4 g (66 % yield) of

( 2S , 4R ) -l-allyloxycarbonyl-2- [ ( E ) - 3 - methanesulfonylamino-l-methylpropenyl]-4-t-butyldimethyl silyloxypyrrolidine.

NMR (CDC1,) δ

0.03(s,6H), 0.83(s,9H), 1.59(ε,3H), 1.79(m,lH), 2.00(m,lH), 2.93(m,3H), 3.50(m,2H), 3.78(t,2H), 4.35(m,2H), 4.51(m,2H), 5.10-5.41(m, 3H) , 5.88(m,lH)

REFERENCE EXAMPLE 2-5

To a εolution of 1.4 g (3.2 mmol) of (2S,4R)-l- allyloxycarbonyl-2-[ (E)-3-methaneεulfonyla ino-1- methylpropenyl]-4-t-butyldimethylεilyloxypyrrolidine in

15 ml of methanol, 0.9 ml of 6N-HC1 was added. The mixture was stirred for 1.5 hours at room temperature.

The mixture was then neutralized with 2N-NaOH and concentrated under reduced pressure. The residue was extracted with ethyl acetate. The organic layer was washed with water and then aqueous εodium chloride and dried over anhydrouε magnesium εulfate and concentrated under reduced preεεure. The reεidue was dissolved in 13 ml of dichloromethane and 0.24 ml (3.1 mmol) of methane sulfonyl chloride and 0.44 ml (3.1 mmol) of triethyl

amine were added to the mixture and the mixture εtirred for 30 minuteε at 0 °C. The reaction mixture waε waεhed with water and then aqueouε εodium chloride, dried over anhydrouε magnesium sulfate, and concentrated under reduced -preεεure to obtain 0.96 g (75 % yield) of

( 2 S , 4R ) -l -a l lyl oxycarbonyl - 2 - [ ( E ) - 3 - me thane εulfonyl amino-1-methyl propenyl ] -4- methaneεulf onyloxypyrrolidine .

NMR (CDC1 ₃ ) δ :

1.61(S,3H), 2.00(m,lH), 2.42(m,lH), 2.93(ε,3H), 3.03(ε,3H), 3.79(t.2H), 4.50(m,3H), 5.40(t,lH),

5.88(m,lH)

REFERENCE EXAMPLE 2-6

To a solution of 0.96 g (2.4 mmol) of (2S,4R)-l- allyloxycarbonyl-2-[ (E)-3-methaneεulfonylamino-1- methylpropenyl]-4-methaneεulfonyloxypyrrolidine obtained in REFERENCE EXAMPLE 2-5 in 10 ml Of DMF and 10 ml Of toluene, 0.55 g (4.8 mmol) of potassium thioacetate were added. The mixture was stirred for 1.5 hours at 90 °C. 300 ml of ethyl acetate waε added to the mixture. The mixture waε washed with water and then an aqueous εodium chloride, dried over anhydrouε magneεium sulfate, and concentrated under reduced preεεure. The reεidue waε diεεoived in 15 ml of methanol, and 2 ml (4 mmol) of 2N- NaOH waε added. The mixture waε stirred for 10 minutes, and then neutralized with 1N-HC1 and concentrated under reduced presεure. The residue was extracted with ethyl acetate. The organic layer was waεhed with water and an aqueouε εodium chloride, dried over anhydrouε magnesium sulfate, and concentrated under reduced pressure. The reεidue waε refined by column chromatography on εilica gel (eluted with ethyl acetate:hexane = 1:1) to obtain

0.33 g (41 % yield) of (2S, 4S)-l-allyloxycarbonyl-2- [ (E) -3-methaneεulf onyl ami no- 1 -methyl propenyl ]-4- mercaptopyrroiidine .

NMR (CDC1 ₃ ) δ :

1.58(ε,3H), 1.69(d,lH), 2.54(m,lH), 2.94(ε,3H), 3.20(m,2H),

3.80(t.2H), 4.58(m,2H), 5.40(t,lH), 5.90(m,lH)

REFERENCE EXAMPLE 3

( 2S_ 4S)-l-allyloxycarbonyl-2-r (E)-3-fluoro-1- propenyl1-4-mercaptopyrrolidine

REFERENCE EXAMPLE 3-1

To a εolution of 1.03 g (3.06 mmol) of (2S,4R)-1- allγloxycarbonyl-2-[ (E)-3-hydroxγ-l-propenyl ]-4-t- butyldimethylεilyloxypyrrolidine obtained in REFERENCE

EXAMPLE 1-2 in 30 ml of dichloromethane, 0.5 ml (3.78 mmol) of diethylaminoεulfur trifluoride (DAST) was added dropwise at 0 °C. The mixture was εtirred for 30 minutes under a nitrogen stream at the same temperature.

5 ml of saturated εodium carbonate aqueouε εoltuion waε added to the mixture. The mixture was stirred for 10 minuteε, and then extracted with chloroform. The organic layer was concentrated under reduced pressure, and refined by column chromatography on εilica gel (eluted with ethyl acetate:hexane = 1:5) to obtain 0.35 g (34 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3-fluoro-1- propenyl]-4-t-butyldimethylεilyloxypyrrolidine.

NMR (CDC1 δ :

0.04(ε,6H), 0.84(ε, 9H), 1.85(m,lH), 2.02(m,lH), 3.47(d,m,2H), 4.34(t,lH), 4.53(m,lH), 4.57(m,2H), 4.70(bε, 1H), 4.94(bd,lH), 5.15(d,lH), 5.21(d,lH), 5.74(bε, 2H), 5.85(m,lH)

REFERENCE EXAMPLE 3-2

0.31 g (0.92 mmol) of (2S, 4R)-l-allyloxγcarbonyl-2- [ (E)-3-fluoro-l-propenyl]-4-t-butyldimethylsilyloxy- pyrrolidine obtained in REFERENCE EXAMPLE 3-1 was treated by the εame proceεε aε that of REFERENCE EXAMPLE 2-5 to obtain a deεired material, 0.26 g (94 % yield) of (2S,4R)-l-allyloxycarbonyl-2-[ (E)-3-fluoro-l-propenyl]- 4-methaneεulfonyloxypyrrolidine.

NMR (CDC1 δ

2.05(m,lH), 2.50(bε,lH), 3.03(ε,3H), 3.63(dd,lH), 3.95(bd, 1H), 4.57(m,3H), 4.71(d,lH), 4.95(d,lH), 5.22(m,3H), 5.78(bε,2H), 5.82(m,lH)

REFERENCE EXAMPLE 3-3

0.24 g (0.79 mmol) of (2S, 4R)-l-allyloxycarbonyl-2- [ (E)-3-fluoro-l-propenyl ]-4-methaneεulfonyloxy pyrrolidine obtained in REFERENCE EXAMPLE 3-2 waε treated by the same process aε that of reference example 1-6 to obtain a desired material, 0.32 g (100 % yield) of ( 2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3-f luoro-1- propenyl ]-4-acetylthiopyrrolidine.

NMR (CDC1 ₃ ) δ :

1.76(m-lH), 2.31(S,3H), 2.57(m,lH), 3.96(m,2H), 4.42(m,lH), 4.55(m,3H), 4.71(d,lH), 4.95(d,lH),

5.19(d,lH), 5.23(d,lH), 5.77(bS,2H), 5.82 (ill, 1H)

REFERENCE EXAMPLE 3-4

0.31 g (1.1 mmol) of (2S,4S)-l-allyloxycarbonyl-2- [ (E)-3-fluoro-l-propenyl ]-4-acetylthiopyrrolidine obtained- in REFERENCE EXAMPLE 3-3 was treated by the εame process aε that of REFERENCE EXAMPLE 1-7, and the crude product obtained aε described above was refined by column chromatography on silica gel (eluted with chloroform:acetone = 8:1) to give 0.12 g (63 % yield) of (2S,4S)-l-allyloxycarbonyl-2-[ (E)-3-fluoro-l-propenyl]- 4-mercaptopyrrolidine.

NMR (CDC1 ₃ ) δ :

1.70(m-lH), 2.60(m,lH), 4.04(bS,2H), 4.38(m,lH),

4.56(m,3H), 4.71(d,lH), 4.95(d,lH), 5.17(d,lH), 5.26(d,lH), 5.80(bs,2H), 5.82(m,lH)

REFERENCE EXAMPLE 4

(2S, 4S)-l-allyloxycarbonyl-2-[ (Z)-3-methanesul fonylamino-1-propenyl1-4-mercaptopyrroiidine

REFERENCE EXAMPLE 4-1

41 g (130.8 mmol) of (2S,4R)-l-allyloxycarbonyl-2- formyl-4-t-butyldimethylεilyloxypyrroiidine obtained by

known proceεε waε diεεolved in 400 ml of methanoi and cooled to -20 °C. 23.9 g (69.1 mmol) of (carbetoxy methylene)triphenylphoεphorane was added dropwise to the mixture. The reaction mixture was stirred overnight at -20°C, and concentrated under reduced pressure. The residue was dissolved in the mixture εolution (1:4 v/v) of ethyl acetate and hexane, and εtirred for 10 minuteε. The inεoluble material waε removed by filtration and the

filtrate waε concentrated under reduced preεεure. The reεidue waε refined by column chromatography on εilica gel (eluted with ethyl acetate:hexane = 1:8) to give 18.lg (72 % yield) of ethyl 3-[ (2S, 4R)-1- allyloxycarbonyl-4-t-butyldimethylεilyloxypyrrolidine-2- yl] acrylate.

NMR(CDC1 ₃ ) δ :

0.05(S,6H), 1.90(S,9H), 1.30(t,3H), 1.80(m,lH),

2.10(m,lH), 3.50(m,2H), 4.15(q,2H), 4.35(m,lH), 4.59(m,2H), 5.20(m,2H), 5.43(m, 1HX2/3 ) , 5.84(m, 1HX7/3) , 6.15(m,lHx2/3), 6.80(dd,lHxl/3)

REFERENCE EXAMPLE 4-2

18 g (46.9 mmol) of ethyl 3-[ (2S,4R)-l-allyloxycar bonyl-4-t-butyldimethylεilyloxypyrrolidin-2-yl] acrylate obtained in REFERENCE EXAMPLE 4-1 was disεolved in 250 ml of anhydrouε tetrahydrofuran, and cooled to -78 °C uεing a dry ice -acetone bath. To the reaction mixture, 96.5 ml (144.8 mmol) of 1.5 M diiεobutylaluminum hydride was added dropwise and the mixture was stirred for 2 hours at the same temperature and warmed to room temperature. 100 ml of methanoi was added and the mixture waε stirred for half an hour at the εame temperature. The solid material was removed by using celite. The filtrate waε concentrated under reduced preεεure, and the reεidue waε refined by column chromatography on silica gel (eluted with ethyl acetate: hexane = 4:1) to give 6 g (60 % yield) of (2S,4R)-1- allyloxycarbonyl-2-[ (Z)-3-hydroxy-l-propenyl ]-4-t- butyldimethyl silyloxypyrrolidine and 5 g (30 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3-hγdroxy-l- propenyl ]-4-t-butyldimethylεilyloxypyrrolidine, respectively.

2

41

(Z) form

NMR(CDC1 ₃ ) δ

0.05(S,6H), 0.90(ε,9H), 1.70(m,lH), 2.05(m,lH),

3.50(m,2H), 3.90(m,lH), 4.30-4.60(m,5H), 5.90(m,lH), 5.30(m,3H), 5.90(m,lH)

(E) form

NMR(CDC1 δ

0.05(ε,6H), 0.95(S,9H), 1.80(m,lH), 2.01(m,lH), 3.50(m,2H), 4.10(d,2H), 4.30-4.60(m, 4H) , 5.20(m,2H), 5.50-5.95(m,3H)

REFERENCE EXAMPLE 4-3

5.03 g (14.73 mmol) of (2S, 4R)-l-allyloxycarbonyl - 2-[ (Z)-3-hydroxy-l-propenyl]-4-t-butyldimethylsilyloxy pyrrolidine obtained in REFERENCE EXAMPLE 4-2 was dissolved in 150 ml of tetrahydrofuran, and cooled to 0 °C. 5.8 g (22.1 mmol) of triphenylphosphine and 3.8 g (25.8 mmol) of phthalimide were added to the mixture. The reaction mixture was stirred for 10 minutes. Then, to the mixture was added 3.49 g (22.2 mmol) of diethyi azodicarboxylate. The mixture was stirred for another 2.5 hours at 0 °C and concentrated under reduced presεure. To the reεidue, ethyl acetate and water were added and the mixture was extracted with ethyl acetate. The organic layer was washed with water and then saturated sodium chloride aqueous εolution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was refined by column chromatography on silica gel (eluted with ethyl acetate.-hexane = 1:7) to give 4.71 g (70 % yield) of

(2S, 4R)-l-allyloxycarbonyl-2-[ ( Z)-3-phthalimido-l- propenyl]-4-t-butyldimethylsilyloxy pyrrolidine.

0.05(ε,6H), 0.90(ε,9H), 1.80(m,lH), 2.10(m,lH), 3.50(m,2H), 4.30-4.63(m, 5H) , 4.90-5.50 (m, 5H) , 5.90(m,lH), 7.60-7.85(m,4H)

REFERENCE EXAMPLE 4-4

To a εolution of 4.7 g (10.3 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[ (Z)-3-phthalimido-l-propenyl]-4-t- butyldimethylεilyloxypyrrolidine obtained in REFERENCE EXAMPLE 4-3 in 250 ml of anhydrσuε ethanol, 1.6 ml (47.4 mmol) of hydrazine hydrate waε added. The mixture waε εtirred overnight at room temperature. The εolid material waε removed by filtration and the filtrate was concentrated under reduced pressure. To the residue, ethyl acetate and water were added, and the organic layer was waεhed in εequence with 3 N-ammonia εolution, εaturated εodium chloride aqueous solution, dried over the anhydrouε εodium εulfate and concentrated under reduced preεεure. The residue waε diεεolved in 100 ml of methylene chloride and cooled to 0 °C. To the mixture were added 7.2 ml (51.5 mmol) of triethylamine and 2.8 ml (36.1 mmol) of methanesulfonyl chloride, and the mixture was εtirred at 0 °C for half an hour. 100 ml of methylene chloride and water were added to the reaction mixture, and the organic layer waε washed with a saturated citric acid εolution and, then εaturated εodium chloride aqueouε εolution, dried over the anhydrous sodium εulfate and concentrated under reduced preεεure. The reεidue waε refined by column chromatography on εilica gel (eluted with ethyl acetate: hexane=l: 1.5) to give 2.21 g (51.3 % yield) of (2S,4R)-

l-allyloxycarbonyl-2-[ ( Z )-3-methaneεulf onylamino-1- propenyl ] -4-t-buryldιmethylειlyioxypyrrolidine .

NMR(CDC1 3' δ

0.05(S,6H), 0.90(s,9H), 1.70(m,lH), 2.05(m,lH), 2.93(ε,3H), 3.40(m,2H), 3.60(m,lH), 4.01(m,lH), 4.30- 4.75(m,3H), 5.10-5.40(m,3H), 5.61-5.98 (m, 3H )

REFERENCE EXAMPLE 4-5

To a solution of 2.2 g (5.26 mmol) of (2S,4R)-l- allyloxycarbonyl-2-[ ( Z )-3-methanesulfonylamιno-1- propenyl]-4-t-butyldimethylεilyloxypyrrolidine obtained in REFERENCE EXAMPLE 4-4 in 150 ml of tetrahydrofuran, 6.6 ml (6.6 mmol) of lM-tetrabutylammonium fluoride waε added at room temperature. The mixture waε εtirred for half an hour and concentrated under reduced preεεure. To the mixture was added ethyl acetate and water and then organic layer was washed with water and then saturated sodium chloride aqueouε solution, dried over the anhydrouε sodium sulfate, and concentrated under reduced pressure. The residue waε diεεolved in 100 ml of methylene chloride and cooled to 0 °C. To the reaction mixture were added 2.8 ml (38.1 mmol) of triethylamine and 1.6 ml (20.7 mmol) of methanesulfonyl chloride and the mixture was stirred at 0 °C for half an hour. Then 100 ml of methylene chloride and water were added to the reaction solution and the organic layer washed with water and then saturated citric acid aqueous εolution, dried over the anhydrouε sodium sulfate and concentrated under reduced pressure. The residue was refined by column chromatography on silica gel (eluted with ethyl acetate:hexane = 30:1) to give 1.3 g (64.6 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (Z)-3-methanesul fonylamιno-1-propenyl]-4-methaneεulfonyloxypyrrolidine.

NMR(CDC1 ₃ ) δ :

1.95(m,lH), 2.50(m,lH), 2.93(s,3H), 3.03(s,3H), 3.62(m,2H), 4.00(m,2H), 4.44(d,2H), 4.80(q,lH), 5.05- 5.41(m,4H), 5.70-5.95(m, 2H)

REFERENCE EXAMPLE 4-6

To a solution of 1.3 g (3.4 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[ (Z )-3-methanesulfonylamino-1- propenyl]-4-methaneεulfonyloxypyrrolidine obtained in

REFERENCE EXAMPLE 4-5 in 100 ml Of acetonitrile, 1.4 g

(12.26 mmol) of potaεεium thioacetate waε added. The reaction εolution waε refluxed for 4 hours, cooled and concentrated under reduced preεεure. Ethyl acetate and water were added to the reεidue. The organic layer was washed with water and then saturated sodium chloride aqueous solution, dried over the anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was refined by chromatography on silica gel

(eluted with ethyl acetate:hexane = 2:1) to give 730 mg

(60 % yield) of (2S,4S)-l-allyloxγcarbonyl-2-[(Z)-3- methanesul f onyiamino-l-propenyl ] -4- acetylthiopyrrolidine .

NMR(CDC1 δ

1.60(m,lH), 2.30(S,3H), 2.60(m,lH), 2.95(S,3H) 3.10(m,lH), 3.95(m,3H), 4.60(m,3H), 5.10-5.50 (m, 3H) , 5.62-5.95(m,3H)

REFERENCE EXAMPLE 4 -7

730 mg (2.01 mmol) of (2S, 4S)-l-allyloxycarbonyl-2- [ ( Z )-3-methanesulf onylamino-1-propenyl ]-4-acetylthio pyrrolidine obtained in REFERENCE EXAMPLE 4-6 was

dissolved in 60 ml of methaneol and cooled to 0 °C. 2.3 ml of 2 N-sodium hydroxide was added to the mixture and the reaction solution was stirred for 3 minutes. Then, the mixture was immediately neutralized with saturated citric acid aqueouε εolution (pH = 3-4). The reaction mixture waε concentrated under reduced preεεure, and the reεidue waε extracted with ethyl acetate. The organic layer was washed with water and the saturated sodium chloride aqueous εolution, dried over anhydrouε εodium εulfate, and concentrated under reduced preεεure. The reεidue waε refined by column chromatography on εilica gel (eluted with ethyl acetate:hexane=2:1) to give 610 mg (94.7 % yield) of (2S,4S)-l-allyloxycarbonyl-2-[ (Z)- 3-methaneεulfonylamino-l-propenyl ] - 4 - mercaptopyrrolidine.

NMR(CDC1 ₃ ) δ :

1.63(m,lH), 1.70(d,lH), 2.60(m,lH), 2.98(s,3H) 3.23(m,lH), 3.60-4.05(m, 3H), 5.54(m,3H), 5.13-5.58 (m,3H), 5.62-5.95(m, 3H)

REFERENCE EXAMPLE 5

(2S.4S)-l-allyloxycarbonyl-2-r(E)-3-methanesulfonyl amino-2-methyl-l-proρenyl1-4-mercaptopyrrolidine

REFERENCE EXAMPLE 5-1

11.5 g (36.7 mmol) of (2S,4R)-l-allyloxycarbonyl-2- formyl-4-t-butyldimethylεilyloxypyrrolidine obtained by known processeε and 26.5 g (73.4 mmol) of (carbetoxy ethylidene)triphenylphosphorane were dissolved in 250 ml of methylene chloride at room temperature and stirred for 20 hours. The mixture was concentrated under reduced presεure. The εolvent mixture (1:4 v/v) of

ethyl acetate and hexane waε added to the mixture and the εolid was removed by filtration. The filtrate waε concentrated under reduced preεεure and the residue waε εubject to column chromatography on εilica gel (eluted with ethyl acetate:hexane = 1:7) to give 9.74 g (66.9 % yield) of ethyl 3-[ (2S, 4R)-l-allyloxycarbonyl-4-t- butyldimethγlεilyloxypyrrolidin-2-yl ]-2-methylacrylate.

NMR(CDC1 ₃ ) δ :

0.05(ε,6H), 0.80(ε,9H), 1.12(t,3H), 1.70(m,lH), 1.89(S,3H), 2.05(m,lH), 3.50(m,2H), 4.20(q,2H), 4.30- 4.80(m,4H), 5.20(m,2H), 5.82(m,lH), 6.55(d,lH)

REFERENCE EXAMPLE 5-2

The same procedure as in REFERENCE EXAMPLE 4-2 was carried out by using 9.74 g (24.5 mmol) of ethyl 3- [ (2S, 4R)-l-allyloxycarbonyl-4-t-buthyldimethylsilyloxy pyrrolidin-2-yl]-2-methylacrylate obtained in REFERENCE EXAMPLE 5-1 and 62 ml (62 mmol) of lM-diiεobutyl aluminum hydride to obtain 8.15 g (93.7 % yield) of (2S,4R)-l-allyloxycarbonyl-2-[ (E)-3-hydroxy-2-methyl-l- propenyl]-4-t-butyldimethylsilyloxypyrrolidine.

0.05(S,6H), 1.81(ε,9H), 1.63(m,lH), 1.70(s,3H), 2.03(m,lH), 3.50(m,2H), 4.01(s,2H), 4.01(s,2H), 4. SO- 4.70(m,4H), 5.20(m,3H), 5.90(m,lH)

REFERENCE EXAMPLE 5-3

The εame procedure as in REFERENCE EXAMPLE 5-2 waε carried out by uεing 8.15 g (22.96 mmol) of (2S,4R)-l- allyloxycarbonyl-2-[ (E)-3-hydroxy-2-methyl-l-propenyl ]-

4-t-butyldimethylsilyloxypyrrolidine obtained in REFERENCE EXAMPLE 5-2, 9.03 g (34.4 mmol) Of triphenyl phosphine, 5.74 g (39.01 mmol) of phthalimide and 5.4 ml (34.3 mmol) of diethyi azodicarboxylate to obtain 7.97 g (73.1 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3- phtalimido-2-methyl-l-propenyl]-4-t-butyldimethylεilyl oxypyrroϋdine.

NMR(CDC1 δ

0.05(ε,6H), 0.83(ε,9H), 1.68(m,lH), 1.70(s,3H), 2.00(m,lH), 3.40(d,2H), 4.20-4.70(m, 6H) , 5.20(m,3H), 5.90(m,lH), 7.70-7.90(m,4H)

REFERENCE EXAMPLE 5-4

The same procedure aε in REFERENCE EXAMPLE 4-4 waε carried out by uεing 7.97 g (16.96 mmol) of (2S,4R)-1- allyloxycarbonyl-2-[ (E)-3-phthaiimido-2-methy1-1- propenyl]-4-t-butyldimethylsilyloxypyrrolidine obtained in REFERENCE EXAMPLE 5-3, 3 ml (61.85 mmol) of hydrazine hydrate, 4.8 ml (62.01 mmol) of methanesulfonyl chloride and 12.1 ml (86.81 mmol) of triethylamine to obtain 4.53 g (61.7 % yield) of (2S,4R)-l-allyloxycarbonyl-2-[ (E)-3- methanesulfonylamino-2-methyl-l-propenyl]-4-t-butyl dimethylsilyloxypyrroiidine.

NMR(CDC1 ₃ ) δ :

0.05(S,6H), 0.80(S,9H), 1.65(m,lH), 1.70(s,3H),

2.03(m,lH), 2.90(S,3H), 3.40(m,2H), 3.60(m,2H),4.30- 4.70(m,4H), 5.10-5.30(m, 3H), 5.83(m,lH)

REFERENCE EXAMPLE 5-5

The same procedure as in REFERENCE EXAMPLE 4-5 was

carried out by using 4.92 g (11.37 mmol) of (2S,4R)-1- allyloxycarbonyl-2- [(E) -3-methanεulf onylamino-2-methγl- 1-propenyl ]-4-t-butyldimethylεilyloxypyrrolidine Obtained in REFERENCE EXAMPLE 5-4, 14.8 ml (14.8 mmol ) of lM-tetrabutyl ammonium fluoride, 3.6 ml (46.51 mmol) of methanesulfonyl chloride and 8.5 ml (60.98 mmol) of triethylamine to obtain 3.07 g (68.1 % yield) of ( 2 S , 4R ) -l-al lyloxycarbonyl -2- [ ( E ) - 3 - methanesulfonyl amino-2-methyl-l-propenyl] -4- methanesulf onyloxypyrrolidine .

NMR(CDC1 ₃ ) δ :

1.70(S,3H), 1.90(m,lH), 2.50(m,lH), 2.95(ε,3H), 3.05(ε,3H), 3.62(m,3H), 3.98(m,lH), 4.50-4.80 (m, 4H) ,

5.25(m,4H), 5.90(m,lH)

REFERENCE EXAMPLE 5-6

The εame procedure as in REFERENCE EXAMPLE 4-6 was carried out by using 3.07 g (7.75 mmol) of (2S,4R)-1- allyloxycarbonyl-2- [(E) -3-methanesulf onylamino-2-methyl- 1 -propenyl ]-4-methaneεulf onyloxypyrrolidine obtained in REFERENCE EXAMPLE 5-5 and 2.91 g (25.6 mmol) Of potassium thioacetate to obtain 2.54 g (87.1 % yield) of

( 2S, 4S ) -l-allyloxycarbonyl-2-[ (E)-3-methaneεulf onyl amino-2-methyl-l-propenyl]-4-acetylthiopyrrolidine.

1.62(m,lH), 1.80(ε,3H), 2.30(ε,3H), 2.54 (m, 1H) , 2.95(ε,3H), 3.23(m,lH), 3.62(m,2H), 4.10-3.85 (m, 2H) , 4.50(m,4H), 5.15-5.42(m,3H) , 5.90(m,lH)

REFERENCE EXAMPLE 5-7

The εame procedure aε in REFERENCE EXAMPLE 4-7 waε

carried out by uεing 2.54 g (6.75 mmol) of (2S,4S)-1- allyloxycarbonyl-[ (E)-3-methaneεulfonylamino-2-methyl-l- propenyl]-4-acetylthiopyrrolidine obtained in REFERENCE EXAMPLE 5-6 and 8.1 ml of 2N-NaOH to obtain 1.89 g (83.1 % yield) of (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3- methanesulfonylamino-2-methyl-l-propenyl]-4-mercapto pyrrolidine.

NMR(CDC1 ₃ ) δ :

1.60(m,2H), 1.70(S,3H), 2.50(m,lH), 2.90(s,3H), 3.20(m,2H), 3.60(m,2H), 4.01(m,lH), 4.50(m,4H), 5.42- 5.10(m,3H), 5.90(m,lH)

REFERENCE EXAMPLE 6

( 2S , 4S ) -1-all yloxycarbonyl-2- r ( E ) -3- ( N , N-dimethyl εul f amoyl amino ) -2 -methyl -l -propenyl l -4 -mercapto pyrrolidine

REFERENCE EXAMPLE 6-1

The εame procedure as in REFERENCE EXAMPLE 5-4 was carried out by using 7.05 g (15 mmol) of (2S,4R)-1- allyioxycarbonyl-2-[ (E)-3-phthalimido-2-methyl-l- propenyl]-4-t-butyldimetylεilyloxypyrrolidine obtained in REFERENCE EXAMPLE 5-3, 2.7 ml (55.66 mmol) Of hydrazine hydrate, 5.4 ml (38.74 mmol) of triethylamine and 2.8 ml (26.07 mmol) of dimethylεulfamoyl chloride to obtain 1.62 g (47 % yield) of (2S, 4R)-l-allyloxy carbonyl-2-[ (E)-3-(N,N-dimethylεulfamoylamino)-2-methyl- 1-propenyl]-4-t-butyldimethylεilyloxypyrrolidine.

NMR(CDC1 ₃ ) δ :

0.05(s,6H), 1.81(ε,9H), 1.65(m,lH), 1.70(ε,3H)

2.05(m,lH), 2.80(s,6H), 3.50(m,4H), 4.30-4.70 (m, 4H) , 5.20(m,3H), 5.90(m,lH)

REFERENCE EXAMPLE 6-2

The same procedure as in REFERENCE EXAMPLE 5-5 waε carried out by using 1.62 g (3.49 mmol) of (2S,4R)-1- allyloxycarbonyl-2- [ ( E ) -3- ( N, N-dimethylsulf amoylamino ) - 2-methyl-l-propenyl]-4-t-butyldimethylsilyloxy pyrrolidine obtained in REFERENCE EXAMPLE 6-1, 4.3 ml

(4.3 mmol) of lM-tetrabutyl ammonium fluoride, 2.6 ml (18.65 mmol) of triethylamine and 1.1 ml (14.21 mmol) of methanesulfonyl chloride to obtain 1.33 g (89.0 % yield) of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3-(N,N-dimethyl sulf amoylamino )-2-methyl-l -propenyl ]-4-methaneεul f onyloxypyrrolidine .

NMR(CDC1 ₃ ) δ :

1.70(ε,3H), 1.90(m,lH), 2.50(m,lH), 2.80(ε,6H),

3.04(ε,3H), 3.50-4.01(m, 4H) , 4.50-4.80 ( m, 3H ) ,

5.20(m,4H), 5.90(m,lH)

REFERENCE EXAMPLE 6-3

The εame procedure aε in REFERENCE EXAMPLE 5-6 waε carried out by using 1.33 g (3.11 mmol) of (2S,4R)-1- allyloxγcarbonyl-2- [(E) -3- ( N, N-dimetylεulf amoylamino ) -2- methyl-l-propenyl ]-4-methanesulf onyloxypyrrolidine obtained in REFERENCE EXAMPLE 6-2 and 1.2 g (10.5 mmol) of potasεium thioacetate to obtain 910 mg (71.7 % yield) of ( 2S, 4S )-l-allyloxycarbonyl-2-[ ( E)-3-(N, - dimethyisulfamoylamino)-2-methyl-l-propenyl]-4- acetylthiopyrrolidine .

NMR(CDC1 ₃ ) δ

1.60(m,lH), 1.75(S,3H), 2.30(ε,3H), 2.58(m,lH), 2.80(ε,6H), 3.23(dd,lH), 3.55(S,2H), 3.90(m,2H),

4.55(m,3H), 5.30(m,3H), 5.90(m,lH)

REFERENCE EXAMPLE 6-4

The same procedure aε in REFERENCE EXAMPLE 5-7 waε carried out by using 910 mg (2.23 mmol) of (2S,4S)-1- allyloxycarbonyl-2-[(E)-3-(N,N-dimethylsulfamoylamino)- 2-methyl-l-propenyl]-4-acetylthiopyrrolidineobtainedin REFERENCE EXAMPLE 6-3 and 2.7 ml Of 2N-NaOH to Obtain 630 mg (77.2 % yield) of (2S, 4S)-l-allyloxycarbonyl-2- [ (E)-3- (N, N-dimethylεuIfamoylamino) -2-methy1-1- propenyl]-4-mercaptopyrrolidine.

1.60(ε,2H), 1.70(ε,3H), 2.52(m,lH), 2.80(ε,6H), 3.10(m,lH), 3.50(ε,2H), 4.00(m,lH), 4.50(m,3H), 5.30(m,3H), 5.90(m,lH)

REFERENCE EXAMPLE 7

(2S, 4S)-l-allyloxycarbonyl-2-r (E)-3 ( 4-morpholino εulfonylamino)-2-methyl-l-propenyll-4-mercapto pyrrolidine

REFERENCE EXAMPLE 7-1

To a εolution of (2S,4R)-l-allyloxycarbonyl-2-[ (E)-

3-phtha1imido-1-propenyl ]-4-t-butyldimethylεilyoxy pyrrolidine (3.53 g, 7.51 mmole) in anhydrouε ethanol (80 ml) waε added hydrazine hydrate (1.4 ml, 41.6 mmol).

The reaction mixture was stirred at room temperature

overnight, filtered to remove insoluble material and the filtrate was concentrated under reduced presεure.

To the above reεidue waε added ethyl acetate and the organic layer was εtirred waεhed with 3N-ammonia εolution and then εaturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was diεεolved in dimethylformamide (50 ml) and triethylamine (1.3 ml, 94 mmol) and catechol sulfate (1.55 g, 9.01 mmol) was added successively thereto, and the reaction mixture waε εtirred for 1 hour. To the above solution was added ethyl acetate and water, and the ethyl actate layer was separated. The water layer was again extracted, a few times, with ethyl acetate. The combined organic layer waε waεhed successively with saturated citric acid and then saturated aqueous εodium chloride,and dried over anhydrous sodium sulfate. The solvent waε removed in vacuo, and the residue was subjected to silica gel column chromatography (eluting with ethyl acetate:hexane = 1:2) to give pure (2S,4R)-l- allyloxycarbonyl-2-[ (E)-3-catecholεulfonylamino-2- methyl-l-propenyl]-4-t-butyldimethylsilyloxypyrrolidine (2.06 g, yield:52.2 %) .

0.05(ε,6H), 0.93(ε, 9H), 1.70(ε,3H), 2.01(m,lH), 3.43(m,2H), 3.70(d,2H), 4.35(m,lH), 4.60(m,3H), 5.30(m,3H), 5.90(m,lH), 6.80-7.30 (m, 4H)

REFERENCE EXAMPLE 7-2

To a εolution of ( 2S , 4R ) -l-allyloxycarbonyl-2- [ ( E ) - 3-catechol sul f onyl amino- 2 -methyl - l -propenyl ] - 4 -t- butyldimethylsilyloxypyrrolidine ( 2 . 06 g , 3 . 92 mmol ) in

dioxane (60 ml) were added triethylamine (1.1 ml, 7.8 mmol) and morpholine (0.6 ml, 6.3 mmol). The reaction mixture waε heated to reflux for 1.5 hour, and cooled to room temperature. The exceεε dioxane waε removed in vacuo. To the reεidue waε added ethyl acetate and the organic layer waε washed with εaturated aqueouε εodium chloride, dried over anhydrouε sodium sulfate and concentrated under reduced presεure. The crude product waε purified by column chromatography on silica gel (eluting with ethyl acetate:hexane = 1:1 ) to give

(2S,4R)-l-allyloxycarbonyl-2-[ (E)-(4-morpholinosulfonyl amino)-2-methyl-l-propenyl]-4-t-butyldimethylsilyloxy pyrrolidine (1.61 g, yield:81.8 %).

NMR(CDC1 ₃ ) δ:

0.05(S,6H), 0.93(S,9H), 1.75(s,3H), 2.01(m,lH), 3.10(m,4H), 3.46(d,2H), 3.60(d,2H), 3.70(m,4H), 4.30- 4.70(m,4H), 5.22(m,3H), 5.90(m,lH)

REFERENCE EXAMPLE 7-3

A meεylation of an amino moiety of (2S,4R)-1- allyloxycarbonyi-2-[ (E)-(4-morphoiinosulfonylamino)-2- methyl-1-propenyl]-4-t-butyldimethylεilyloxypyrrolidine (1.61 g, 3.21 mmol) obtained in REFERENCE EXAMPLE 7-2 waε conducted in the εame manner aε described in REFERENCE EXAMPLE 4-4 to REFERENCE EXAMPLE 4-7 to give (2S,4S)-l-allyloxycarbonyl-2-[(E)-3( 4-morpholi nosulfonylamino)-2-methyl-l-propenyl]-4-mercaptopyrroli dine (0.8 g, yield:61.6%) .

NMR(CDC1 ₃ ) δ:

2.59(m,2H), 2.70(s,3H), 2.52(m,lH), 3.20(m,4H),

3.69(brs,2H), 3.70(m,4H), 4.01(m,lH), 4.50(m,4H), 5.15-

5 . 42 ( m, 3H ) , 5 . 90 ( m, lH )

REFERENCE EXAMPLE 8

( 2S, 4S )-l-allyloxycarbonyl-2-r (E)-2-chloro-3- methanesulfonylamino-1-propenyl1-4-mercaptopyrrolidine

REFERENCE EXAMPLE 8-1

To an ice cooled suεpenεion of εodium hydride (2.09g of 60 % mineral oil, 52.27 mmol) in anhydrouε tetrahydrofuran (135 ml) waε added, succeεsively and dropwise triethyl 2-chloro-2-phosphonoacetate (13.5 g, 52.27 mmole) and (2S,4S)-l-allyloxycarbonyl-2-formyl-4- t-butyldimethylsilyloxypyrrolidine (13.65 g, 43.56 mmol) obtained by known procesεeε. The mixture was stirred at the same temperature for 1 hour, and citric acid waε added thereto to acidify the εolution to pH 4.5. The excess organic εolvent waε evaporated in vacuo, and the resulting residue was diluted with methylene chloride, washed with water and then εaturated aqueouε εodium chloirde, dried over magenesium sulfate and concentrated under reduced presεure. The crude product waε purified by flash chromatography on silica gel, eluting with a mixture of ethyl acetate:hexane (= 1:4) to give ethyl-3- [ (2S, 4R)-l-allyloxycarbonyl-4-t-butyldimethylsilyloxy pyrroiidin-2-yl]-2-chloroacrylate (10.4 g, yield:57.1 %)

0.05(ε,6H), 0.80(S,9H), 1.30(t,3H), 1.80(m, 1H) , 2.20(m,lH), 3.50(m,2H), 4.05-4.40(m, 3H) , 4.59(m,2H), 4.90(m,lH), 5.20(m,2H), 5.90(m,lH), 7.01(t,lH)

REFERENCE EXAMPLE 8-2

A crude product (8.7 g) obtained by the εame process as that of REFERENCE EXAMPLE 1-2 from ethyl 3- [ (2S, 4R)-l-allyloxycarbonyl-4-t-butyldimethylsilyloxy pyrrolidin-2-yl]-2-chloloacrylate (6.0 g, 14.35 mmol) obtained in REFERENCE EXAMPLE 8-1 and diiεobutylaluminum hydride (43 ml in 1.0 M toluene εolution, 43 mmol) in anhydrouε tetrahydrofuran (100 ml) waε purified by column chromatography on εilica gel (eluting with ethyl acetate:hexane = 1:2) to give (2S,4R)-1- allyloxycarbonyl-2-[(E)-chloro-3-hydroxy-l-propenyl]-4- t-butyldimethylεilyoxypyrroiidine (2.2g, yield:40.8%) and (2S, 4R)-l-allyloxycarbonyl-2-t-[ (Z)-2-chloro-3- hydroxy-l-propenyl]-4-t-butyldimethylεilyloxypyrrolidine (0.8 g, yield:l4.8 %) .

(E) form

NMR(CDC1 ₃ ) δ:

0.05(ε,6H), 0.80(s,9H), 1.70(m, 1H), 2.20(m,lH),

3.50(m,2H), 4.10(S,2H), 4.30(m,lH), 4.55(m,2H), 4.80(q,lH), 5.20(m,2H), 5.90(m,2H)

(Z) form

NMR(CDCl-) δ

0.06(S,6H), 0.82(S,9H), 1.65(m,lH), 2.17(m,lH), 3.47(m,2H), 4.05(S,2H), 4.25(m,lH), 4.50(m,2H), 4.72(q,lH), 5.15(m,2H), 5.85(m,2H)

REFERENCE EXAMPLE 8-3

To an ice cooled solution of (2S, 4R)-l-allyloxy carbonyl-2-[ (E)-chloro-3-hydroxy-1-propenyl ]-4-t- butyldimethylεilyloxypyrroiidine (0.8 g, 2.13 mmol)

obtained in REFERENCE EXAMPLE 8-2 in anhydrous tetrahydrofuran (10 ml) was added dropwise triphenylphospine (0.84 g, 32.02 mmol) and phthalimide (0.55 g, 37.38 mmol), and diethyi azodicarboxylate (0.5 g, 31.75 mmole). The mixture waε εtirred for 1 hour, concentrated under reduced preεεure to evaporate the organic solvent, and methylen chloride (200 ml) waε added thereto. The organic layer was stirred, washed successively with water and saturated aqueouε εodium chloirde, dried over anhydrouε magnesium εulfate and concentrated in vacuo. The crude product was subjected to silica gel column chromatography (eluting with the mixture of .ethyl acetate:hexane = 1:3) to give (2S,4R)- l-allyloxycarbonyl-2-[ (E)-chloro-3-phthalimido-l- propenyl]-4-t-butyldimethylεilyloxypyrrolidine (1.0 g, yield:92.6%) .

NMR(CDC1 ₃ ) δ:

0.04(S,6H), 0.83(s,9H), 1.74(m,lH), 2.19(m,2H),

3.43(m,2H), 4.30-4.60(m,4H), 4.80(q,lH), 5.00- 5.40(m,2H), 5.90(m,2H), 7.70-7.90(m, 4H)

REFERENCE EXAMPLE 8-4

A crude product (0.8 g) obtained by the εame proceεs as that of REFERENCE EXAMPLE 1-4 from (2S,4R)-1- allyloxycarbonyl-2-[ (E)-chloro-3-phthalimido-l- propenyl]-4-t-butyldimethylεilyloxypyrrolidine (1.0 g, 19.80 mmol), obtained in REFERENCE EXAMPLE 8-3, and ethanol (15 ml), hydrazine hydrate (0.27 ml, 55.3 mmol), triethylamine (0.7ml, 50.4 mmol), and methanesulfonyl chloride (0.215 ml, 7.72 mmole) was purified by column chromatography on silica gel (eluting with ethyl acetate :hexane = 1:1.5) to give (2S,4R)-l-allyloxycarbonyl-2- [ (E)-chloro-3-methaneεulfonylamino-l-propenyl]-4-t-butyi

dimethylsilyloxypyrrolidme (0.61 g, yield: 82.4 %).

NMR(CDC1 ₃ ) δ:

0.03(s,6H), 0.83(ε,9H), 1.70(m,lH), 2.18(m,lH),

3.00(m,2H) 3.43(m,2H), 4.30(m,lH), 4.50(m,2H), 4.80(q,lH), 4.90(m,2H), 5.20(m,2H), 5.90(m,2H)

REFERENCE EXAMPLE 8-5

( 2S, 4R)-l-allyloxycarbonyl-2-[ (E)-chloro-3- methanesulfonylammo-1-propenyl]-4-t-butyldιmethylsιlyl oxypyrroiidme (0.61 g, 13.46 mmol), obtained in REFERENCE EXAMPLE 8-4, waε dissolved m methanoi (50 ml), stirred at room temperature, and then HCl solution (1.2 ml) was added dropwiεe thereto. The mixture waε εtirred for 4 hour and concentrated under reduced pressure. The residual material waε dissolved in methylene chloride, waεhed with water and then εaturated aqueous εodium chloride, and the organic layer waε dried over anhydrouε sodium sulfate and filtered. The filtrate waε concentrated in vacuo. The resulting crude product waε subjected to column chromatography on silica gel (eluting with chloroform:methanoi = 20:1) to afford (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-chloro-3- methanesulfonyl mino-1-propenγl ]-4-hydroxypyrrolιdιne (0.36 g, yιeld:78.9 %) .

1.70(m,lH), 2.30(m,lH), 3.01(ε,2H), 3.50(m,2H), 3.90(m,2H), 4.40-4.60(m, 2H), 4.80(q,lH), 5.20(m,2H), 5.90(m, 2H)

REFERENCE EXAMPLE 8-6

To a solution of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)- c 1oro-3-methaneεulfonylamino-1-propenyl]-4-hydroxy pyrrolidine (1.8 g, 5.32 mmol) obtained m REFERENCE EXAMPLE 8-5 in methylene chloride (60 ml) was added successively triethylamine (1.7 ml, 12.19 mmol) and methanesulfonyl chloride (0.49 ml, 6.33 mmol) at room temperature and the mixture was stirred for 40 minutes. The reaction mixture was waεhed succesεively with 1N- HC1, water, εaturated εodium bicarbonate aqueouε solution and finally distilled water, and dried over anhydrous magneεium εulfate, and then concentrated under reduced preεεure to give (2S, 4R)-l-allyloxycarbonyl-2- [ (E)-chloro-3-methanesulfonylamιno-l-propenyl]-4-methane sulfonyloxypyrrolidine (2.1 g, yield:95.0%) , which waε uεed at the next εtage without purification.

NMR(CDC1 ₃ ) δ:

1.90(m,lH), 2.80(m,lH), 2.95(ε,3H), 3.08(s,3H), 3.90(m,2H), 4.55(m,lH), 4.80(q,lH), 5.20(m,2H), 5.90(m,2H)

REFERENCE EXAMPLE 8-7

To a solution of (2S, 4R)-l-allyloxycarbonyl-2-[ (E)- chloro-3-methaneεulfonylammo-1-propenyl]-4-methanεul fonyloxypyrrolidine (2.1 g, 5.04 mmol) prepared m REFFERENCE EXAMPLE 8-6 in anhydrouε acetonitrile (250 ml) was added potassium thioacetate (0.91 g, 7.95 mmol). The mixture waε heated to reflux for 4 hourε, cooled, and filtered to remove mεoluble material. The filtrate was concentrated under reduced presεure. The reεidual material was disεolved m methylene chloride, waεhed with water and the organic layer waε dried over anhydrous sodium sulfate, concentrated again under reduced pressure. The residue was purified by column

chromatography on silica gel (eluting with chloroform :methanoi = 20:1) to give (2S,4S)-l-allyloxycarbonyl-2- [ (E)-chloro-3-methanesulfonylamino-l-propenyl ]-4- thioacetylpyrrolidine (1.4 g, yield:67 %).

NMR(CDC1 ₃ ) δ:

1.70(m,lH), 2.32(ε,3H), 2.80(m,lH), 3.00(ε,3H), 3.30(m,lH), 3.90(m,2H), 4.50(m,2H), 4.80(q,lH), 5.20(m,2H), 5.90(m,2H)

REFERENCE EXAMPLE 8-8

To an ice cooled solution of (2S,4S)-l-allyloxy carbonyl-2-[ ( E)-chloro-3-methanesulfonylamino-l- propenyl]-4-thioacetylpyrroiidine (1.4 g, 3.53 mmol) obtained in REFERENCE EXAMPLE 8-7 in methanoi (140 ml) waε added 2N-NaOH (3.5 ml, 7.06 mmol) over the courεe of 1 minute and then an aqueouε citric acid solution was added to adjust the pH of the solution 4.0. The mixture waε concentrated under reduced preεsure and diluted with methylene chloride. The organic layer was washed with water, dried over anhydrouε magneεium εulfate, concentrated in vacuo to give (2S,4S)-l-allyloxy carbonyl-2-[ (E)-chloro-3-methaneεulfonylamino-l- propenyl]-4-mercaptopyrrolidine (1.16 g, yield:92.8 %), which was used at the next stage without purification.

1.69(m,lH), 2.79(m,lH), 3.03(s,3H), 3.25(m,lH), 3.91(m,2H), 4.49(m,2H), 4.82(q,lH), 5.19(m,lH), 5.89(m, 2H)

REFERENCE EXAMPLE 9

(2S, 4R)-l-allyloxycarbonyl-2-r ( E ) -3 -methane sulfonyl-l-propenvil-4-t-butyldimeτ.hylsilyloxy pyrrolidine

REFERENCE EXAMPLE 9-1

To an ice cooled solution of 2-aminoethanol 5 g (81.9 mmol) in dichloromethane (200 ml) waε added dropwise triethylamine (28.5 ml, 204.8 mmol) and methanesulfonyl chloride (14.6 ml, 188.4 mmol). The mixture waε εtirred at 0 °C for 30 minutes, and additional dichloromethane (100 ml) was added thereto. The organic layer was waεhed with a small amount of diluted-HCl solutionε and then εaturated aqueouε εodium chloride, dried over anhydrouε εodium εulfate, and filtered. The filtrate waε concentrated under reduced preεsure to give N, O-dimethaneεulfonylethanoiamine (14.5 g, yield: 81.5 %) .

NMR(CDC1 ₃ ) δ:

3.02(ε,3H), 3.09(ε,3H), 3.51(q,2H), 4.35(t,3H), 5.10(bε,lH)

REFERENCE EXAMPLE 9-2

A solution of N,O-dimethanesulfonylethanolamine

(8.3 g, 38.2 mmol) obtained in REFERENCE EXAMPLE 9-1 and lithium bromide (4.0 g, 46.1 mmol) in acetone (120 ml) waε heated to relux for 2 hourε, and then chloroform (50 ml) was added thereto. The resulting precipitate was

filtered off and the filtrate was concentrated in vacuo. The crude product obtained waε purified by εilica gel chromatography (eluting with mixture of chloroform: acetone = 10:1) to give N-methaneεulfonyl-2- bro oethy1amine (8.3 g, yield:89 %).

NMR(CDC1 ₃ ) δ:

3.03(ε,3H), 3.54-3.57(m,4H), 4.90(bs,lH)

REFERENCE EXAMPLE 9-3

N-methaneεulfonyl-2-bromoethylamine (8.0 g, 39.6 mmol) obtained in REFERENCE EXAMPLE 9-2 and triphenyl phophine (11.5 g, 43.8 mmol) were dissolved in toluene (80 ml). The solution was heated to reflux for 9 hourε, cooled to room temperature and concentrated under reduced pressure. The crude material was disεolved in dichloromethane and ether to crystallize the desired product. The solid which formed was obtained by filtration and dried under vacuum to give [(2- methaneεulfonylamino)ethyl]triphenylphoεphonium bromide (11.5 g, yield:62.5 %) .

NMR(DMSO-d.) δ:

2.93(s,3H), 3.29(m,2H), 3.85(m,2H), 7.50(t,lH),

7 . 70-8 . 01 ( m, 15H )

REFERENCE EXAMPLE 9-4

To a dry-ice (-78 °C) cooled solution of [(2- methaneεulfonylamino)ethyl]triphenylphosphonium bromide (0.39 g, 0.84 mmol) obtained in REFERENCE EXAMPLE 9-3 in tetrahydrofuran (4 ml) was added dropwise and carefully n-butyllithium (1.26 ml of hexane solution, 1.26 mmol) solution. The reaction solution waε warmed to -40 °C, stirred for 10 minutes, and cooled to -78 °C again. Hexamethylphosphorouε triamide (0.37 ml, 2.13 mmol) waε added thereto. The mixture was stirred for 10 minutes, and to the above solution was added a solution of (2S, 4R)-l-allyloxycarbonyl-2-formγl-4-t-butyldimethγl εilyloxypyrrolidine (0.131 g, 0.418 mmol), obtained by known procesεeε, in tetrahydrofuran (4 ml). The reaction mixture waε warmed εlowly to room temperature, concentrated under reduced preεsure, and the reεidue waε diεεolved in ethyl acetate, waεhed with water, dried over anhydrouε εodium sulfate, and filtered. The filtrate was concentrated in vacuo. The concentrate was subjected to column chromatography on silica gel (eluting with ethyl acetate:hexane = 1:2) to give (2S, 4R)-l-allyloxycarbonyl-2-[ (E)-3-methanesul fonylamino-1-propenyl ]-4-t-butyldimethylsilyloxy pyrrolidine (0.106 g, yield: 60.4 %).

NMR(CDC1 ₃ ) δ:

0.03(ε,6H), 0.85(ε,9H), 1.68-1.78(m, 1H) , 1.98-

2.06(m,lH), 2.93(ε,3H), 3.45-3.47 (d, 2H) , 3.70- 3.74(m,2H), 4.31-4.43 (m, 2H) , 4.51-4.54 (m, 3H) , 5.14- 5.30(m,2H), 5.62(bs,2H), 5.81-5.98 (m, 1H)

Detailed Description of the Preferred Embodiments

EXAMPLE 1

(lR,5S,6S)-2-r(2S,4S)-2-r ( E ) -3-methanesulf onyl amino-l-propenyl 1 pyrrol idin-4-ylthiol -6- r ( R ) -1- hydroxyethyi l-l-methyl-l-carbapen-2-em-3-carboχylic acid

EXAMPLE 1-1

To an ice-cooled solution of allyl ( IR, 5S, 6S)-2- diphenoxyphoεphoryioxy-6-[ (R)-l-hydroxyethyl ]-l-methyl- l-carbapen-2-em-3-carboxylate (4.12 g, 9.17 mmol) obtained by known proceεεeε, in anhydrous acetonitrile (50 ml) was added diisopropylethylamine (1.6 ml, 9.18 mmol). The above solution waε εtirred for 5 minutes. And then the solution of ( 2S, 4S)-l-allyloxycarbonyl-2- [ ( E ) -3-methaneεulfonγlamino-l-propenγl ]-4- mercaptopyrrolidine (2.45 g, 7.01 mmol) obtained in REFERRENCE EXAMPLE 1-7 in anhydrous acetonitrile (10 ml)

was added dropwise thereto. The reaction mixture waε stirred for 4 hourε and concentrated under reduced pressure. The residue was diluted with ethylacetate, washed with distilled water, dried over anhydrous magnesium sulfate and the solvent waε removed in vacuo. The residue was subjected to column chromatography on silica gel (eluting with ethylacetate:hexane = 2:1, and chloroform:methanoi = 50:1) to give pure allyl (1R,5S, 6S)-2-[ (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3-methanesul fonylamino-l-propenyl ]pyrrolidin-4-ylthio]-6-[ (R)-l- hydroxyethyl ]-l-methyl-l-carbapen-2-em-3-carboxylate (2.8 g, yield:65 %) .

NMR(CDCl-)

1.19-1.24(d,3H), 1.30-1.33 (d, 3H) , 1.78-1.84(m, 1H) , 2.52-2.59(m,lH) , 2.43(s,3H), 3.20-3.42(m, 3H) , 3.70- 3.75(m,3H), 3.84-4.05(m,lH), 4.41-4.52(m, 1H) , 4.53- 4.55(m,2H), 4.61-4.70(m,lH), 4.77-4.81(m, 1H) , 5.17- 5.30(m,3H), 5.37-5.47 (m, 2H) , 5.55-5.68(m, 2H) , 5.84- 5.95(m,2H)

EXAMPLE 1-2

To an ice cooled mixture of ally ( IR, 5S, 6S)-2- [ (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3-methanesulfonyl amino-l-propenyl ]pyrrolidin-4-ylthio]-6-[ (R)-1-hydroxy

ethyl]-l-methyl-l-carbapen-2-em-3-carboxylate (2.8 g, 4.91 mmol), obtained in EXAMPLE 1-1, and diεtilled water (0.4 ml, 22.2 mmol) in anhydrouε dichloromethane (59 ml) waε added successively bis(triphenylphosphine) palladium(II) chloride (0.03 g, 0.043 mmol) and tributyltin hydride (2.9 ml, 10.78 mmol). The solution was stirred for 10 minutes, extracted with distilled water (100 ml x 3), washed with chloroform (100 ml x 2), and the aqueous layer was freeze-dried to give (1R,5S, 6S)-2-[(2S,4S)-2-[(E)-3-methaneεulfonylamino-l- propenyl]-pyrrolidin-4-ylthio]-6-[ (R)-l-hydroxyethyl]-l- methyl-l-carbapen-2-em-3-carboxylic acid (1.31 g, yield: 60 %) .

NMR(DMSO-d ₆ + D ₂ 0)δ:

1.02-1.05(d, 3H), 1.10-1.13(d,3H), 1.45-1.63(m, 1H), 2.41-2.50(m,lH), 2.86(S,3H), 3.01-3.12(m, 3H), 3.45- 3.57(m,3H), 3.87-4.05(m,.2H), 5.77-5.88(m,2H)

EXAMPLE

(lR,5S.6S)-2-r(2S.4S)-2-r (E)-3-methaneεulfonyl amino-1-methylpropenyl1-pyrrolidin-4-ylthio1-6-r (R)-l- hydroxyethyl1-l-methyl-l-carbapen-2-em-3-carboχylic acid

EXAMPLE 2-1

To an ice cooled solution of allyl ( IR, 5S, 6S)-2- diphenoxyphoεphoryloxy-6- [ ( R ) -l-hydroxyethyl ] -l-methyl- l-carbapen-2-em-3-carboxylate (0.5 g, 1.0 mmol), obtained in known proceεεeε, and diiεopropylethylamine (0.21 ml, 1.2mmol) in acetonitrile (10 ml) waε added dropwiεe a εolution of (2S, 4S)-l-allyloxycarbonyl-2-

[ ( E ) -3-methanesulfonylamino-l-propenyl ] - 4 - mercaptopyrrolidine (0.33 g, 1.0 mmol), obtained in

REFERRENCE EXAMPLE 2-6 in acetonitrile (5 ml). The above reaction solution waε stirred for 3 hours and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel

(eluting with chloroform: acetone = 3:1) to give allyl

(IR, 5S, 6S)-2-[ (2S, 4S ) -l-allyloxycarbonyl-2- [ (E)-3- methanesulf onylamino-1-methyl-propenyl ]pyrroiidin-4- ylthio ] -6- [ ( R ) -l-hydroxyethyl ] -l-methyl-l-carbapen-2-em-

3-carboxylate (0.16 g, yield: 28 %).

NMR ( CDC 1,

1.22(d,3H), 1.34(d,3H), 1.64(s,3H), 1.84(m,lH), 2.51(m,lH), 2.94(ε,3H), 3.30(m,2H), 3.61(m,lH),

3.81(m,2H), 4.21(m,2H), 4.52(m,2H), 5.19-5.50 (m, 5H) , 5.90(m,2H)

EXAMPLE 2-2

To a solution of allyl (IR,5S, 6S)-2-[ (2S, 4S)-1- allyloxycarbonyl-2-[(E)-3-methaneεulfonylamino-1-methyl- propenyl]pyrrolidin-4-ylthio]-6-[ (R)-l-hydroxyethyl]-1- methyl-l-carbapen-2-em-3-carboxylate (0.16 g, 0.28 mmol) obtained in EXAMPLE 2-1, and diεtilled water (0.22 ml) in dichloromethane (5.5 ml) waε added of biε(triphenyl phosphine) palladium (II) chloride (10 mg, 0.014 mmol) and tributyltin hydride (0.15 ml, 0.55 mmol). The solution-waε stirred for 20 minutes, and then extracted with distilled water (10 ml x 3). The aqueous layer was washed with chloroform and freeze-dried to give (IR,5S, 6S)-2-[ (2S, 4S)-2-[ (E)-3-methaneεulfonylamino-1- methylpropenyl ]-pyrrolidin-4-ylthio]-6- [ (R) -1- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylic acid (55 mg, yield:43 %) .

NMR(D ₂ 0) δ:

1.08(d,3H), 1.15(d,3H), 1.68(S,3H), 1.82(m,lH), 2.60(m,lH), 2.95(S,3H), 3.22(m,3H), 3.54(m,lH), 3.68(d,2H), 3.90(m,lH), 4.00-4.21(m, 3H) , 5.60(t,lH)

EXAMPLE 3

(IR, 5S, 6S)-2-r (2S, 4S)-2-f (E)-3-fluoro-1-propenyl1 pyrroiidin-4-yl hio1-6-f (R)-l-hydroxyethyl1-l-methyl-l- carbapen-2-em-3-carboχylic acid

EXAMPLE 3-1

Allyl (IR,5S,6S)-2-diphenoxyphoεphoryloxγ-6-[ (R)-l- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylate (0.23 g, 0.5 mmol), obtained by known proceεεeε, and (2S,4S)-l-allyloxycarbonyl-2-[ (E)-3-fluoro-l-propenyl]- 4-mercaptopyrrolidine (0.12 g, 0.5 mmol), obtained in REFERENCE EXAMPLE 3-4, were treated by the same proceεε aε that of EXAMPLE 1-1 to give crude material (87 mg), which waε subjected to silica gel chromatography (eluting with chloroform:acetone = 4:1) to give allyl (IR, 5S, 6S)-2-[ (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3- fluoro-l-propenyl]pyrrolidin-4-ylthio]-6-[ (R)-1-hydroxy ethyl]-l-methγl-l-carbapen-2-em-3-carboxylate (24 mg, yield:11 %) .

NMR(CDC1 ₃ ) δ:

1.23(d,3H), 1.38(d,3H), 1.75(m,lH), 2.57(m,lH), 3.10(dd,lH), 4.20(m,2H), 4.95(d,lH), 5.10-5.50(m,5H), 5.80(m,4H)

EXAMPLE 3-2

Allyl (lR,5S,6S)-2-[ (2S,4S)-l-allyloxycarbonyl-2- [(E)-3-fluoro-l-propenyl]pγrrolidin-4-ylthio]-6-[ (R)-1- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylate (22

mg, 0.05 mmol), obtained in EXAMPLE 3-1, waε treated by the εame proceεε as that of EXAMPLE 1-2 to give (1R,5S, 6S)-2-[(2S, 4S)-2-[ (E)-3-fluoro-l-propenyl]pyrrolidin-4- ylthio]-6-[ (R)-l-hydroxyethyl]-l-methyl-l-carbapen-2-em- 3-carboxylic acid (12 mg, yield: 73 %).

NMR(D ₂ 0) δ:

1.07(d,3H), 1.16(d,3H), 2.50(m,2H), 3.20(m,lH), 3.30(dd,lH), 3.43-3.70(m,3H), 3.83(m,lH), 3.92- 4.13(m,2H), 5.05(d,lH), 5.10(d,lH), 5.62(m,lH)

EXAMPLE 4

(lR,5S,6S)-2-r(2S.4S)-2-f (Z)-3-methaneεulfonvi amino-1-propenyllpyrrolidin-4-ylthio1-6-f (R)-l-hydroxy ethyl1-l-methyl-l-carbapen-2-em-3-carboχylic acid

EXAMPLE 4-1

To a solution of allyl (IR,5S, 6S)-2-diphenoxyphos phoryloxy-6-[ (R)-l-hydroxyethyl]-l-methyl-l-carbapen-2- em-3-carboxylate (980 mg, 1.96 mmol), obtained by known proceεεes, and (2S, 4S)-allyloxycarbonyl-2-[ (Z)-3-methane sulfonylamino-l-propenyl]-4-mercaptopyrrolidine (640mg, 2.0 mmol) , obtained in REFERENCE EXAMPLE 4-7, in anhydrous acetonitrile (60 ml) waε added N,N-

diisopropylethylamine (0.37 ml, 2.12 mol). The above mixture waε εtirred for 6 hours and concentrated under reduced pressure at room temperature. Ethyl acetate was added thereto and the organic layer waε washed with water and then saturated aqueous sodium chloride, dried over anhydrous εodium sulfate and concentrated in vacuo. This crude product waε purified by column chromatography on εilica gel (eluting with chloroform:methanoi = 30:1) to give pure allyl (IR,5S,6S)-2-[(2S,4S)-l-allyloxy carbonyl-2-[ (Z)-3-methaneεulfonylamino-l-propenyl ] pyrrolidin-4-ylthio]-6-[ (R)-l-hydroxyethyl]-1-methγl-l- carbapen-2-em-3-carboxylate (560 mg, yield:49.0 %).

NMR(CDC1, 3)' δ:

1.25(d,3H), 1.33(d,3H), 1.70(m,lH), 2.60(m,lH), 2.95(ε,3H), 3.30(m,3H), 3.63(m,2H), 3.95(m,2H), 4.20(m,2H), 4.52(d,2H), 4.60-4.83 (m, 3H) , 5.20-6.02(m, 8H)

EXAMPLE 4-2

To an ice cooled εolution of allyl (IR,5S,6S)-2- [ (2S, 4S)-l-allyloxycarbonyl-2-[ (Z)-3-methaneεulfonyl amino-1-propenyl] yrrolidin-4-ylthio ]-6-[ (R)-1- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylate (1.58 g, 2.27 mmol) in methylenechloride (54 ml) were added succesεively diεtilled water (0.196 ml),

biε(tripenyiphoεphine)palladium (II) chloride (90 mg, 0.14 mmol) and tributyltin hydride (1.39 ml, 5.18 mmol). The above εolution was εtirred for 20 minutes at 0°C, extracted with distilled water and the aqueous layer waε waεhed with chloroform εeveral times, and filtered through 0.5 μm filter paper. The trace chloroform in the aqueous layer was removed thoroughly under reduced presεure and the aqueouε reεidue waε freezedried to give (1R,5S, 6S)-2-[ (2S, 4S)-2~[ (Z)-3-methanesulfonyl amino-1- propenyl]pyrrolidin-4-ylthio]-6-[ (R)-l-hydroxyethyl]-l- methyl-l-carbapen-2-em-3-carboxylic acid (1.1 g, yield: 89 %).

NMR (DMSO-d ₆ ) δ:

1.05(m,6H), 1.55(m,lH), 2.50(m,lH), 2.90- 3.30(m,4H), 3.50-3.80(m,4H),3.90 (s, 3H), 4.01 (m, 1H), 4.05(m,lH), 4.30(m,lH), 5.70(m,2H), 7.10(brs.lH)

EXAMPLE 5

(1R,5S,6S)-2-Γ(2S, 4S)-2-r (E)-3-methanesulfonyl amino-2-methyl-l-propenyl1pyrrolidin-4-ylthio1-6-[(R)-1- hydro yethyl ]-l-methyl-l-carbapen-2-em-3-carboxylic acid.

EXAMPLE 5-1

Allyl (IR, 5S, 6S)-2-[ ( 2S, 4S ) -l-allyloxycarbonyl-2-

[(E) -3 -methanesulf onylamino-2-methyl-l-propenyl ] pyrrolidin-4-ylthio ] -6- [ ( R ) -l-hydroxyethyl ] -1-methyl-l- carbapen-2-em-3-carboxylate (1.58 g, yield: 47.8 %) was obtained from allyl (IR, 5S, 6S) -2-diphenoxyphosphoryloxy-

6-[ (R)-l-hydroxyethyl ]-l-methyl-l-carbapen-2-em-3- carboxylate (2.97 g, 5.94 mmol) obtained by known processeε, and (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3- methaneεulfonylamino-2-methyl-l-propenyl]-4-mercapto pyrrolidine (1.89 g, 5.66 mmol) obtained in REFERENCE

EXAMPLE 5-7 by the same process aε that of EXAMPLE 4-1.

NMR(CDC1 ₃ ) δ

1.25(d,3H), 1.30(d,3H), 1.65(m,1H), 1.70(ε,3H),

2.60(m,2H), 1.90(ε,3H), 3.30(m,3H), 3.60 (m, 3H), 3.98(m,lH), 4.20(m,2H), 4.50(d,2H), 4.58-4.82 (m, 3H) , 5.20-5.50(m,5H), 5.90(m,2H)

EXAMPLE 5-2

(lR,5S,6S)-2-[(2S, 4S)-2-(E)-3-methanesulfonyl amino-2-methyl-l-propenyl]pγrroiidin-4-ylthio]-6-[ (R)-l- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylic acid (1.1 g, yield:89 %) was obtained from allyl (1R,5S,6S)- 2-[ (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3-methanesulfonyl amino-2-methyl-l-propenyl]pyrrolidin-4-ylthio]-6-[ (R)-l-

hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylate (1.58 g, 2.71 mmol) obtained in EXAMPLE 5-1, bis (triphenylphospine)palladium (II) chloride (90 mg, 0.14 mmol), and tributyltinhydride (1.39 ml, 5.18 mmol) by the same procesε aε that of EXAMPLE 4-2.

NMR(DMSO-d ₆ ) δ :

1.10(m,6H), 1.50(m,lH), 1.70(s,3H), 2.40mε,lH), 2.87(s,3H), 3.01-3.30(m,4H), 3.50(ε,3H), 3.70-4.10 (m,3H), 5.48(d,lH), 7.23(t.lH)

EXAMPLE 6

(IR.5S, 6S)-2-r (2S, 4S)-2-r (E)-3-(N, N-dimethylsul famoylamino-2-methyl-l-propenyl , pyrrol idin-4-yl thio 1-6- [ (R) -l-hydroxyethyl 1-l-methyl-l-carbapen-2-em-3- carboxylic acid

EXAMPLE 6-1

Allyl ( IR, 5S, 6S ) -2- [ ( 2S, 4S ) -l-allyloxycarbonyl-2- [ (E ) -3- ( N, N-d i me thylεu If amoylamino ) -2-methyl-l- propenyl ] yrrol idin-4-yl thio ] -6- [ ( R ) -l-hydroxyethyl ] -l- methyl-l-carbapen-2-em-3-carboxylate (360 mg, yield:

34.1 %) waε obtained from allyl (IR, 5S, 6S)-2- diphenoxyphosphoryloxy-6-[ (R ) -l-hydroxyethyl ]-l-methyl-

l-carbapen-2-em-3-carboxylate (630 mg, 1.72 mmol) obtained by known processeε, (2S, 4S)-l-aliyloxycarbonyl- 2-[(E)-3-(N, N-dimethylεulfamoylamino)-2-methy1-1- propenyl]-4-mercaptopyrrolidine (902 mg, 1.81 mmol) obtained in REFERENCE EXAMPLE 6-4, and N,N-diisopropyl ethylamine (0.33 ml, 1.89 mmol) by the same process as that Of EXAMPLE 4-1.

NMR(CDC1 ₃ ) δ:

1.25(d,3H), 1.30(d,3H), 1.70(m,lH), 1.72(s,3H),

2.50(m,2H), 2.80(s,6H), 3.30(m,3H), 3.58(d,2H),

4.00(m,lH), 4.22(m,2H), 4.50-4.87(m, 3H), 5.19-5.45 (m,5H), 5.90(m,2H)

EXAMPLE 6-2

(lR,5S,6S)-2-[ (2S, 4S)-2~[ (E)-3-(N,N-dimethylsul famoylamino-2-methy1-1-propenyl-4-ylthio]-6-[ (R)-l- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylic acid (164 mg, 56.9 mmol) waε obtained from allyl (1R,5S,6S)- 2-[ (2S,4S)-1-allyloxycarbonyl-2-[ (E)-3-( -N-dimethylsul famoylamino-2-methyl-l-propenyl]pyrrolidin-4-ylthio]-6- [ (R)-l-hydroxyethyl ]-l-methyl-l-carbapen-2-em-3- carboxylate (360 mg, 0.59 mmol) obtained in EXAMPLE 6-1, distilled water (0.043 ml), bis(triphenylphosphine) palladium(II) chloride (20 mg, 0.03 mmol), and

tributyltin hydride (0.303 ml, 1.13 mmol) by the same process aε that of EXAMPLE 4-2.

NM (CDCl-) δ:

1.07(d,3H), 1.15(d,3H), 1.40(m,lH), 1.62(ε,3H), 2.40(m,lH), 2.80(ε,6H), 2.95-3.50(m, 4H) , 3.70-4.10 (m,4H), 5.50(d,lH), 7.40(t,lH)

EXAMPLE 7

. IR, 5S, 6S)-2-r (2S, 4S)-2-f ( E ) -3- ( 4-morphOlinoεul f onylamino ) -2-methyl-l-propenyl 1 pyrrolidin-4-ylthio 1 -6- T (R)-l-hvdroχyethyl 1-l-methyl-l-carbapen-2-em-3- carboxylic acid

EXAMPLE 7-1

Allyl (IR, 5S, 6S)-2-[ (2S, 4S)-l-allyloxycarbonyl-2- [ (E)-3-(4-morpholinoεulfonylamino)-2-methyl-l- propenyl ] pyrrolidin-4-ylthio ] -6- [ ( R ) -l-hydroxyethyl ] -1- methyl-l-carbapen-2-em-3-carboxylate 583 mg (yield: 45%) waε obtained from allyl (IR, 5S, 6S)-2-diphenoxyphoε phoryloxy-6-[ (R ) -l-hydroxyethyl ]-l-methy l-l-carbapen-2- em-3-carboxylate (1.04 g, 2.08 mmol) obtained by well known proceεεeε, (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-3-

( 4-morpholinosulf onylamino)-2-methyl-l-propenyl ]-4-

mercaptopyrrolidine (800 mg, 1.98 mmol) obtained in REFERENCE EXAMPLE 7-3, and N,N-diisopropylethylamine (0.38 ml, 2.18 mmol) by the εame proceεε aε that of EXAMPLE 4-1.

NMR(CDCl-) δ:

1.24(d,3H), 1.34(d,3H), 1.69(m,lH), 1.70(ε,3H), 2.50(m,lH), 3.20(m,7H), 3.58(d,2H), 3.75(m,4H), 3.99(m,lH), 4.20(m,2H), 4.40-4.80 (m, 5H) , 5.20-5.40 (m,5H), 5.89(m,2H)

EXAMPLE 7-2

(IR, 5S, 6S)-2-[ (2S, 4S)-2-[ ( E ) -3- ( 4-morpholinoεul fonγlamino)-2-methyl-l-propenyl] pyrrol idin-4-yl thio] -6- [ (R) -l-hydroxyethyl ]-l-methyl-l-carbapen-2-em-3- carboxylic acid (354 mg, yield: 75 %) waε obtained from allyl ( IR, 5S , 6S ) -2- [ ( 2S , 4S ) -l-allyloxycarbonyl-2- [(E) -3- ( 4 -mo lpolynoεul fonyl amino ) -2-methyl-l-propenyl ] pyrrolidin-4-yl thio ] -6- [ ( R ) -l-hydroxyethyl ] -1-methyl-l- carbapen-2-em-3-carboxylate (580 mg, 0.89 mmol) obtained in EXAMPLE 7-1, diεtilled water (0.065ml), biε

(triphenylphoεphine) palladium(II) chloride 30 ml (0.045 mmol), and tributyltin hydride (0.46 ml, 1.71 mmol) by the εame process as that of EXAMPLE 4-2.

NMR(DMSO-d ₆ ) δ:

1.06(d,3H), 1.14(d,3H), 1.40(m,lH), 1.61(S,3H), 2.40(m,lH), 2.90-3.50(m,8H), 3.69(m,4H), 3.7-4.09 (m, 4H) , 5.4(d,lH), 7.39(t,lH)

EXAMPLE 8

.IR,5S,6S) -2-f .2S, 4S)-2-f (E)-2-chloro-3-methaneεul fonyiamino)-1-methyl-l-propenyl1pyrrolidin-4-vithio1-6- T (R)-l-hydroxyethyl 1-l-methyl-l-carbapen-2-em-3- carboxylic acid

EXAMPLE 8-1

Allyl (IR, 5S, 6S)-2-[ (2S, 4S )-l-allyloxycarbonyl-2- [(E) -2 -chl oro-3 -methane εulf onylamino-l-propenyl ] pyrrolidin-4-ylthio ] -6- [ ( R ) -l-hydroxyethyl ] -1-methyl-l- carbapen-2-em-3-carboxylate (0.706 g, yield: 42 %) waε obtained from allyl ( IR, 5S, 6S) -2-diphenoxyphosphoryloxy-

6-[ (R)-l-hydroxyethyl ]-l-methyl-l-carbapen-2-em-3- carboxylate (1.25 g, 2.78 mmol) obtained by well known processeε, (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-2-chloro-3- methaneεulfonylamino)-l-propenyl ]-4-mercaptopyrroiidine (0.96g, 2.7mmθl) obtained in REFERENCE EXAMPLE 8-8, and

N,N-diisopropylethylamine (0.28 ml, 2.9 mmol) by the same procesε aε that of REFERENCE EXAMPLE 4-1.

NMR(CDC1 ₃ ) δ:

1.21(d,3H), 1.30(d,3H), 1.70(m,lH), 2.59(m,lH), 2.90(S,3H), 3.30(m,3H), 3.60(m,3H), 4.20(m,2H), 4.45- 4.80(m,5H), 5.10-5.45(m,5H), 5.90(m,2H)

EXAMPLE 8-2

(IR,5S, 6S)-2-[ (2S,4S)-2-[(E)-2-chloro-3-methanesul fonylamino)-l-propenyl]pyrrolidin-4-ylthio]-6-[ (R)-l- hydroxyethyl]-l-methyl-l-carbapen-2-em-3-carboxylic acid (0.36 g, yield:64.7 %) waε obtained from allyl (1R,5S, 6S)-2-[ (2S, 4S)-l-allyloxycarbonyl-2-[ (E)-2- chloro-3-methane εulfonylamino)-1-propenyl]pyrrolidin-4- ylthio]-6-[ (R)-l-hydroxyethyl]-l-methyl-l-carbapen-2-em- 3-carboxylate (0.7 g, 1.16 mmol) obtained in EXAMPLE 8- 1, bis(triphenylphosphine)palladium(II) chloride (42 mg, 0.06 mmol), and tributyltin hydride (0.73 ml, 2.32 mmol) by the same proceεε aε that of EXAMPLE 4-2.

NMR(DMSO-d ₆ ) δ:

1.05(m,6H), 1.50(m,lH), 2.40(m,lH), 2.84(s,3H), 3.02-3.40(m,4H), 3.80-4.10 (m, 3H) , 5.59(d,lH), 7.30(t,lH)