Background of the Invention Field of the Invention

The present invention relates to novel carbapenem

( 3-pyrrolidinylthio-l-azabicyclo[3,2,0 ]hept-2-ene-7-one-

2-carboxylic acid compounds) derivatives, processes for preparing the derivitiveε and antibiotics containing such compounds as active ingredients.

Description of the Related Art

Thienamycin, (J. Am. Chem. Soc. Vol. 100, p.6491( 1978 ) ) isolated from the fermentation of the soil

Actinomycets, Streptomyces cattleya has antibacterial activity against gram negative gram positive bacteria.

Thienamycin has strong antibacterial activities, however, thienamycin itself is chemically unstable and has been reported to be decomposed in vivo by enzymes such as renal dehydropeptidase I (hereinafter referred to DHP-I), whereby the antibacterial activities decrease, and the urinary recovery is low (Antimicrob.

Agents Chemother., vol 22, p.62(1982); ditto, vol. 23, p.300(1983) ) .

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. No. 4,194,047). However, like thienamycin, imipenem is

likely to be decomposed by DHP-I in the human kidney. 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), p. 1(1983) ) .

However, in the 1980'ε, there has been a growing demand for otable carbapenem compounds and, various carbapenem compounds having a 1 β-methyl carbapenem basic skeleton have been developed. Sumitomo Co. has synthesized meropenem which is much more stable against DHP-I than imipenem and has a broad antibacterial spectrum (EP No.126587; Antimicrobial Agents and Chemotherapy vol. 33, No. 7, 1009(1989); Journal of Antibiotics, vol. 43, No. 5, p.519(1990) ) . Further Lederle has synthesized L.J.C. 10627 which shows improved antibacterial activities and stabilities against DHP-I.

Prior art disclosed in EP No. 182213, EP No. 243686, and EP No. 411664 is well known. These references disclose carbapenem derivatives having an amidinoalkyl, ureiren alkyl, carbamoyl alkyl, alkyloxy iminoalkyl and iminoalkyl at the 2' -position of pyrrolidine whose 4' position is linked as S( sulfur) to the carbapenem skeleton. β-lactam antibiotics exhibit selective antibacterial activity and show little or no toxic

effects against animal cells. Therefore, they are widely used in treatment of infectious disease caused by bacteria. Especially useful one carbapenem compounds which have a broad antibacterial spectrum against gram positive and gram negative, and lower toxicity than other antibacterial agents.

However, it is still desirable to improve antibacterial activities and stability against DHP-I, and to reduce the toxicity against the kidneys and side effects against the central nervous system.

The present invention provides novel carbapenem derivatives having an alkyloxyimino, a hydroxyimino or a hydrazono group at 2' position of pyrrolidine, which show having excellent antibacterial activities particularly against gram positive and gram negative together with high stability against DHP-I.

Summary of the Invention The present invention provides a compound of the formula (I)

M

wherein Ri is a hydrogen atom or a methyl group, R _j is a hydrogen atom, a metal or a nonmetal salt group, or a carboxy protecting group.

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 aliphatic salt such aε triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N',N'- dibenzylethyleneamine salt, dibenzylamine salt; an acid addition salt, for example, an inorganic salt such aε hydrochloride, hydrobromide, sulfate, phosphate; an organic salt such as formate, acetate, trifluoracetate, malate, tartrate, methanesulfonate, benzenesulfonate, toluenesul onate; or intermolecular quaternary salt.

The carboxy protecting group may, for example, be a lower alkyl 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 group such aε an acetoxymethyl group, a propionyloxymethyl group, a butyryloxymethyl group, a valeryloxymethyl group, a pivaloyloxymethyl group, a hexanoyloxymethyl group; a lower alkanesulfonyl(lower)alkyl group such aε a 2-methylethyl group; a mono(or di , or tri )halo(lower)alkyl group such as a 2-iodomethyl group, a 2,2,2-trichloroethyl group; a lower alkoxycarbonyloxy(lower)alkyl group such as a methoxyca rbonyloxyme thyl group , an e t h ox y ca r bo n yl o xym e t h yl g r o up , a p o po yc a r bony 1 oxyme t hy 1 group , a t - butoxycarbonyloxymethyl group, a l-(or 2-)

methoxycarbonyioxyethyl group, an l- (or 2- )ethoxycarbonyloxyethyl group; a lower alkyl group capable of having phthalidenyl(lower)alkyl group such aε a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a t- butyl group, a pentylgroup, a hexyl group; a lower alkenyl group εuch aε a vinyl group, an allyl group, an iεopropenyl group; a lower alkynyl group such as an ethynyl group, propynyl group; an al( lower)alkyl group capable of having at least more than one substituent such as a benzyl group, a 4-methoxybenzyl group, a 4- nitrobenzyl group, a 2-nitrobenzyl group, a trityl group, a benzhydryl group, a bis(methoxyphenyl)methyl group, a 3, -dimethoxybenzyl group, a 4-hydroxy-3, 5-di- t-butylbenzyl group; an aryl group capable of having at least more than one substituent such as a phenyl group, a -chlorophenyl group, a tolyl group, and a t- butylphenyl group, a xylyl group, a meεityl group, a guanyl group; a phthalidyl group. "Lower" means that the number of carbon is 1 to 6. The "Lower alkyl" includes a normal or a side alkyl such aε methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, and hexyl. "Lower alkoxy" also includes a normal alkoxy or a side alkoxy such aε methoxy, ethoxy, n-propoxy, iεopropoxy, n-butoxy, sec-butoxy, t-butoxy.

R3 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

aiiphaticacyl subεtituted with an aromatic group, an aliphaticacyl substituted with a heterocyclic group, all of which are derived from a carboxylic acid, a carbonic acid, a sulfonic acid, or a carbamic acid. The aliphatic acyl includes a saturated or unsaturated acyclic or cyclicacyl, for example, a lower alkanoyl such as formyl, acetyl, propionyl, butyl, isobutyryl, valeryl, isovaleryl, pivaroyl, and hexanoyl ; a lower alkylsulfonyl such aε meεyl, ethylεulfonyl , propylsulfonyl, isopropylsulfonyl, butylsulfonyl, isobutylsulfonyl, pentylsulfonyl, and hexylsulfonyl; a carbamoyl; an N-alkylcarbamoyl such as methylcarbamoyl, and ethylcarbamoyl; a lower alkoxycarbonyl such aε methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, and t-butoxycarbonyl; a lower alkenyloxycarbonyl such as vinyloxycarbonyl , allyloxycarbonyl, and isoprophenyloxycarbonyl; a lower alkenoyl such as acryloyl, metacryloyl, and crotonoyl; a cyclo( lower )alkanecarbonyl such as cyclopropanecarbonyl, cyclopentanecarbony1, and cyclohexanecarbonyl .

The aromatic acyl includes an aroyl such aε benzoyl, toluyl, and xyloyl; an N-arylcarbamoyl such aε N- phenylcarbamoyl, N- tolylcarbamoyl , and N- naphtylcarbamoyl; an arenεulfonyl εuch aε benzensulfonyl, and tosyl. The heterocyclic acyl includes a heterocyclic acyl such aε proyl , nicotinoyl, isonicotinoyl, thiazolylcarbonyl, thiadiazolylcarbonyl , and tetrazolylcarbonyl .

The aliphatic acyl substituted with an aromatic group includes an aralkanoyl, for example, a phenyl (lower )alkanoyl such aε phenylacetyl , phenyl propionyl , and pheny1 hex anoyl ; an a ra l ko xyc arb ony l , fo r exa mpl e , a phenyl(lower)alkoxycarbonyl εuch aε a benzyloxycarbonyl, and penetyloxycarbonyl; an aryloxyalkanoyl, for example, a phenoxy(lower)alkanoyl εuch aε phenoxyacetyl and phenoxypropinoyl. The aliphatic acyl substituted with a heterocyclic group includes a heterocyclic(lower)alkanoyl , for example, a heterocyclic(lower)alkanoyl εuch aε thienylacetyl, i idazolylacetyl, furylacetyl, tetrazolylacetyl, thiazolylacetyl, thiadiazolylacetyl, thienylpropionyl, and thiadiazolylpropionyl.

The above-mentioned acyl group can be substituted with one or more substitutents selected from the group consisting of a lower alkyl such as methyl, ethyl, propyl, isopropyl, butyl, pentyl , and hexyl; a halogen such aε chlorine, bromine, iodine, and fluorine; a lower alkoxy εuch aε methoxy, ethoxy, propoxy, iεopropoxy, butoxy, pentyloxy, and hexyloxy; a lower alkylthio such aε methylthio, ethylthio, propylthio, iεopropylthio, butylthio, pentylthio, and hexylthio; nitro. The preferable acyl group having εuch subεtituteε are selected from the group consiεting of a mono(or di , or tri)halo alkanoyl such aε chloroacetyl, bromoacetyl, dichloroacetyl, and trifluoroacetyl; a mono(or di or tri )haloalkoxycarbonyl such aε chloromethoxycarbonyl ,

di chl o romethoxyca rbonyl , and 2 , 2 , 2 - trichloroethoxycarbonyl ; a nitro(or halo, or lower alkoxy) ; an a r a 1 k ox yc a bo ny 1 such aε nitrobenzyloxylcarbonyl , chlor obenzyloxycarbonyl , methoxybenzyloxycarbonyl , monofor di , or tr i ) halo (lower )aklyl εulf onyl such aε fluoromethylsulfonyl , difluoromethylεulfonyl, trif luoromethylsulf onyl , and trichloromethylsulfonyl .

The "imino protecting group" iε preferably (C _j -C^) alkenyloxycarbonyl, phenyl ( C _j -C,, )alkoxycarbonyl , o- nitro(or m-nitro, or p-nitro)benzyloxycarbonyl , and o- methoxy(or m-methoxy, or p-methox )benzyloxycarbonyl .

Rι_ represents a hydrogen atom, lower alkyl group, a hydroxy group, a cyano group, a halogen group such aε chlorine, bromine, iodine, and fluorine. _j represents a hydroxy group, a lower alkoxy group, a protected or unprotected amino group, or one of the following general formula (l)-(4);

wherein R _j and R ₇ are independently either a hydrogen atom or a lower alkyl group. R _j is a hydroxy group, a cyano group, a halogen atom such as chlorine, bromine, iodine, fluorine, or a heterocyclic group of a 5-or 6- membered ring containing 1 to 4 heteroatoms which may be optionally substituted with an appropriate substituent, a protected or unprotected amino group, a the following general formula,

NH R ₉

(where R _j is a lower alkylsulfonyl such as methylsulfonyl, halo(CJ-C )alkylsulfonyl, a phenyl(C _j -

C4)alkylsulfonyl such as a p-toluenesulfonyl , and a N,N- ( lower)dialkylεulfamoyl such as a N,N-dimethylεulfamoyl.

0

!

(2) — 0(CH ₂ ) _n — C — OR ₆ wherein _j is the same as defined above.

^R 7 wherein R _j , R7 are the same as defined above.

| ⁶

(4) — N — R _1D wherein R _j is the same as defined above, R _j is a lower alkyl group, a lower alkylsulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C^-C^ )alkylsulfonyl group, an N- ( lower) alkylsulfamoyl group, an N,N- (lower)dialkylsulfamoyl group, a heterocyclic group of a 5-or 6-membered ring containing 1 to 4 heteroatoms which may be optionally substituted by an appropriate substituent, a protected or unprotected amino group, of the following general formula, x=^Rx A 0 ORr wherein R^ is a — C 1 — OR^ , a — CINI — 7 , a halogen atom, a hydroxy group, a cyano group, the group of which can be substituted at one of the o-, m- , or p-positions of a phenyl group, and R _j , R7 are the same as defined

above .

The heterocyclic group of a 5-or 6-membered ring containing 1 to 4 heteroatoms includes an unsaturated 5 or 6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolidiyl, imidazolyl(εuch as 2-imidazole) , imidazolinyl(such as 2- imidazolinyl) , pyrazolyl, pyrazolinyl, pyridyl, pyridyl N- oxide, pyri din i o, di hyd ropyridyl , tetrahydropyridyl(such aε 1,2,3,6-tetrahydropyridyl) , pyrimidinyl, pyrimidinio, pyrazinyl, pyrazinio, pyridazinyl(such as 1,3,5-triazinyl- , 1,2,4-triazinyl and 1,2,3-triazinyl) , tetrahydrotriazinyl(such as l,2,5,6-tetrahydro-l,2,4-triazinyl, 1,4,5,6-tetrahydro- 1,2,4-triazinyl) , triazinio, triazolyl(such aε 1H-1,2, - triazolyl, 1H-1 , 2 ,3-triazolyl, and 2H-1,2,3-triazolyl) , triazolio, tetrazinyl, tetrazinio, tetrazolyl(such as lH-tetrazolyl and 2H-tetrazolyl ) , tetrazolio; an unsaturated 5-or 6-membered heteromonocyclic group containing 1 to 3 nitrogen atom and 1 to 2 sulfur atoms, for example, thiazolyl, thiazolyl, isothiazolyl, thiadizolyl(such aε 1,2, 3-thiadiazolyl, 1,2,4- thiadiazolyl , 1, 3 ,4 -thiadiazolyl, and 1,2,5- thiadiazolyl) , thiadiazolio, thiazolinyl(such as 2- thiazolinyl) , and dihydrothiazinyl; an unsaturated 5-or 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxadiazolyl( such aε 1,2, 4-oxadiazolyl, 1,3,4- oxadiazolyl, and 1,2,5-oxadiazolyl) ; a saturated 5-or 6- membered heteromonocyclic group containing 1 to 4 oxygen

atoms, for example, dioxolanyl(such aε 1,3-dioxolanyl) , dioxanyl(such aε 1,3-dioxanyl, and 1,4-dioxanyl) ; a saturated 5-or 6-membered heteromonocyclic group containing 1 to 4 sulfur atoms, for example, dithiolanyl(such as 1,3-dithiolanyl) , dithianyl(such as 1,3-dithianyl and 1, -dithianyl) ; and a "quaternized" group of the above-mentioned group of an unsaturated 5- or 6-membered heteromonocyclic group containing nitrogen atoms. 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(such as methylamino, ethylamino, propylamino, isopropylamino, butylamino, and hexylamino); ureido(lower)alkyl(such as ureidomethyl, ureidoethyl, ureidopropyl, ureidohexyl); carbamoyl; a lower alkyl as defined above; an amino(lower)alkyl(such as aminomethyl, aminoethyl, aminopropyl, aminobutyl, and aminohexyl); a ,hydroxy(lower)alkyl and protected hydroxy(lower)alkyl; an azido(lower)alkyl(such aε azidomethyl, azidoethyl, and azidohexyl); a halo(lower)alkyl( such aε chloromethyl, bromomethyl, iodoethyl, bromopropyl, and bromohexyl). The "protected hydroxy( lower)alkyl" includes a phenyl(C _j -C^ )alkoxycarbonyloxy(C -C^ )alkyl having a nitro group; a triphenyl(C _j -C^ )alkoxy(C _j -C^ )alkyl having a nitro group; a tri(C _j -C^ )alkylsilyloxy(Ci-C. )alkyl having a nitro group.

If a thiazolyl has the group of an amino or protecting amino group at the 2-position, or an 1,2,4- oxadiazolyl having the group of an amino or protecting amino group at the 3 position, the above-mentioned heterocyclic groups have "tautomeric isomerε" aε shown in the following formula;

wherein R^ is an amino or a protected amino group, ^ is an imino or a protected imino group.

The scope of the present invention includes all the tautomeric isomers mentioned above. However, the desired product and the intermediate products containing "tautomeric isomers" are, for example, 2-amino(or protected amino) -thiazolyl is shown in the following formula,

3-amino(or protected amino) -1,2,4-oxadiazolyl is shown in the following formula,

wherein the "protected amino group" includes the amino group which has one of the group of C1-C4 alkoxycarbonyl

such as t-butyloxycarbonyl; a halo(Cl-C3)alkoxycarbonyl such as 2-iodoethyloxycarbonyl, and 2,2,2- trichloroethyloxycarbonyl ) ; a substituted or unsubstituted al(lower)alkyloxycarbonyl; a substituted or unsubstituted phenyl(Cl-C3)alkyloxycarbonyl such aε benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o- nitrobenzyloxycarbonyl, and p-nitorbenzyloxycarbonyl; tri(Cl-C4)alkylsilyl such as trimethylsilyl and t- butyldimethylsilyl attached to an amino group, wherein n is an integer of 1 to 4.

Also the present invention provides a process for preparing a compound of the following (IV),

(IV) wherein R _j , R _j , R _j , R^ , and R _j are the same as defined above, R^ is not difined above, reacting a compound of the following formula (II),

(II) wherein R _j and R _j is the same as defined above, R^ is hydrogen or a hydroxyl protecting group, with a compound

of the following formula (III)

(III) wherein R3, R^ and R5 are the same aε defined above, and provide useful steps to synthesize the above compound III. Further, the present invention confirms that a compound of the general formula (I) shows effective antibacterial activities and low toxicity (Tables 1, 2, 3).

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

which is systematically referred to aε 7-oxo-l- azabicyclo[ 3,2,0]hept-2-ene-e-carboxylic acid or 1- azabicyclof 3,2,0]hept-2-ene-7-one-2-carboxylic acid. In thiε εpecification, this basic structure will be referred to as a 1-carbapen-2-em-3-carboxylic acid, the numbers based on a commonly used carbapenem of the following formula;

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

Accordingly, among compounds of the formula (I), a group of compoundε having preferred stereo- configurations are represented by the following formula

wherein R _j , R _j , R _j , R^ and R _j are as defined above.

The 2' - (N-substituted)pyrrolidin-4' -yl-thio group also includes all prossible isomers based on the asymmetrical carbon atoms at the 2- and 4-positionε of the pyrrolidine structure. Among these isomers, preferred compoundε are of a (2'S,4'S) configuration and a (2'R,4'S) configuration.

Among the compounds of the formula (I-a), a group of compounds wherein R, is a methyl group, _j is a

( lower)alkoxide

/ group R ₆ of N group is a hydrogen atom or a

\

MO

(lower)alkylgroup, O i s lowe r ) alkyl or

( lowe r ) alkylsulfonyl , phenyl ( Cl - C4 ) alkylsul f onyl or (wherein Rll is the same aε defined above), have particularly excellent antibacterial activities.

Further, with respect to the double bond of the 2- (N-substituted imino) group, cis(Z) and tranε(E) geometric isomers are present. These isomers are also included in the present invention. Of these isomers, the (E)-isomer haε particularly excellent antibacterial activitieε.

The compound of formula (II) should be converted to its reactive derivativeε before reacting with the formula (III). That is, the compound of formula (II) iε added to the inert organic εolvent and reacted with activating agents under alkali conditions to obtain the activating derivativeε of the formula (Il-a),

(Il-a) wherein R, , R _j , R^ iε the same aε defined above and A iε a leaving group.

The activating reagent to be used for the reaction may, for example, be an acid anhydride such aε methanesulfonic anhydride, trifluromethanesulfonic anhydride, p -tolueneεulfonic anhydride, and trifluoroacetic anhydride; or an acid chloride such aε methanesulfonyl chloride, p-toluenesulfonyl chloride or diphenyl chlorophosphate. Particulary preferred iε diphenylchlorophosphate.

In the formula (Il-a), A is a leaving group such as tri fluoroacetoxy, methanesulfonyl oxy , trifluoromethanesulfonyloxy, p-toluenesulfonyloxy or diphenoxyphosphoryloxy. Particularly preferred iε a diphenoxyphosphoryloxy group.

The inert organic solvent to be used for the reaction may, for example, be methylene chloride, chloroform, carbon tetrachloride, dichloroethane, trichloroethylene, diethylether, tetrahydrofuran, dioxane, benzene, toluene, chlorobenzene, acetone, ethylacetate, acetonitrile, N,N-dimethylformamide, hexamethylphosphorictriamide or a mixture of such solvent. Particulary preferred is acetonitrile, benzene, toluene, the mixture of toluene and benzene, or the mixture of toluene and ethylacetate.

The baεe to be used for the reaction may, for example, be trimethylamine, triethylamine, N,N- di iεopropylethylamine, N -methylmorpholine, N- methylpyrrolidine , N -methylpiperidine , N,N- dimethylaniline, 1 , 8- diazabicyclo [ 5 ,4 ,0 ]endec -7 - ene(DBU), or 1, 5-diazabicyclo[ ,3,0] -non-5-ene(DBU) , or

pyridine 4-dimethylaminopyridine, picoline, lutidine, quinoline or isoquinoline. Particularly preferred is N,N-diisopropylethylamine and triethylamine.

With respect to the compound of the formula (II), it is well known that the 3,7-dioxo-l- azabicyclof 3,2,0]heptane ring system of the following formula (II) has a tautomeric relation with 3-hydroxy-7- oxo-1-azabicyclof3,2,0 ]hept-2-end ring system and these two ring systems are substantially the same. 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 the formula (II) .

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

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

The compound of the formula (II-a) may be reacted with the compound of the formula (III) without isolation. The reaction iε conducted using the above-

mentioned inert organic solvent and the base and from 1 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 the formula (III) are used per mol of the compound of the formula(II-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 75 hours.

Further, the compound of the formula (IV) can be prepared in one pot reaction from the compound of the formula (II), namely, without isolating the reactive derivative of the formula (Il-a). Thus, from 2 to 5 mol, preferably from 2.5 to 3.8 mol, the base is employed per mol of the compound of the formula (II). After completion of the reaction, usual treatment iε conducted to obtain a crude product of the 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, e.g., silica gel.

From the compound of the formula (IV), a compound of the formula (I) can be obtained, if necessary, by removing a protecting group for a hydroxyl group, an 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 are, for example,

by addition of a εolvent for decomposition; by chemical reduction using salt of an amine, a metal such as zinc amalgam, a chromic compound such as chloro chromouε, a formyl chromous together with an organic or inorganic acid such as acetic acid, propionic acid, hydrochloric acid, hydrosulfuric acid; or by catalytic hydrogenation using a platinum or palladium compound. For example, in the formula (IV), the protecting group of the hydroxyl group, amino group or the imino group iε an aralkyloxycarbonyl group such aε a benzyloxycarbonyl group or a p-nitrobenzyloxycarbonyl group, and the protecting group for the carboxyl group iε an aralkyl group such as a benzyl group, a p-nitrobenzyl group or a benzhydryl group. Such protecting groups can be removed by catalytic hydrogenation by means of a platinum catalyst such as platinum oxide, platinum wire or platinum black, or a palladium catalyst such as palladium black, 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, allylisopropenyl, such a protecting group can be removed by catalytic hydrogenation using a palladium ligand complex catalyst such aε palladium-carbon, palladium black, palladium hydroxide -carbon, palladium (II) chloride, tetrakis (triphenylphosphine) palladium ( 0) , biε(dibenzylidenylacetone) -palladium (0), di(l,2- bis(diphenylp oεpino (ethane) palladium, and tetrakis(triphenylphosphine)palladium (0) .

The εolvent useful for the catalytic hydrogenation includes, for example, water, methyl alcohol, ethyl alcohol, propylalcohol, tetrahydrofuran, dioxane, acetonitrile, acetic acid and the above 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 amino 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, ethylacetate, acetonitrile, methylenechloride, chloroform and the solvent mixture thereof. The allyl group-capturing agent may be, for example, sodium 2- ethylhexanoate, potassium 2-ethylhexanoate, pyridine, piperidine.

The reaction iε conducted usually within a temperature range of from -10° to 50°C, preferably from 0° to 30 C° using from O.ϋl 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 reactions for removing the protecting group, the compound of the formula (I) can be isolated by column chromatography loading on silica gel, adεorptive resin such as Diaion HP-29, or freeze drying or crystallization.

The compound of the formula (II) as the starting material, can be obtained by the Salzmann method (J. Am. Chem. Soc. Vol 102, 6161-6163, 1980) which iε incorporated herein by reference in the case that R ₁ iε a hydrogen atom, and by Shih method (Heterocycleε, Vol. 21, 29-40, 1984 or EP No. 272,455) which are incorporated herein by reference in the case where _j iε a methyl group.

The compound of the formula (III) as the starting material, can be obtained by the following scheme 1 or scheme 2.

-Rs

HS- \

Me

R3

(III -α)

The compound (6) of scheme 1 and compound (13) of scheme 2 are known and the method for preparing therefore was described in EP No. 182213, EP No. 243686 and U.S. Pat. No. 4,921,352 and are incorporated herein by reference. The thiol derivativeε of the formula

(III) or (III-a) can be obtained from the compound (6) or (13), and the methodε for preparing thereof are described in the following examples. Abreaction used are aε followε: Ac : acetyl

Boc : t-butoxycarbonyl

Cbz : benzyloxycarbony

Et : ethyl

Me : methyl Mε : methanesulfonyl

PMB : p-methoxybenzyl

PNB : p-nitrobenzyl

PNZ : p-nitrobenzyloxycarbonyl

TBDMS : t-butydimethylsilyl ε : p-toluene εulfonyl

Ph : phenyl

E : tranε z : cis

I. Antibacterial Testε The antibacterial activitieε were tested by an agar dilution method in accordance with the standard method of the Japan Society of Chemotherapy [Chemotherapy, Vol.

29,76-79 (1981)] which iε incorporated herein by reference. Imipenem was used as internal standard

material. After 10 ml of Mueller Hinton Broth was poured into the sterilized test tubes, one platinum loop of each test microorganism was inoculated and incubated overnight at 37°C. Staphylococcus aureuε was cultured in Trypticase Soy Broth inεtead of Mueller Hinton Broth. The antibacterial agent εolutionε were prepared by dissolving 5 mg of each antibacterial agent in sterilized distilled water to give the concentration of 1 mg/ml, and by preparing a two-fold dilution series to concentration of 0.25 μg/ml. After 1 ml of each of the antibacterial solutions was poured into each petri dish and sterilized, 9 ml of Mueller Hinton agar media which had been sterilized and cooled was added, mixed well, and solidified to prepare the agar plate media the for Minimal Inhibitory Concentration (MIC) Test. 0.11 ml of bacterial culture was poured into a sterilized test tube containing 10 ml of buffered saline gelatin (BSG) solution and thoroughly mixed. The agar plate containing the antibacterial agent was then inoculated with a bacterial suspension using a stamp, and cultured at 37°C for 18 hourε. After observing the growth of bacteria, MIC was conεidered to be the loweεt drug concentration at which there iε no growth. The reεultε are shown in table 1.

Table. 1-1 Minimal Inhibitory Concentration (MIC, μg/ml)

Table. 1-2 Minimal Inhibitory Concentration (MIC, μg/ml)

Table. 1-3 Minimal Inhibitory Concentration (MIC, μg/ml)

II. Stability to renal dehydropeptidase-I from porcine 300g of the swine kidney was homogenized and εuεpended in a Tris buffer (10mM Tris, 50mM NaCl, O.OlmM ZnCl ₂ , pH 8.0), and 10% Triton X-100 waε 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 diabiltered to remove Triton X-100. The diabiltered solution was brought to, 75% saturation of ammonium sulfate the resultant precipitates was collected by.

The above precipitateε were dissolved in a Tris buffer, and loaded on DEAE-Sepharose fast flow, and anion exchange chromatography was carried out to give the swine DHP-I. The above DHP-I was divided into 1ml portion of the concentration of 1 unit/ml and stored at 0°-70°C.

Glycyldehydrophenylalanie (GDP) and imipenem were employed as control compounds. Accordingly to Campbell's method (Methods Engymol . 19:722-729, 1970) incorporated herein -by reference, the decrease in absorbence due to enzyme reaction is observed, and can be used to determine the maximum hydrolysiε velocity. The stability to DHP-I is represented as the comparative hydrolysiε velocity to that of GDP.

Table 2. Stability to renal dehydropeptidaεe-1 from porcine

III. Toxicity Test After disεolving 300 mg of each antibacterial agent in the sterilized saline to 200 mg/ml, the antibacterial agent solution was prepared by a four-fold dilution series to 0.781 mg/ml using the sterilized test tube. After 0.2 ml of the antibacterial solution was administered to the tail vein of a 4-5 weeks old aged ICR mouse with 20 + lg weight and observed for 2 weeks. The LD _jβ was determined by probit analyεiε. The numberε of both male and female are five, reεpectively.

Table 3. LD^ mg/Kg

The compoundε of the preεent invention have excellent antibacterial activitieε against various gram positive bacteria and gram negative bacteria and are useful aε antibacterial agentε for treatment and prevention of human infectious diseases caused by such bacteria. Because of the broad antibacterial spectrum the compounds of the present invention may be used in the form of additives for animal food, preserving agents, and other sterilization and disinfection agentε for induεtrial use as well as medical use.

The compound of the present invention may be used in the form of drug formulation suitable for nonoral administration, oral administration, external administration; liquid formulation such as injection solutionε, syrups or emulsionε; solid formulation such aε tablets, capsules or granules; and external application formulations such aε ointments or εuppoεitionε.

Dosage varies 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 dose of the active ingredient to an adult iε from about 5 to 50 mg/kg, and a preferred daily dose to a child is 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 the present invention may be administered in combination with a DHP-I inhibiting agent such as cilastatin.

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

Examples. However, it should be understood that the present invention is by no means restricted by such specific examples.

In the Examples and Reference Examples, for the thin layer chromatography, silica gel 60 F _jj ^ (Merck) was uεed as the plate, and an ultraviolet detector or ninhydrin color development method KM _n O _j waε uεed aε a detecting means. As the silica gel for a column, silicagel 60 (Merck) was used, and UV spectrophotometer DMS 100S (Varian) waε used for detecting the UV absorbency. For high speed liquid chromatography an M- 352 (ACS) model was used. When the NMR spectrum waε measured in heavy dimethyl sulfoxide-d^ or heavy chloroform-d _j solution, tetramethylsiland (TMS) was used as the internal standard material, and when it waε measured in a deuterium oxide solution, 2,2-dimethyl-2- silapentane-5-sulfonate (DSS) was used. The measurement was conducted by Ac 2-00P (200 MHz:Bruker) model, and all δ values were represented by ppm.

The meaning of the abbreviation used in NMR measurement are as follows : s : singlet DHSO-d^ : dimethylsulfoxide-d^

CDCI3 : chlorform-di Dτθ : deuterium oxide J : coupling integer Hz : hertz

dd : double doublet m : multiplet REFERENCE EXAMPLE 1

(4R,5R,6S,8R) -p-nitrobenzyl- 4-methyl-6- ( 1- hydro yethyl )-l-azabicyclo[3,2,0]-hept-3,7-dione-2- carboxylate

REFERENCE EXAMPLE 1-1

To 100 ml of the mixture solution of n-comprised hexane/ethylacetate(l/3 ) waε added 0.982 g (2.51 mmol ) of (3S,4R) -3-[ (1R) - ( 1 -hydroxyethyl ) ] -4-[ (R) -l-methyl-3- diazo-3-p-nitrobenzyloxycarbonyl-2-oxo-propyl ] - azetidine-2-one , Rhodium (II) acetate dimer aε a catalyst, and the solution waε refluxed for 2 hourε.

After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the organic solvent, and the material produced in the form of a syrup was dried in a vacuum to give 0.867 g (95%) of (4R,5R,6S,8R) -p-nitrobenzyl -4 -methyl -6- ( 1- hydro yethyl ) -1 -azabicyclo [ 3 ,2 , 0 ] - hept - 3 ,7 -dione -2 - carboxylate. This compound is unεtable it should be used immediately after preparation.

NMR(CDC1 ₃ ) δ:

1.10(d,3H), 1.32(d,3H), 2.80(m,lH), 3.22(dd,lH), 4.20(dd,lH), 4.25(m,lH), 4.70(s,lH), 5.25(m,2H), 7.50(d,2H), 8.20(d,2H)

REFERENCE EXAMPLE 1-2

To 100 ml of the mixture solution of comprised toluene/ethylacetate(l/l) was added 0.982 g (2.51 mmol) of (3S,4R) -3-[ (1R) -(1-hydroxyethyl) ] -4-[ (R) -1-methyl-3-

diazo-3-p-nitrobenzyloxycarbonyl-2-oxo-propyl] - azetidine-2-one, Rhodium(II )acetate dimer aε a catalyst, and the solution was refluxed for 1.5 hourε. After the reaction, the reaction mixture was treated, as discribed in REFERENCE EXAMPLE 1-1, to give 0.611 g (67%) of (4R,5R,6S,8R) -p-nitrobenzyl-4-methyl-6-(1- hydroxyethyl )-l-azabicyclo[3,2,0]-hept-3,7-dione-2- carboxylate.

REFERENCE EXAMPLE 2 (4R,5R,6S,8R) -allyl-4-methyl-6- (1-hydroxyethyl ) -1- azabicyclo[3,2,0] -hept-3,7-dione-2-carboxylate

REFERENCE EXAMPLE 2-1

To 120 ml of the mixture solution of comprised n- hexane/ethylacetate(l/3) waε added 0.982 g (2.51 mmol) of (3S,4R) -3-[ (1R) - (1-hydroxyethyl) ] -4- [ (R) -l-methyl-3- diazo-3-allyloxycarbonyl-2-oxo-propyl ] -azetidine-2-one,

Rhodium (II) acetate dimer aε a catalyst, and the solution was refluxed for 2.5 hours. After the reaction, the mixture solution waε concentrated under reduced pressure to evaporate the organic solvent, and the produced material in the form of a syrup waε dried in a vacuum to give 0.632 g (87.3%) of (4R,5R,6S,8R) -allyl-4- methyl-6- (1-hydroxyethyl ) -1 -azabicyclo[ 3,2,0] -hept-3,7- dione-2-carboxylate. This compound is also unstable so it must be uεed immediately after preparation.

NMR(CDC1 ₃ ) δ:

1.10(d,3H), 1.32(d,3H), 2.81(m,lH), 3.21(dd,lH), 4.21(dd,lH), 4.23(m,lH), 4.66(d,2H), 4.69(ε,lH), 5.29- 6.20(m,3H)

REFERENCE EXAMPLE 3

( 5R ,6S, 8R ) - p- ni trobenzyl- 6- (1 -hydroxyethyl ) -1 - azabicyclo [3,2,0] -hept-3 ,7-dione-2-carboxylate REFERENCE EXAMPLE 3-1 To 140 ml of benzene waε added 7 g (18.6 mmol) of (3S,4R)-3-[ (1R) -(1 -hydroxyethyl ) ] -4 - [ 3- diazo- 3- (p - nitrobenzyloxycarbony1 ) -2-oxo-propyl ] -azetidine-2-one and Rhodium( II )acetate dimer aε a catalyεt, and the solution was refluxed for 2 hourε. After the reaction, the mixture solution was concentrated under reduced presεure to evaporate the organic εolvent, a εraall volumn of methylene chloride waε added to the reεidue material and stirred at room temperature to dissolve. Diethylether was added dropwise to the reaction mixture to cryεtallize and the mixture waε stirred for 15 minutes. The material waε filtrated and dried in a vacuum to give 3.95 g (61.0%) of ( 5R,6S,8R) -p- nitrobenzyl-6- (1-hydroxyethyl ) -1-azabicyclo[ 3,2,0] -hept- 3 ,7 -dione-2-carboxylate . Since, this compound is unεtable in the εucceεsive procesε it iε should be used immediately after preparation. NMR(CDCl) δ:

1.35(d,3H, =6.lHz) , 2.44(m,lH), 2.92(m,lH), 3.19(dd,lH,J=2.0Hz, J=7.0Hz), 4.15(m,lH), 4.30(m,lH), 4.75(ε,lH) , 5.28(m,2H), 7.48 ( d , 2H , J=8.7Hz ) , 8.20(d,2H,J=8.9Hz)

Detailed Description of the Preferred Embodiments EXAMPLE 1 (E) and (Z) isomers of ( 4R, 5S,6S,8R,2 ' S, 4 'S) -3- [ (2-

methoxyiminopyrro1 idin ) - 4 - y1- hio } - -methy1 - 6 - ( 1 - hydro yethyl ) -1 -azabi cyclo[ 3,2,0] -hept- 2-ene- 7-one- 2- carboxylic acid EXAMPLE 1-1 1.197 g (3.3 mmol) of ( 4R, 5R, 6S,8R) -p-nitrobenzyl - 4-methyl- 6- ( 1-hydroxyethyl ) -1 -azabicyclo[3,2,0]-hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 100 ml of anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using ice bath. At the same temperature, 750 μl (4.3 mmol) of N,N- diiεopropylethylamine was added dropwise, 830μl(4.0 mmol) of diphenylchlorophosphate added and εtirred for 1 hour. And then 750 μl (4.3 mmol) of N,N- diiεopropylethylamine waε added and the εolution of 1.36 g (4.0 mmol) of ( 2S, 4S) -1- (p-nitrobenzyloxycarbonyl ) -2- methoxyimino-4-mercaptopyrrolidine obtained in EXAMPLE 18-5 in 20 ml of anhydrouε acetonitrile was added dropwise to the reaction mixture. After the addition, the ice bath waε removed, and the reaction mixture waε εtirred at room temperature for 5 hourε. After the reaction, the reaction mixture waε concentrated under reduced presεure, and ethylacetate waε added to the residue to extract the product. The extract waε waεhed successively with water and saturated aqueous sodium chloride, and the organic layer dried over anhydrous magnesium sulfate. After the filtration, the filtrate waε concentrated under reduced preεεure and the εolvent evaporated to obtain 2.2 g of crude material. Thiε crude material waε subjected to the column

chromatography on silica gel (eluted with n- hexane:ethylacetate=l : 4 ) and purified to give 0.236 g (10.4%) of (Z)-(4R,5S,6S,8R,2'S,4'S) -p-nitrobenzyl - 3 -[ 1 - (p-nit robenzyloxycarbony 1 ) -2-methoxyiminopyrrolidin-4 - yl - thi o ] - 4 -methyl - 6 - ( 1 -hyd roxyethyl ) - 1 - azabicyclof 3,2,0]-hept-2-ene-7 -one -2 -carboxylate , 0.202 g (8.9%) of (E) - (4R,5S,6S,8R, 2 ' S , 4 ' S ) -p-nitrobenzyl - 3 - [1 - (p-nitrobenzyloxycarbonyl ) - 2 -methoxyiminopyrrolidin- 4 -yl - thio ] - 4 -methyl -6 - ( 1 -hydroxyethyl ) - 1 - azabicyclof 3 , 2 , 0 ] -hept- 2 -ene- 7 -one- 2 -carboxylate and

0.570 g (25.2%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R, 2'S,4'S) -p-nitrobenzyl-3- [l-(p- nitrobenzyloxycarbonyl ) - 2 -methoxyiminopyrrolidin- -yl - thio] - 4 -methyl -6- ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0]- hept-2-ene-7 -one- 2 -carboxylate .

NMR( Acetone-d _j )

(1) Z form: δ:

1.12 (dd, 6H ) , 2.01 (m ,lH) , 2.80 (m ,lH) ,

3.32(dd, J=2.4Hz, J=6.4Hz) , 3.55 - 3.80 ( m, 2H ) , 3.75(s,3H), 3.95-4.32(m,2H) , 4.35(m,lH) , 4.60(m,lH) , 5.20-

5.60(m,5H), 7.43 ( d, 1H , J=6.6Hz ) , 7.63 ( d , 2H , J=8.4Hz ) ,

7.79(d,2H,J=8.5Hz) , 8.20(dd,4H)

(2) E form: δ:

1.23(d,3H, J=5.9Hz) , 1.26 ( d , 3H, j=7.2Hz ) , 2.01(m,lH) , 2.80(m,lH), 3.33(dd,lH) , 3.55 - 3.80 ( m, 2H ) , 3.81(s,3H),

3.95-4.32(m,3H) , 4.31(m,lH) , 4.80 - 5.05(m, 1H ) , 5.20-

5.60(m,4H) , 6.90(m, lH) , 7.65 ( , 2H , J = 8.5Hz ) ,

7.80(d,2H,J=8.9Hz) , 8.20(dd,4H)

EXAMPLE 1-2

To the mixture of 10 ml of tetrahydrofuran and 10 ml of 0.1 M 4-morpholinepropanesulfonic acid buffer(pH 7.0) waε added 0.316 g (0.462 mmol) of the (E)- (4R,5S, 6S ,8R, 2' S, 4' S) -p -nit robenzyl -3 - [ 1- ( p - nitrobenzylo ycarbonyl ) - 2-methoxyiminopyrrolidin-4-yl - thio] - 4-methyl-6- ( 1-hydroxyethyl ) -1-azabicyclo[ 3,2,0] - hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 1-1 and stirred to disεolve. 0.594 g of 10% palladium/carbon catalyεt waε added and εtirred at room temperature for 3 hourε under a hydrogen gas stream. After the reaction, the catalyst waε removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. After the residue waε washed with ethylacetate, separated, and the water layer concentrated under reduced pressure at 10° - 20°C, then the extracted impurity waε removed by filtration. The clarified filtrate was subjected to polymerchromatography (Diaion HP-20 resin:previouεly waεhed with methylalcohol and water). After loading the εompie an the resin, the resin waε waεhed first with water to remove 4- morpholinepropansulfonic acid and then eluted with 5% aqueous acetone solution to collect, the fractions containing the desired product. The fractions were combined and concentrated at 10°-20°C under reduced presεure. The residue waε lyophilized to give 0.101 g (59.2%) of ( E ) - ( 4R, 5S , 6S , 8R , 2 ' S ,4 ' S ) - 3- [ ( 2 - rαethoxyiminopyrrolidin) -4-yl-thio]-4-methyl-6-(l- hydroxyethyl ) -1 -azabi cyclo[ 3,2,0] -hept- 2-ene- -one- 2-

carboxylic acid. NMR(D ₂ 0) 6:

0.96(d,3H,J=7.2Hz) , 1.08 (d, 3H,J=6.3Hz ) , 1.60(m,lH) , 2.45(m,lH), 2.92(m,lH), 3.21(m,3H), 3.63(ε,3H), 3.24(m,lH) , 4.02(m,2H) , 4.42(m,lH), 6.83- 7.40(dd,lH,J=5.3Hz,J=5.9Hz) EXAMPLE 1-3

To the mixture of 17 ml of tetrahydrofuran and 17 ml of 0.1 M 4-morpholinepropanesulfonic acid buffer(pH 7.0) waε added 0.521 g (0.764 mmol) of the (Z)- (4R,5S,6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 - [ 1- (p- nitrobenzyloxycarbonyl ) - 2-methoxyiminopyrrolidin-4-yl - thio] - 4-methyl-6- ( 1-hydroxyethyl ) -1-azabicyclof 3,2,0]- hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 1-1 and εtirred to dissolve. 0.980 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 3 hours under a hydrogen gas stream. After the reaction, the catalyst waε removed by filtration and the filtrate waε concentrated under reduced preεεure to evaporate the organic εolvent. After the reεidue waε washed with ethylacetate, separated, and the water layer concentrated under reduced pressure at 10° - 20°C, then the extracted impurity was removed by filtration. The clarified filtrate was treated according to the same procedure aε in EXAMPLE 1-2 to give 0.135 g (47.8%) of (Z)- ( 4R,5S ,6S,8R,2'S,4 'S) -3- [ ( 2-methoxyiminopyrrolidin) -4- yl - thi o ] - 4 -methyl - 6 - ( 1 -hyd roxyethyl ) - 1 - azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylic acid.

NMR ( D ₂ 0 ) 6 :

0.98(d,3H,J=7.2Hz) , 1.08(d, 3H,J=6.3Hz ) , 1.65( ,1H) , 2.53(m,lH), 3.01-3.42(m,4H) , 3.70(d,6H), 4.02(m,3H), 4.40(m,lH), 6.90 and 7.40(dd, 1H,J=5.2Hz ,J=5.5Hz ) EXAMPLE 2

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ ( 2 - ethoxycarbon lmethoxyimino pyrrolidin)-4-ylthio]-4- methyl -6- ( 1-hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept-2- ene-7-one-2-carboxylic acid EXAMPLE 2-1

1.39 g (3.84 mmol) of ( 4R, 5R,6S, 8R) -p-nitrobenzyl - 4-methyl - 6- ( 1 -hydroxyethyl ) -1-azabicyclof 3,2,0) -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 80 ml of anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C using ice bath. At the same temperature, 803 μl (4.61 mmol) of N,N- diisopropylethylamine was added dropwise, 916 μl (4.42 mmol) of diphenylchlorophosphate waε added and the mixture stirred for 1 hour. And then 803 μl (4.61 mmol) of N,N-diisopropylethylamine waε added and the solution of 1.58 g (3.84 mmol) of ( 2S, 4S ) -1- (p- nitrobenzyloxycarbony1 ) -2-ethoxycarbonylmethoxyimino-4 - mercaptopyrrolidine obtained in EXAMPLE 19-5 in 20 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath waε removed, and the reaction mixture waε stirred at room temperature for 4 hourε. After the reaction, the reaction mixture waε concentrated under reduced pressure, and ethylacetate waε added to the reεidue to extract the

product. The extract waε washed εuccesεively with water and saturated aqueous sodium chloride, and the organic layer dried over anhydrouε magneεium εulfate. After the filtration, the filtrate waε concentrated under reduced preεεure and the εolvent evaporated to obtain 2.55 g of crude material. This crude product was subjected to column chromatography on silica gel (eluted with chlorof orm:methylalcohol=40 : 1 ) and purified to give 1.32 g (45.5%) of the mixtures of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p -nit robenzyl - 3 - [ 1- (p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ethoxycarbonylmethoxyiminopyrrolidin- 4-yl -thio] -4 - methyl -6- ( 1 -hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2- ene-7 -one -2 - carboxylate . NMR(CDCl ₃ ) δ:

1.10(m,9H), 2.10(m,lH), 2.70(m,lH), 3.20(m,lH), 3.21(m,lH), 4.01(m,lH), 4.20(m,5H), 4.56(m,2H), 5.10- 5.50(m,5H), 7.51(d,2H, J=8.3Hz) , 7.62 ( d , 2H, J=8.5Hz ) , 8.20(d,4H, =8.7Hz) EXAMPLE 2-2

To the mixture of 14 ml of tetrahydrofuran and 14 ml of 0.1 M 4 -morpholinepropanesulf onic acid buffer (pH 7.0) was added 0.439 g (0.581 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S) -p-nitrobenzyl - 3 -[ 1 - ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - etho ycarbonylmethoxyiminopyrrolidin- 4-yl -thio] -4 - methyl -6- ( 1 -hyd roxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2- ene-7 -one-2-carboxylate obtained in EXAMPLE 2-1 and was stirred to disεolve. 0.280 g of 10% palladium/carbon

catalyst waε added and stirred at room temperature for 3 hourε under a hydrogen gas stream. After the reaction, the catalyst waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic εolvent. The resultant residue waε washed with ethylacetate, separated, and the water layer waε concentrated under reduced pressure at 10° - 20°C and then the extracted impurity was removed by filtration. The clarified filtrate waε subjected to polymerchromatography (Diaion HP-20 resin:previously washed with methylalcohol and water). After loading the somple on the resin, the resin waε waεhed first with water to remove 4-morpholinepropanesulfonic acid and then eluted with 5% aqueous acetone solution to collect the fractions containing the desired product. The fractionε were combined and concentrated at 10° - 20°C under reduced pressure. The residue was lyophilized to give 0.125 g (48.8%) of the mixture of (E) and (Z) of ( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ ( 2 - ethoxycarbonylmethαxyiminopyrrolidin)-4-yl-thio] -4 - methyl -6- (1-hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylic acid. NMR(D ₂ 0) 6:

1.10(m,9H), 1.70(m,lH), 2.53(m,lH), 3.05- 3.60(m,3H), 3.60-4.00(m,2H) , 4.20(m,2H), 4.30- 4.80(m,5H), 6.99-7.55( ,2H) EXAMPLE 3

( 4R , 5 S , 6 S , 8R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N , N - dimethyla inocarbonylmethoxyimino)pyrrolidin-4-yl-thio] -

-methyl-6- (1-hydroxyethyl ) -1-azabicyclof3,2,0] -hept-2- ene-7-one-2-carboxylic acid EXAMPLE 3-1

0.696 g (1.92 mmol) of (4R,5R,6S,8R) -p-nitrobenzyl- 4-methyl- 6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 40 ml of anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C uεing an ice bath. At the same temperature, 402 μl (2.31 mmol) of N,N- diisopropylethylamine waε added dropwiεe, 458 μl (2.21 mmol) of diphenylchlorophosphate was added and stirred for 1 hour. Then 402 μl (2.31 mmol) of N,N- diiεopropylethylamine waε added and the solution of 0.866 g (2.11 mmol) of ( 2S , 4S ) -1 - ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( N , N - dimethylaminocarbonylmethoxyimino) -4-mercaptopyrrolidine obtained in EXAMPLE 20-5 in 20 ml of anhydrouε acetonitrile was added dropwiεe to the mixture solution. After the addition, the ice bath waε removed, and the reaction mixture waε. εtirred at room temperature for 5 hourε. After the reaction, the reaction mixture waε concentrated under reduced preεεure, ethylacetate waε added to the residue to extract the product. The extract waε treated with the εame operation aε in EXAMPLE 1-1 to obtain 1.4 g of crude material. Thiε crude material waε subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=40:1 ) and purified to give 0.825 g (56.9%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-p-nitrobenzyl-3-fl-(p-

nitrobenzyloxycarbonyl ) - 2- ( N,N -dimethylaminoca bonyl metho yimino )pyrrolidin - 4-yl- thio ] - 4 -methyl- 6 - ( 1 - hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept-2-ene- 7-one- 2- carboxylate. NMR(Acetone-d ₆ ) 6:

1.20(dd,6H), 1.25(m,lH), 2.25(m,lH), 2.85(ε,3H),

3.01(s,3H), 3.32(dd,lH,J=2.6Hz,J=6.4Hz) , 3.60(m,2H),

4.01-4.40(m,3H) , 4.70(m,3H), 4.80-5.20(m,1H) , 5.20-

5.56(m,4H), 7. 6(d,2H,J=8.5Hz) , 7.80(d,2H,J=8.6Hz ) , 8.20(m,4H)

EXAMPLE 3-2

To a mixture of 10 ml of tetrahydrofuran and 10 ml of 0.1 M -morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.520 g (0.689 mmol) of the mixtures of (E) and (Z) of ( 4R,5S,6S,8R, 2'S, 'S) -p-nitrobenzyl-3- [ 1 - ( p - ni t r obenzyl oxyca rbonyl ) - 2 - ( N , N - dimethy1aminocarbonylmethoxyimino) pyrrolidin-4-yl- thio] - -methyl-6- ( 1-hydroxyethyl ) -1-azabicyclof 3,2,0]- hept-2-ene-7 -one-2-carboxylate obtained in EXAMPLE 3-1 and εtirred to diεεolve. 0.320 g of 10% palladium/carbon catalyεt waε added and stirred at room temperature for 3 hourε a under hydrogen gaε stream. After the reaction, the catalyst waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic solvent. The residue waε treated aε diεcribed in EXAMPLE 2-2 to give 0.162 g (53.5%) of the mixtureε of (E) and (Z) of ( 4R,5S,6S,8R, 2'S, 4'S) - 3- [2- (N,N- dimethylamino carbonylmethoxyimino)pyrrolidin-4-y1- thio] - 4-methyl-6- ( 1-hydroxyethyl ) -1-azabicyclof 3, 2,0] -

hept - 2 - ene - 7 - one - 2 - carboxyli c acid . NMR(DMSO-d _{ ) 6:

1.10(d,3H,J=6.5Hz) , 1.12 ( d, 3H, J=6.2Hz ) , 1.40(m,lH) , 2.40(m,lH), 2.80(ε,3H), 2.90(s,3H), 2.90 - 3.40 (m, 4H) , 3.80-4.10(m,3H) , 4.64(s,2H), 4.90(m,lH)

EXAMPLE 4

( 4 R , 5 S , 6 S , 8 R , 2 - S , 4 ^f S ) - 3 - f ( 2 - carboxymethoxyimino)pyrrolidin-4 -yl -thio] -4 -methyl -6 - ( 1 - hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2 -ene- 7 -one- 2- carboxylic acid

EXAMPLE 4-1

0.627 g (1.71 mmol) of ( 4R, 5R, 6S , 8R) -p-nitrobenzyl - 4- methyl- 6- ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 70 ml of anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 320 μl (1.82 mmol) of N,N- diisopropylethylamine was added dropwise, 377 μl (1.82 mmol) of diphenylchlorophosphate added and εtirred for 1 hour. And then 320 μl (1.82 mmol) of N,N- diiεopropylethylamine waε added and the εolution of 0.755 g ( 1.5 mmol ) of ( 2S , 4 S ) - 1 - ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( P - methoxybenzyloxyca rbonylmethoxyimino ) - 4 - mercaptopyrrolidine obtained in EXAMPLE 21-2 in 20 ml of anhydrouε acetonitrile waε added dropwiεe to the mixture solution. After the addition, the ice bath waε removed, and the reaction mixture waε stirred at room temperature for 5 hourε. After the reaction, the reaction mixture

waε concentrated under reduced pressure, and ethylacetate was added to the residue to extract the product. The extract was treated with the same operation as in EXAMPLE 1-1 to obtain 1.02 g of crude material. This crude product waε subjected to the column chromatography on silica gel (eluted with chlorof orm:methylalcohol = 40 : 1 ) and purified to give 0.647 g (44.1%) of the mixtures of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 - [ 1- ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( P - me t hox ybe nzyl ox ycarbonylmethoxy imino) pyrrol id in- 4 -yl - thio] - -methyl-6- ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0]- hept-2-ene-7 -one -2 -carboxylate . NMR(Acetone-d ₆ ) δ: 1.22(dd,6H) , 2.05 (m,lH) , 2.80 (m,lH) ,

3.32(dd,lH,J=2.5Hz, J=6.4Hz) , 3.60(m,lH), 3.65(ε,3H), 4.01-4.20(m,4H) , 4.30(m,lH), 4.60(s,2H), 5.21-

5.54(m,7H), 7.60 - 7.80 (m, 6H) , 8.20(m,6H) EXAMPLE 4-2 To a mixture of 12 ml of tetrahydrofuran and 12 ml of 0.1 M 4-morpholinepropaneεulf onic acid buffer (pH 7.0) was added 0.410g (0.484 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S, 8R,2 ' S , 4 ' S ) -p-nitrobenzyl - 3 - [ 1 - ( p - ni t r ob en zy lo xy ca rb onyl ) - 2 - ( P - methoxybenzyloxycarbonylmethoxyimino)py rrolidin-4 -yl - thio] - 4 -methyl -6 - ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0]- hept-2-ene-7 -one-2-carboxylate obtained in EXAMPLE 4-1 and stirred to dissolve. 0.2 g of 10% palladium/carbon catalyst waε added and εtirred at room temperature for

3 hourε under a hydrogen gaε stream. After the reaction, the catalyst waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic εolvent. The residue waε treated with the εame operation aε in EXAMPLE 2-2 to give 0.09 g (45%) of the mixtureε of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3-[ (2- carboxymethoxyimino pyrrolidin)-4-yl-thio]-4-methyl-6 - (1-hydrox ethyl) - 1- azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylic acid. NMR(DMSO-d _j ) δ:

1.12(d,3H,J=7.2Hz) , 1.15(d, 3H,J=6.2Hz) , 1.42(m,lH) , 2.30 ( m , 1 H ) , 2.62 ( m, lH) , 3.10 ( m , 1 H ) , 3.11(dd,lH,J=2.3Hz,J=6.3Hz) , 3.20-4.20(m, H) , 4.40(m,3H)

EXAMPLE 5 ( 4 R , 5 S , 6 S , 8 R , 2 ^, S , 4 ^, S ) - 3 - [ ( 2 - aminocarbonylmetho yimino)pyrrolidin-4-yl-thio]-4- methyl -6- (1-hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylic acid

EXAMPLE 5-1 0.714 g (1.97 mmol) of ( 4R,5R,6S,8R) -p-nitrobenzyl-

4-methyl- 6- (1-hydroxyethyl ) -1-azabicyclof 3,2, 0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 40 ml of anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C using ice bath. At the εame temperature, 390 μl (3.24 mmol) of N,N- diiεopropylethylamine was added dropwise, 445 μl (2.15 mmol) of diphenylchlorophosphate added and εtirred for 1 hour. And then 390 μl (3.24 mmol) of N,N- diisopropylethylamine was added and the solution of

0.782 g (2.05 mmol) of ( 2S , 4S ) -1 - ( p- nitrobenzylo ycarbonyl) - 2-aminocarbonylmethoxyimino- - mercaptopyrrolidine obtained in EXAMPLE 22-4 in 20 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath waε removed, and the reaction mixture was stirred at room temperature for 5 hourε. After the reaction, the reaction mixture waε concentrated under reduced pressure, and ethylacetate was added to the residue to extract the product. The extract was treated, is discribed in

EXAMPLE 1-1, to obtain 1.25 g of crude material. Thiε crude material waε subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=40:l) and purified to give 0.8 g (56%) of the mixtures of (E) and (Z) of

(4R,5S, 6S,8R, 2'S, 4' S) -p-nitrobenzyl -3 - [ l- (p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ami nocar bony lmethoxy iminopyrroli din- 4 -yl - thio] -4 -methyl -

6- ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept -2- ene -7- one - 2 -carboxylate .

NMR(CDC1 ₃ ) δ:

1.12(d,3H, J- 7.2Hz) , 1.15 ( d, 3H , J=6.1Hz ) , 1.80(m,lH) ,

2.60(m,lH), 3.23(dd,lH,J=2.5Hz,J=6.4Hz) , 3.50(m,2H),

3.90(m,2H), 4.22(m,lH), 4.50(m,3H), 5.10 - 5.45 ( m, 5H) , 5.50-6.20(m,2H) , 6.80 -7.05 ( m, 1H ) , 7.40 - 7.64 ( m, 4H ) ,

8.20(dd,4H)

EXAMPLE 5-2

To the mixture of 18 ml of tetrahydrofuran and 18 ml of 0.1 M 4 -morpholinepropanesulf onic acid buffer (pH

7.0) was added 0.515 g (0.71 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S, 8R,2'S, 4 'S) -p-nitrobenzyl-3- [ 1 - ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - aminocarbonylmethoxyiminopyrrolidin-4-yl-thio] -4-methyl- 6- ( 1 -hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-2-ene-7-one- 2-carboxylate obtained in EXAMPLE 5-1 and εtirred to diεεolve. 0.31 g of 10% of the palladium/carbon catalyst waε added and εtirred at room temperature for 3 hourε under a hydrogen gaε stream. After the reaction the catalyst waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic solvent. The reεidue waε treated, aε diεcribed in EXAMPLE 2-2, to give 0.17 g (58.2%) of the mixtures of (E) and (Z) of ( 4R, 5S,6S,8R, 2' S, 4 'S ) -3 - [ ( 2- aminocarbonylmethoxyimino pyrrolidin) -4-yl- thio] - 4- methyl -6- (1-hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylic acid. NMR(D ₂ 0) δ:

1.05(d,3H,J=7.2Hz) , 1.08(d, 3H,J=6.2Hz ) , 1.60(m,lH) , 2.40(m,lH), 2.95(m,lH), 3.05-3.40(m, 3H) , 3.70- 4.10(m,3H), 4.40(m,lH), 4.64(s,2H) EXAMPLE 6

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ^f S ) - 3 - [ 2 - ( N - methylaminocarbonylmethoxyimino) yrrolidin-4-yl-thio] -4 - methyl -6- (1 -hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylic acid EXAMPLE 6-1

0.601 g (1.66 mmol) of ( R, 5R,6S,8R) -p-nitrobenzyl - 4-methyl- 6- ( 1 -hydroxyethyl ) -1-azabicyclof 3 ,2,0]-hept-

3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 40 ml of anhydrous acetonitrile, εtirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 348 μl (2.00 mmol) of N,N- diisopropylethylamine waε added dropwise, 396 μl (1.91 mmol) of diphenylchlorophosphate added and stirred for 1 hour. Then 348 μl (2.00 mmol) of N,N- diiεopropylethylamine waε added and the εolution of 0.723 g (1.83 mmol) of ( 2S , 4 S ) -1 - ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( N - me thy laminocar bony lmethoxy imino) - 4 -mercaptopyrrolidine obtained in EXAMPLE 23-2, 15 ml of anhydrouε acetonitrile was added dropwise to the mixture solution. After the addition and the ice bath removed, and the reaction mixture was stirred at room temperature for 6 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, ethylacetate waε added to the residue to extract the product. The extract was treated aε discribed in EXAMPLE 1-1 to obtain 1.15 g of crude material. This crude material was subjected to the column chromatography on silica gel(eluted with chlorof orm:methylalcohol=40 : 1 ) and purified to give 0.706 g (57.7%) of the mixtures of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 -[ 1- ( p - nitrobenzyloxycarbonyl ) -2- (N-methylaminocarbonyl methoxyimino )pyrrolidin - 4-yl- thio ] - 4 -me thyl- 6 - ( 1 - hy roxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2 -ene- 7 -one- 2- carboxylate .

NMR(CDC1 ₃ ) δ:

1.11 (d, 3H,J=7.2Hz) , 1.15(d,3H,J=6.3Hz) , 1.80(m,lH),

2.60(m,lH), 2.80(d,3H), 3.22(dd,1H, =2. Hz ,J=6.4Hz ) ,

3.45(m,2H), 3.80(m,2H), 4.22(m,lH), 4.50(m,3H), 5.10-

5.50(m,5H), 7.44 (d,2H,J=8.4Hz ) , 7.60(d,2H,J=8.5Hz ) , 8.20(dd,4H)

EXAMPLE 6-2

To the mixture of 17 ml of tetrahydrofuran and 17 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.490g (0.662 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S, 8R,2'S, 4 'S) -p-nitrobenzyl-3- [ 1 - ( p - ni t r ob en zy lo xy ca rb onyl ) - 2 - ( N - methylaminocarbonylmethoxyiminopyrrolidin) -4 -yl -thio] -4 - methyl -6- ( 1-hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylate obtained in EXAMPLE 6-1 and stirred to dissolve. 0.30 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 4 hours under hydrogen gas stream. After the reaction the catalyst waε removed by filtration and the filtrate waε concentrated under reduced preεsure to evaporate the organic solvent. The residue was treated, as discribed in EXAMPLE 2-21 to give 0.173 g (61.3%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3- [2- (N-methyla inocarbonyl methoxyimino )pyrrolidin - -yl- thio ] - 4 -methyl- 6 - ( 1 - hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept-2-ene-7 -one- 2- carboxylic acid. NMR(DMSO-d ₆ ) δ:

1.03(d,3H, =7.3Hz) , 1.05(d, 3H,J=6.1Hz ) , 1.42(m,lH) , 2.42(m,lH), 2.81(d,3H), 2.91-3.39(m, 4H) , 3.81-

4.11(m,3H) , 4.65(d,2H) , 4.91(m,lH) EXAMPLE 7

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - f ( 2 - hydroxyiminopyrrolidin) - 4-yl- thio ] - -methyl- 6 - ( 1 - hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2-ene- 7-one- 2- carboxylic acid EXAMPLE 7-1

0.975 g (2.69 mmol) of ( 4R,5R,6S,8R) -p-nitrobenzyl - 4-methyl- 6- (1-hydroxyethyl ) -1-azabicyclof 3,2, 0 ] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 80 ml anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 550 μl (3.08 mmol) of N,N- diisopropylethylamine was added dropwiεe, 613 μl (2.96 mmol) of diphenylchlorophosphate added and stirred for 1 hour. And then 550 μl (3.08 mmol) of N,N- diisopropylethylamine was added and the solution of 0.878 g (2.70 mmol) of ( 2S , 4S ) -1 - ( p - nitrobenzyloxycarbonyl ) - 2 -hydroxyimino- 4 - mercaptopyrrolidine obtained in EXAMPLE 24-6, 20 ml of anhydrous acetonitrile waε added dropwiεe to the mixture εolution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 5 hourε. After the reaction, the reaction mixture waε concentrated under reduced pressure, and ethylacetate waε added to the reεidue to extract the product. The extract waε treated, aε diεcribed in EXAMPLE 1-1, to obtain 0.987 g of crude material. This crude product was subjected to the column chromatography

on εilica gel(eluted with chlorof orm:methylalcohol = 35 :1 ) and purified to give 0.756 g (42.0%) of the mixtures of (E) and (Z) of ( 4R, 5S , 6S , 8R, 2 ' S , 4 ' S ) -p-nitrobenzyl - 3 -[ 1 - ( p-nit robenzyloxycarbonyl ) -2-hydroxyiminopyrrolidin- - yl - thi o] - 4 -methyl - 6 - ( 1 -hydroxyethyl ) - 1 - azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 -carboxylate . NMR ( DMSO -d _j ) δ:

1.10(d,3H,J=7.2Hz) , 1.12 ( d , 3H, J=6.2Hz ) , 1.90(m,lH) , 1.65(m,lH), 3.30(dd,lH), 3.52(m,lH), 3.95(m,3H), 4.22 (m,lH) , 4.49( m, lH ) , 5.01 - 5.50 ( m , 5 H ) ,

7.60(d,2H,J=8.7Hz) , 7.72 ( d, 2H , =8.7Hz ) , 8.20(d,4H,), 10.8-11.2(br s,lH) EXAMPLE 7-2 To the mixture of 15 ml of tetrahydrofuran and 15 ml of 0.1 M morpholinepropaneεulf onic acid buffer (pH

7.0) waε added 0.470 g (0.720 mmol) of the mixtureε of (E) and (Z) of (4R,5S,6S, 8R, 2 ' S , ' S ) -p-nitrobenzyl - 3 - [1 - ( p-nitrobenzyloxycarbonyl ) -2- hydroxy iminopyrrolidin- 4 -yl - thio ] - 4 -methyl - 6 - ( 1 -hydroxyethyl ) - 1 - azabicyclo[3,2,0] τhept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 7-1 and εtirred to disεolve. 0.240 g of 10% of the palladium/carbon catalyεt waε added and εtirred at room temperature for 3 hours under an atmospheric pressure of hydrogen. After the reaction the catalyεt waε removed by filtration and the filtrate was concentrated under reduced presεure to evaporate the organic εolvent. The reεidue waε treated with the εame operation aε in EXAMPLE 2-2 to give 0.132 g (52.8%) of the mixtureε of (E) and (Z) of ( 4R, 5S , 6S,8R, 2 ' S, 4 ' S ) - 3 -

f (2 -hydroxyimino)pyrrolidin -4-yl- thio] - 4-methyl-6- ( 1- hydroxyethyl) -1 -azabicyclof 3,2,0] -hept-2-ene-7-one- 2- carboxylic acid. NMR(DMS0-d ₆ ) δ: 1.05(d,3H,J=7.2Hz) , 1.08(d,3H,J=6.2Hz) , 1.40(m,lH) ,

2.40(m,lH), 2.70(m,lH), 3.10-3.30(m,3H) , 3.40- 3.80(m,2H), 3.80-4.10(m,2H) , 10.63(br ε, 1H) EXAMPLE 8 (4R,5S,6S,8R,2'S,4'S)-3-f2-( N-methaneεulfonyl-N- methylhydrazono)methylpyrrolidin-4-yl -thio] -4-methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylic acid EXAMPLE 8-1 0.804 g (2.22 mmol) of (4R,5R,6S,8R) -p-nitrobenzyl- 4-methyl- 6- (1-hydroxyethyl) -1-azabicyclof3,2, 0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 100 ml anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C uεing an ice bath. At the εame temperature, 520 μl (2.98 mmol) of N,N- diiεopropylethylamine waε added dropwiεe, and 600 μl (2.89 mmol) of diphenylchlorophoεphate waε added and stirred for 2 hours. And then 520 μl (2.98 mmol) of N,N-diiεopropylethylamine was added and the εolution of 1.0 g (2.40 mmol) of ( 2 S , 4S ) - 1 - ( p - nitrobenzyloxycarbonyl ) - 2 - (N-methaneεulfonyl-N- methylhydrazono) methyl-4-mercaptopyrrolidine obtained in EXAMPLE 25-5, 25 ml of anhydrous acetonitrile waε added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction

mixture waε εtirred at room temperature for 72 hourε. After the reaction, the reaction mixture waε concentrated under reduced preεεure, and methylenechloride waε added to the reεidue to extract the product. The extract was waεhed with water and saturated with aqueous sodium chloride and the organic layer dried over anhydrous magnesium sulfate. After the filtration, the filtrate waε concentrated under reduced pressure and the solvent evaporated to obtain 1.32 g of crude material. This crude material was εubjected to the column chromatography on εilica gel(eluted with chloroform:acetone=20:l ) and purified to give 0.88 g (52.1%) of the mixtureε of (E) and (Z) of ( R, 5S, 6S,8R, 2'S,4 'S) -p-nitrobenzyl-3- [ 1 - (p-nitrobenzyloxycarbonyl ) - 2- (N-methanesulfonyl-N-methylhydrazono)methylpyrrolidin- 4-yl-thio ] - -methyl- 6- (1 -hydroxyethyl) -1 -azabi cyclo [3,2,0] -hept-2-ene-7-one-2 -carboxylate. NMR(CDC1 ₃ ) δ:

1.23(d,3H,J=7.4Hz) , 1.31(d, 3H, =6.2Hz) , 2.35(m,lH) , 2.64 ( m , 1 H ) , 3. 01 ( s , 3 H ) , 3.10 ( s , 3H ) , 3.30(dd,lH,J=2.6Hz,J=6.7Hz) , 3.32(m,lH), 3.50(m,1H) , 3.80-4.10(m,2H) , 4.24(m,2H), 4.73(m,lH), 5.13- 5.22(m,4H) , 7.10(m,lH), 7.43 ( d , 2H , J=8.5Hz ) , 7.60(d,2H,J=8.7Hz) , 8.20(dd,4H) EXAMPLE 8-2

1.76 g (2.31 mmol) of the mixtures of (E) and (Z) (4R,5S,6S, 8R,2'S,4'S) -p-nitrobenzyl-3-[l- (p- nitrobenzyloxycarbonyl ) -2- (N-methanesulfonyl-N- ethylhydrazonomethyl)pyrrolidin-4-yl-thio] -4 -methyl -6-

( 1 -hydroxyethyl) - 1 -azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 - carboxylate obtained in EXAMPLE 8-1 was subjected to the preparatory thin layer chromatography on silica gel 60F- 254 (eluted with chlorof orm:methylalcohol = 20 : 1 ) to separate the isomers and purified to give 0.22 g (12.5%) of (E) - (4R,5S,6S,8R, 2 ' S , 4 ' S ) -p-nitrobenzyl - 3 -[ 1 -( p- nitrobenzyloxycarbonyl ) -2- (N-methaneεulf onyl-N- methylhydrazonomethyl )pyrrolidin-4-yl-thio] -4 -methyl -6- ( 1 -hydroxyethyl) -1 -azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 - carboxylate and 0.088 g (5.0%) of (Z)-

( 4R ,5S, 6S ,8R, 2' S, 4 ' S) -p -nitrobenzyl -3 - [ l- (p- nitrobenzyloxycarbonyl ) -2- (N-methaneεulfonyl-N- methylhydrazonomethyl ) pyrrolidin- 4-yl -thio] -4 -methyl -6- ( 1 -hydroxyethyl) -1 -azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 - carboxylate.

NMR(CDC1 ₃ ) δ:

(E) : 1.23(d,2H,J=7.3Hz) , 1.37 ( d, 2H, J=6.3Hz ) , 2.35(m,lH) , 2.64( m ,lH ) , 3.01 ( s , 3 H ) , 3.20 ( s , 3H ) ,

3.30 (dd, 1H , J=2.7Hz , J=6.8Hz) , 3.31(m,lH) , 3.51(m,lH), 4.90(m,2H), 4.23(m,2H) , 4.72(m,lH) ,

5.10-5.50(m,2H) , 7.50 ( d , 2H , J=8.5Hz ) , 7.60(d,2H, J=8.7Hz), 8.20(d,2H,J=8.7Hz) (Z) : 1.24(d,2H, =7.2Hz) , 1.36 ( d, 2H , J=6.3Hz ) , 2.37(m,lH) , 2.65( m ,lH ) , 3.03 ( ε, 3H) , 3.21 ( ε , 3H ) , 3.29 (dd, 1H,J=2.8Hz , J=6.9Hz) , 3.40(m,2H) ,

4.29(m,3H) , 4.89(m,2H) , 5.11 - 5.51 ( m , 2H ) ,

7.50(d ,2H ,J=8.5Hz ) , 7.61(d ,2H ,J=8.7Hz ) ,

8.21(d,2H,J=8.6Hz) EXAMPLE 8-3

To the mixture of 12 ml of tetrahydrofuran and 12 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) waε added 0.37 g (0.486 mmol) of (E)- (4R,5S, 6S,8R, 2' S, 4' S) -p-nitrobenzyl -3 ^■ [ 1- (p - nitrobenzyloxycarbonyl ) -2- (N-methaneεulfonyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylate obtained in EXAMPLE 8-2 and εtirred to diεεolve. 0.27 g of 10% of the palladium/carbon catalyεt waε added and stirred at room temperature for 15 hourε under a hydrogen gaε εtream. After the reaction the catalyεt waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic solvent. After the residue was washed with ethylacetate, separated, and the water layer concentrated under reduced presεure at 10-20°C, then the extracted impurity waε removed by filtration. The clarified filtrate waε subjected to polymerchromatography(Diaion HP-20 resin:previouεly waεhed with methylalcohol and water). During the chromatography, at first it was eluted with water to remove 4-morpholinepropane sulfonic acid and then eluted with 5% aqueous acetone solution to collect the fractions containing the desired product. The fractions were combined and concentrated at 10-20°C under reduced pressure. The residue waε lyophilized to give 0.140 g (64.5%) of ( E) -( 4R,5S, 6S ,8R, 2' S, 4' S) -3 - [ 2- (N- methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio] - 4-methyl-6- ( 1-hydroxyethyl ) -1-azabicyclof3,2,0]-

hept-2-ene-7-one-2-carboxylic acid. NMR(DMSO-d _{ ) δ: (E): 1.10(dd,6H), 1.64(m,lH), 2.45(m,lH), 2.90(m,lH),

2 . 9 9 ( ε , 3 H ) , 3 . 0 8 ( s , 3 H ) , 3.20 ( dd, 1H, J =2.4Hz , J=6.4 Hz ) , 3.32(m,2H) ,

3.63(m,lH), 3.90(m,2H), 4.10(m,lH)

EXAMPLE 8-4

To the mixture of 11 ml of tetrahydrofuran and 11 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.33 g (0.434 mmol) of (Z)-

(4R,5S, 6S ,8E, 2' S, 4' S) -p-nitrobenzyl -3 - [ 1- (p- nitrobenzyloxycarbonyl ) -2- (N-methanesulfonyl-N- methylhydrazonomethyl )pyrrolidin-4-yl-thio]-4-methyl-6-

(1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylate obtained in EXAMPLE 8-2 and stirred to dissolve. 0.23 g of 10% of the palladium/carbon catalyεt waε added and εtirred at room temperature for 17 hourε under a hydrogen gaε stream. After the reaction, the catalyst waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic solvent. After the reεidue was waεhed with ethylacetate, εeparated, and the water layer concentrated under reduced preεεure at 10-20°C, then the extracted impurity waε removed by filtration. The clarified filtrate was treated, as discribed in EXAMPLE

8-3, to give 0.116 g (59.8%) of (Z)-

(4R,5S,6S,8R,2'S,4'S) -3- [ ( 2 - ( N- me thane sulf onyl- N - me thy lhydrazonome thyl ) pyrrolidin- 4-yl -thio] -4 -me thyl -6 -

( 1 -hydroxyethyl) -1 -azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 -

carboxylic acid. NMR(DMSO-d ₆ ) δ:

(Z): 1.09(dd,6H), 1.64(m,lH), 2.46(m,lH), 2.91(m,lH), 2 . 9 8 ( ε , 3 H ) , 3 . 0 5 ( s , 3 H ) , 3.19 ( dd , 1H , J =2.3Hz , J=6. Hz ) , 3.31(m,2H) , 3.62(m,lH), 3.89(m,2H), 4.08(m,lH) EXAMPLE 8-5

To the mixture of 20 ml of tetrahydrofuran and 20 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) waε added 0.88 g (1.16 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S) -p-nitrobenzyl-3- f l-(p- nitrobenzyloxycarbonyl ) -2- (N-methanesulfonyl-N- methylhydrazonomethyl )pyrrolidin-4-yl-thio] - -methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylic acid obtained in EXAMPLE 8-1 and εtirred to dissolve. 0.5 g of 10% palladium/carbon catalyst was added and εtirred at room temperature for 22 hours under hydrogen gas stream. After the reaction the catalyεt waε removed by filtration and the filtrate waε concentrated under reduced preεεure to evaporate the organic εolvent. After the reεidue waε washed with ethylacetate, separated, and the water layer concentrated under reduced pressure at 10-20°C, then the formed impurity waε removed by filtration. The clarified filtrate was subjected to polymerchromatography Diaion HP-20 resin:previouεly washed with methylalcohol and water). During the chromatography, at first it waε eluted with water to remove 4-morpholinepropanesulfonic acid and then eluted with 5% aqueouε acetone εolution to collect

the fractions containing the desired product. The fractions were combined and concentrated at 10-20°C under reduced pressure. The residue was lyophilized to give 0.30 g (58.0%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3-[2-(N-methanesulfonyl-N- methylhydrazonoraethyl)pyrrolidin-4-yl-thio] -4-methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylic acid. NMR(DMSO-d _{ ) δ: l.ll(d,3H,J=7.lHz) , 1.13(d,3H, =6.1Hz) , 1.63(m,lH) , 2.20(m,lH), 3.75(m,2H), 2.93(s,3H), 3.05(s,3H), 3.15(dd,lH), 3.30(m,2H), 3.60-3.90(m,2H) , 4.10(m,lH)

EXAMPLE 9

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( 2 - pyridiny lhydrazonome thyl ) pyrrolidin- 4 -yl- thio] -4 -methyl -

6- (1 -hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept -2- ene -7- one - 2-carboxylic acid

EXAMPLE 9-1

1.81 g (5.0 mmol) of (4R, R,6S,8R) -p-nitrobenzyl-4- methyl-6- (1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-3,7- dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 110 ml anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 1.1 ml(6.31 mmol) of N,N- diisopropylethylamine waε added dropwiεe, 1.2 ml(5.79 mmol) of diphenylchlorophoεphate added and εtirred for 2 hourε. And then 1.1 ml (6.31 mmol) of N,N- diiεopropylethylamine was added and the solution of 1.9 g (4.73 mmol) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl ) -2-

(2 -pyridinylhydrazono )methyl- 4-me reaptopyrrolidine obtained in EXAMPLE 26-5 in 30 ml of anhydrous acetonitrile waε added dropwiεe to the mixture εolution. After the addition, the ice bath waε removed, and the reaction mixture was stirred at room temperature for 5 hourε. After the reaction, the reaction mixture waε concentrated under reduced preεεure, and methylenechloride waε added to the residue to extract the product. The extract waε treated with the εame operation as EXAMPLE 8-1 to obtain 2.5 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=20:1 ) and purified to give 2.08 g (55.8%) of the mixtures of (E) and (Z) of ( 4R, 5S, 6S , 8R, 2 ' S , 4 ' S ) - p - ni trobenzy1 - 3 - f 1 - ( p - nitrobenzyloxycarbonyl ) - 2- ( 2-pyridinylhydrazonomethyl) pyrrolidin-4-yl-thio] -4-methyl -6- (1- hydroxyethyl ) -1 - azabicyclof 3,2,0] -hept-2-ene-7 -one-2-carboxylate . NMR(CDC1 ₃ ) δ: 1.31(d,3H,J=7.3Hz) , 1.38(d, 3H,J=6.2Hz ) , 2.10(m,lH) , 2.64(m,lH), 2.7(dd, 1H,J=2.6Hz,J=6.8Hz ) , 3.50(m,5H), 3.75(m,lH), 4.01(m,lH), 4.12(m,2H), 4.62(m,lH), 5.05- 5.50(m,5H), 6.75(m,lH), 7.10(m,2H), 7.30-7.65(m, 5H) , 7.90-8.35(m,5H) EXAMPLE 9-2

To the mixture of 25 ml of tetrahydrofuran and 25 ml of 0.1 M morpholinepropaneεulfonic acid buffer (pH 7.0) was added 1.3 g (1.82 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S,8R, 2'S, 4 'S ) -p-nitrobenzyl -3-[ 1-(p-

n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( 2 - pyridinylhydrazonomethyl)pyrrolidin-4-yl-thio] -4-methyl- 6- (1-hydroxyethyl) -1-azabicyclof 3,2, 0 ] -hept-2-ene-7-one- 2-carboxylic acid obtained in EXAMPLE 9-1 and stirred to dissolve. 0.5g of 10% of the palladium/carbon catalyεt waε added and stirred at room temperature for 5 hourε under an atmospheric presεure of hydrogen. After the reaction, the catalyεt waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic solvent. After the residue was washed with ethylacetate, separated, and the water layer concentrated under reduced presεure at 10-20°C, then the formed impurity waε removed by filtration. The clarified filtrate waε treated, aε diεcribed in EXAMPLE 8-5, to give 0.422 g (53.6%) of the mixtureε of (E) and ( Z ) of ( 4R, 5S , 6S , 8R, 2 ' S , 4 ' S ) - 3 - [ 2 - ( 2 - pyridinylhydrazonomethyl )pyrrolidin-4-yl-thio] -4-methyl- 6- ( 1-hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept-2-ene-7-one- 2-carboxylic acid. NMR(DMSO-d ₆ ) δ:

1.04(d,3H,J=7.2Hz) , 1.06(d,3H,J=6.2Hz ) , 1.64(m,lH) , 2.50(m,lH), 2.90(m,lH), 3.19(dd,1H,J=2.5Hz ,J=6.7Hz ) , 3.10-3.60(m,lH) , 3.80(m,2H), 4.10(dd,lH), 6.70(m,lH), 7.05(d,lH,J=8.3Hz) , 7.33(d,1H,J=5.4Hz ) , 7.56(m,lH), 8.03(m,lH)

EXAMPLE 10

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( p - carbo yphenylhydrazono ethyl )pyrrolidin -4-yl- thio] - 4- methyl -6- ( 1-hydroxyethyl ) -1 -azabi cyclof 3,2,0 ] -hept- 2-

ene-7-one-2-carboxylic acid EXAMPLE 10-1

0.672 g (1.85 mmol) of ( 4R, 5R,6S,8R) -p-nitrobenzyl- 4-methyl- 6- ( 1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 100 ml anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 0.38 ml (2.30 mmol) of N,N- diisopropylethylamine was added dropwise, and 0.44 ml (2.12 mmol) of diphenylchlorophosphate was added and stirred for 2 hourε. Then 0.38 ml (2.30 mmol) of N,N- diiεopropylethylamine waε added and the solution of 1.0 g (1.77 mmol) of ( 2S,4S) -1- (p-nitrobenzyloxycarbonyl ) -2- (p-methoxybenzyloxycarbonylphenyl hydrazono)methyl-4- mercaptopyrrolidine obtained in EXAMPLE 27-3 in 15 ml of anhydrous acetonitrile waε added dropwiεe to the mixture solution. After the addition, the ice bath waε removed, and the reaction mixture waε εtirred at room temperature for 10 hourε. After the reaction, the reaction mixture εolution waε concentrated under reduced presεure, and methylenechloride waε added to the reεidue to extract the product. The extract waε treated with the same operation as EXAMPLE 8-1 to obtain 1.42 g of crude material. Thiε crude product was subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=20:l ) and purified to give 0.855 g (50.7%) of the mixtures of (E) and (Z) of ( 4R, 5S,6S , 8R,2'S, 4'S) - p-nitrobenzyl-3 - f 1 - ( p- ni trobenzyloxycarbonyl ) - 2- (p - methoxybenzyloxycarbonylphenylhydrazonomethyl )pyrrolid

in- 4-yl -thio] -4 -methyl - 6- ( 1 -hydroxyethyl ) - 1 - azabicyclof 3,2,0] -hept-2-ene-7 -one- 2 - carboxylate . NMR(CDC1 ₃ ) 8:

1.23(d,3H, J=7.4Hz) , 1.27 ( d, 3H, J=6.2Hz ) , 2.05(m,lH) , 2.70(m,lH), 3.22 ( dd, 1H, =2.5Hz , J=6.7Hz ) , 3.40(m,2H),

3.78(s,3H), 4.01(m,lH), 4.22(m,2H), 4.64(m,lH), 5.05- 5.45(m,7H), 6.91 ( d, 2H , J=8.7Hz ) , 6.93 ( d , 2H, J=8.2Hz ) , 7.33 ( d ,2H, J = 8.5Hz ) , 7.45 ( m , 1H ) , 7.90(m, 4H) ,

8.20(d,4H,J=8.7Hz) EXAMPLE 10-2

To the mixture εolution of 10 ml of tetrahydrofuran and 10 ml of O.lM morpholinepropaneεulfonic acid buffer(pH 7.0) waε added 0.38 g (0.418 mmol) of the mixtureε of (E) and (Z) of (4R,5S,6S, 8R,2'S,4'S) -p- nitrobenzyl-3-[l-(p-nitrobenzyloxycarbonyl)-2-(p- methoxybenzyloxycarbonylphenylhydrazonomethyl)pyrrolid in- 4-yl -thio] -4 -methyl- 6- (1 -hydroxyethyl) - 1 - azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylic acid obtained in EXAMPLE 10-1 and εtirred to diεεolve. 0.15 g of 10% palladium/carbon catalyεt waε added and εtirred at room temperature for 6 hourε under atmoεpheric preεsure of hydrogen. After the reaction, the catalyεt waε removed by filtration and the filtrate waε concentrated under reduced presεure to evaporate the organic εolvent. After the reεidue waε waεhed with ethylacetate, separated, and the water layer concentrated under reduced presεure at 10-20°C, then the extracted impurity waε removed by filtration. The clarified filtrate waε treated with the same operation

aε EXAMPLE 8-5 to give 0.102 g (51.5%) of the mixtureε of (E) and (Z) of ( 4R, 5S ,6S ,8R,2 ' S , 4 ' S ) - 3 - [ 2- (p- carboxypheny lhydrazonome thyl ) pyrrolidin-4-yl-thio]-4- methyl -6- ( 1 -hyd roxyethyl ) -1 -azabicyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylic acid.

NMR(D ₆ 0) δ:

1.22(d,3H, J=7.2Hz) , 1.28 ( d , 3H, J=6.3Hz ) , 1.43(m,lH) , 2.35(m,lH) , 2.80(m,2H), 3.15 ( dd, 1H , J=2.5Hz , =6.5Hz ) , 3.30 (m,2H) , 3.61 - 3.85 (m , 2H ) , 4.10 (m ,lH) , 6.90(d,2H,8.5Hz) , 7.30 ( , 2H, 8.3Hz )

EXAMPLE 11

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - f 2 - ( N - me thy lhydrazonome thyl ) pyrrolidin- 4 -yl -thio] -4 -methyl -6 - ( 1 -hydroxyethyl) -1 -azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 - carboxylic acid

EXAMPLE 11-1

1.16 g (3.20 mmol) of ( 4R,5R,6S,8R) -p-nitrobenzyl- 4-methyl- 6- (1-hydroxyethyl ) -1-azabicyclof 3,2, 0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 120 ml .anhydrous acetonitrile, εtirred at room temperature and cooled to 0°C uεing an ice bath. At the same temperature, 0.66 ml (3.78 mmol) of N,N- diiεopropylethylamine waε added dropwise, and 0.72 ml (3.49 mmol) of diphenylchlorophosphate was added and εtirred for 2 hourε. Then 0.66 ml (3.78 mmol) of N,N- diiεopropylethylamine waε added and the solution of 1.51 g (2.91 mmol) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2- fN- (p-nitrobenzyloxycarbonyl ) -N-methylhydrazonomethyl ] - 4-mercaptopyrrolidine obtained in EXAMPLE 28-4 in 20 ml

of anhydrous acetonitrile was added dropwiεe to the reaction mixture εolution. After the addition, the ice bath waε removed, and the reaction mixture εolution waε stirred at room temperature for 72 hours. After the reaction, the reaction mixture εolution was concentrated under reduced pressure, and methylenechloride waε added to the reεidue to extract the product. The extract was treated with the same operation as EXAMPLE 8-1 to obtain 2.45 g of crude material. Thiε crude material waε subjected to the column chromatography on silica gel(eluted with chloroform: methylalcohol=20 :1 ) and purified to give 1.6 g (58%) of the mixtures of (E) and (Z) of (4R,5S,6S, 8R,2'S,4'S) -p-nitrobenzyl-3- [1- (p- nitrobenzyloxycarbonyl ) -2- [N- (p-nitrobenzyloxycarbonyl ) - N-methylhydrazonomethyl]pyrrolidin-4-yl-thio] -4-methyl- 6- (1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-2-ene-7-one- 2-carboxylic acid. NMR(CDC1 ₃ ) δ:

1.12(d,3H, =7.3Hz) , 1.18(d, 3H, =6.3Hz ) , 2.10(m,lH) , 2.70(m,lH), 3.10(m,4H), 3.40(m,2H), 3.75(m,lH), 4.01(m,lH), 4.22(m,lH), 4.70(m,lH), 5.05-5.45(m,6H) , 7.50(d,2H,J=8.7Hz) , 7.60(d,2H,J=8.9Hz ) , 8.15(d,4H) EXAMPLE 11-2 To the mixture of 30 ml of tetrahydrofuran and 30 ml of 0.1 M morpholinepropanesulfonic acid buffer(pH 7.0) was added 1.6 g (1.85 mmol) of the mixtureε of (E) and (Z) of (4R,5S,6S,8R, 2'S, 4 'S ) -p-nitrobenzyl -3- f 1-(p- nitrobenzyloxycarbonyl ) -2- fN- (p-nitrobenzyloxycarbonyl ) - N-methylhydrazonomethyl ]pyrrolidin-4-yl-thio] -4-methyl -

6- (1-hydroxyethyl ) -l-azabicyclo[3,2,0] -hept-2-ene-7-one- 2-carboxylate obtained in EXAMPLE 11-1 and εtirred to diεεolve. 0.8 g of 10% palladium/carbon catalyεt waε added and εtirred at room temperature for 5 hours under an atmospheric preεsure of hydrogen. After the reaction, the catalyst was removed by filtration and the filtrate waε concentrated under reduced presεure to evaporate the organic εolvent. After the reεidue waε waεhed with ethylacetate, separated, and the water layer concentrated under reduced preεεure at 10-20°C, then the extracted impurity waε removed by filtration. The clarified filtrate waε treated with the same operation aε EXAMPLE 8-5 to give 0.325 g (51.5%) of the mixtures of (E) and (Z) of ( 4R, 5S,6S,8R,2'S,4'S) -3- [2- (N- methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylic acid. NMR(DMSO-d ₆ ) δ:

1.07(d,2H,J=6.lHz) , 1.09(d,2H,7.2Hz) , 1.62(m,lH), 2.19(m,lH), 3.05(br,ε,3H) , 3.15(dd,1H,J=2.3Hz,J=6. Hz) , 3.30(m,2H), 3.61-3.85(m,2H) , 3.75(m,2H), 4.05(m,lH)

EXAMPLE 12

( 4 S , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N , N - dimethylhydrazonomethyl ) pyrrolidin-4-yl-thio] -4-methyl- 6- ( 1-hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept-2-ene-7-one- 2-carboxylic acid

EXAMPLE 12-1

2.83 g (7.91 mmol) of ( 4R, 5R, 6S, 8R) -p-nitrobenzyl - 4-methyl-6-(l -hydroxyethyl ) -1 -azabicyclof 3,2, 0 ] -hept-

3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 260 ml anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C using an ice bath. At the εame temperature, 1.61 ml (9.22 mmol) of N,N- diisopropylethylamine was added dropwise, and 1.76 ml (8.50 mmol) of diphenylchlorophosphate was added and εtirred for 2 hourε. Then 1.61 ml (9.22 mmol) of N,N- diisopropylehtylamine was added and the solution of 2.7 g (7.66 mmol) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2- (N, N-dimethylhydrazono )methyl- 4-mercaptopyrrolidine obtained in EXAMPLE 29-2 in 35 ml of anhydrous acetonitrile was added dropwise to the reaction mixture solution. After the addition, the ice bath was removed, and the reaction mixture solution was stirred at room temperature for 7 hours. After the reaction, the reaction mixture εolution was concentrated under reduced presεure, and methylenechloride was added to the residue to extract the product. The extract waε treated, aε diεcribed in EXAMPLE 8-1, to obtain 3.5 g of crude material. Thiε crude material was subjected to the column chromatography on silica gel (eluted with chloroform:acetone=20:1 ) and purified to give 3.2 g (58.8%) of the mixtureε of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 -f l- (p- nitrobenzyloxycarbonyl ) -2- (N,N-dimethylhydrazonomethyl ) pyrrolidin-4 -yl -thio] -4-methyl -6- (1- hydroxyethyl ) -1 - azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylate. NMR(CDC1 ₃ ) δ:

1.13(d,3H, =7.3Hz) , 1.17(d,3H,J=6.3Hz) , 2.01(m,lH) ,

2.60(m,lH), 2.75(s,6H), 3.23(dd, 1H,J=2.7Hz,J=6.8Hz ) , 3.27(m,lH), 3.64(m,lH), 4.01(m,lH), 4.22(m,2H), 4.55(m,lH), 5.10- 5.50(m , 4H) , 7.55( d,2H,J=8.8Hz ) , 7.62(d,2H,J=8.7Hz) , 8.20(m,4H) EXAMPLE 12-2

To the mixture of 30 ml of tetrahydrofuran and 20 ml of 0.1 M morpholinepropaneεulfonic acid buffer (pH 7.0) waε added 1.0 g (1.43 mmol) of the mixtureε of (E) and (Z) of (4R,5S,6S,8R, 2'S, 4'S ) -p-nitrobenzyl-3- [ 1- (p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( N , N - dimethylhydrazonomethyl )pyrrolidin-4-yl-thio] -4-methyl- 6- (1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-2-ene-7-one- 2-carboxylate obtained in EXAMPLE 12-1 and εtirred to dissolve. 0.45 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 6 hourε under an atmospheric presεure of hydrogen. After the reaction, the catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. After the residue was washed with ethylacetate, separated, and the water layer concentraed under reduced presεure at 10-20°C, Then the extracted impurity waε removed by filtration. The clarified filtrate was treated, aε diεcribed in EXAMPLE 8-5, to give 0.22 g (40.0%) of the mixtureε of (E) and (Z ) of ( 4R , 5S , 6S , 8R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N, N - dimethylhydrazonomethyl )pyrrolidin-4-yl-thio]-4-methyl - 6- (1-hydroxyethyl ) -l-azabicyclo[3,2,0] -hept-2-ene-7-one- 2-carboxylic acid. NMR(DMSO-d _j ) δ:

1.01(m,6H), 1.59(m,lH), 2.10(m,lH), 2.40(m,lH), 2.64(m,lH), 2.70(s,6H), 2.84(m,lH), 3.10-3. 0 (m, 3H) , 3.60-4.13(m,3H) EXAMPLE 13 ( 4R , 5 S , 6 S , 8R , 2 ' S , 4 ' S ) - 3 - f 2 - ( N . N - diethylhydrazonomethyl) pyrrolidin-4-yl-thio] -4-methyl- 6- (1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-2-ene-7-one- 2-carboxylic acid EXAMPLE 13-1 1.12 g (3.09 mmol) of ( 4R, 5R,6S,8R) -p-nitrobenzyl-

4-methyl- 6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 100 ml anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 0.74 ml (4.25 mmol) of N,N- diisopropylethylamine was added dropwise, and 0.70ml(3.4 mmol) of diphenylchlorophosphate was added and stirred for 2 hourε. Then 0.74 ml (4.25 mmol) of N,N- diiεopropylethylamine waε added and the εolution of 1.24 g (3.26 mmol) of ( 2S,4S)-1-(p-nitrobenzyloxycarbonyl ) -2- (N,N-diethylhydrazono)methyl-4-mercaptopyrrolidine obtained in EXAMPLE 30-2 in 20 ml anhydrouε acetonitrile waε added dropwiεe to the mixture εolution. After the addition, the ice bath waε removed, and the reaction mixture was stirred at room temperature for 5 hourε. After the reaction, the reaction mixture waε concentrated under reduced presεure, and methylenechloride was added to the residue to extract the product. The extract was treated, aε discribed in

EXAMPLE 8-1, to obtain 2.5 g of crude material. Thiε crude material waε εubjected to the column chromatography on εilica gel (eluted with chloroform:acetone=20:1 ) and purified to give 1.35 g (60.2%) of the mixtureε of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p -nit robenzyl - 3 - [ 1- ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( N , N - diethylhydrazonomethyl)pyrrolidin-4-yl-thio] -4-methyl-6- (1-hydroxyethyl) -1-azabicyclo[ 3,2,0]-hept-2-ene-7-one-2- carboxylate. NMR(CDC1 ₃ ) δ:

1.00(t,6H) , 1.24(d,3H,J=7.3Hz) , 1.32(d, 3H,H=6.3Hz) ,

2.01 ( m , 1 H ) , 2.58 ( m , 1 H ) , 3 .10 ( q , 4 H ) ,

3.23(dd,lH,J=2.6Hz,J=6.8Hz) , 3.37(m,2H), 3.63(m,lH), 4.01(m,lH), 4.20(m,2H), 4.33(m,lH), 5.10-5.30(m, 4H) ,

7.50(d,2H,J=8.8Hz) , 7.61(d,2H, =8.8Hz) , 8.20(m,4H)

EXAMPLE 13-2

To the mixture of 30 ml of tetrahydrofuran and 20 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was added 1.0 g (1.38 mmol) of the mixtureε of (E) and (Z) of (4R,5S,6S,8R, 2'S , 4 ' S) -p-nitrobenzyl- 3- f 1-(p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( N , N - diethylhydrazonomethyl )pyrrolidin-4-yl-thio] - -methyl -6 - ( 1-hydroxyethyl) -1 -azabicyclof 3,2,0] -hept-2-ene-7 -one-2- carboxylate obtained in EXAMPLE 13-1 and εtirred to diεεolve. 0.4 g of 10% of the palladium/carbon catalyεt waε added and εtirred at room temperature for 2 hourε under an atmospheric presεure of hydrogen. After the reaction the catalyεt waε removed by filtration and the

filtrate waε concentrated under reduced pressure to evaporate the organic εolvent. After the reεidue was treated, as discribed in EXAMPLE 12-2, to give 0.26 g (46%) of the mixtureε of (E) and (Z) of 5 ( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N , N - diethylhydrazonomethyl)pyrrolidin-4-yl-thio] -4-methyl-6- ( 1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylic acid. NMR(DMSO-d _j ) δ:

10 0.93(t,6H), 1.13(m,6H), 1.53(m,lH), 2.10(m,lH),

2.40(m,lH), 2.64(m,lH), 2.84(m,lH), 3.01(q,4H), 3.10- 3.35(m,3H), 3.61-4.15(m,3H) EXAMPLE 14 (4R,5S,6S,8R,2 ^, S,4'S) -3-[2- [ -methyl-N- (N' ,N' -

-- dimethylsulfamoyl )hydrazonomethyl]pyrrolidin-4-yl-thio] -

4-methyl-6- ( 1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-2- ene-7-one-2-carboxylic acid EXAMPLE 14-1 0.468 g (1.29 mmol) of ( 4R, 5R,6S,8R) -p-nitrobenzyl -

20 4-methyl-6-(l-hydroxyethyl ) -1-azabicyclo[3,2, 0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 50 ml anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 0.271 ml (1.55 mmol) of N,N-

25 diiεopropylethylamine waε added dropwiεe, and 0.30 ml (1.45 mmol) of diphenylchlorophosphate waε added and εtirred for 2 hourε. Then 0.271 ml (1.55 mmol) of N,N- diiεopropylethylamine waε added and the εolution of 0.69 g (1.55 mmol) of (2S, 4S) -1-(p-nitrobenzyloxycarbonyl ) -2-

[N-methyl-N- (N ^f ,N' -dimethylsulfamoyl )hydrazono]methyl -4- mercaptopyrrolidine obtained in EXAMPLE 31-4 in 10 ml anhydrouε acetonitrile waε added dropwiεe to the mixture solution. After the addition, the ice bath waε removed, and the reaction mixture was εtirred at room temperature for 36 hourε. After the reaction, the reaction mixture waε concentrated under reduced preεεure, and ethylacetate waε added to the reεidue to extract the product. The extract was treated, aε diεcribed in EXAMPLE 8-1, to obtain 1.2 g of crude material. Thiε crude material waε εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=40 :1 ) and purified to give 0.152 g (50.2%) of the mixtureε of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 - [ l- (p- nitrobenzyloxycarbonyl) -2- [N-methyl -N- ( N ' ,N' - dimethylεulfamoyl )hydrazonomethylpyrrolidine-4-yl-thio] - -methyl-6- ( 1-hydroxyethyl ) -1-azabicyclof 3,2, 0] -hept-2- ene-7-one-2-carboxylate . NMR(CDC1 ₃ ) δ:

1.12(d,3H,J=7.4Hz) , 1.31 (d, 3H,J=6.3Hz ) , 2.10(m,lH) , 2.70 ( m , 1 H ) , 2 .90 ( ε , 6 H ) , 3 .10 ( s , 3H ) , 3.23(dd,lH,J=2.6Hz,J=6.9Hz) , 3.37(m,2H), 3.63(m,lH), 4.20(m,2H), 4.70(m,lH), 5.10-5.30(m, 4H) , 7.50(m,2H), 7.59(d,2H,J=8.7Hz) , 8.20(m,4H) EXAMPLE 14-2

To the mixture of 15 ml of tetrahydrofuran and 10 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.5 g (0.63 mmol) of the mixtureε of (E)

and (Z) of (4R,5S,6S,8R, 2 ' S , 4 ' S ) -p-nitrobenzyl - 3 -[ 1 -( p- nitrobenzyloxycarbonyl) -2- [N-methyl -N- (N' ,N' - dime thy lεulf amoyl )hydrazonome thyl] pyrrolidin -4 -yl-thio] - 4 -me thyl -6- ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept- 2- ene-7-one-2-carboxylate obtained in EXAMPLE 14-1 and stirred to dissolve. 0.25 g of 10% palladium/carbon catalyst waε added and stirred at room temperature for 3 hourε under an atmospher iε preεεure of hydrogen. After the reaction, the catalyεt waε removed by filtration and the filtrate waε concentrated under reduced pressure to evaporate the organic solvent. After the residue was treated, as discribed in EXAMPLE 12-2, to give 0.16 g (53.1%) of the mixtureε of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S) -3-[ (2-[N-methyl-N-(N' ,N' - dimethylsulf amoyl ) hydrazonomethyl ] py rrolidin-4-yl- thio] - -methyl -6- ( 1 -hydroxyethyl ) -1 -azabicyclof 3,2,0]- hept - 2 - ene - 7 - one - 2 - carboxyl i c acid . NMR(D ₆ 0) δ:

1.01(dd,6H), 1.59(m,lH), 2.10(m,lH), 2.40(m,lH), 2.60(m,lH), 2.84(m,lH), 2.90(s,6H), 3.10(s,3H), 3.10-

3.40(m,3H), 3.60 - 4.10 ( m, 3H ) EXAMPLE 15

(5R, 6S,8R,2 ^f S,4 ^* S)-3-[2-(N-me thane ulf onyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(l- hydroxyethyl ) -1 -azabi cyclof 3,2,0] -hept- 2 -ene- 7 -one- 2 - carboxylic acid

EXAMPLE 15-1

3.49 g (10.0 mmol) of ( 5R,6S,8R) -p-nitrobenzyl-6- (1-hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept-3,7-dione-2-

carboxylate obtained in REFERENCE EXAMPLE 3-1 was added to 200 ml anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 2.26 ml (14.0 mmol) of N,N- diisopropylethylamine was added dropwise, and 2.49 ml (12.0 mmol) of diphenylchlorophosphate was added and εtirred for 2 hours. Then the solution of 4.65 g (11.1 mmol) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2-(N- methaneεulfonyl-N-methylhydrazono) methyl- 4 - mercaptopyrrolidine obtained in EXAMPLE 25-5, 80 ml of anhydrous acetonitrile was added dropwiεe and 2.26 ml (13.0 mmol) of N,N-diisopropylethylamine was added to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture waε εtirred at room temperature for 20 hourε. After the reaction, the reaction mixture waε cooled to 0°C, stirred, and the precipitate was filtrated and washed with water and small amounts of acetone. The precipitate was dried in a vacuum desiccator to give 5.0 g (67.0%) of the mixtureε of (E) and (Z) of ( 5S,6S,8R,2'S,4 'S) -p- nitrobenzyl-3- [1- (p-nitrobenzyloxycarbonyl)-2-(N- methaneεulfonyl-N-methylhydrazonomethyl)pyrrolidin- -yl- thio] -6- (1-hydroxyethyl ) -1-azabicyclof 3,2,0] -hept-2-ene- 7-one-2-carboxylate. NMR(DMSO-d ₆ ) 6:

1.10(d,3H,J=6.3Hz) , 2.03(m,lH), 2.64(m,lH), 3.01(s,3H), 3.28(ε,3H), 3.32(m,3H), 3.82-4.20(m, 4H) , 4.58(m,lH), 5.02-5.50(m,3H) , 7.62(m,4H), 8.20(m,4H) EXAMPLE 15-2

5.0 g (6.70 mmol) of the mixtures of (E) and (Z) of ( 5R, 6S , 8R, 2 ' S , 4 ' S ) -p-nitrobenzyl - 3 - [ l - ( p- nitrobenzyloxycarbonyl) -2- (N-methansulfonyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(l- hydroxyethyl) -1 -azabi cyclof 3,2,0] -hept- 2 -ene- 7 -one- 2- carboxylate obtained in EXAMPLE 15-1 waε subjected to the preparatory thin layer chromatography on silica gel (eluted with chlorof orm:methylalcohol=20 :1 ) to separate the isomers and purified to give 0.7 g (14.0%) of (E)- ( 5R, 6S , 8R,2 ' S , 4 ' S ) -p-nitrobenzyl - 3 - [ l - (p- nitrobenzyloxycarbonyl ) -2- (N-methanesulfonyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(l- hyd roxyethyl) -1 -azabicyclof 3,2,0] -hept- 2 -ene- 7 -one- 2- carboxylate and 0.2 g (4.0%) of ( Z ) - ( 5R,6S ,8R,2'S, 4'S ) - p-nitrobenzyl- 3 - [ 1 - ( p- ni tr obenzyloxycarbonyl ) - 2- (N- methanesulf onyl-N-methylhydrazonomethyl)pyrrolidin-4 -yl - thio] -6- (1 -hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept -2- ene - 7 -one -2 -carboxylate . NMR ( DMSO -d _j ) δ: (E): 1.10(d,3H,J=6.3Hz) , 2.03(m,lH), 2.62(m,lH),

3.02(s,3H), 3.29(s,3H). 3.32(s,3H), 3.82-

4.19(m,4H), 4.58(m,lH), 5.01-5.48(m,3H) ,

7.62(m,4H), 8.20(m,4H) (E): 1.10(d,3H,J=6.2Hz) , 2.03(m,lH), 2.62(m,lH), 3.02(s,3H), 3.25(s,3H), 3.33(m,3H), 3.82-

4.20(m,4H), 4.59(m,lH), 5.02 - 5.46 (m , 3H ) ,

7.63(m,4H), 8.21(m,4H) EXAMPLE 15-3 To the mixture of 15 ml of tetrahydrofuran and 8 ml

of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) waε added 0.5 g (0.67 mmol) of (E) of ( 5R, 6S , 8R, 2 ' S, 4 ' S ) -p-nitrobenzyl - 3 - [ l - (p- nitrobenzyloxycarbonyl ) -2- (N-methaneεulfonyl-N- methylhydrazonometh l)pyrrolidin-4-yl-thio]-6-(l- hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept- 2-ene- 7-one- 2- carboxylate obtained in EXAMPLE 15-2 and εtirred to diεsolve. 0.2 g of 10% of the palladium/carbon catalyst waε added and stirred at room temperature for 5 hourε under an atmoεpheric preεεure of hydrogen. After the reaction, the catalyεt waε removed by filtration and the filtrate waε concentrated under reduced presεure to evaporate the organic εolvent. After the extracted impurity waε filtrated, the filtrate was washed with ethylacetate, and the water layer was concentraed under reduced presεure at 10-20°C to evaporate the residual organic solvent completely. The clarified filtrate was εubjected to polymerchromatography (Diaion HP-20 reεin; previouεly waεhed with methylalcohol and water). During the chromatography, it waε firεt eluted with water to remove 4-morpholinepropane sulfonic acid and then eluted with 5% aqueous acetone solution to collect the fractions containing the desired product. The fractions were combined and concentrated at 10-20°C under reduced presεure. The reεidue waε lyophilized to give 0.12g (41.3%) of ( E ) - ( 5R, 6S,8R, 2 ' S, 4 ' S) - 3 - [ (2 - (N- methaneεulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio]-6- (1-hydroxyethyl) -1-azabicyclof3,2,0] -hept-2-ene- 7-one-2-carboxylic acid.

NMR(DMSO-d ₆ ) 6:

1.05(d,2H,J=6.0Hz) , 1.62(m,lH), 2.40(m,lH),

2.65(m,lH), 2.91(s,3H), 2.92(m,lH), 3.04(s,3H),

3.22(m,3H), 3.60(m,lH), 3.82(m,2H), 4.03(m,lH) EXAMPLE 15-4

To the mixture of 8 ml of tetrahydrofuran and 4 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.2 g (0.26 mmol) of (Z) - (5R,6S,8R,2'S,4'S) -p- nitrobenzyl-3- [1- (p-nitrobenzyloxycarbonyl)-2-(N- methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio] -6- (1-hydroxyethyl) -1-azabicyclof3,2,0] -hept-2-ene- 7-one-2-carboxylate obtained in EXAMPLE 15-2 and stirred to dissolve. 0.1 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 6 hourε under a hydrogen gas stream. After the reaction, the catalyst was removed by filtration and the filtrate was concentrated under reduced presεure to evaporate the organic εolvent. The extracted impurity was removed by filtration and the filtrate was treated, as discribed in EXAMPLE 15-3, to . give 0.08 g (35.6%) of (Z)- (5R,6S,8R,2 'S, 4 ' S ) -3 - f2 - (N -me thanesulfonyl-N - methylhydrazonometh l)pyrrolidin-4-yl-thio]-6-(l- hydroxyethyl ) -1 -azabicyclof 3,2,0] -hept- 2-ene- 7-one- 2- carboxylic acid. NMR(DMSO-d ₆ ) δ:

1.04(d,2H,J=6.lHz) , 1.62(m,lH), 2.40(m,lH), 2.64(m,lH), 2.90(s,3H), 3.03(ε,3H), 3.21(m,3H), 3.59(m,lH), 3.81(m,2H), 4.01(m,lH) EXAMPLE 15-5

To the mixture of 150 ml of tetrahydrofuran and 50 ml of 0.1 M morpholinepropaneεulfonic acid buffer (pH

7.0) waε added 5.0 g (6.7 mmol) of (5R,6S,8R,2'S,4'S) -p- nitrobenzyl-3- fl- (p-nitrobenzyloxycarbonyl)-2-(N- methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio] -6- (1-hydroxyethyl ) -1-azabicyclof3,2,0] -hept-2-ene-

7-one-2-carboxylate obtained in EXAMPLE 15-2 and stirred to dissolve. 2.0 g of 10% of the palladium/carbon catalyεt waε added and stirred at room temperature for 5 hourε under an atmospheric presεure of hydrogen. After the reaction, the catalyεt was removed by filtration and the filtrate was concentrated under reduced preεεure to evaporate the organic solvent. After the extracted impurity waε removed and the filtrate waε treated, aε discribed in EXAMPLE 15-3, to give 1.24 g (42.7%) of

(5R,6S,8R,2'S, 4'S )-3 -[2-(N-methaneεulfonyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(l- hyd roxyethyl) -1 -azabicyclof 3,2,0] -hept- 2 -ene- 7 -one- 2- carboxylic acid. NMR(DMSO-d ₆ ) δ:

1.05(d ,2H,J=6.0Hz) , 1.62(m,lH), 2.39(m,lH),

2.64(m,lH), 2.90(ε,3H), 3.00(ε,3H), 3.25(m,3H), 3.59- 3.83(m,3H), 4.02(m,lH)

EXAMPLE 16 (4R,5S,6S,8R,2 ^, S,4'S)-3-f2- ( N-methanεulfonyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio] - -methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylic acid

EXAMPLE 16-1

1.520 g (4.19 mmol) of ( 4R,5R,6S,8R) -p-nitrobenzyl- 4-methyl- 6- ( 1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 180 ml anhydrouε acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 0.737 ml (4.23 mmol) of N,N- diiεopropylethylamine waε added dropwise, and 0.877 ml (4.23 mmol) of diphenylchlorophoεphate waε added and εtirred for 2 hourε. Then 0.737 ml (4.23 mmol) of N,N- diiεopropylethylamine was added and the solution of 1.80 g (4.18 mmol) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl ) -2- [1 - (N-methanesulfonyl -N-methylhydrazono )methyl] -4 - mercaptopyrrolidine obtained in EXAMPLE 32-5, 25 ml of anhydrouε acetonitrile was added dropwise to the mixture εolution. After the addition, the ice bath waε removed, and the reaction mixture waε stirred at room temperature for 24 hours. After the reaction, the reaction mixture was concentrated under reduced presεure, ethylacetate waε added to the residue to extract the product. The extract waε treated, aε diεcribed in EXAMPLE 8-1, to obtain 3.25 g of crude material. Thiε crude material waε εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=30:1 ) and purified to give 1.74 g (53.4%) of the mixtureε of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 -f l- (p- nitrobenzyloxycarbonyl) -2- (N-methaneεulfonyl-N- methylhydrazonomethyl)pyrrolidin-4-yl-thio] - -methyl-6- (1-hydroxyethyl) -1-azabicyclof 3,2,0] -hept-2-ene-7-one-2- carboxylate.

NMR(CDC1 ₃ ) δ:

1.18(d,3H), 1.30(d,3H), 2.02(s,3H), 2.03(m,lH),

2.65(m,lH), 2.83(m,3H), 3.29(dd,1H,J=6.8Hz,J=2.3Hz ) ,

3.38(m,2H), 3.70(m,lH), 4.10(dd,lH, J=6.8Hz,J=2.6Hz ) , 4.15 (m,lH) , 4.60( t, lH ) , 5.05 - 5.50 ( m , 4 H ) ,

7.52(d,2H,J=8.7Hz) , 7.60(d,2H, =8.7Hz) , 8.20(m,4H)

EXAMPLE 16-2

To the mixture of 20 ml of tetrahydrofuran and 25 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.68 g (0.878 mmol) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 - [ l- (p- ni trobenzyloxycarbonyl ) -2 - [ 1- (N -methaneεuIfonyl -N - methylhydrazonomethyl )pyrrolidin-4-yl-thio] -4-methyl-6- ( 1-hydroxyethyl) -1-azabicyclof 3 ,2,0 ] -hept-2-ene-7-one-2- carboxylate obtained in EXAMPLE 16-2 and εtirred to diεsolve. 0.3 g of 10% of the palladium/carbon catalyεt waε added and εtirred at room temperature for 5 hours under an atmospheric pressure of hydrogen. After the reaction, the catalyst waε removed by filtration and the filtrate was washed with a small amount of 0.5 M morpholineproparesulfonic acid and concentrated under reduced preεεure to evaporate the organic εolvent. After the reεidue waε waεhed with ethylacetate, separated, and the water layer was concentrated under reduced presεure below 20°C, then the formed impurity waε removed by filtration. The clarified filtrate was treated, aε diεcribed in EXAMPLE 8-5, to give 0.218 g (53.9%) of the mixtures of (E) and (Z) of ( 4R, 5S,6S,8R,2 'S, 4'S) -3 - f 2 - (N-methaneεulfonyl-N-methylhydrazonomethyl )pyrrolidin-4 -

yl - thi o ] - 4 -methyl - 6 - ( 1 -hyd roxyethyl ) - 1 - azabi cyclof 3,2,0]-hept-2-ene-7 -one -2 -carboxylic acid. NMR(CDC1 ₃ ) δ:

1.10(m,6H), 1.62(m,lH), 2.01(s,3H), 2.40(m,lH), 2.70 ( m , 1 H ) , 2 .80 ( ε , 3 H ) , 2 .98 ( s , 3H ) ,

3.10(dd,lH,J=6.4Hz,J=2.5Hz) , 3.12- 3.64 (m, 3H) , 3.80- 4.02(m,2H) , 4.10(dd, 1H, J=9.3Hz , J=2.4Hz )

EXAMPLE 17

(4R,5S,6S,8R,2 ^f S,4'S)-3-[2-( N-e thane εulf onyl -N - me thy lhydrazonome thyl ) pyrrolidin- 4 -yl-thio] -4 -methyl -6-

( 1 -hydroxyethyl) -1 -azabicyclof 3,2,0] -hept- 2 -ene -7 -one -2 - carboxylic acid

EXAMPLE 17-1

2.84 g (7.83 mmol) of (4R,5R,6S,8R) -p-nitrobenzyl- -methyl- 6- (1-hydroxyethyl) -1-azabicyclof3,2, 0] -hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 waε added to 250 ml anhydrouε acetonitrile, εtirred at room temperature and cooled to 0°C uεing an ice bath. At the εame temperature, 1.38 ml (7.92 mmol) of N,N- diisopropylethylamine was added dropwise, and 1.64 ml (7.91 mmol) of diphenylchlorophosphate waε added and εtirred for 2 hourε. Then 1.38 ml (7.92 mmol) of N,N- diiεopropylethylamine waε added and the εolution of 3.3 g (7.67 mmol) of (2S,4S)-1- (p-nitrobenzyloxycarbonyl ) -2- ( - ethaneεulfonyl -N-methylhydrazono ) methyl -4- mercaptopyrrolidine obtained in EXAMPLE 33-4 in 50 ml of anhydrouε acetonitrile waε added dropwiεe to the mixture εolution. After the addition, the ice bath waε removed, and the reaction mixture was stirred at room temperature

for 48 hourε. After the reaction, the reaction mixture waε concentrated under reduced presεure, and methylenechloride was added to the residue to extract the product. The extract was treated, as diεcribed in EXAMPLE 8-1, to obtain 5.35g of crude material. This crude material waε εubjected to the column chromatography on εilica gel (eluted with chlorof orm:acetone=30 : 1 ) and purified to give 3.42 g (57.5%) of the mixtureε of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 - f 1 - ( p - nitrobenzyloxycarbonyl ) -2- (N-ethanesulfonyl-N- methylhydrazonomethyl ) pyrrolidin- 4 -yl-thio] -4 -methyl -6- ( 1- hydroxyethyl) -1 -azabicyclof 3,2,0] -hept- 2 -ene -7 -one- 2- carboxylate . NMR(CDC1 ₃ ) δ:

1.30(m,9H), 2.10(m,lH), 2.70(m,lH), 3.10(m,lH), 3.50(dd,lH,J=4.4Hz,J=llHz) , 3.80(m,lH), 4.05(m,lH), 4.12(m,2H), 4.70(m,lH), 5.10 - 5.50 (m, 4H ) , 7.05(m,lH), 7.45(d,2H, =8.lHz) , 7.62 ( d , 2H, J=8.7Hz ) , 8.20(m,4H) EXAMPLE 17-2

To the mixture of 20 ml of tetrahydrofuran and 20 ml of 0.5 M morpholinepropaneεulfonic acid buffer (pH 7.0) was added 0.70g (0.90 mmol) of

(4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 -[ l- (p- nitrobenzyloxycarbonyl ) -2- (N-ethanesulfonyl-N- methylhydrazonomethyl ) pyrrolidin -4-yl -thio] - 4 -methyl -6 - ( 1- hydroxyethyl) -1 -azabicyclof 3,2,0] -hept -2 -ene -7 -one -2 - carboxylic acid obtained in EXAMPLE 17-1 and εtirred to diεsolve. 0.280 g of 10% of the palladium/carbon

catalyst waε added and stirred at room temperature for 7 hourε under an atmospheric pressure of hydrogen. After the reaction, the catalyεt was removed by filtration and the filtrate was concentrated under reduced presεure to evaporate the organic solvent. After the residue waε washed with ethylacetate, separated, and the water layer was concentraed under reduced pressure at 20°C, then the extracted impurity waε removed by filtration. The clarified filtrate waε treated, aε iε discribed in EXAMPLE 8-5, to give 0.227 g (54.5%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S) -3-[2-(N- ethaneεulfonyl-N-methylhydrazonomethyl )pyrrolidin-4 -yl- thio] - 4-methyl-6- ( 1-hydroxyethyl ) -1 -azabicyclof 3,2,0]- hept-2-ene-7-one-2-carboxylic acid. NMR(DMSO-d ₆ ) δ:

1.10(m,6H), 1.13(m,3H), 1.67(m,lH), 2.44(m,lH), 2.79 ( m , lH ) , 3.05 ( ε , 3 H ) , 3.15 ( m , 2 H ) , 3.18 ( dd, lH , J =2.3Hz ,J = 6.3Hz ) , 3.52 ( m , 2 H ) , 4.10(dd,lH,J=2.2Hz, J=9.3Hz), 7.15(d,1H,J=5.3Hz) EXAMPLE 18

( 2 S , 4 S ) - 1- ( p-nitrobenzyloxycarbonyl ) - 2 - methoxyimino-4-mercaptopyrrolidine

EXAMPLE 18-1

After 2.6 g (31.14 mmol) of methoxylamine hydrochloride salt was added to 180 ml of water and εtirred to diεεolve, 1.92 g (18.12 mmol) of εodium bicarbonate waε added dropwiεe and stirred at room temperature for 1 hour. After the reaction, the solution of 10.56 g (25.8 mmol) of ( 2S, 4R) -1- (p-

nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert - butyldi ethylεilyloxypyrrolidine in 360 ml of methylalcohol waε added dropwiεe to the reaction mixture solution. After the addition, the reaction mixture solution was stirred overnight. After the reaction, the mixture εolution waε concentrated under reduced preεsure to evaporate the εolvent, and then extracted with ethylacetate. After the extracted solution was washed several times with water and saturated with sodium chloride, the organic layer was dried over anhydrous magnesium sulfate, filtered and then the filtrate was concentrated under reduced pressure to give 11.5 g of the residue. The residue was subjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate=5:l ) and purified to 7.45 g (66.9%) of (E)- (2S,4R) -l-(p-nitrobenzyloxycarbonyl)-2- methoxyimino-4- tert-butyldime thyl εilyloxypyrr ol idine and 1.7 g (15%) of (Z )- (2S,4R) -1- (p-nitrobenzyloxycarbonyl ) - 2-methoxyimino-4 - tert-butyldimethylεilyloxypyrrolidine . NMR(Acetone-d _j )

(1) E form: δ:

0.03(ε,6H), 0.85(ε,9H), 2.10(m,2H), 3.50(m,2H), 3.75(s,3H), 4.55(m,2H), 5.22(m,2H), 7.38(d,lH), 7.65(d,2H), 8.21(d,2H) (2) Z form: δ:

0.03(s,6H), 0.85(ε,9H), 2.10(m,2H), 3.65(m,2H), 3.85(S,3H), 4.60(m,2H), 5.30(dd,2H), 7.38(d,lH), 7.65(d,2H), 8.21(d,2H) EXAMPLE 18-2

To 100 ml of tetrahydrofuran waε added 4.85 g ( 11.09 mmo l ) o f ( E ) - ( 2 S , 4R ) - l - ( p - nitrobenzyloxycarbonyl ) -2 - methoxyimino -4-tert- butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 18- 1 and stirred at room temperature to disεolve. Then 13.2 ml (13.2 mmol) 1 M te trabutylam onium fluoride( tetrahydrofuran εolution) was added dropwiεe and stirred at room temperature for 30 minutes. After the reaction, the mixture εolution waε concentrated under reduced pressure to evaporate the solvent, and the residue waε disεolved in ethylacetate and washed succesεively with water and εodium chloride. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 5.5 g of the residue.

The residue was subjected to the column chromatography on silica gel (eluted with chlorof orm:methanol= 10:1) and purified to give 3.36 g(93.8%) of (E) - (2S , 4R) -1 - (p- nitrobenzyloxycarbonyl ) - 2 -methoxyimino- 4 - hydroxypyrrolidine.

NMR(Acetone-d ₆ ) δ:

2.03-2.40(m,2H) , 3.45 - 3.73 ( m, 2H ) , 3.79(ε,3H),

4.23(m,lH), 4.43(m,lH), 7.39(d,lH), 7.70(d,2H),

8.23(d,2H) EXAMPLE 18-3

To 150 ml of methylene chloride was added 3.36 g ( 11.07 mmo l ) o f ( E ) - ( 2S , 4R ) - l - ( p - nitrobenzyloxycarbonyl ) -2 -methoxyimino- 4 - hydroxypyrrolidine obtained in EXAMPLE 18-2 and stirred

to dissolve. And then 3.4 ml (24.4 mmol) of triethyamine and 1.12 ml (14.4 mmol) of methanesulfonylchloride was added and stirred at room temperature for 30 minutes. After the reaction, the mixture εolution waε concentrated under reduced preεεure to evaporate the solvent, and the residue waε extracted with ethylacetate and waεhed εuccessively with water and εodium chloride. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 5.7 g of the reεidue. The residue was subjected to the column chromatography on εilica gel (eluted with n- hexane:ethylacetate=l .5:1 ) and purified to give 3.96 g (94.9%) of (E) - (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- methoxyimino-4-methanesulfonyloxypyrrolidine. NMR(Acetone-d _{ ) δ:

2.39-2.64(m,2H) , 3.19(s,3H), 3.74(m,3H), 3.75- 4.05(m,2H), 4.62(m,lH), 5.25(m,3H), 7.41(d,lH), 7.64(d,2H), 8.22(d,2H) EXAMPLE 18-4

In 200 ml of the solution of dimethylformamide and toluene (dimethylformamide:toluene=l :1 ) waε disεolved 3.31 g (8.68 mmol) of (E) - (2S, 4R) -1 - ( p- nitrobenzyloxycarbonyl ) -2 -methoxyimino- 4 - methaneεulfonyloxypyrrolidine obtained in EXAMPLE 18-3. After 16.2 g (14.2 mmol) of potasεium thioacetate waε added and was refluxed for 3 hourε, the reaction εolution was concentrated under reduced pressure and the residue was extracted with ethylacetate. The organic

layer was washed with water and εodium chloride, and the separated organic layer waε dried over anhydrouε magnesium sulfate and filtered. The filtrate waε concentrated under reduced preεsure to give 3.51 g of the residue. The residue was subjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate=l .5:1 ) and purified to give 2.4 g ( 72 . 5 % , E : Z = 3 : 2 ) o f ( 2 S , 4 S ) - 1 - ( p ^■ nitrobenzyloxycarbonyl ) -2-methoxyimino- 4 - acethylthiopyrrolidine. NMR(Acetone-d ₆ ) 6:

2.32(ε,3H), 2.52- 2.82 (m, 2H ) , 3.14(m,lH) , 3.75(ε,3Hx3/5) , 3.81( s, 3Hx2/5) , 3.94- .10(m,2H) , 4.40(m,lH), 5.22(m,2H), 6.82(dd,1HX2/5) , 7. 1(d,1HX3/5) , 7.62(d,2H), 8.21(d,2H) EXAMPLE 18-5

In 70ml of methyl alcohol waε diεεolved 2.4 g (6.29 mmol) of ( 2S, 4S) -1 - (p- nitrobenzyloxycarbonyl )- 2- methoxyimino-4 -acetylpyrrolidine obtained in EXAMPLE 18- 4. After the reaction εolution waε cooled to 0°C using the ice bath, 8.4 ml of 2 N sodium hydroxide aqueous solution was added dropwise and stirred for 3 minutes. Then, at the same temperature, the pH of the reaction εolution waε adjusted to pH 4.5 using saturated citric acid and concentrated under reduced presεure to evaporate the organic εolvent. To the reεidue waε added ethylacetate to extract the product from the εolution, washed with water and saturated εodium chloride, εeparated the organic solvent and dried over anhydrouε

magnesium sulfate. After filtration, the filtrate waε concentrated to give 3.0 g of crude material. Thiε crude material waε subjected to the column chromatography on silica gel (eluted with n-hexane : ethylacetate = 2 : 1) and purified to give 1.4 g (65.6%) of (2S,4S) -1- (p- nitrobenzyloxycarbonyl ) • 2 -methoxyimino- ■ mercaptopyrrolidine. NMR(Acetone-d ₆ ) δ:

2.01(m,lH), 2.65(m,lH), 3.19- 3.62 (m, 2H) , 3.76(ε,3Hx3/5) , 3.81 ( ε,3Hx2/5) , 3.85-4.20(m,1H) , 4.40- 5.02(m,lH), 5.23(m,2H), 6.90(dd,lHx2/5) , 7.44(d,lHx3/5) , 7.65(d,2H), 8.22(d,2H) EXAMPLE 19

(2S , 4S) -l- (p-nitrobenzyloxycarbonyl )-2 - ethoxycarbonylmethoxyimino-4-mercaptopyrrolidine EXAMPLE 19-1

After 1.97 g (18.58 mmol) of sodium bicarbonate and 2.46 g (34.4 mmol) of hydroxylamine hydrochloride salt were added to 380 ml of water and stirred to dissolve (the reaction solution A). In another container 14 g (34.2 mmol) of (2S ,4R) -1(p-nitrobenzyloxycarbonyl) -2- formyl-4-tert-butyIdimethylεilyloxypyrrolidinewaε added in 400 ml of methyl alcohol and diεεolved (the reaction solution B). After solution B was added to the εolution B, the reaction mixture εolution was εtirred at room temperature for 20 hourε, the precipitate waε filtered, and the filtrate was concentrated under reduced presεure. After 30% methylalcohol (aq) was added to the residue and stirred for 20 minutes, it filtered the

precipitate. The above two precipitates were combined and dried in vacuum to give 14.18 g (98.9%) of (2S,4R)- 1- ( p-nitrobenzyloxycarbonyl) -2-hydrox imino-4-tert- butyldimethylεilyloxypyrrolidine. NMR(Acetone-d _{ ) δ:

0.10(d,6H), 0.89(m,9H), 2.15-2.40(m,lH) , 2.82(m,lH), 3.52(m,2H), 4.55(m,2H), 5.23(m,2H), 6.78(m,lHxl/3) , 7.40(m,1HX2/3) , 7.68(d,2H), 8.21(d,2H) EXAMPLE 19-2 To 450 ml of acetone was added 9.38 g (22.16 mmol) of (2S,4R)-l-(p-nitrobenzyloxycarbonyl) -2-hydroxyimino- 4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 19-1 and stirred to disεolve. Then 15.31 g (110.8 mmol) of potaεεium carbonate and 4.91 ml (44.32 mmol) of ethylbromoacetate were added and refluxed at room temperature for 3 day . After the reaction, the mixture εolution was concentrated under reduced presεure to evaporate the εolvent, the reεidue waε diεεolved in water and ethylacetate, and the reaction solution waε adjuεted to pH 5.5 uεing εaturated citric acid εolution. Then the exceεε ethylacetate was added to the reaction solution and shaken, the mixture solution was waεhed successively with water and sodium chloride, the organic layer waε dried over anhydrouε magnesium sulfate, filtered, and then the filtrate was concentrated to give 13.5 g of the crude material. This crude material waε εubjected to the column chromatography on εilica gel (eluted with n-hexane:ethylacetate=2.5:1 ) and purified to 8.83 g ( 78.2%) of ( 2 S , 4R ) - 1 - ( p -

nitrobenzyloxycarbonyl ) -2-ethoxycarbonyl methoxyimino -4 - tert-butyldime thy lsilyloxypyrroli dine . NMR(Acetone-d _j ) δ:

0.03(d,6H), 0.83(ε,9H), 1.12(t,3H), 2.07(m,2H), 3.38 -3.60(m,2H) , 4.20(q,2H), 4.30 - 4.65 (m , 3H) ,

5.22(m,2H), 6.79 (m, lHxl/3 ) , 7.41 (d, 1HX2/3 ) , 7.50(m,2H), 8.20(d,2H)

EXAMPLE 19-3

To 120 ml of tetrahydrofuran waε added 8.08 g (15.86 mmol) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- e t h ox y ca r bo n yl m e th o x y i mi n o - 4 - t e r t - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 19- 2 and εtirred to diεεolve. Then 19.8 ml (19.8 mmol) of 1 M-tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwise and εtirred at room temperature for 30 minutes. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the solvent, and the residue waε extracted with ethylacetate and dried over anhydrouε magnesium sulfate. In the reaction εolution were added dropwiεe 5.27 ml (39.65 mmol) of triethylamine and 1.85 ml (23.9 mmol) of methaneεulfonyl chloride, and after the removal of the ice bath, εtirred at room temperature for 1 hour. After the reaction, the mixture εolution waε concentrated under reduced presεure to evaporate the εolvent, and the reεidue waε extracted with ethylacetate. The extract waε waεhed εuccesεively with water and εaturated εodium chloride, and the organic layer waε dried over anhydrouε magnesium sulfate,

filtered and then concentrated under reduced preεεure to give 8.4 g of the crude material. Thiε crude material waε subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate =1:1.5). After filtration, the filtrate waε concentrated under reduced preεεure and the reεidue waε diεsolved in 160 ml of methylene chloride, and cooled to 0°C uεing an ice bath and purified to 5.84 g (77.8%) of (2S,4R) -1- (p- nitrobenzyloxycarbony1) -2-ethoxycarbonylmethoxyimino-4- methanesulfonyloxypyrrolidine. NMR(Acetone-dg) δ:

1.10(m,3H), 2.44(m,2H), 2.80(m,2H), 3.20(s,3H), 3.64-4.01(m,2H) , 4.12(q,2H), 4.51-4.72(m,3H) , 5.21- 5.42(m,3H), 7.01(dd,lHxl/3 ) , 7.58(d,lHx2/3 ) , 7.63(d,2H), 8.21(d,2H)

EXAMPLE 19-4

In 300 ml of the solution of acetonitrile waε diεεolved 3.31 g (8.68 mmol) of (2S,4R) -1- (p- nitrobenzyloxycarbony1) -2-ethoxycarbonylmethoxyimino-4- methaneεulfonylpyrrolidine obtained in EXAMPLE 19-3. After 5.64 g (49.4 mmol) of potasεium thioacetate waε added and refluxed for 3 hourε, the reaction εolution waε concentrated under reduced preεεure and the reεidue was extracted with ethylacetate. The organic layer waε washed with water and sodium chloride and the separated organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate waε concentrated under reduced presεure to give 3.51 g of the reεidue. The residue was subjected to the column chromatography on

silica gel (eluted with n-hexane:ethylacetate=l.5:1 ) and purified to 4.16 g (72.8%) of (2S,4S ) -1- (p- nitrobenzyloxycarbony1) -2-ethoxycarbonylmethoxyimino-4- acethylthiopyrrolidine. NMR(Acetone-d ₆ ) δ:

1.10(m,3H), 2.01(m,lH), 2.30(ε,3H), 2.70(m,lH), 3.20-3.62(m,2H) , 3.92- .22(m, 4H) , 4.50-4.62(m,2H) , 5.25(ε,2H), 6.92(dd,lHxl/3) , 7.54(d,1HX2/3 ) , 7.64(d,2H), 8.23(d,2H) EXAMPLE 19-5

In 50 ml of ethyl alcohol was disεolved 4.16 g (9.18 mmol) of ( 2S,4S) -1- (p-nitrobenzyloxycarbonyl ) -2- ethoxycarbony1 methoxyimino-4-acetylthiopyrrolidine obtained in EXAMPLE 19-4. After the reaction εolution waε cooled to 0°C uεing the ice bath, 6 ml of 2 N εodium hydroxide aqueous solution was added dropwiεe and stirred at 0°C for 3 minutes. Then, the pH of the reaction solution was adjusted to pH 3.5 using saturated citric acid and concentrated under reduced preεεure to evaporate the organic εolvent. The reεidue was extracted with ethylacetate, and the organic layer washed with water and εaturated sodium chloride, and dried over anhydrous magnesium sulfate. After filtration, the filtrate waε concentrated under reduced preεεure to give 5.78 g of the reεidue. The reεidue was subjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate =1:1) and purified to 3.83 g (75%) of (2S, 4S ) - 1- (p-nitrobenzyloxycarbonyl) - 2 - ethoxycarbonylmethoxyimino-4-mercaptopyrrolidine.

NMR(Acetone-d ₆ ) δ:

1.12(m,3H), 2.03(m,lH), 2.65(m,lH), 3.25-

3.75(m,2H) , 3.95 - 4.25 ( m, 2H ) , 4.43 - 4.70 (m ,2H) ,

5.30(ε,2H), 6.98(dd,lHxl/3) , 7.51 (d, lHx2/3 ) , 7.65(d,2H), 8.21(d,2H)

EXAMPLE 20

(2S,4S)-l-(p-nitrobenzyloxycarbonyl ) - 2 - ( N, N- dimethylaminocarbonylmethoxyimino) -4-mercaptopyrrolidine

EXAMPLE 20-1 1.85 g (3.63 mmol) of (2S,4R)-l(p- nitrobenzyloxycarbonyl) -2-ethoxycarbonylmethoxyimino-4- tert-butyldimethylεilyloxypyrrolidine was added to 30 ml of acetone and stirred to disεolve. At room temperature, 2.8 ml of 2 N Sodium hydroxide aqueouε εolution was added dropwiεe and εtirred for 3 hourε, and at the εame temperature the pH of the εolution waε adjuεted to pH 2.0 using saturated citric acid. The layer was washed several times with water and εaturated sodium chloride after separation, the organic layer waε dried over anhydrous magneεium sulfate, filtered, and then the filtrate waε concentrated under reduced preεsure. The solid material produced was dried under reduced pressure 1.73 g (99%) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl ) -2-carboxymethoxyimino -4-tert- butyldimethylsilyloxypyrrolidine.

NMR(CDC1 ₃ ) δ:

0.03(d,6H), 0.81(ε,9H), 2.10(m,2H), 3.50(m,2H), 4.38(m,lH), 4.58(m,3H), 5.20(m,2H), 6.80(m,lHxl/3) , 7.40(d,lHx2/3) , 7.50(d,2H), 8.19(d,2H)

EXAMPLE 20 - 2

To 70 ml of tetrahydrofuran was added 4.13 g (8.58 mmol) of ( 2S, 4R) -1- (p- nitrobenzyloxycarbonyl )- 2- c a r b o x y m e t h o x y i m i n o - 4 - t e r t - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 20- 1 and cooled to 0°C uεing an ice bath. Then, 1.56 ml (11.19 mmol) of triethylamine and 0.93 ml (10.35 mmol) of ethylchloroformate waε added dropwiεe and εtirred at the εame temperature for 30 minuteε. 0.93 ml (10.3 mmol) of 50% of the dimethylamine aqueous εolution waε added dropwiεe, removed from the ice bath and εtirred at room temperature for 1 hour. After the reaction, the reaction mixture waε concentrated under reduced pressure, and extracted with ethylacetate. The organic layer was washed succeεεively with water and εaturated aqueouε εodium chloride. After separation, the organic layer was dried over anhydrouε magnesium sulfate, filtered, and concentrated to give 4.14 g (95%) crude material of ( 2S, 4R) -1 - (p-nitrobenzyloxycarbonyl ) -2- (N,N- dimethylaminocarbonylmethoxyimino ) - 4 -tert - butyldimethylεilyloxypyrrolidine . This crude material waε uεed at the next reaction without purification. NMR(CDC1 ₃ ) δ:

0.03(ε,6H), 0.80(ε,9H), 2.03(m,2H), 2.95(d,6H), 3.50(m,2H), 4.30-4.75(m, 4H) , 5.20(m,2H), 6.80(m,lHxl/3 ) , 7.50(d,lHx2/3) , 8.19(d,2H) EXAMPLE 20-3

To 70 ml of tetrahydrofuran waε added 4.3 g (8.46 mmol) of (2S, 4R) -1- (p-nitrobenzyloxycarbonyl ) -2- (N,N-

dimethylaminocarbonyl methoxyimino) - 4-tert - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 20- 2 and added dropwiεe 10.5 ml (10.5 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran εolution) which waε stirred at room temperature for 30 minutes. After the reaction, the reaction mixture waε concentrated under reduced preεεure and extracted with ethylacetate. The organic layer waε dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced preεεure to give 3.29 g of the crude material. The crude material waε diεεolved in 70 ml of methylenechloride. After the temperature of the reaction εolution was adjusted to 0°C using an ice bath, 2.9 ml (21.02 mmol) of triethylamine and 0.98 ml (12.66 mmol) of methanesulfonyl chloride was added dropwiεe, and raised the temperature up to room temperature by removal of the ice bath. At the same temperature, after stirring for 1 hour, the reaction mixture was concentrated under reduced presεure and extracted with ethylacetate. The orgainc layer was washed succeεsively with water and saturated aqueous εodium chloride. After εeparation, the organic layer waε dried over anhydrouε magneεium sulfate, filtered, and concentrated to give 5.6 g of the reεidue. The reεidue waε εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=20:1 ) and purified to 3.4 g (85%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- (N,N- dimethylaminoca rbonyl methoxyimino ) -4 - methaneεulfonyloxypyrrolidine .

NMR(Acetone-d ₆ ) δ :

1.80-2.20(m,2H) , 2.80 - 3.05 ( d, 6H) , 3.09(ε,3H), 3.50- 3.70(m,2H), 4.50 - 4.80 (m, 3H ) , 5.10(m,lH), 5.20(m,2H), 6.90(dd,lHxl/3) , 7.50(d,lHx2/3 ) , 7.65(d,2H), 8.21(d,2H) EXAMPLE 20-4

In 100 ml of acetonitrile waε diεεolved 1.55 g

(32.84 mmol) of ( 2S , R) -1 -( p-nitrobenzyloxycarbonyl ) -2 -

(N,N-dimethylaminocarbonylmethoxyimino ) -4 - methaneεulf onyloxypyrrolidine obtained in EXAMPLE 20-3. After 1.1 g (96.3 mmol) of potaεεium thioacetate waε added and refluxed for 4 hourε, as discribed in EXAMPLE

19-4, and waε carried out to give 1.2 g of crude material. This crude material solution was concentrated under reduced presεure to give 3.51 g of the reεidue. The reεidue was subjected to the column chromatography on silica gel (eluted with acetone :ethylacetate=l :9 ) and purified to 0.82 g (55%) of ( 2S,4S ) -1 - (p- nitrobenzyloxycarbonyl ) - 2- ( N,N -dimethylaminocarbonyl methoxyimino) -4 - ace tyl thiopyrrol idine . NMR(CDC1 ₃ ) δ :

2.05(m,2H), 2.12(s,3H), 2.90(d,6H), 3.55(m,lH), 3.95(m,2H), 4.50-4.80(m,3H) , 5.19(s,2H), 6.80 ( , lHxl/3 ) , 7.50(d,lHx8/3) , 8.18(d,2H)

EXAMPLE 20-5 in 30 ml of methyl alcohol waε disεolved 0.68 g

(0.503 mmol) of ( 2S, 4S ) -1 -( p-nitrobenzyloxycarbonyl ) -2 - ( N,N-dimethylaminocarbonylmethoxyimino ) - 4 - acetylpyrrolidine obtained in EXAMPLE 20-4 and cooled to 0°C uεing an ice bath. 1.5 ml of 2 N sodium hydroxide

aqueous solution waε added dropwiεe and εtirred for 3 minuteε. Then, at the εame temperature, the pH of the reaction solution was adjusted to pH 4.2 using saturated citric acid. The reaction mixture was concentrated under reduced preεεure, aε discribed in EXAMPLE 18-5, and was carried out to give 0.82 g of crude material. This crude material waε εubjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=30:1 ) and purified to 0.524 g (85%) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2- (N,N- dimethylaminocarbonylmethoxyimino) -4-mercaptopyrroldine. NMR(CDC1 ₃ ) δ :

2.0(m,2H), 2.40-2.60(m,2H) , 2.82(s,6H), 3.32(m,lH), 3.95(m,2H), 4.44(m,lH), 4.62(m,2H), 5.10(m,2H), 7.42(d,2H,J=8.6Hz) , 8.59 ( d, 2H, J=8.1Hz )

EXAMPLE 21

(2S,4S) -1- (p-nitrobenzyloxycarbonyl ) -2- ( p - methoxybenzyloxyca rbonylmethoxyimino ) - 4 - mercaptopyrrolidine EXAMPLE 21-1

The εolution of 6.6 g (13.7 mmol) of (2S,4R) -1- (p- nitrobenzyloxycarbony1 ) -2- ( carboxymethoxyimino) -4-tert- butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 20- 1 in 150 ml of N,N-dimethylformamide waε added to the reaction mixture, 4.9 g (35.45 mmol) of potassium carbonate added, and 2.3 g (14.7 mmol) of p- methoxybenzylchloride was added with vigorous stirring. At the same temperature, the reaction mixture waε εtirred for a day, concentrated under reduced preεsure

by a high vacuum pump, and then extracted with ethylacetate. After the extracted εolution waε waεhed with water and saturated aqueous εodium chloride, the organic layer waε dried over anhydrouε magneεium sulfate, filtered, and then the filtrate waε concentrated under reduced presεure to give 6.04 g (73.3%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- (p- methoxybenzyloxyca bonyl methoxyimino) -4-tert- butyldimethylεilyloxypyrrolidine NMR(CDC1 ₃ ) δ:

0.03(d,6H), 0.80(ε,9H), 2.00(m,2H), 3.50(m,2H), 3.80(ε,3H), 4.40(m,lH), 4.60(m,3H), 5.10(m,2H), 5.20(m,2H), 6.83(d,lHx7/3) , 7.23(d,2H), 7.50(m,1HX8/3) , 8.19(d,2H) EXAMPLE 21-2

(2S,4R) -1- (p-nitrobenzyloxycarbonyl ) -2- ( p- methoxybenzyloxycarbonylme thoxyimino) - 4 -tert - butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 21- 1 was treated with the same operation as EXAMPLE 20 to give the desired product of ( 2S , 4S ) -1- (p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( p - methoxybenzyloxyca rbonylmethoxyimino ) - 4 - mercaptopyrrolidine . NMR(CDC1 ₃ ) δ: 1.80(t,lH), 2.02(m,2H), 2.39-2.90(m,1H) , 3.30-

3.70(m,2H), 3.78(s,3H), 4.00(m,lH), 4.42-4.64(m,2H) , 4.70- 5.05 (m, 1H ) , 5.15(m,4H), 7.00 ( d, lHx2/3 ) , 7.56(d,lHxl/3) , 8.22(d,4H) EXAMPLE 22

( 2 S , 4 S ) -1- ( p-nitrobenzyloxycarbonyl ) - 2 - aminocarbonylmethoxyimino-4-mercaptopyrrolidine EXAMPLE 22-1

4.29 g (8.91 mmol) of (2S, R) -1- (p- nitrobenzyloxycarbonyl) -2-carboxymethoxyimino -4-tert- butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 20- 1 waε disεolved in 100 ml of tetrahydrofuran and cooled to 0°C uεing an ice bath. Then 1.61 ml (11.6 mmol) of triethylamine and 1.02 ml (10.7 mmol) of ethylchloroformate was added dropwise, and stirred at the same temperature for 1 hour. 2 ml of 28% of aqueous ammonia was added, removed from the ice bath, and stirred at room temperature for 1 hour. After the reaction, the reaction mixture was concentrated under reduced pressure and then extracted with ethylacetate. After the extract was washed εucceεεively with water and εaturated aqueouε sodium chloride, the organic layer waε dried over anhydrous magnesium sulfate, Filtered and then the filtrate waε concentrated under reduced presεure to give 6.-7 g of crude material. The crude material was εubjected to the column chromatography on εilica gel (eluted with chloroform: methylalcohol=20:1 ) and purified to 3.25 g (76%) of (2S,4R) -1- (p- nitrobenzylo ycarbonyl ) - 2-aminocarbonylmethoxyimino-4 - tert-butyldimethylεilyloxypyrrolidine. NMR(CDC1 ₃ ) δ:

0.03(d,6H), 0.83(ε,9H), 1.70- 2.15 (m, 2H ) , 3.46(m,2H), 4.35-4.65(m,4H) , 5.20(m,2H), 6.60(d,lHxl/3 ) , 7.49(d,lHx8/3) , 4.35-8.20(d,2H)

EXAMPLE 22-2

In 80 ml of tetrahydrofuran waε diεεolved 2.52 g (5.26 mmol) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl ) -2- am in oc a r bo ny l m et ho xy im in o - 4 - te rt - butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 22- 1. Then, 6.29 ml (6.29 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwiεe and εtirred at room temperature for 30 minutes. After the reaction, the reaction mixture was concentrated under reduced presεure. The reεidue waε diεεolved in ethylacetate and dried over anhydrouε magneεium εulfate, and then concentrated under reduced preεεure. The reεidue waε diεεolved in 80 ml of methylenechloride and cooled to 0°C using an ice bath 1.46 ml (10.84 mmol) of triethylamine and 0.53 ml (6.81 mmol) of methanesulfonylchloride which waε added dropwiεe, removed from the ice bath, and εtirred at room temperature for 1 hour. After the reaction, the mixture εolution waε concentrated under reduced preεεure, extracted with ethylacetate and waεhed εucceεεively with water and brine. After the εeparation of the organic layer, it waε dried over anhydrous magnesium sulfate and filtrate, and then the filtrate waε concentrated to give 2.8 g of the reεidue. The reεidue waε subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=25:l) and purified to 1.56 g (66.9%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl ) -2- a m i n o c a r b o n y l m e t h o x y i m i n o - 4 - methanesulfonyloxypyrrolidine.

NMR(CDC1 ₃ ) δ:

2.01-2.80(m, 2H) , 3.03(ε,3H) , 3.70(m,lH) , 4.01(m,lH), 4.42-4.70(m,3H) , 5.20(m,3H), 6.62(d,lHxl/3 ) , 7.49(d,lHx8/3), 8.20(d,2H) EXAMPLE 22-3

1.28 g (2.89 mmol) of (2S, 4R) -1- (p- nitrobenzyloxycarbonyl ) - 2-aminocarbonylmethoxyimino-4- methaneεulfonyloxypyrrolidine obtained in EXAMPLE 22-2 was disεolved in 100 ml of acetonitrile. Then, 0.825 g (7.23 mmol) of potassium thioacetate waε added and refluxed for 4 hourε. After the reaction, the mixture εolution waε concentrated under reduced presεure, and the reεidue was treated, aε diεcribed in EXAMPLE 19-4, to obtain 2.2 g of the crude material. The crude material waε subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=25:l ) and purified to 1.0 g (81.7%) of (2S, 4S) -1- (p- nitrobenzyloxycarbonyl ) - 2-aminocarbonylmethoxyimino-4- acetylthiopyrrolidine. NMR(CDC1 ₃ ) δ:

2.03(m,lH), 2.28(s,3H), 2.60(m,lH), 3.34(m,lH), 3.98(m,2H), 4.43(m,H), 5.19(s,2H), 6.01-6.43(m,2H) , 6.70(d,lHxl/3) , 7.50(d,lHx8/3) , 8.19(d,2H) EXAMPLE 22-4 In the mixture εolvent of 50 ml of methylalcohol and 10ml of tetrahydrofuran waε diεεolved 1 g (2.36 mmol) of ( 2S,4S) -1- (p- nitrobenzyloxycarbonyl )- 2- aminocarbonylmethoxyimino- 4 -acetylthiopyrrolidine obtained in EXAMPLE 22-3 and cooled to 0°C using an ice

bath. After 2.4 ml 2 N εodium hydroxide waε added dropwise and stirred for 3 minutes. The pH of the εolution was adjusted to pH 3.5 using a εaturated citric acid εolusion. The reaction solution was concentrated under reduced presεure, the reεidue waε extracted with ethylacetate, and then treated, as discribed in EXAMPLE 19-1, to give 0.95 g of crude material. The crude material was εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=18:1) and purified to 0.77 g (85.6%) of (2S,4S) -1- (p- nitrobenzyloxycarbonyl ) - 2-aminocarbonylmethoxyimino-4- mercaptopyrrolidine. NMR(CDC1 ₃ ) δ:

1.60-2.00(m,2H) , 2.40(m,2H), 3.20-3.60(m,2H) , 3.90(m,2H), 4.40(m,2H), 5.09(m,lH), 5.12(ε,2H), 5.85- 6.80(m,2H), 6.80(d,lHxl/3) , 7.42(d,lHx8/3) , 8.05(d,2H) EXAMPLE 23

(2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2- ( N- methylaminocarbonylmethoxyimino) -4-mercaptopyrrolidine EXAMPLE 23-1

To 90 ml of tetrahydrofuran cooled to 0°C uεing an ice bath waε added 5.31 g (11.03 mmol) of (2S,4R) -1- (p- nitrobenzyloxycarbony1 ) -2-carboxymethoxyimino -4-tert- butyldimethylεilyloxypyrrolidine and εtirred to diεsolve. 2 ml(14.36 mmol) of triethylamine and 1.27 ml (13.28 mol) of ethyl chloroformate was added and the reaction mixture solution εtirred at the same temperature for 30 minuteε. Then, 1.07 ml (14.3 mmol) of 40% methylamine aqueouε εolution added and stirred

for 1 hour without an ice bath. After the reaction, the mixture solution waε concentrated under reduced presεure, and then extracted with ethylacetate. The extract was treated aε diεcribed in EXAMPLE 20-2, to give 5.3 g (97.2% ) of ( 2S , 4 R ) - 1 - ( p - n i t r o b e n z y l o x y c a r b o n y l ) - 2 - ( N - methylaminocarbonylme thoxyimino ) - 4 - tert - butyldimethylεilyloxypyrrolidine . The product was used for the next reaction without purification. NMR(CDC1 ₃ ) δ:

0.03(s,6H), 0.80(ε,9H), 2.05(m,2H), 2.92(d,3H), 3.48(m,2H), .29 - 4.72 ( m, 4H) , 5.21(m,2H), 6.80 ( m, lHxl/3 ) , 7.51(m,lHx8/3) , 8.20(d,2H) EXAMPLE 23-2 (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2-(N- methylaminocarbonylme thoxyimino ) - 4 - tert - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 23- 1 waε treated, aε discribed in EXAMPLE 20, to give ( 2 S , 4 R ) - 1 - ( p-nitrobenzyloxycarbonyl ) -2 - ( N - methylaminocarbonylmethoxyimino ) -4-mercaptopyrrolidine .

NMR(CDC1 ₃ ) δ:

2.10(m,lH), 2.55(m,lH), 2.60(d,3H), 3.20-

3.70(m,2H), 4.05(m,lH), 4.42(d,2H), 4.50 - 4.80 (m, 1H) , 5.32(d,2H), 7.01-7.50(m,lH) , 7.70(d,2H), 8.22(d,2H) EXAMPLE 24

( 2S,4S ) -l-(p- nitrobenzyloxycarbonyl ) -2-hydroxyimino -mercaptopyrrolidine EXAMPLE 24-1 In 200 ml of methylenechloride, cooled to 0°C uεing

an ice bath, waε diεεolved 19.9 g (61.36 mmol) of (E)-l- (p-nitrobenzylo ycarbonyl) - -hydroxy-L-prolinemethy1 ester. 11.12 ml (79.78 mmol) of triethyl amine and 4.99 ml (64.45 mmol) of methaneεulfonylchloride waε added and εtirred at room temperature for 3 hourε after removing the ice bath. After the reaction, 200 ml of water waε added, εhaken vigorouεly, and the water layer εeparated from the organic layer. To the water layer waε added 250 ml of methylenechloride which waε shaken vigorously to extract the product. The organic layer was combined with the previous organic layer and waεhed εucceεεively with water and brine. The organic layer was εeparated again, dried over anhydrouε magneεium εulfate, filtered, and then the filtrate waε concentrated under reduced preεεure to give 24.45 g (99%) of crude material of (E) -1-(p-nitrobenzyloxycarbonyl) -4-methaneεulfonyl-L- prolinemethyl eεter. Thiε material waε used for the next reaction without purification. NMR(CDC1 ₃ ) δ: 2.02(m,lH), 2.35(m,lH), 3.01(ε,3H), 3.50- 3.70(m,2H), 3.60(s,3H), 4.10(m,lH), 5.20(ε,2H), 5.22(m,lH), 7.43(d,2H), 8.15(d,2H) EXAMPLE 24-2 After 2.16 g (19.46 mmol) of anhydrouε calcium chloride waε added to 30 ml of anhydrouε ethylalcohol, εtirred and diεpersed the mixture solution waε cooled to 0°C uεing an ice bath and 1.07 g (28.28 mmol) εodium borohydride (εolution A) was added. After stirring at the εame temperature, the εoiution of 2.85 g (7.08mmol)

of (E) -1-( -nitrobenzyloxycarbonyl) -4-methanesulfonyl-L- prolinemethyl ester obtained in EXAMPLE 24-1 in 11 ml of anhydrous ethylalcohol waε added dropwise to εolution A. After removal of the ice bath, the reaction mixture solution waε added and stirred at room temperature for 3 hours, saturated citric acid solution was added to remove the excesε sodium borohydride, and the mixture waε concentrated under reduced preεεure. The reεidue waε extracted with ethylacetate and waεhed εucceεεively with water and brine. After the separation of the organic layer, it waε dried over anhydrous magnesium sulfate, filtred and then the filtrate waε concentrated to give 2.59 g (7.7%) of nearly pure material of (2S,4R)-1- (p-nitrobenzyloxycarbonyl ) -2-hydroxymethyl-4- methaneεulfonyloxypyrrolidine. This material was uεed for the next - reaction without purification. NMR(CDC1 ₃ ) δ:

2.03(m,lH), 2.32(m,lH), 3.02(s,3H), 3.50- 4.20(m,5H), 5.19(ε,2H), 5.21(m,lH), 7.22(d,2H), 8.15(d,2H)

EXAMPLE 24-3

In 10 ml of anhydrouε methylenechloride, cooled to -10°C uεing a dry ice bath, waε diεεolved 298 μl (3.49 mmol) of oxalylchloride, the reaction mixture εolution cooled to -78°C. then, 454 μl (6.40 mmol) of anhydrouε dimethylsulfoxide was added dropwiεe, εtirred at the εame temperature for 10 minutes, and the εolution of 1.09 g (2.91 mmol ) of ( 2S , 4 R ) - 1 - ( p - nitrobenzyloxycarbonyl ) - 2-hydroxymethyl -4 -

methanesulfonyloxypyrrolidine obtained in EXAMPLE 24-2 in 5 ml of anhydrous methylenechloride was added dropwise. After the addition, the solution was stirred at -60°C for 30 minutes 2.03 ml (11.64 mmol) of diisopropylethylamine was added, and the solution from the dry ice bath to naturally raise the temperature to room temperature. At room temperature, the solution waε stirred for 1 hour, concentrated under reduced preεεure, and the reεidue waε extracted with ethylacetate and waεhed εuccesεively with water and brine. After the εeparation of the organic layer, it waε dried over anhydrouε magneεium εulfate, filtred and then the filtrate waε concentrated to give 1.3 g of crude material. The crude material waε εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=40:1) and purified to 0.95 g (87.7%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- formyl-4-methaneεulfonyloxypyrrolidine. NMR(CDC1 ₃ ) 6: 2.10-2.60(m,2H) , 3.01(d,3H), 3.60-4.01(m,2H) , 4.45(m,lH), 5.19(m,lH), 5.22(ε,2H), 7.43(d,2H), 8.15(d,2H), 9.50(d,lH) EXAMPLE 24-4 After 86.8 ml (1.25 mmol) of εodium bicarbonate and 164 mg (1.55 mmol) of hydroxylamine hydrochlorate εalt waε added to 10 ml of water, the solution of 442 mg (1.19 mmol of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- formyl-4-methaneεulfonyloxypyrrolidine obtained in EXAMPLE 24-3 in 10 ml of ethylalcohol was added and then

εtirred at room temperature for 1.5 hourε. The reaction solution was concentrated under reduced pressure and the residue was extracted with ethylacetate. The organic layer was waεhed with water and brine and the εeparated organic layer waε dried over anhydrouε magneεium εulfate and filtred. 640 mg of the obtained crude material waε concentrated under reduced preεεure to give 350mg of the reεidue. The residue waε subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=25:l) and purified to 350 mg (75.9%) of (2S,4R)-l-(p-nitrobenzyloxycarbonyl) -2-hydroxyimino-4- methanesulfonyloxy pyrrolidine. NMR(CDC1 ₃ ) δ:

2.01- 2.90 (m, 2H) , 3.00(d,3H) , 3.65(m,lH) , 3.95(m,lH), 4.63(m.lH), 5.20(m,3H), 6.80(d,lHxl/3) , 7.15(d,lHx2/3) , 7.50(d,2H), 8.15(d,2H) EXAMPLE 24-5

In 10 ml of acetonitrile was disεolved 255 mg (0.66 mmol) of ( 2S,4R) -1- (p- nitrobenzyloxycarbonyl)- 2- hydroxyimino-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 24-4. After 188 mg (1.65 mmol) of potasεium thio acetate waε added and refluxed at room temperature for 2 hourε, it waε concentrated under reduced preεsure to evaporate the organic solvent. The reεidue was extracted with ethylacetate, washed with water and brine, εeparated from the organic εolvent and dried over anhydrous magnesium εulfate. After filtration, the filtrate waε concentrated to give 118 mg of crude material. Thiε crude material was εubjected to the

column chromatography on silica gel (eluted with chloroform:methylalcohol«=50 :1 ) and purified to 87.1 mg (36%) of ( 2S, 4S ) -1 - (p- nitrobenzyloxycarbonyl )- 2- hydr oxy imino- 4 -acetylthiopyrrol idine . NMR(CDC1 ₃ ) 6:

1.80(m,lH), 2.60(m,lH), 3.30(m,lH), 4.0(m,2H), 4.5- 5.0(ε,lH), 5.20(s,2H), 6.75, 7.30 (d,dd, 1H) , 7.60(d,2H), 8.20(d,2H), 10.8-11.0(m,lH) EXAMPLE 24-6 After the solution of methyl alcohol and 2 ml of tetrahydrofuran was cooled to 0°C using an ice bath, 100 mg of (2S, S ) - 1- (p-nitrobenzyloxycarbonyl) -2 - hydroxyimino-4-acetylthiopyrrolidine obtained in EXAMPLE 24-5 was added and εtirred to dissolve 0.32 ml of 2 N εodium hydroxide solution at the same temperature for 3 minutes, and the pH of the solution was adjusted to pH 3.5 uεing εaturated citric acid. The reaction εolution was concentrated under reduced preεεure and the reεidue waε extracted with ethylacetate. The organic layer waε waεhed succeεsively . with water and brine and the separated organic layer was dried over anhydrouε magnesium sulfate and filtrend. The filtrate was concentrated under reduced preεεure to give 101 mg of the crude material. Thiε crude material was εubjected to the column chromatography on silica gel (eluted with chloroform: methylalcoholacetate=30:1) and purified to 79 mg (89.3%) of (2S,4S)-1- (p-nitrobenzyloxycarbonyl) -2- hydroxyimino-4-mercaptopyrrolidine. NMR(CDC1 ₃ ) δ:

2.01(m,lH), 2.30(d,2H), 2.71(m,2H), 3.21-

3.59(m,2H), 4.01(m,lH), 4.91 - 5.01 ( , 1H) , 5.31(ε,2H), 7.71(d,2H), 8.20(d,2H), 10.01(m,lH) EXAMPLE 25 (2S,4S) -l-(p-nitrobenzyloxycarbonyl)-2-(N- methaneεulfonyl -N-methylhydrazono )me thy 1 - 4 - mercaptopyrrolidine EXAMPLE 25-1

After 8.16 g (19.97 mmol) of ( 2S, 4R) -1 - (p- nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert - butyldimethylεilyloxypyrrolidine waε added to 200 ml of methylenechloride, 1.5 ml (28.20 mmol) of methylhydrazine waε added and εtirred for 2 hourε.

After the reaction, the mixture εolution waε waεhed with 200 ml of water and the organic layer waε dried over anhydrouε εodium εulfate, filtred, and then the filtrate waε concentrated under reduced preεεure and carried out in vacuum drying to give 8.7 g (99.7%) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl ) - 2- (N-methylhydrazono)methyl-4- tert-butyldimethylεilyloxypyrrolidine.

NMR(CDC1 ₃ ) δ:

0.02(d,6H), 0.81(ε,9H), 1.95- 2.10 ( , 2H ) ,

2.75(s,3H), 3.5(m,2H), 4.39 ( br , s , 1H) , 4.59(m,lH), 5.20(m,2H), 7.49(d,2H), 8.16(d,2H) EXAMPLE 25-2

To 150 ml of tetrahydrofuran cooled to 0°C using an ice bath, was added 8.7 g (19.93 mmol) of (2S,4R)-1- (p-nitrobenzyloxycarbonyl) -2- ( -methylhydrazono) methyl - 4 - tert-butyldimethylεilyloxypyrrolidine obtained in

EXAMPLE 25-1. And then 27 ml (27.0 mmol) of 1 M tetrabutylammonium fluoride(tetrahydrofuran solution) was added dropwiεe and εtirred for 30 minuteε. After the ice bath waε removed, the reaction mixture waε stirred at room temperature for 1 hour, and concentrated under reduced pressure, and 200 ml of methylenechloride waε added to the reεidue and waεhed with water and brine. After the separation of the organic layer, it was dried over anhydrous εodium εulfate, filtred, and then the filtrate waε concentrated to give 7.5 g of crude material. Thiε crude material waε subjected to the column chromatography on silica gel (eluted with chloroform:methanol>=10:1) and purified to 5.8 g (90.2%) of (2S.4R) -1- (p-nitrobenzyloxycarbonyl) -2- ( N- methylhydrazono) methyl-4-hydroxypyrrolidine. NMR(CDC1 ₃ ) δ:

2.10(rα,lH), 2.56(m,lH), 2.73(s,3H), 3.58(m,2H), 4.44(brs,lH) , 4.62(m,lH), 5.19(m,2H), 7.49(d,2H), 8.18(d,2H) EXAMPLE 25-3

To 100 ml of methylenechloride waε added 5.8 g (17.97 mmol) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- (N-methylhydrazono) methyl-4-hydroxypyrrolidine obtained in EXAMPLE 25-2 and 5.5 ml (39.5 mmol) of triethylamine and 3.17 ml (40.96 mmol) of methaneεulfonylchloride waε added and εtirred at room temperature for 6 hourε. After the reaction, the mixture solution waε waεhed with water and dried over anhydrouε εodium εulfate, filtered and the filtrate concentrated under reduced, preεεure to

give 7.6 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol = 20 : 1 ) and purified to 6.12 g (71%) of ( 2S , R) -1 - (p- nitrobenzyloxycarbonyl) -2- (N-methanesulfonyl-N- methylhydrazono) me thyl -4 -methaneεulf onyloxypyrrolidine . NMR(CDC1 ₃ ) δ:

2.40(m,2H), 2.83(ε,3H), 2.94(ε,3H), 3.04(m,3H), 3.63(m,lH), 3.89(m,lH), 4.70(m,2H), 5.19(ε,2H), 5.21(m,lH), 7.40(d,2H), 8.16(d,2H)

EXAMPLE 25-4

To 150 ml anhydrouε acetonitride was dissolved 5 g (10.44 mmol) of ( 2S, 4R) -1- (p-nitrobenzyloxycarbonyl ) -2- (N-methaneεulf onyl -N -methylhydrazono )methyl - 4 - methaneεulf onyloxypyrrolidine obtained in EXAMPLE 25-3.

After 1.67 g (4.62 mmol) of potaεεium thioacetate added and was refluxed for 4 hours, the reaction mixture waε concentrated under reduced presεure, and methylenechloride was added dissolved, and dried over anhydrous sodium εulfate . The filtrate waε concentrated under reduced preεεure to give 7.7 g of crude material. The crude material waε εubjected to the column chromatography on εilica gel (eluted with n- hexane:ethylacetate=l :1.5) and purified to 3.0 g (62.6%) of (2S,4S)-1- (p-nitrobenzyloxycarbonyl) -2-(N- methaneεulfonyl -N-methylhydrazono (methyl- 4 - acetylthiopyrrolidine . NMR(CDC1 ₃ ) δ:

2.10(m,lH), 2.64(m,lH), 2.98(ε,3H), 3.16(ε,3H),

3.42(m,lH), 4.04(m,lH), 4.62(m,2H), 5.23(ε,2H),

7.10(m,lH), 7.52(d,2H), 8.11(d,2H) EXAMPLE 25-5

In 80 ml of methylalcohol, cooled to 0°C uεing an ice bath, waε added 1.5 g (3.27 mmol) of (2S,4S) -1-(p- nitrobenzyloxycarbonyl) -2- (N-methaneεulfonyl-N- methylhydrazono)methyl-4-acetylthiopyrrolidine obtained in EXAMPLE 25-4, and εtirred to diεεolve, 3.3 ml of 2 N εodium hydroxide waε added dropwiεe and εtirred for 2 minuteε. Then, at the εame temperature, the pH of the reaction εolution waε adjuεted to pH 4.0 uεing εaturated citric acid and concentrated under reduced pressure to evaporate the organic solvent. In the reεidue methylenechloride was dissolved and the extrate waε waεhed with water. The organic layer waε dried over anhydrouε εodium sulfate. After the filtration, the filtrate was concentrated under reduced presεure, and the produced cryεtal waε dried in a vacuum to give 1.0 g (73.4%) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2-(N- methaneεulfonyl -N-methylhydrazono (methyl- 4 - mercaptopyrrolidine .

NMR(CDC1 ₃ ) δ:

2.05(m,lH), 2.81-3.03(m,6H) , 3.41 - 3.75(m, 3H) , 4.60- 4.80(m,2H), 5.10(m,2H), 7.52(dd,2H), 8.20(m,2H) EXAMPLE 26

(2S,4S)-l-(p-nitrobenzyloxycarbonyl ) -2-N- ( 2 - pyridinylhydrazono)methyl -4 -mercaptopyrrolidine EXAMPLE 26-1 After 4.9 g (11.99 mmol) of ( 2S , 4R) - 1 - ( p-

nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert - butyldimethylsilyloxypyrrolidine was added to 100 ml of methylenechloride, 1.37 g (12.55 mmol) of 2- hydrazonopyridine was added and stirred for 4 hourε. After the reaction, the organic layer obtained by waεhing with water waε dried over anhydrous sodium εulfate, filtered, and then the filtrate waε concentrated under reduced preεεure to give 6.8 g of crude material. The crude material waε εubjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate=l :1 ) and purified to 4.9 g (81.8%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) - 2- N - ( 2 - p y r i d i n y l h y d r a z o n o ) m e t h y l - 4 - t e r t - butyldime thyl silyloxypyrrol idine . NMR(CDC1 ₃ ) 6:

0.03(d,6H), 0.83(ε,9H), 2.19(m,2H), 3.52(m,2H), 4.42(m,lH), 4.63(m,lH), 5.19(m,2H), 6.72(m,lH), 7.01- 7.30(m,2H) , 7.31 -7.60 ( m, 3H ) , 7.90 - 8.20 (m ,2H) ,

9.20(brε,lH) EXAMPLE 26-2

To 100 ml of tetrahydrofuran, cooled to 0°C uεing an ice bath, waε added 4.9 g (9.8 mmol) of ( 2S , 4R) -1 - (p- n i t r o b e n z y l o x y,c a r b o n y l ) - 2 - N - ( 2 - py r i d i n y l h yd r a z o n o ( me th y l - 4 - t e r t - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 26-

1 and εtirred at room temperature to dissolve. Then, 13.01 ml (13.0 mmol) of 1 M tetrabutylammonium fluoride( tetrahydrofuran solution) waε added dropwiεe and εtirred for 30 minuteε. After the removal of the ice

bath, the mixture εolution waε εtirred at room temperature for 1 hour. The mixture εolution waε concentrated under reduced preεεure, 100 ml of methylenechloride waε added, εtirred to diεεolve and treated, aε discribed EXAMPLE 25-2, to give 7.1 g of crude material. The crude material was εubjected to the column chromatography on εilica gel (eluted with chloroform:methanol=10:1) and purified to 3.67 g (97.1%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) - 2-N- ( 2 - pyridinylhydrazono)methyl-4-hydroxypyrrolidine. NMR(CDC1 ₃ ) δ:

2.20(m,2H), 3.64(m,2H), 4.39(m,lH), 4.75(m,lH), 5.20(m,2H), 6.72(m,lH), 6.90-7.19(m,2H) , 7.15- 7.60(m,3H), 7.89-8.14(m,3H) , 9.01(br, s,1H) EXAMPLE 26-3

To 100 ml of methylenechloride was added 3.67 g (9.52 mmol) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl ) -2-N- ( 2-pyridinylhydrazono) methyl- 4-hydroxypyrrolidine obtained in EXAMPLE 26-2 and stirred to disεolve. Then 2.9 ml (20.9 mmol) .of triethyamine and 0.9 ml (11.63 mmol) of methaneεulfonylchloride waε added and εtirred at room temperature for 1 hour. After the reaction, the mixture εolution waε washed with water and the organic layer was dried over anhydrous sodium sulfate, filtered and then the filtrate was concentrated to give 5.1 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=20 :1 ) and purified to 3.9 g (88.4%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2-N- (2-

p y r i d i n y l h y d r a z o n o ) m e t h y 1 - 4 - methanesulf onyloxypyrrolidine . NMR(CDC1 ₃ ) δ:

2.35-2.80 (m, 2H) , 3.75(m,lH) , 4.02(m,lH) , 4.80(m,lH), 5.22(m,3H), 6.80(m,lH), 7.05(m,2H), 7.40-

7.60(m,3H), 7.90 -8. 2 (m, H)

EXAMPLE 26-4

To the solution of 100 ml of anhydrouε acetonitrile and 5 ml of dimethylformamide waε added 3.9 g (8.4 mmol) of ( 2S , 4R ) -1 - (p -nitrobenzyloxycarbonyl) - 2-N- ( 2 - p y r i d i n y l h y d r a z o n o ) m e t h y l - 4 - methanesulfonyloxypyrrolidine obtained in EXAMPLE 26-3 and stirred to dissolve. After 2.11 g (18.5 mmol) of potaεεium thioacetate waε added and refluxed for 7 hourε, the reaction solution waε concentrated under reduced pressure and methylenechloride was added to disεolve, dried over anhydrouε sodium sulfate, and filtered. The filtrate waε concentrated under reduced preεεure to give 5.3 g of crude material. The crude material was εubjected to the column chromatography on εilica gel(eluted with n-hexane :ethylacetate=2:l) and purified to 2.87 g (76.9%) of (2S.4S) -1- (p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - N - ( 2 - pyridinylhydrazono)methyl-4-acethylthiopyrrolidine. NMR(CDC1 ₃ ) δ:

2.05(m,lH), 2.32(ε,3H), 3.41(m,lH), 4.05(m,2H), 4.62(m,lH), 5.20(m,2H), 6.74(m,lH), 7.12(m,2H), 7.38- 7.62(m,3H), 7.89-8.23(m,3H) , 8.98(m,lH)

EXAMPLE 26-5

In 100 ml of methylalcohol cooled to 0°C using an ice bath, was added 2.8 g (6.31 mmol) of (2S,4S) -1- (p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - N - ( 2 - pyridinylhydrazono )methyl- 4 -acetylthiopyrrolidine obtained in EXAMPLE 26-4 and stirred to dissolve. 6.31 ml of 2 N sodium hydroxide was added dropwise and εtirred for 3 minutes. At the same temperature, the pH of the reaction solution was adjusted to pH 4.2 using saturated citric acid and concentrated under reduced preεεure to evaporate the organic εolvent. The reεidue waε diεεolved in methylenechloride, waεhed with water, and the organic εolvent εeparated and dried over anhydrous εodium εulfate. After filtration, the filtrate waε concentrated under reduced preεεure to give 4.3 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroforπ methyl alcohol=30:1) and purified to 1.9 g

(75%) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2-N-(2- pyridinylhydrazono)methyl-4-mercaptopyrrolidine. NMR(CDC1 ₃ ) δ:

1.70-2.20(m,3H) , 3.30-3.90(m,2H) , 4.52-5.40(m,4H) , 6.59-7.15(m,3H) , 7.20-7.62( ,3H) , 7.80-8.40(m, H)

EXAMPLE 27

(2S,4S)-l-(p-nitrobenzyloxycarbonyl ) -2-N- (p- me thox yb enzylox yc a rbo nyl phe nylhyd razono (methyl- - mercaptopyrrolidine

EXAMPLE 27-1

After 5.16 g (12.63 mmol) of (2S,4R)-l(p- nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert -

butyldimethylsilyloxypyrrolidine was added to 100 ml of methylene chloride and stirred to dissolve, 2.092 g (13.75 mmol) of 4-hydrazonobenzoic acid and 3.5 ml (25.1 mmol) of triethylamine was added and εtirred at room temperature for 15 hours. After the reaction, the mixture solution was waεhed with 100 ml of water, the pH of the organic layer waε adjuεted to pH 5.0 uεing εaturated citric acid. After the mixture εolution waε waεhed with water and εaturated sodium chloride, the organic layer waε dried over anhydrouε sodium sulfate,filtered, and then the filtrate waε concentrated under reduced preεεure. Until the cryεtal waε produced, the mixture εolution waε dried, and dried in vacuum to give 5.0 g (73.0% ) of ( 2S , 4 R ) - 1 - ( p- n i t r o b e n z y l o x y c a r b o n y l ) - 2 - N - ( 4 - carboxyphenylhydrazono ( methyl - 4 - te rt - butyldimethylεilyloxy pyrrolidine. NMR(CDC1 ₃ ) δ:

0.03(d,6H), 0.82(ε,9H), 2.01(m,2H), 3.21(m,2H), 4.39(m,lH), 4.45(m,lH), 5.05(m,2H), 6.75(d,2H), 7.30(m,2H), 7.70(m,3H), 7.98(d,lH), 9.10(d,lH)

EXAMPLE 27-2

To 30 ml of dimethylf ormamide waε added 5.0 g (9.21 mmol) of ( 2S,4R) -1 - (p-nitrobenzyloxycarbonyl ) -2-N- ( 4 - ca rboxyphenyl hyd r a z ono ) me thyl - 4 - te rt - butyldimethylεilyloxy pyrrolidine obtained in EXAMPLE 27-1 and εtirred to diεεolve. Then 2.54 g (18.4 mmol) of 1 M potaεεium carbonate and 1.729 g ( 11.04mmol ) of p- methoxybenzylchloride waε added and raiεed the

temperature to 50°C. At thiε temperature the mixture solution was stirred for 4 hourε, 150 ml of methylene chloride waε added and shaken vigorouεly, and the mixture εolution was washed with water (100 ml x 3). After separation of the organic layer, it waε dried over anhydrouε εodium εulfate and filtered, and then the filtrate was concentrated to give 6.7 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate =2:1) and purified to 3.8 g (62.3%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) - 2-N- ( 4 - methoxybenzyloxycarbonylphenylhydrazono)methyl-4-tert- butyldimethylsilyloxypyrrolidine. NMR(CDC1 ₃ ) 6: 0.03(d,6H), 0.82(s,9H), 2.01- 2.40 (m, 2H ) , 3.50(m,2H), 3.80(ε,3H), 4.43(m,lH), 4.64(m,lH), 5.20(m,4H), 6.90(dd,4H), 7.40(dd,4H), 7.90(m,2H), 8.20(m,lH)

EXAMPLE 27-3 The ester compound obtained in EXAMPLE 27-2 waε treated, as diεcribed in EXAMPLE 25, to give the desired product of (2S,4R) -1- (p-nitrobenzyloxycarbonyl ) -2-N- ( 4- methoxybenzylox yea rbonylphenylhyd razono (me thy 1 - 4 - mercaptopyrrolidine . NMR(CDC1 ₃ ) δ:

1.95-2.10(m,2H) , 3.20 - 3.80 ( m, 2H ) , 3.80(ε,3H), 4.50- 4.90(m,2H), 5.20(m,4H), 6.90(m,4H), 7.25 - 7.50 ( m, 4H ) , 7.90(m,3H), 8.19(m,2H) EXAMPLE 28

(2S,4S)-1- (p-nitrobenzyloxycarbonyl ) - 2 - ^" N - ( p - nit robenzylo ycarbonyl ) - N- me thyl hydr az ono ] me thyl - 4 - mercaptopyrrolidine EXAMPLE 28-1 After 8.7 g (19.93 mmol) of ( 2S, 4R) -1- (p- nit robenzyloxycarbonyl ) - 2- (N-methylhydrazono) methyl -4 - tert-butyldimethylsilyloxypyrrolidine obtained in

EXAMPLE 25-1 was added to 200 ml of methylenechloride and 4.72g(21.89 mmol) of 4 -nitrobenzylchlorof ormate sequentially, 3.9 ml (27.96 mmol) of triethylamine was added and stirred at room temperature for 24 hourε. The reaction mixture waε waεhed with water and the organic layer waε dried over anhydrouε εodium εulfate, filtered, and then the filtrate waε concentrated under reduced presεure. The residue waε subjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate=l :1 ) and purified to 6.62 g (54%) of ( 2S , 4R ) - 1 - ( p -nit robenz yl oxyc arbonyl ) - 2 - m e t h o x y i m i n o m e t h y l - 4 - t e r t - butylmethylsilyloxypyrrolidine .

NMR(CDC1 ₃ ) 6:

0.03(d,6H), 0.82(ε,9H), 2.07(m,2H), 3.11(d,3H), 3.52(m,2H), 4.40(m,lH), 4.75(m,lH), 5.10 - 5.40 (m, H ) , 7.50(m,4H), 8.10(m,4H) EXAMPLE 28-2

To 50 ml of tetrahydrofuran, cooled to 0°C using an ice bath, was added 5.31 g (8.62 mmol) of ( 2S , 4R) -1 - (p- nit robenzyloxycarbonyl ) -2- fN- (p-nitorbenzyloxycarbonyl ) - N - m e t h y l h y d r a z o n o ] m e t h y l - 4 - t e r t -

butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 28- 1 and stirred to dissolve. Then, 11.5 ml (11.5 mmol) of 1 M tetrabutylammonium fluoride(tetrahydrofuran solution) was added dropwise and stirred for 30 minutes. After the ice bath was removed, the mixture solution was εtirred at room temperature for 1 hour. After the reaction, the mixture εolution waε concentrated under reduced preεεure, and methylenechloride waε added to the reεidue. The reaction mixture εolution waε treated, as described in EXAMPLE 25-2, to give 5.8 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform: acetone=20:l) and purified to 2.3 g (53.2%) of (2S,4R)- 1 - ( p - nit robenzyloxyca rbonyl ) - 2 - [ N - ( p - nitorbenzyloxycarbonyl ) -N-methylhydrazono]methyl -4 - hydroxypyrrolidine. NMR(CDC1 ₃ ) δ:

2.20(m,2H), 3.29(d,3H), 3.60(m,2H), 4.50(m,lH), 4.80(m,lH), 5.10-5.30(m,4H) , 7.20(m,4H), 8.10(m,4H) EXAMPLE 28-3

To 100 ml of methylenechloride was added 2.3 g (4.58 mmol) of (2S,4R) -1-(p-nitrobenzyloxycarbonyl ) -2- fN- (p-nitorbenzyloxycarbonyl) -N-methylhydrazono]methyl- 4-hydroxypyrrolidine obtained in EXAMPLE 28-2 and stirred to dissolve. Then, 1.4 ml (10.1 mmol) of triethyamine and 0.4 ml (5.55 mmol) of methanesulfonylchloride waε added and εtirred at room temperature for 1 hour. After the reaction, the mixture εolution was treated, aε deεcribed in EXAMPLE 25-3, to

give 2.7 g of crude material. The crude material was disεolved in 100 ml of anhydrouε acetonitrile and 0.732g(6.43 mmol) of potassium thioacetate waε added and refluxed for 5 hours. After the reaction, the mixture solution was concentrated under reduced presεure, and 100 ml of methylenechloride was added and dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 2.6 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with n- hexane:ethylacetate=l:2) and purified to 1.8 g (70.3%) of (2S, 4S ) - 1- (p-nitrobenzyloxycarbonyl) -2 - f N- (p- nitorbenzyloxycarbonyl ) -N-methylhydrazono]methyl -4 - acetylthiopyrrolidine. NMR(CDC1 ₃ ) δ:

2.10(m,lH), 2.30(s,3H), 2.62(m,lH), 3.20(ε,3H), 3.40(m,lH), 4.03(m,2H), 4.63(m,lH), 5.20(m,2H), 5.30(s,2H), 7.01(m,lH), 7.44(m,4H), 8.14(m,4H) EXAMPLE 28-4 To the mixed solvent 70 ml of methylalcohol and 30 ml of tetrahydrofuran cooled to 0°C using an ice bath, waε added 1.8 g (3.21 mmol) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl ) -2- [N- (p-nitorbenzyloxycarbonyl) - N-methylhydrazono ]methyl -4 -acetylthiopyrrolidine obtained in EXAMPLE 28-3 and stirred to dissolve, 3.2 ml of 2 N sodium hydroxide was added and stirred for 3 minutes. Then the pH of the reaction mixture waε adjusted to pH 4.0 and concentrated under reduced presεure. The residue waε diεεolved in 100 ml of

methylenechloride, washed with water and the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate waε concentrated under reduced preεεure and the produced cryεtal waε dried in a vacuum to give 1.51 g (90.7%) of ( 2S, 4S) -1- (p- nitrobenzyloxycarbonyl ) -2- [N- (p-nitorbenzyloxycarbony1 ) - N-methylhydrazono]methyl-4-mercaptopyrrolidine. NMR(CDC1 ₃ ) δ:

2.03(m,2H), 3.12(d,3H), 3.30(m,2H), 4.54(m,2H), 5.20(m,4H), 7.54(m,4H), 8.19(m,4H)

EXAMPLE 29

(2S,4S)-l-(p-nitrobenzyloxycarbonyl ) - 2 - ( N , N - dimethylhydrazono)methyl-4-mercaptopyrrolidine

EXAMPLE 29-1 After 5.72 g (14.0 mmol) of ( 2S,4R) -1- (p- nitrobenzyloxycarbonyl ) - 2 - formyl - 4 - tert - butyldimethylsilyloxypyrrolidine waε added to 150 ml of methylenechloride at room temperature, 4.31 ml (30.8 mmol) of triethylamine and 1.22 g (16.8mmol) of dimethylhydrazinehydrochloride waε added and stirred for 3 hourε. After the reaction,. the mixture εolution waε waεhed with water and the organic layer was dried over anhydrouε εodium sulfate, filtered, and then the filtrate waε concentrated under reduced presεure to give 3.2 g of the reεidue. 150 ml of tetrahydrofuran, cooled to 0°C uεing an ice bath, waε added to the reεidue, and 12.14 ml (12.14 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran solution) waε added dropwiεe and εtirred for 40 minutes. After removal of the ice bath,

the mixture εolution waε εtirred at room temperature for 1.5 hourε, washed with water and saturated sodium chloride, and the organic layer waε drier over anhydrouε sodium εulfate. After filtration, the filtrate waε concentrated under reduced presεure to give 6.8 g of crude material.

The crude material waε subjected to the column chromatography on silica gel (eluted with chloroform:acetone=2 :1 ) and purified to 4.21 g (89.5%) of ( 2S , 4R ) -1 - (p- ni trobenzyloxycarbonyl ) - 2- ( N, - dimethylhydrazono)methyl-4-hydroxypyrrolidine. NMR(CDC1 ₃ ) δ:

1.92- 2.60 ( , 2H ) , 2.70(ε,6H) , 3.58(m,2H) , 4.42 ( br , s, lH) , 4.62(m,lH) , 5.19 ( m , 2 H ) , 7.42(d,2H,J=8.6HZ) , 7,17(d,2H, =8.5Hz) EXAMPLE 29-2

(2S,4R)-l-(p-nitrobenzyloxycarbonyl ) - 2 - ( N ,N- dimethylhydrazono)methyl -4 -hydroxypyrrolidine obtained in EXAMPLE 29-1 waε treated, aε deεcribed in EXAMPLE 26, with a result of (2S,4R) -1 -(p-nitrobenzyloxycarbonyl ) -2- (N,N-dimethylhydrazono)methyl -4-hydroxypyrrolidine . NMR(CDC1 ₃ ) δ:

2.01(m,lH), 2.61(m,lH), 2.75(s,6H), 3.40(m,2H), 4.50(m,lH), 5.20(m,3H), 7.40(d,2H, J=8.7Hz), 8.19(m,2H) EXAMPLE 30

(2S,4S)-l-(p-nitrobenzyloxycarbonyl) -2-(N,N- diethylhydrazono(methyl -4-mercaptopyrrolidine EXAMPLE 30-1 5.72 g (14.00 mmol) of (2S, 4R) -1 - (p-

nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert - butyldimethylεilyloxypyrrolidine waε added to 150 ml of methylenechloride and εtirred at room temperature, 4.3ml(30.8 mmol) of triethylamine and 2.09g(16.8 mmol) of diethylhydrazine hydrochloride εalt were added and εtirred for 4 hourε. After the reaction, the mixture εolution waε concentrated under reduced preεsure, and then extracted with ethylacetate. After the extracted solution was washed several times with water and saturated aqueouε εodium chloride, the organic layer waε dried over anhydrouε magneεium εulfate, filtered, and then the filtrate waε concentrated under reduced preεεure to give 7.2 g of the reεidue. The reεidue waε subjected to the column chromatography on εilica gel (eluted with n-hexane :ethylacetate=2:l ) and purified to

5.6 g (83.3%) of ( 2S , 4R) -1 - (p-nitrobenzyloxycarbonyl ) -2 - ( N , N - d i e th ylhyd r a z ono ) methyl - 4 - te r t - butyldime thyl εilyloxypyrrol idine . NMR(CDC1 ₃ ) δ: 0.03(ε,6H), 0.80(ε,9H), 1.01(t,6H), 1.85-2.15(2,

2H), 3.05(q,4H), 3.23(m,2H), 4.40(m,lH), 4.55(m,lH), 5.18(m,2H), 7.44 ( d, 2H, J=8.9Hz ) , 8.15 (d.2H , =8.1Hz ) EXAMPLE 30-2 (2S,4R)-l-(p-nitrobenzyloxycarbonyl ) - 2 - ( N , N - d i e t h y l h y d r a z o n o ) m e t h y l - 4 - t e r t - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 30- 1 was treated with the same separation of EXAMPLE 26 to give the desired product ( 2S , 4 S ) - 1 - ( p - nitrobenzyloxycarbonyl ) -2- (N,N-diethylhydrazono)methyl -

4-mercaptopyrrolidine. NMR(CDC1 ₃ ) δ:

1.00(m,6H), 2.01(m,lH), 2.50(m,2H), 3.08(q,4H), 3.20-3.60(m,2H) , 4.50(m,lH), 5.20(m,lH), 7.44(m,2H), 8.17(m,2H)

EXAMPLE 31

( 2S,4S ) -1 -( p-nitrobenzyloxycarbonyl ) -2- [N- me thyl -N- ( N ' , N ' -dimethylsulfamoyl)hydrazono]methyl -4 - mercaptopyrrolidine EXAMPLE 31-1

After 6.4 g (14.66 mmol) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl) - 2- ( N-methylhydrazono)methyl - tert-butyldimethylsilyloxypyrrolidine waε added to 80 ml of methylene- chloride and 5.1 ml (36.59 mmol) of triethylamine, and 2.3 ml (21.42 mmol) of dimethylsulfamoylchloride waε added dropwiεe and refluxed for 6 hourε. After the reaction, the mixture εolution waε concentrated under reduced preεsure, and then extracted with ethylacetate. After the extracted εolution was waεhed. with water and εaturated εodium chloride, the organic layer waε dried over anhydrouε magneεium εulfate, filtered, and then the filtrate waε concentrated under reduced preεεure to give 8.4 g of crude material. The crude material waε subjected to the column chromatography on εilica gel (eluted with n- hexane:ethylacetate =5:1) and purified to 7.35 g (92.2%) of (2S,4R)-l-(p-nitrobenzyloxycarbonyl ) -2- [N-methyl-N- (N' ,N' -dimethylεulfamoyl ) hydrazono]methyl -4-tert- butyldimethylεilyloxypyrrolidine.

NMR(CDC1 ₃ ) δ:

0.02(d,6H), 0.82(5,9H), 2.02(m,lH), 2.85(ε.3H), 2.90(5,6H), 3.01(m,lH), 3.41(m,2H), 4.40(m,lH), 4.60(m,lH) , 5.20(m,2H), 7.44 ( d , 2H , =8.7Hz ) , 8.20(d,2H,J=8.6Hz)

EXAMPLE 31-2

To 120 ml of tetrahydrofuran waε added 7.3 g (13.43 mmol) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- [N- methyl -N- (N' ,N' -dimethylsulfamoyl )hydrazono]methyl- 4- tert-butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 31-1, stirred to disεolve, and cooled to 0°C using an ice bath. Then 16 ml (16.0 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran εolution) waε added dropwiεe and εtirred for 20 minuteε. After removal of the ice bath, the mixture solution was εtirred for 30 minuteε and concentrated under reduced preεεure, and the residue was diεsolved in ethylacetate and waεhed εucceεsively with water and εaturated aqueouε εodium chloride. After the separation of the organic layer, the organic, layer waε dried over anhydrous magnesium sulfate, filtered, and then the filtrate waε concentrated to give 7.5 g of crude material. The crude material was dissolved in 120 ml of methylenechloride, and 4,7 ml (33.60 mmol) of triethylamine and 1.6 ml (20.67 mmol) of methanesulfonylchloride were added and εtirred at room temperature for 30 minutes. After the reaction, the reaction mixture solution was washed with water and saturated aqueous sodium chloride, and the organic layer was dried over anhydrouε sodium sulfate,

filtered, and then the filtrate was concentrated to give 5.7 g of crude material. The crude material waε εubjected to the column chromatography on silica gel(eluted with chloroform:methanol=300:1 ) and purified to 4.2 g ( 61.6% ) of ( 2S , R ) - 1 - ( p - nitrobenzyloxycarbonyl) -2- [N-methyl -N- (N' ,N' - dimethylεulfamoyl)hydrazono]methyl- -methanesulfonyloxy hydroxyhydroxyoxypyrrolidine. NMR(CDC1 ₃ ) δ: 2.30(m,2H), 2.84(ε,6H), 3.02(s,3H), 3.13(s,3H), 3.60 - 4.01 ( m, 2H ) , 4.75(m,lH) , 5.20 (m,3H) , 7.50(d,2,J=8.6Hz) , 8.20(d,2H,J=8.7Hz) EXAMPLE 31-3 To 80 ml of anhydrouε acetonitrile waε added 2.0 g (3.94 mmol) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- fN-methyl-N- (N',N' -dimethylεulfamoyl)hydrazono]methyl-4- methaneεulfonyloxypyrrolidine obtained in EXAMPLE 31-2 and εtirred to dissolve. Then, 1.37 g .(12.0 mmol) of potassium thioacetate waε added and refluxed for 3 hourε. After the reaction, the mixture εolution was concentrated under reduced presεure, and the residue was dissolved in ethylacetate and washed εucceεεively with water and εaturated aqueous sodium chloride. After the separation of the organic layer, it waε dried over anhydrouε magneεium εulfate, filtered, and then the filtrate waε concentrated to give 2.5 g of crude material. The crude material waε εubjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=50:1 ) and purified to 1.39 g

(72.4%) of (2S,4S) -I- (p-nitrobenzyloxycarbonyl) -2- [N- methyl -N- (N' ,N' -dimethylsulfamoyl )hydrazono]methyl- 4- acetylthiopyrrolidine. NMR(CDC1 ₃ ) δ: 2.10(m,lH), 2.30(S,3H), 2.60(m,lH), 2.90(s,6H), 3.12(ε,3H), 3.40(m,H), 4.00(m,2H), 4.60(m,lH), 5.20(br ε,2H), 7.48(d,2H,J=8.3Hz) , 8.20(d,2H, =8.7Hz ) EXAMPLE 31-4 To the mixed εolvent of 50 ml of methylalcohol and 5 ml of tetrahydrofuran waε added 1.39 g (2.85 mmol) of (2S,4S)-1 -(p-nitrobenzyloxyearbonyl) -2 - f N-methyl -N - (N' ,N' -dimethylεulfamoyl ) hydrazono]methyl-4- acetylthiopyrrolidine obtained in EXAMPLE 31-3 waε εtirred to diεεolve. Then 3.0 ml of 2 N εodium hydroxide waε added dropwiεe and εtirred for 3 minuteε. After the reaction, the pH of the εolution waε adjuεted to pH 4.2 uεing εaturated citric acid, and the reaction mixture waε treated, aε deεcribed in EXAMPLE 19-5, to give 1.1 g of crude material. The crude material waε εubjected to the column chromatography on εilica gel (eluted with n-hexane: ethylacetate=l :1.5) and purified to 0.69 g (54.3%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- [N- methyl -N- (N' ,N' -dimethylεulfamoyl )hydrazono)methyl- 4- mercaptopyrrolidine . NMR(CDC1 ₃ ) δ:

2.05(m,2H), 2.50- 2.79 (m, 2H ) , 2.83 ( ε , 3H ) , 2.93(ε,3H), 3.10(ε,3H), 3.30-3.90(m,2H) , 5.20(m,2H), 7.40(m,2H), 8.20(m,2H) EXAMPLE 32

( 2S,4S) -1- (p-nitrobenzyloxycarbonyl ) -2- ( N - methaneεulfonyl -N-methylhydrazono (methyl- 4 - mercaptopyrrolidine EXAMPLE 32-1 6.91 g (16.35 mmol) of ( 2S , 4R) -1 - (p- nitrobenzyloxycarbonyl ) - 2 - acetyl - 4 - tert - butyldimethylεilyloxypyrrolidine waε added to 100 ml of methylenechloride at room temperature. And 1.92 ml (36.09 mmol) of methylhydrazine and 2 g of anhydrouε magneεium εulfate waε added and refluxed for 5 hourε.

After the reaction, the mixture εolution waε cooled, filtered, and then the filtrate was concentrated under reduced preεεure to give 8.47 g of the reεidue. The reεidue waε subjected to the column chromatography on silica gel (eluted with chlorof orm:methylalcohol 30:1) and purified to 7.15 g (97.0%) of ( 2S , R) -1 - (p- nit robenzyloxycarbonyl ) - 2- (N-methylhydrazono (methyl -4- ter t-butyldime thy lεilyloxypyrroli dine. NMR(CDC1 ₃ ) δ: 0.02(s,6H) , _. 0.82(ε,9H) , 1.52 ( ε , 1.75H ) ,

1.63(s,1.25H) , 1.70-2.10(m,2H) , 2.90(s,2H), 3.40- 3.80(m,2H), 4.40(m,lH), 4.60(m,lH), 5.03 - 5.30 ( m, 2H) , 7.43(d,2H, =8.9Hz) , 8.19 ( d, 2H , J=8. Hz ) EXAMPLE 32-2 To 80 ml of methylenechloride waε added 6.8 g

(15.09 mmol) of (2S,4R)-l-(p-nitrobenzyloxycarbonyl)-2- ( N - m e t h y l h y d r a z o n o ) m e t h y l - 4 - t e r t - butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 32- 1, εtirred to diεεolve, and cooled to 0°C. Then, 13.2 ml

(22.8 mmol) of triethylamine and 1.41 ml (18.2 mmol) of methanesulfonylchloride was added and stirred for 30 minuteε. After the reaction, the reaction mixture solution was washed with satuεatedcitric acid and εaturated aqueouε εodium chloride. The organic layer waε dried over anhydrouε magneεium εulfate and filtered. The filtrate was concentrated under reduced presεure, and 100 ml of tetrahydrofuran waε added to the reεidue and stirred to dissolve. Then, 18 ml (18 mmol) of 1 M- tetrabutylammonium fluoride (tetrahydrofuran solution) waε added dropwise and stirred at room temperature for 30 minuteε. After the reaction, the mixture εolution waε concentrated under reduced preεεure, the reεidue waε diεsolved in ethylacetate and waεhed εucceεsively with water and saturated aqueouε sodium chloride. After the separation of the organic layer, it was dried over anhydrous magneεium εulfate, filtered, and then the filtrate waε concentrated to give 6.4 g of the reεidue. The residue waε subjected to the column chromatography on silica gel (eluted with chloroform:acetone=2:1) and purified to 4.56 g (72.9%) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl ) - 2 - (N-methaneεulfonyl-N- methylhydrazono)methyl-4-hydroxypyrrolidine. NMR(CDC1 ₃ ) δ: 1.95(5,1.8H) , 1.98(m,1,2H) , 2.10-2,64(m,2H) , 2.74- 2.76(ε,ε,3H) , 2.79-2.83(ε,s,3H) , 3.50-3.80(m,2H) , 4.50(ε,lH), 4.63(m,lH), 5.10(m,2H), 7.4 (d,2H,J=8.7Hz) , 8.18(dd,2H,J=8.6Hz,J=8.6Hz) EXAMPLE 32-3

To 70 ml of methylenechloride waε added 4.5 g (10.86 mmol) of (2S,4R) -1-( -nitrobenzyloxycarbonyl) -2- (N-methaneεulfonyl - -methylhydra zono (methyl - 4 - hydroxypyrrolidine obtained in EXAMPLE 32-2 and εtirred to diεεolve. The reaction mixture εolution waε cooled to 0°C uεing an ice bath, and 2.23 ml (16.0 mmol) of triethyamine and 0.95 ml (12.3 mmol) of methanesulfonylchloride waε added and stirred at the same temperature for 30 minutes. After the reaction, the organic layer waε washed with water, and washed succeεεively with citric acid εolution and εodium chloride εolution. After the filtration, the filtrate was concentrated to give crude material of 4.94 g (92.4%) of (2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2-(N- methaneεulfonyl -N- ethylhydrazono (methyl- - methaneεulfonyloxypyrrolidine.

Thiε material itεelf waε uεed for next reaction. NMR(CDC1 ₃ ) 6:

1.99(s.l.6H) , 2.05(ε.l.4H) , 2.20(m,lH), 2.60(m,lH), 2.78-2.79(s,ε,3H) , ■ 2.80-2.87( ε,ε,3H) , 3.73(m,lH), 4.01(m,lH) , 4.68( t, lH) , 5.05 - 9.32 ( m , 3 H ) , 7.42(d,2H,J=8.2Hz) , 8.20(dd,2H,J=7.2,8.5Hz)

EXAMPLE 32-4

To 80 ml of acetonitrile waε added 4.9 g (9.95 mmol) of (2S,4R) -1-(p-nitrobenzyloxycarbonyl) -2- (N- methaneεulfonyl -N-methylhydrazono (methyl- 4 - methaneεulfonyloxypyrrolidine obtained in EXAMPLE 32-3. After 2.8 g (24.52 mmol) of potasεium thioacetate waε added and refluxed for 5 hourε, the reaction εolution

was concentrated under reduced presεure, and the reεidue waε diεεolved in ethylacetate. The organic layer waε waεhed with water and saturated aqueouε sodium chloride, dried over anhydrouε magnesium εulfate, and filtered. The filtrate waε concentrated under reduced preεεure to give 5.1 g of the crude material. The reεidue waε subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=l:1 ) and purified to 3.21 g (68.3%) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) - 2- ( _N -methanesulfonyl - _N -methylhydrazono (methy1-4- acetylthiopyrrolidine. NMR(CDC1 ₃ ) δ:

1.99-2.02(ε,s,3H) , 2.30(ε,3H), 2.70(m,lH), 2.80- 3.01(m,6H), 2.90(m,lH), 3.30(m,lH), 3.95(m,lH), 4.16(m,lH), 4.52(t,3H), 5.12(m,2H), 7.43(d,2H, =8.3Hz) 8.18(d,2H,J=5.4Hz) ,

EXAMPLE 32-5

To 60ml of methyl alcohol waε added 3.21 g (6.79 mmol) of (2S,4S) -1- (p-nitrobenzyloxycarbonyl) -2- (N- methaneεulfonyl -N-methylhydrazono (methyl- 4 - acetylpyrrolidine obtained in EXAMPLE 32-4 and εtirred to diεεolve. After the reaction εolution waε cooled using the ice bath, 5 ml of 2 N sodium hydroxide aqueouε εolution was added dropwise and εtirred for 4 minuteε. Then, the pH of the reaction solution was adjusted to pH 3.5 using saturated citric acid and concentrated under reduced pressure to evaporate the organic solvent. To dissolve the reεidue ethylacetate was added, washed εucceεεively with water and εaturated aqueous sodium

chloride, and dried over anhydrous magneεium sulfate. After the filtration, the filtrate was concentrated to give 3.7 g of crude material. This crude material waε subjected to the column chromatography on silica gel 5 (eluted with n-hexane :ethylacetate=l : 3 ) and purified to

2.31 g (79%) of ( 2S , 4S )- 1 -( p-nitrobenzyloxycarbonyl )- 2 - (N-methaneεulfonyl-N-methylhydrazono)methyl- 4- mercaptopyrrol idine . NMR(CDC1 ₃ ) δ:

10 1.75(d,lH), 2.01(ε,1.5H), 2.03(ε,1.5H), 2.70(m,2H),

2.80-2.90(m,6H) , 3.30(m,lH), 3.70(m,lH), 4.10(m,lH), 4.50(m,lH), 5.09(m,2H), 7.42 ( d, 2H, J=8.6Hz ) , 8.19(m,2H) EXAMPLE 33 (2S, 4S) -1- (p-nitrobenzyloxycarbonyl ) -2- ( N-

-- ethaneεulfonyl -N-methylhydrazono (methyl - 4 - mercaptopyrrolidine EXAMPLE 33-1

12.26 g (30.01 mmol) of (2S, 4R) -1- (p- nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert -

20 butyldimethylεilyloxypyrrolidine waε added to 120 ml of methylenechloride and stirred to disεolve. 2.4 ml (45.11 mmol) of methylhydrazine and 3 g of anhydrouε magneεium εulfate was added, stirred at room temperature for 1.5 hour, and cooled to 0°C using an ice bath. 8.4 ml (60.22

25 mmol) of triethylamine and 4 ml (35.3 mmol) of ethanesulfonylchloride waε added, and εtirred for 2 hourε, washed εucceεεively with water and εaturated aqueouε εodium chloride and the organic layer was dried over anhydrouε magneεium εulfate, filtered, and then the

filtrate waε concentrated under reduced preεεure to give 15.8 g of crude material. The crude material waε εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=30:1 ) and purified to 14.7 g ( 92.7%) of ( 2 S , 4R ) - 1 - ( p - nitrobenzyloxycarbonyl) -2- (N-ethaneεul fonyl-N- m e t h y l h y d r a z o n o ) m e t h y l - 4 - t e r t - butyldimethylεilyloxypyrrolidine . NMR(CDC1 ₃ ) 6: 0.03(d,6H), 1.83(ε,9H), 1.13(t,3H), 2.10(m,2H), 3.10(m,5H), 3.50(m,2H), 4.40(m,lH), 4.70(m,lH), 5.10(m,2H), 7.48(d,2H, =8.5Hz) , 8.20(d,2H,J=8.6Hz ) EXAMPLE 33-2 To 160 ml of tetrahydrofuran waε added 14.7 g (27.80 mmol) of (2S, R) -1- (p-nitrobenzyloxycarbonyl ) -2- (N-ethaneεulfonyl-N-methylhydrazono (methy1-4- tert- butyldimethylεilyloxypyrrolidine obtained in EXAMPLE 33- 1 and εtirred to diεεolve. Then, 37 ml (3.7 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran εolution) was added dropwiεe and εtirred at room temperature for 3 hourε. After the reaction, the mixture εolution waε concentrated under reduced preεεure, and the reεidue waε diεεolved in methylenechloride and waεhed εuccesεively with water and saturated aqueous εodium chloride. The organic layer waε dried over anhydrouε magneεium sulfate and filtered. The filtrate waε concentrated under reduced preεsure and 160 ml of methylenechloride was added to the crude material produced, cooled to 0°C using an ice bath, and then 8.53 ml (61.2 mmol) of

triethylamine and 2.58 ml (33.3 mmol) of methanesulfonylchloride waε added dropwiεe, and stirred at 0°C for 1 hour. The reaction mixture εolution waε waεhed εuccesεively with citric acid εolution and εaturated aqueouε εodium chloride. After the εeparation of the organic layer, it waε dried over anhydrous magnesium sulfate, filtered, and then the filtrate waε concentrated to give 13.7 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methanol=20:1 ) and purified to 12.6 g (92.0%) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl) -2- (N-ethaneεulfonyl-N- methylhydrazono)methyl-4-methaneεulfonyloxypyrrolidine. NMR(CDC1 ₃ ) δ: 1.14(t,3H), 2.50(m,2H), 3.03(s,3H), 3.10-

3.25(m,5H), 3.80(m,2H), 4.80(m,lH), 5.10(m,3H), 7.50(d,2H,J=8.5Hz) , 8.20(d,2H,J=8.3Hz) EXAMPLE 33-3 To 400 ml of acetonitrile waε added 12.6 g (11.07 mmol) of (2S,4R) rl-(p-nitrobenzyloxycarbonyl )-2- (N- ethaneεulfonyl -N-methylhydrazono ) methyl - 4 - methanesulfonyloxypyrrolidine obtained in EXAMPLE 33-2 and εtirred to diεsolve. Then, 4.73 g (41.4 mmol) of potasεium thioacetate waε added and refluxed for 5 hourε. After the reaction mixture εolution waε cooled to room temperature, the mixture εolution waε concentrated under reduced preεεure to evaporate the εolvent, and the reεidue was diεεolved in 300 ml of methylenechloride and waεhed εuccessively with water and

εaturated aqueouε εodium chloride. After the separation of the organic layer, it waε dried over anhydrouε magneεium εulfate, filtered, and then the filtrate waε concentrated to give 9.8 g of crude material. The crude material waε εubjected to the column chromatography on εilica gel (eluted with chloroform:methylalcohol=20:1) and purified to 8.2 g (62.9%) of (2S,4R) -1- (p- nitrobenzyloxycarbonyl) -2- (N-ethaneεulfonyl-N- methylhydrazono)methyl-4-acetylthiopyrrolidine. NMR(CDC1 ₃ ) δ:

1.30(m,3H), 2.10(m,lH), 2.30(ε,3H), 2.60(m,14), 3.10(m,5H), 4.05(m,2H), 4.65(m,lH), 5.10(ε,2H), 7.44(d,2H,J=8.4Hz) , 8.19(d,2H,J=8.6Hz) EXAMPLE 33-4 To 120 ml of methylalcohol was added 7.4 g (15.66 mmol) of (2S, R) -1- (p-nitrobenzyloxycarbonyl) -2- (N- ethaneεulfonyl - -methylhydrazono ) methyl - 4 - acetylthiopyrrolidine obtained in EXAMPLE 33-3, εtirred to diεεolve, and cooled to 0°C uεing an ice bath. After 11.7 ml of 2 N sodium hydroxide solution was added and εtirred for 3 minuteε, the pH of the εolution waε adjuεted to pH 3.7 using citric acid solution. The reaction εolution waε concentrated under reduced pressure and 200 ml of ethylacetate waε added, washed succeεεively with water and εaturated aqueous sodium chloride, and the separated organic layer was dried over anhydrouε magneεium sulfate and filtered. The filtrate was concentrated under reduced presεure to give 5.7 g of crude material. The crude material waε εubjected to the

column chromatography on silica gel (eluted with chlorof orm:methylacohol =20:1) and puri ied to 4.1 g (60.8% ₎ of (2S,4S) -1- (p-nitrobenzyloxycarbonyl)-2-(N- ethaneεulfonyl-N-methylhydrazono) methyl-4 - mercaptopyrrlidine .

NMR(CDC1 ₃ ) δ:

1.14(m,3H), 2.10(m,2H), 3.01(ε,3H)/ 3.10(m,5H), 3.50(m,3H), 3.70(m,lH), 4.65(m,lH), 5.10(m,2H),

7.50(m,2H), 8.20(m,2H)