WO1994005667A1

WO1994005667A1 - Carbapenem derivatives and processes for preparing the same

Info

Publication number: WO1994005667A1
Application number: PCT/KR1993/000079
Authority: WO
Inventors: Min Sun Chang; Jon In Lim; Nam Sik Kim; Hee Chan Shin; Gye Won Kim; Ji Young Kim; Jae Keol Rhee; Chon Woo Lee; Weon Bin Im; Dong Sung Kim
Original assignee: Dong-A Pharmaceutical Co., Ltd.
Priority date: 1992-09-02
Filing date: 1993-09-02
Publication date: 1994-03-17
Also published as: TW255892B; KR940007029A; CN1090283A; KR950702989A; JPH08502732A; EP0658162A1

Abstract

The present invention relates to a compound of formula (I) wherein R1 is selected from the group consisting of a hydrogen atom or a methyl group, R2 is selected from the group consisting of a hydrogen atom, a metal or a nonmetal salt group, or a carboxy protecting group, R3 is selected from the group consisting of a hydrogen atom or an imino protecting group, R4 is selected from the group consisting of a hydrogen atom, a lower alkyl group, a hydroxy group, a cyano group, or a halogen atom, and R5 is defined as in claim 1; or a pharmaceutically acceptable salt or ester thereof. The compounds of the present invention have excellent antibacterial activities against various gram positive bacteria and gram negative bacteria and are useful as antibacterial agents for treatment and prevention of the human infectious diseases caused by such bacteria.

Description

CARBAPENEM DERIVATIVES AND PROCESSES

FOR PREPARING THE SAME

Background of the Invention

Field of the Invention

The present invention relates to novel carbapenem

(3-pyrrolidinylthio-1-azabicyclo[3,2,0]hept-2-ene-7-one- 2-carboxylic acid compounds) derivatives, processes for preparing the derivitives 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's, 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)

wherein R₁ is a hydrogen atom or a methyl group, R₂ 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 as 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 as hydrochloride, hydrobromide, sulfate, phosphate; an organic salt such as formate, acetate, trifluoracetate, malate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate; 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 as 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 as 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 methoxycarbonyloxymethyl group, an ethoxyca rbonyloxymethyl group , a propoxycarbonyloxymethyl group, a t-butoxycarbonyloxymethyl group, a 1-(or 2-) methoxycarbonyioxyethyl group, an 1-(or 2-)ethoxycarbonyloxyethyl group; a lower alkyl group capable of having phthalidenyl(lower)alkyl group such as 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 such as a vinyl group, an allyl group, an isopropenyl 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 3,4-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 4 -chlorophenyl group, a tolyl group, and a t-butylphenyl group, a xylyl group, a mesityl 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 as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, and hexyl. "Lower alkoxy" also includes a normal alkoxy or a side alkoxy such as methoxy, ethoxy, n-propoxy, isopropoxy, 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 aliphaticacyl substituted with an aromatic group, an aliphaticacyl substituted with a heterocyclic group, all of which are derived from a carboxylic acid, a carbonic acid, 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 as mesyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, isobutylsulfonyl, pentylsulfonyl, and hexylsulfonyl; a carbamoyl; an N-alkylcarbamoyl such as methylcarbamoyl, and ethylcarbamoyl; a lower alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, and t-butoxycarbonyl; a lower alkenyloxycarbonyl such as vinyloxyca rbonyl, allyloxycarbonyl, and isoprophenyloxycarbonyl; a lower alkenoyl such as acryloyl, metacryloyl, and crotonoyl; a cyclo(lower)alkanecarbonyl such as cyclopropanecarbonyl, cyclopentanecarbonyl, and cyclohexanecarbonyl.

The aromatic acyl includes an aroyl such as benzoyl, toluyl, and xyloyl; an N-arylcarbamoyl such as N-phenylcarbamoyl, N-tolylcarbamoyl, and N-naphtylcarbamoyl; an arensulfonyl such as benzensulfonyl, and tosyl. The heterocyclic acyl includes a heterocyclic acyl such as 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 as phenylacetyl, phenylpropionyl, and phenylhexanoyl; an aralkoxycarbonyl , for exampl e , a phenyl(lower)alkoxycarbonyl such as a benzyloxycarbonyl, and penetyloxycarbonyl; an aryloxyalkanoyl, for example, a phenoxy(lower)alkanoyl such as phenoxyacetyl and phenoxypropinoyl.

The aliphatic acyl substituted with a heterocyclic group includes a heterocyclic(lower)alkanoyl, for example, a heterocyclic(lower)alkanoyl such as thienylacetyl, imidazolylacetyl, 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 , i sopropyl , butyl , pentyl , and hexyl ; a halogen such as chlorine, bromine, iodine, and fluorine; a lower alkoxy such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentyloxy, and hexyloxy; a lower alkylthio such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, pentylthio, and hexylthio; nitro. The preferable acyl group having such substitutes are selected from the group consisting of a mono(or di, or tri)halo alkanoyl such as chloroacetyl, bromoacetyl, dichloroacetyl, and trifluoroacetyl; a mono(or di or tri)haloalkoxycarbonyl such as chloromethoxycarbonyl, di chl o romethoxyca rbonyl , and 2 , 2 , 2 -trichloroethoxycarbonyl; a nitro(or halo, or lower alkoxy) ; an aralkoxycarbonyl such as nitrobenzyloxylcarbonyl, chlor obenzyloxycarbonyl, methoxybenzyloxycarbonyl, mono(or di, or tr i ) halo (lower )aklyl sulf onyl such as fluoromethylsulfonyl, difluoromethylsulfonyl, trifluoromethylsulfonyl, and trichloromethylsulfonyl.

The "imino protecting group" is preferably (C₁-C₄) alkenyloxycarbonyl, phenyl(C₁-C₄)alkoxycarbonyl, o-nitro(or m-nitro, or p-nitro)benzyloxycarbonyl, and o-methoxy(or m-methoxy, or p-methoxy)benzyloxycarbonyl.

R₄ represents a hydrogen atom, lower alkyl group, a hydroxy group, a cyano group, a halogen group such as chlorine, bromine, iodine, and fluorine.

R₅ represents a hydroxy group, a lower alkoxy group, a protected or unprotected amino group, or one of the following general formula (1)-(4);

wherein R₆ and R₇ are independently either a hydrogen atom or a lower alkyl group. R₈ 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,

(where R₉ is a lower alkylsulfonyl such as methylsulfonyl, halo(C₁-C₄)alkylsulfonyl, a phenyl(C₁- C₄)alkylsulfonyl such as a p-toluenesulfonyl, and a N,N-(lower)dialkylsulfamoyl such as a N,N-dimethylsulfamoyl.

(2)

wherein R₆ is the same as defined above.

(3)

wherein R₆, R₇ are the same as defined above.

(4)

wherein R₆ is the same as defined above, R₁₀ is a lower alkyl group, a lower alkylsulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄)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,

wherein R₁₁ is a

, a

, 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 R₆, 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(such as 2-imidazole), imidazolinyl(such as 2-imidazolinyl), pyrazolyl, pyrazolinyl, pyridyl, pyridyl N-oxide, pyridinio, dihydropyridyl , tetrahydropyridyl ( such as 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 1,2,5,6-tetrahydro-1,2,4-triazinyl, 1,4,5,6-tetrahydro-1,2,4-triazinyl), triazinio, triazolyl(such as 1H-1,2,4-triazolyl, 1H-1, 2 ,3-triazolyl, and 2H-1,2,3-triazolyl), triazolio, tetrazinyl, tetrazinio, tetrazolyl (such as 1H-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 as 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 as 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 as 1,3-dioxolanyl), dioxanyl(such as 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 1,4-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 as azidomethyl, azidoethyl, and azidohexyl); a halo(lower)alkyl(such as chloromethyl, bromomethyl, iodoethyl, bromopropyl, and bromohexyl).

The "protected hydroxy(lower)alkyl" includes a phenyl(C₁-C₄)alkoxycarbonyloxy(C₁-C₄)alkyl having a nitro group; a triphenyl(C₁-C₄)alkoxy(C₁-C₄)alkyl having a nitro group; a tri(C₁-C₄)alkylsilyloxy(C₁-C₄)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 isomers" as shown in the following formula;

OR

wherein R₁₂ is an amino or a protected amino group, R₁₃ 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-oxadi,zolyl 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(C1-C3)alkoxycarbonyl such as 2-iodoethyloxycarbonyl, and 2,2,2-trichloroethyloxycarbonyl); a substituted or unsubstituted al(lower)alkyloxycarbonyl; a substituted or unsubstituted phenyl(C1-C3)alkyloxycarbonyl such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, and p-nitorbenzyloxycarbonyl; tri(C1-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),

wherein R₁, R₂, R₃, R₄, and R₅ are the same as defined above, R₁₄ is not difined above, reacting a compound of the following formula (II),

wherein R₁ and R₂ is the same as defined above, R₁₄ is hydrogen or a hydroxyl protecting group, with a compound of the following formula (III)

wherein R₃, R₄ and R₅ are the same as 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 as 7-oxo-1-azabicyclo[3,2,0]hept-2-ene-e-carboxylic acid or 1-azabicyclo[3,2,0]hept-2-ene-7-one-2-carboxylic acid. In this specification, 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-position takes 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 compounds having preferred stereo-configurations are represented by the following formula (I-a):

wherein R₁, R₂, R₃, R₄ and R₅ 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-positions of the pyrrolidine structure. Among these isomers, preferred compounds 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 R₁ is a methyl group, R₅ is a

(lower)alkoxide group R₆ of group is a hydrogen atom or a

(lower)alkylgroup, R₁₀ i s lowe r ) alkyl or

( lowe r ) alkylsulfonyl , phenyl ( C1 - C4 ) alkylsul fonyl or group (wherein Rll is the same as defined

above), have particularly excellent antibacterial activities.

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

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

wherein R₁, R₂, R₁₄ is the same as defined above and A is a leaving group. The activating reagent to be used for the reaction may, for example, be an acid anhydride such as methanesulfonic anhydride, trifluromethanesulfonic anhydride, p-toluene sulfonic anhydride, and trifluoroacetic anhydride; or an acid chloride such as methanesulfonyl chloride, p-toluenesulfonyl chloride or diphenyl chlorophosphate. Particulary preferred is diphenylchlorophosphate.

In the formula (II-a), A is a leaving group such as trifluoroacetoxy, methanesulfonyloxy, trifluoromethanesulfonyloxy, p-toluenesulfonyloxy or diphenoxyphosphoryloxy. Particularly preferred is 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 base to be used for the reaction may, for example, be trimethylamine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, N,N-dimethylaniline, 1,8-diazabicyclo[5,4,0]endec-7-ene(DBU), or 1,5-diazabicyclo[4,3,0]-non-5-ene(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-1- azabicyclo[3,2,0]heptane ring system of the following formula (II) has a tautomeric relation with 3-hydroxy-7- oxo-1-azabicyclo[3,2,0]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 is not important, the reaction is conducted usually 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 is 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 is 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 is conducted usually 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 (II-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 is 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 is 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 solvent for decomposition; by chemical reduction using salt of an amine, a metal such as zinc amalgam, a chromic compound such as chloro chromous, 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 is an aralkyloxycarbonyl group such as a benzyloxycarbonyl group or a p-nitrobenzyloxycarbonyl group, and the protecting group for the carboxyl group is an aralkyl group such as a benzyl group, a p-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 as palladium-carbon, palladium black, palladium hydroxide-carbon, palladium (II) chloride, tetrakis (triphenylphosphine) palladium (O) , bis(dibenzylidenylacetone)-palladium (O), di(1,2-bis(diphenylphospino(ethane)palladium, and tetrakis(triphenylphosphine)palladium (O). The solvent 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)propanesulfonic 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 is conducted usually within a temperature range of from -10° to 50°C, preferably from 0° to 30 C° using from 0.01 to 0.5 mol of the palladium ligand complex catalyst and from 0.5 to 5 mol of the nucleophilic agent relative to 1 mol of the compound of the formula (IV), and the reaction is completed usually in from 0.5 to 5 hours.

After completion of the reactions for removing the protecting group, the compound of the formula (I) can be isolated by column chromatography loading on silica gel, adsorptive 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 is incorporated herein by reference in the case that R₁ is a hydrogen atom, and by Shih method (Heterocycles, Vol. 21, 29-40, 1984 or EP No. 272,455) which are incorporated herein by reference in the case where R₁ is a methyl group.

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

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 derivatived of the formula

(III) or (III-a) can be obtained from the compound (6) or (13), and the methods for preparing thereof are described in the following examples. Abreaction used are as follows:

Ac : acetyl

Boc : t-butoxycarbonyl

Cbz : benzyloxycarbony

Et : ethyl

Me : methyl

Ms : methanesulfonyl

PMB : p-methoxybenzyl

PNB : p-nitrobenzyl

PNZ : p-nitrobenzyloxycarbonyl

TBDMS : t-butydimethylsilyl

s : p-toluene sulfonyl

Ph : phenyl

E : trans

z : cis

I. Antibacterial Tests

The antibacterial activities 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 is 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 aureus was cultured in Trypticase Soy Broth instead of Mueller Hinton Broth. The antibacterial agent solutions 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 hours. After observing the growth of bacteria, MIC was considered to be the lowest drug concentration at which there is no growth. The results are shown in table 1.

II. Stability to renal dehydropeptidase-I from porcine 300g of the swine kidney was homogenized and suspended in a Tris buffer (10mM Tris, 50mM NaCl, 0.01mM ZnCl₂, pH 8.0), and 10% Triton X-100 was added and stirred at 4°C for 24 hours to extract DHP-I. A supernatant, prepared by ultracentrifugation of the above extract at 186,000 × 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 precipitates 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 hydrolysis velocity. The stability to DHP-I is represented as the comparative hydrolysis velocity to that of GDP.

III. Toxicity Test

After dissolving 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 + 1g weight and observed for 2 weeks. The LD₅₀ was determined by probit analysis. The numbers of both male and female are five, respectively.

The compounds of the present invention have excellent antibacterial activities against various gram positive bacteria and gram negative bacteria and are useful as antibacterial agents 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 agents for industrial 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 solutions, syrups or emulsions; solid formulation such as tablets, capsules or granules; and external application formulations such as ointments or suppositions.

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 is 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 F₂₅₄ (Merck) was used as the plate, and an ultraviolet detector or ninhydrin color development method KM_nO₄ was used as a detecting means. As the silica gel for a column, silicagel 60 (Merck) was used, and UV spectrophotometer DMS 100S (Varian) was used for detecting the UV absorbency. For high speed liquid chromatography an M-352 (ACS) model was used. When the NMR spectrum was measured in heavy dimethyl sulfoxide-d₆ or heavy chloroform-d₁ solution, tetramethylsiland (TMS) was used as the internal standard material, and when it was 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-d₆ : dimethylsulfoxide-d₆

d : doublet CDCl₃ : chlorform-di t : triplet D₂O : deuterium oxide

quarter J : coupling integer br : broad Hz : hertz dd : double doublet

m : multiplet

REFERENCE EXAMPLE 1

(4R,5R,6S,8R) -p-nitrobenzyl- 4-methyl-6- ( 1-hydroxyethyl)-1-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(1/3) 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 as a catalyst, and the solution was refluxed for 2 hours.

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-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-3,7-dione-2-carboxylate. This compound is unstable it should be used immediately after preparation.

NMR(CDCl₃) δ:

1.10(d,3H), 1.32(d,3H), 2.80(m,1H), 3.22(dd,1H), 4.20(dd,1H), 4.25(m,1H), 4.70(s,1H), 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(1/1) 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 as a catalyst, and the solution was refluxed for 1.5 hours. 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)-1-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(1/3) was added 0.982 g (2.51 mmol) of (3S,4R)-3-[(1R)-(1-hydroxyethyl)]-4-[(R)-1-methyl-3-diazo-3-allyloxycarbonyl-2-oxo-propyl]-azetidine-2-one,

Rhodium (II) acetate dimer as a catalyst, and the solution was refluxed for 2.5 hours. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the organic solvent, and the produced material in the form of a syrup was 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 used immediately after preparation.

NMR(CDCl₃) δ:

1.10(d,3H), 1.32(d,3H), 2.81(m,1H), 3.21(dd,1H), 4.21(dd,1H), 4.23(m,1H), 4.66(d,2H), 4.69(s,1H), 5.29-6.20(m,3H) REFERENCE EXAMPLE 3

(5R,6S,8R)-p-nitrobenzyl-6-(1-hydroxyethyl)-1-azabicyclo [3,2,0]-hept-3,7-dione-2-carboxylate

REFERENCE EXAMPLE 3-1

To 140 ml of benzene was 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 as a catalyst, and the solution was refluxed for 2 hours. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the organic solvent, a sraall volumn of methylene chloride was added to the residue material and stirred at room temperature to dissolve. Diethylether was added dropwise to the reaction mixture to crystallize and the mixture was stirred for 15 minutes. The material was 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 unstable in the successive process it is should be used immediately after preparation.

NMR(CDCl) δ:

1.35(d,3H, J=6.1Hz), 2.44(m,1H), 2.92(m,1H), 3.19(dd,1H,J=2.0Hz, J=7.0Hz), 4.15(m,1H), 4.30(m,1H), 4.75(s,1H), 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- methoxyiminopyrrolidin)-4-yl-thio}-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-diisopropylethylamine was added dropwise, 830μl(4.0 mmol) of diphenylchlorophosphate added and stirred for 1 hour. And then 750 μl (4.3 mmol) of N,N-diisopropylethylamine was added and the solution 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 anhydrous acetonitrile was added dropwise to the reaction mixture. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and ethylacetate was added to the residue to extract the product. The extract was washed successively with water and saturated aqueous sodium chloride, and the organic layer dried over anhydrous magnesium sulfate. After the filtration, the filtrate was concentrated under reduced pressure and the solvent evaporated to obtain 2.2 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1:4) and purified to give 0.236 g (10.4%) of (Z)-(4R,5S,6S,8R,2'S,4'S)-p-nitrobenzyl-3-[1-(p-nitrobenzyloxycarbonyl)-2-methoxyiminopyrrolidin-4-yl - thi o ] - 4 -methyl - 6 - ( 1 -hyd roxyethyl ) -1 -azabicyclo[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 -azabicyclo[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- [1-(p-nitrobenzyloxycarbonyl)-2-methoxyiminopyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR( Acetone-d₆)

(1) Z form: δ:

1.12 (dd, 6H ) , 2.01 (m ,1H ) , 2.80 (m ,1H ) ,

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,1H) , 4.60(m,1H) , 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,1H), 2.80(m,1H), 3.33(dd,1H) , 3.55-3.80(m,2H), 3.81(s,3H),

3.95-4.32(m,3H), 4.31(m,1H) , 4.80-5.05(m,1H), 5.20- 5.60(m,4H) , 6.90(m,1H) , 7.65(d,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) was added 0.316 g (0.462 mmol) of the (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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 1-1 and stirred to dissolve. 0.594 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 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 concentrated under reduced pressure at 10° - 20°C, then the extracted impurity was removed by filtration. The clarified filtrate was subjected to polymerchromatography (Diaion HP-20 resin:previously washed with methylalcohol and water). After loading the somple an the resin, the resin was washed 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 pressure. The residue was lyophilized to give 0.101 g (59.2%) of ( E)-(4R,5S,6S,8R,2'S,4'S)-3-[ (2-me thoxyiminopyrrolidin)-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2- carboxylic acid.

NMR(D₂O) δ:

0.96(d,3H,J=7.2Hz), 1.08(d,3H,J=6.3Hz), 1.60(m,1H), 2.45(m,1H), 2.92(m,1H), 3.21(m,3H), 3.63(s,3H), 3.24(m,1H), 4.02(m,2H), 4.42(m,1H), 6.83-7.40(dd,1H,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) was 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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 1-1 and stirred 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 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 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 as 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- thio] -4 -methyl- 6- (1 -hydroxyethyl) -1 -azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid. NMR(D₂O) δ:

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

EXAMPLE 2

( 4 R , 5 S , 6 S , 8R , 2 ' S , 4 ' S ) - 3 - [ ( 2 -ethoxycarbonylmethoxyimino pyrrolidin)-4-ylthio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-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-azabicyclo[3,2,0)-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 80 ml of anhydrous acetonitrile, stirred 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 was added and the mixture stirred for 1 hour. And then 803 μl (4.61 mmol) of N,N-diisopropylethylamine was added and the solution of 1.58 g (3.84 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-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 was removed, and the reaction mixture was stirred at room temperature for 4 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and ethylacetate was added to the residue to extract the product. The extract was washed successively with water and saturated aqueous sodium chloride, and the organic layer dried over anhydrous magnesium sulfate. After the filtration, the filtrate was concentrated under reduced pressure and the solvent evaporated to obtain 2.55 g of crude material. This crude product was subjected to column chromatography on silica gel (eluted with chloroform: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-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 -ethoxycarbonylmethoxyiminopyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.10(m,9H), 2.10(m,1H), 2.70(m,1H), 3.20(m,1H), 3.21(m,1H), 4.01(m,1H), 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,J=8.7Hz)

EXAMPLE 2-2

To the mixture of 14 ml of tetrahydrofuran and 14 ml of 0.1 M 4-morpholinepropanesulfonic 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 -ethoxycarbonylmethoxyiminopyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 2-1 and was stirred to dissolve. 0.280 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 was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. The resultant residue was washed with ethylacetate, separated, and the water layer was concentrated under reduced pressure at 10° - 20°C and then the extracted impurity was removed by filtration. The clarified filtrate was subjected to polymerchromatography (Diaion HP-20 resin:previously washed with methylalcohol and water). After loading the somple on the resin, the resin was washed 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 fractions 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 -ethoxycarbonylmethoxyiminopyrrolidin)-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(D₂O) 6:

1.10(m,9H), 1.70(m,1H), 2.53(m,1H), 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 (m, 2H )

EXAMPLE 3

( 4R , 5S , 6S , 8R , 2 ' S, 4 ' S ) - 3 - [ 2 - (N ,N -dimethylaminocarbonylmethoxyimino)pyrrolidin-4-yl-thio]- 4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-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-azabicyclo[3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 40 ml of anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 402 μl (2.31 mmol) of N,N-diisopropylethylamine was added dropwise, 458 μl (2.21 mmol) of diphenylchlorophosphate was added and stirred for 1 hour. Then 402 μl (2.31 mmol) of N,N-diisopropylethylamine was added and the solution of 0.866 g (2.11 mmol) of (2S,4S)-1-(p-ni t r o b en z y l o xy c a r bony l ) - 2 - ( N , N -dimethylaminocarbonylmethoxyimino)-4-mercaptopyrrolidine obtained in EXAMPLE 20-5 in 20 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was. stirred at room temperature for 5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, 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 1.4 g of crude material. This crude material was 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-[1-(p- nitrobenzyloxycarbonyl)-2-(N,N-dimethylaminocarbonylmethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR (Acetone-d₆) δ:

1.20(dd,6H), 1.25(m,1H), 2.25(m,1H), 2.85(s,3H),

3.01(s,3H), 3.32(dd,1H, 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), 7.46(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 4 -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,4'S)-p-nitrobenzyl-3-[1 - ( p-nitrobenzyloxycarbonyl ) - 2 - ( N,N-dimethylaminocarbonylmethoxyimino) pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 3-1 and stirred to dissolve. 0.320 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 3 hours a under hydrogen gas stream. After the reaction, the catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. The residue was treated as discribed in EXAMPLE 2-2 to give 0.162 g (53.5%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3-[2-(N,N-dimethylamino carbonylmethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0] - hept-2-ene-7-one-2-carboxylic acid.

NMR(DMSO-d₆) δ:

1.10(d,3H,J=6.5Hz), 1.12(d,3H,J=6.2Hz), 1.40(m,1H), 2.40(m,1H), 2.80(s,3H), 2.90(s,3H), 2.90-3.40(m,4H), 3.80-4.10(m,3H), 4.64(s,2H), 4.90(m,1H)

EXAMPLE 4

( 4 R , 5 S , 6 S , 8 R , 2 - S , 4 ' S ) - 3 - [ ( 2 -carboxymethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof 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-azabicyclof3,2,0]-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 stirred for 1 hour. And then 320 μl (1.82 mmol) of N,N-diisopropylethylamine was added and the solution of 0.755 g ( 1.5 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 - ( P -methoxybenzyloxyca rbonylmethoxyimino ) - 4 -mercaptopyrrolidine obtained in EXAMPLE 21-2 in 20 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction, the reaction mixture was 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 was subjected to the column chromatography on silica gel (eluted with chloroform: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 -methoxybenzyloxycarbonylmethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(Acetone-d₆) δ:

1.22(dd,6H) , 2.05 (m,1H ) , 2.80 (m,1H ) ,

3.32(dd,1H,J=2.5Hz, J=6.4Hz), 3.60(m,1H), 3.65(s,3H), 4.01-4.20(m,4H), 4.30(m,1H), 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-morpholinepropanesulf 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)pyrrolidin-4-yl- thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 4-1 and stirred to dissolve. 0.2 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 3 hours under a hydrogen gas stream. After the reaction, the catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. The residue was treated with the same operation as in EXAMPLE 2-2 to give 0.09 g (45%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3-[(2-carboxymethoxyiminopyrrolidin)-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid. NMR(DMSO-d₆) δ:

1.12(d,3H,J=7.2Hz), 1.15(d,3H,J=6.2Hz), 1.42(m,1H), 2.30(m,1H) , 2.62 (m, 1H) , 3.10 (m, 1H) , 3.11(dd,1H,J=2.3Hz,J=6.3Hz), 3.20-4.20(m,4H), 4.40(m,3H)

EXAMPLE 5

( 4 R , 5S , 6 S , 8R , 2 ' S , 4 ' S ) - 3 - [ ( 2 -aminocarbonylmethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-azabicyclo[3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 40 ml of anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using ice bath. At the same temperature, 390 μl (3.24 mmol) of N,N-diisopropylethylamine was added dropwise, 445 μl (2.15 mmol) of diphenylchlorophosphate added and stirred 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-nitrobenzyloxycarbonyl)-2-aminocarbonylmethoxyimino-4-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 was removed, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction, the reaction mixture was 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. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=40:1) 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-[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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.12(d,3H,J-7.2Hz), 1.15(d,3H,J=6.1Hz), 1.80(m,1H),

2.60(m,1H), 3.23(dd,1H,J=2.5Hz,J=6.4Hz), 3.50(m,2H),

3.90(m,2H), 4.22(m,1H), 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-morpholinepropanesulfonic 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 obe n zy l ox yc a r bo nyl ) - 2 -aminocarbonylmethoxyiminopyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 5-1 and stirred to dissolve. 0.31 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 3 hours under a hydrogen gas stream. After the reaction the catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. The residue was treated, as discribed 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-azabicyclo[3,2,0] -hept-2-ene-7-one-2-carboxylic acid.

NMR(D₂O) δ:

1.05(d,3H,J=7.2Hz), 1.08(d,3H,J=6.2Hz), 1.60(m,1H), 2.40(m,1H), 2.95(m,1H), 3.05-3.40(m,3H), 3.70-4.10(m,3H), 4.40(m,1H), 4.64(s,2H)

EXAMPLE 6

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

EXAMPLE 6-1

0.601 g (1.66 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 40 ml of anhydrous acetonitrile, stirred 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 was added dropwise, 396 μl (1.91 mmol) of diphenylchlorophosphate added and stirred for 1 hour. Then 348 μl (2.00 mmol) of N,N-diisopropylethylamine was added and the solution of 0.723 g (1.83 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 -methylaminocarbonylmethoxyimino) -4-mercaptopyrrolidine obtained in EXAMPLE 23-2, 15 ml of anhydrous 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 was added to the residue to extract the product. The extract was treated as 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 chloroform: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-methylaminocarbonylmethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ: 1.11(d,3H,J=7.2Hz), 1.15(d,3H,J=6.3Hz), 1.80(m,1H),

2.60(m,1H), 2.80(d,3H), 3.22(dd,1H,J=2.4Hz,J=6.4Hz),

3.45(m,2H), 3.80(m,2H), 4.22(m,1H), 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 trobenzyloxyca rbonyl ) - 2 - (N -methylaminocarbonylmethoxyiminopyrrolidin)-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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 was removed by filtration and the filtrate was concentrated under reduced pressure 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-methylaminocarbonylmethoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(DMSO-d₆) δ:

1.03(d,3H,J=7.3Hz), 1.05(d,3H,J=6.1Hz), 1.42(m,1H), 2.42(m,1H), 2.81(d,3H), 2.91-3.39(m,4H), 3.81- 4.11(m,3H) , 4.65(d,2H) , 4.91(m,1H)

EXAMPLE 7

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ ( 2 -hydroxyiminopyrrolidin)-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-azabicyclo[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 dropwise, 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 was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and ethylacetate was added to the residue to extract the product. The extract was treated, as discribed in EXAMPLE 1-1, to obtain 0.987 g of crude material. This crude product was subjected to the column chromatography on silica gel(eluted with chloroform: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-nitrobenzyloxycarbonyl)-2-hydroxyiminopyrrolidin-4-yl - thi o] - 4 -methyl - 6 - ( 1 -hydroxyethyl ) -1 -azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR ( DMSO -d₆) δ:

1.10(d,3H,J=7.2Hz), 1.12(d,3H,J=6.2Hz), 1.90(m,1H), 1.65(m,1H), 3.30(dd,1H), 3.52(m,1H), 3.95(m,3H), 4.22 (m,1H ) , 4.49( m, 1H ) , 5.01- 5.50 (m,5H) ,

7.60(d,2H,J=8.7Hz), 7.72(d,2H,J=8.7Hz), 8.20(d,4H,), 10.8-11.2(br s,1H)

EXAMPLE 7-2

To the mixture of 15 ml of tetrahydrofuran and 15 ml of 0.1 M morpholinepropanesulf onic acid buffer (pH

7.0) was added 0.470 g (0.720 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S, 8R,2'S,4'S)-p-nitrobenzyl-3-[1-(p-nitrobenzyloxycarbonyl)-2-hydroxy iminopyrrolidin-4 -yl - thio ] - 4 -methyl - 6 - ( 1 -hydroxyethyl ) - 1 -azabicyclo[3,2,0] -hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 7-1 and stirred to dissolve. 0.240 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 3 hours under an atmospheric pressure of hydrogen. After the reaction the catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. The residue was treated with the same operation as in EXAMPLE 2-2 to give 0.132 g (52.8%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3- [(2-hydroxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(DMSO-d₆) δ:

1.05(d,3H,J=7.2Hz), 1.08(d,3H,J=6.2Hz), 1.40(m,1H),

2.40(m,1H), 2.70(m,1H), 3.10-3.30(m,3H), 3.40-3.80(m,2H), 3.80-4.10(m,2H), 10.63(br s, 1H)

EXAMPLE 8

(4R,5S,6S,8R,2'S,4'S)-3-[2-(N-methanesulfonyl-N-methylhydrazono)methylpyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-azabicycl o [3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 100 ml anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 520 μl (2.98 mmol) of N,N-diisopropylethylamine was added dropwise, and 600 μl (2.89 mmol) of diphenylchlorophosphate was added and stirred for 2 hours. And then 520 μl (2.98 mmol) of N,N-diisopropylethylamine was added and the solution of 1.0 g (2.40 mmol) of ( 2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono) methyl-4-mercaptopyrrolidine obtained in EXAMPLE 25-5, 25 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was washed with water and saturated with aqueous sodium chloride and the organic layer dried over anhydrous magnesium sulfate. After the filtration, the filtrate was concentrated under reduced pressure and the solvent evaporated to obtain 1.32 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform:acetone=20:1) and purified to give 0.88 g (52.1%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-p-nitrobenzyl-3-[1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono)methylpyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.23(d,3H,J=7.4Hz), 1.31(d,3H,J=6.2Hz), 2.35(m,1H), 2.64(m,1H ) , 3. 01 ( s, 3H) , 3.10 (s , 3H ) , 3.30(dd,1H,J=2.6Hz,J=6.7Hz), 3.32(m,1H), 3.50(m,1H), 3.80-4.10(m,2H), 4.24(m,2H), 4.73(m,1H), 5.13-5.22(m,4H), 7.10(m,1H), 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-[1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclo[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 chloroform: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-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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 - [ 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.

NMR(CDCl₃) δ:

(E): 1.23(d,2H,J=7.3Hz), 1.37(d,2H,J=6.3Hz), 2.35(m,1H), 2.64( m ,1H ) , 3.01 ( s, 3H) , 3.20 ( s , 3H ) ,

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

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,J=7.2Hz) , 1.36(d,2H,J=6.3Hz), 2.37(m,1H), 2.65( m ,1H ) , 3.03 ( s, 3H) , 3.21 (s , 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) was 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-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 8-2 and stirred to dissolve. 0.27 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 15 hours under a hydrogen gas stream. 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 concentrated under reduced pressure at 10-20°C, then the extracted impurity was removed by filtration. The clarified filtrate was subjected to polymerchromatography(Diaion HP-20 resin:previously washed 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 was 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-azabicycl o [3,2,0]- hept-2-ene-7-one-2-carboxylic acid.

NMR ( DMSO-d₆) δ:

(E): 1.10(dd,6H), 1.64(m,1H), 2.45(m,1H), 2.90(m,1H),

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

3.63(m,1H), 3.90(m,2H), 4.10(m,1H)

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-azabicyclo[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 catalyst was added and stirred at room temperature for 17 hours under a hydrogen gas stream. 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 concentrated under reduced pressure at 10-20°C, then the extracted impurity was 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-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2- carboxylic acid.

NMR( DMSO-d₆) δ:

(Z): 1.09(dd,6H), 1.64(m,1H), 2.46(m,1H), 2.91(m,1H), 2 . 9 8 ( s , 3 H ) , 3 . 0 5 ( s , 3 H ) , 3.19(dd,1H,J=2.3Hz, J =6.4Hz), 3.31(m,2H), 3.62(m,1H), 3.89(m,2H), 4.08(m,1H)

EXAMPLE 8-5

To the mixture of 20 ml of tetrahydrofuran and 20 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH 7.0) was 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-[1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid obtained in EXAMPLE 8-1 and stirred to dissolve. 0.5 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 22 hours under hydrogen gas stream. 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 concentrated under reduced pressure at 10-20°C, then the formed impurity was removed by filtration. The clarified filtrate was subjected to polymerchromatography Diaion HP-20 resin:previously washed with methylalcohol and water). During the chromatography, at first it was eluted with water to remove 4-morpholinepropanesulfonic 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 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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR( DMSO-d₆) δ:

1.11(d,3H,J=7.1Hz), 1.13(d,3H,J=6.1Hz), 1.63(m,1H), 2.20(m,1H), 3.75(m,2H), 2.93(s,3H), 3.05(s,3H), 3.15(dd,1H), 3.30(m,2H), 3.60-3.90(m,2H), 4.10(m,1H)

EXAMPLE 9

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( 2 -pyridinylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl- 6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylic acid

EXAMPLE 9-1

1.81 g (5.0 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 110 ml anhydrous acetonitrile, stirred 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 was added dropwise, 1.2 ml(5.79 mmol) of diphenylchlorophosphate added and stirred for 2 hours. And then 1.1 ml (6.31 mmol) of N,N-diisopropylethylamine was added and the solution of 1.9 g (4.73 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2- (2-pyridinylhydrazono)methyl-4-mereaptopyrrolidine obtained in EXAMPLE 26-5 in 30 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was treated with the same 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-nitrobenzy1-3-[1-(p-nitrobenzyloxycarbonyl)-2-(2-pyridinylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.31(d,3H,J=7.3Hz), 1.38(d,3H,J=6.2Hz), 2.10(m,1H), 2.64(m,1H), 2.7(dd,1H,J=2.6Hz,J=6.8Hz), 3.50(m,5H), 3.75(m,1H), 4.01(m,1H), 4.12(m,2H), 4.62(m,1H), 5.05-5.50(m,5H), 6.75(m,1H), 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 morpholinepropanesulfonic 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-azabicyclo[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 catalyst was added and stirred at room temperature for 5 hours under an atmospheric pressure 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 concentrated under reduced pressure at 10-20°C, then the formed impurity was removed by filtration. The clarified filtrate was treated, as discribed in EXAMPLE 8-5, to give 0.422 g (53.6%) of the mixtures 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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR( DMSO -d₆) δ:

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

EXAMPLE 10

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( p - carboxyphenylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-azabicyclof3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 100 ml anhydrous acetonitrile, stirred 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 hours. Then 0.38 ml (2.30 mmol) of N,N-diisopropylethylamine was 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 was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 10 hours. After the reaction, the reaction mixture solution was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was treated with the same operation as EXAMPLE 8-1 to obtain 1.42 g of crude material. This crude product was subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=20:1) 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-[1-(p-nitrobenzyloxycarbonyl)-2-(p-methoxybenzyloxycarbonylphenylhydrazonomethyl)pyrrolid in- 4-yl -thio] -4 -methyl - 6- ( 1 -hydroxyethyl ) -1 -azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

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

3.78(s,3H), 4.01(m,1H), 4.22(m,2H), 4.64(m,1H), 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 solution of 10 ml of tetrahydrofuran and 10 ml of 0.1M morpholinepropanesulfonic acid buffer(pH 7.0) was added 0.38 g (0.418 mmol) of the mixtures of (E) and (Z) of (4R,5S,6S, 8R,2'S,4'S)-p-nitrobenzyl-3-[1-(p-nitrobenzyloxycarbonyl)-2-(p-methoxybenzyloxycarbonylphenylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl) -1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid obtained in EXAMPLE 10-1 and stirred to dissolve. 0.15 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 6 hours under atmospheric pressure 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 concentrated under reduced pressure at 10-20°C, then the extracted impurity as removed by filtration. The clarified filtrate was treated with the same operation as EXAMPLE 8-5 to give 0.102 g (51.5%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-3-[2-(p-carboxyphenylhydrazonomethyl) pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylic acid.

NMR(D₆O) δ:

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

EXAMPLE 11

( 4 R , 5 S , 6 S , 8 R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N -methylhydrazonomethyl) pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-azabicyclo[3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 120 ml .anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 0.66 ml (3.78 mmol) of N,N-diisopropylethylamine was added dropwise, and 0.72 ml (3.49 mmol) of diphenylchlorophosphate was added and stirred for 2 hours. Then 0.66 ml (3.78 mmol) of N,N-diisopropylethylamine was added and the solution of 1.51 g (2.91 mmol) of ( 2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2- [N-(p-nitrobenzyloxycarbonyl)-N-methylhydrazonomethyl]-4-mercaptopyrrolidine obtained in EXAMPLE 28-4 in 20 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 72 hours. After the reaction, the reaction mixture solution was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was treated with the same operation as EXAMPLE 8-1 to obtain 2.45 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 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-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(CDCl₃) δ:

1.12(d,3H,J=7.3Hz), 1.18(d,3H,J=6.3Hz), 2.10(m,1H), 2.70(m,1H), 3.10(m,4H), 3.40(m,2H), 3.75(m,1H), 4.01(m,1H), 4.22(m,1H), 4.70(m,1H), 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 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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 11-1 and stirred to dissolve. 0.8 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 5 hours under an atmospheric pressure 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 concentrated under reduced pressure at 10-20°C, then the extracted impurity was removed by filtration. The clarified filtrate was treated with the same operation as 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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR( DMSO-d₆) δ:

1.07(d,2H,J=6.1Hz), 1.09(d,2H,7.2Hz), 1.62(m,1H), 2.19(m,1H), 3.05(br,s,3H), 3.15(dd,1H,J=2.3Hz,J=6.4Hz), 3.30(m,2H), 3.61-3.85(m,2H), 3.75(m,2H), 4.05(m,1H)

EXAMPLE 12

( 4S , 5S , 6S , 8R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N ,N -dimethylhydrazonomethyl) pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-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-(1-hydroxyethyl) -1-azabicyclo[3,2,0]-hept- 3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 260 ml anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same 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 stirred for 2 hours. Then 1.61 ml (9.22 mmol) of N,Ndiisopropylehtylamine 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 solution was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was treated, as discribed in EXAMPLE 8-1, to obtain 3.5 g of crude material. This 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 mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S)-p-nitrobenzyl-3-[1-(p-nitrobenzyloxycarbonyl)-2-(N,N-dimethylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.13(d,3H,J=7.3Hz), 1.17(d,3H,J=6.3Hz), 2.01(m,1H), 2.60(m,1H), 2.75(s,6H), 3.23(dd,1H,J=2.7Hz,J=6.8Hz), 3.27(m,1H), 3.64(m,1H), 4.01(m,1H), 4.22(m,2H), 4.55(m,1H), 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 morpholinepropanesulfonic acid buffer (pH 7.0) was added 1.0 g (1.43 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 o b en z y l o xy c a r b on y L ) - 2 - ( N , N -dimethylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 12-1 and stirred to dissolve. 0.45 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 6 hours under an atmospheric pressure 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 pressure at 10-20°C, Then the extracted impurity was removed by filtration. The clarified filtrate was treated, as discribed in EXAMPLE 8-5, to give 0.22 g (40.0%) of the mixtures 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)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR( DMSO-d₆) δ: 1.01(m,6H), 1.59(m,1H), 2.10(m,1H), 2.40(m,1H), 2.64(m,1H), 2.70(s,6H), 2.84(m,1H), 3.10-3.40(m,3H), 3.60-4.13(m,3H)

EXAMPLE 13

( 4R , 5S , 6S , 8R , 2 ' S , 4 ' S ) - 3 - [ 2 - ( N ,N -diethylhydrazonomethyl) pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-azabicycl o [3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 100 ml anhydrous acetonitrile, stirred 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 hours. Then 0.74 ml (4.25 mmol) of N,N-diisopropylethylamine was added and the solution 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 anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was treated, as discribed in 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:acetone=20:1) and purified to give 1.35 g (60.2%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S) -p-nitrobenzyl-3-[1- (p-ni t r o b en z y l o xy c a r b on 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(CDCl₃) δ:

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

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

3.23(dd,1H,J=2.6Hz,J=6.8Hz), 3.37(m,2H), 3.63(m,1H), 4.01(m,1H), 4.20(m,2H), 4.33(m,1H), 5.10 - 5.30 (m, 4H) ,

7.50(d,2H,J=8.8Hz), 7.61(d,2H,J=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 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 en z y l o xy c a r b on 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 obtained in EXAMPLE 13-1 and stirred to dissolve. 0.4 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 2 hours under an atmospheric pressure 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 treated, as discribed in EXAMPLE 12-2, to give 0.26 g (46%) of the mixtures of (E) and (Z) of ( 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-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR( DMSO-d₆) δ:

0.93(t,6H), 1.13(m,6H), 1.53(m,1H), 2.10(m,1H),

2.40(m,1H), 2.64(m,1H), 2.84(m,1H), 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-[N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazonomethyl]pyrrolidin-4-yl-thio]- 4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[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-4-methyl-6-(1-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-diisopropylethylamine was added dropwise, and 0.30 ml (1.45 mmol) of diphenylchlorophosphate was added and stirred for 2 hours. Then 0.271 ml (1.55 mmol) of N,N-diisopropylethylamine was added and the solution of 0.69 g (1.55 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2- [N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazono]methyl-4-mercaptopyrrolidine obtained in EXAMPLE 31-4 in 10 ml anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 36 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and ethylacetate was added to the residue to extract the product. The extract was treated, as discribed in EXAMPLE 8-1, to obtain 1.2 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=40:1) and purified to give 0.152 g (50.2%) of the mixtures 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' -dimethylsulfamoyl)hydrazonomethylpyrrolidine-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.12(d,3H,J=7.4Hz), 1.31(d,3H,J=6.3Hz), 2.10(m,1H), 2.70( m,1H ) , 2 .90 ( s, 6H) , 3 .10 ( s , 3H ) , 3.23(dd,1H,J=2.6Hz,J=6.9Hz), 3.37(m,2H), 3.63(m,1H), 4.20(m,2H), 4.70(m,1H), 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 mixtures 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' -dimethylsulfamoyl)hydrazonomethyl]pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 14-1 and stirred to dissolve. 0.25 g of 10% palladium/carbon catalyst was added and stirred at room temperature for 3 hours under an atmospher is pressure 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 treated, as discribed in EXAMPLE 12-2, to give 0.16 g (53.1%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,2'S,4'S) -3-[ (2-[N-methyl-N-(N' ,N' -dimethylsulfamoyl)hydrazonomethyl] pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(D₆O) δ:

1.01(dd,6H), 1.59(m,1H), 2.10(m,1H), 2.40(m,1H), 2.60(m,1H), 2.84(m,1H), 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'S,4'S)-3-[2-(N-methaneεulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclof 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-azabicyclo[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 stirred for 2 hours. Then the solution of 4.65 g (11.1 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono) methyl-4-mercaptopyrrolidine obtained in EXAMPLE 25-5, 80 ml of anhydrous acetonitrile was added dropwise 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 was stirred at room temperature for 20 hours. After the reaction, the reaction mixture was 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 mixtures of (E) and (Z) of (5S,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-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(DMSO-d₆) δ:

1.10(d,3H,J=6.3Hz), 2.03(m,1H), 2.64(m,1H), 3.01(s,3H), 3.28(s,3H), 3.32(m,3H), 3.82-4.20(m,4H), 4.58(m,1H), 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 - [ 1 - ( p-nitrobenzyloxycarbonyl) -2- (N-methansulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclof 3,2,0] -hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 15-1 was 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 - [ 1 - (p-nitrobenzyloxycarbonyl) -2- (N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-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-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR ( DMSO -d₆) δ:

(E): 1.10(d,3H,J=6.3Hz) , 2.03(m,1H), 2.62(m,1H),

3.02(s,3H), 3.29(s,3H). 3.32(s,3H), 3.82- 4.19(m,4H), 4.58(m,1H), 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,1H), 2.62(m,1H), 3.02(s,3H), 3.25(s,3H), 3.33(m,3H), 3.82- 4.20(m,4H), 4.59(m,1H), 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) was added 0.5 g (0.67 mmol) of (E) of (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-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 15-2 and stirred to dissolve. 0.2 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 5 hours under an atmospheric pressure 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 extracted impurity was filtrated, the filtrate was washed with ethylacetate, and the water layer was concentraed under reduced pressure at 10-20°C to evaporate the residual organic solvent completely. The clarified filtrate was subjected to polymerchromatography (Diaion HP-20 resin; previously washed with methylalcohol and water). During the chromatography, it was first 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 was lyophilized to give 0.12g (41.3%) of ( E)-(5R,6S,8R,2 'S,4'S)-3-[(2-(N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid. NMR(DMSO-d₆) δ:

1.05(d,2H,J=6.0Hz) , 1.62(m,1H), 2.40(m,1H),

2.65(m,1H), 2.91(s,3H), 2.92(m,1H), 3.04(s,3H),

3.22(m,3H), 3.60(m,1H), 3.82(m,2H), 4.03(m,1H)

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-azabicyclo[3,2,0]-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 hours under a hydrogen gas stream. After the reaction, the catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to evaporate the organic solvent. 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-[2-(N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(DMSO-d₆) δ:

1.04(d,2H,J=6.1Hz), 1.62(m,1H), 2.40(m,1H), 2.64(m,1H), 2.90(s,3H), 3.03(s,3H), 3.21(m,3H), 3.59(m,1H), 3.81(m,2H), 4.01(m,1H)

EXAMPLE 15-5 To the mixture of 150 ml of tetrahydrofuran and 50 ml of 0.1 M morpholinepropanesulfonic acid buffer (pH

7.0) was added 5.0 g (6.7 mmol) of (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-azabicyclo[3,2,0]-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 catalyst was added and stirred at room temperature for 5 hours under an atmospheric pressure 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 extracted impurity was removed and the filtrate was treated, as discribed in EXAMPLE 15-3, to give 1.24 g (42.7%) of

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

NMR(DMSO-d₆) δ:

1.05(d,2H,J=6.0Hz) , 1.62(m,1H), 2.39(m,1H),

2.64(m,1H), 2.90(s,3H), 3.00(s,3H), 3.25(m,3H), 3.59-3.83(m,3H), 4.02(m,1H)

EXAMPLE 16

(4R,5S,6S,8R,2^,S,4'S)-3-[2-(N-methansulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclo[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-azabicyclof3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 180 ml anhydrous 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-diisopropylethylamine was added dropwise, and 0.877 ml (4.23 mmol) of diphenylchlorophosphate was added and stirred for 2 hours. Then 0.737 ml (4.23 mmol) of N,N-diisopropylethylamine 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 anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, ethylacetate was added to the residue to extract the product. The extract was treated, as discribed in EXAMPLE 8-1, to obtain 3.25 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=30:1) and purified to give 1.74 g (53.4%) of the mixtures of (E) and (Z) of (4R,5S,6S,8R,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-azabicycl o [3,2,0]-hept-2-ene-7-one-2- carboxylate. NMR(CDCl₃) δ:

1.18(d,3H), 1.30(d,3H), 2.02(s,3H), 2.03(m,1H),

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

3.38(m,2H), 3.70(m,1H), 4.10(dd,1H, J=6.8Hz,J=2.6Hz), 4.15(m,1H) , 4.60(t,1H), 5.05-5.50(m,4H) ,

7.52(d,2H,J=8.7Hz), 7.60(d,2H,J=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-[1- (p-nitrobenzyloxycarbonyl)-2-[1-(N-methanesuIfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylate obtained in EXAMPLE 16-2 and stirred to dissolve. 0.3 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 5 hours under an atmospheric pressure of hydrogen. After the reaction, the catalyst was removed by filtration and the filtrate was washed with a small amount of 0.5 M morpholineproparesulfonic acid and concentrated under reduced pressure to evaporate the organic solvent. After the residue was washed with ethylacetate, separated, and the water layer was concentrated under reduced pressure below 20°C, then the formed impurity was removed by filtration. The clarified filtrate was treated, as discribed 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-[2- (N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4- yl - thi o ] - 4 -methyl - 6 - ( 1 -hyd roxyethyl ) -1 -azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid. NMR(CDCl₃) δ:

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

3.10(dd,1H,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,2fS,4'S)-3-[2-(N-ethaneεulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylic acid

EXAMPLE 17-1

2.84 g (7.83 mmol) of (4R,5R,6S,8R)-p-nitrobenzyl-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-3,7-dione-2-carboxylate obtained in REFERENCE EXAMPLE 1 was added to 250 ml anhydrous acetonitrile, stirred at room temperature and cooled to 0°C using an ice bath. At the same temperature, 1.38 ml (7.92 mmol) of N,N-diisopropylethylamine was added dropwise, and 1.64 ml (7.91 mmol) of diphenylchlorophosphate was added and stirred for 2 hours. Then 1.38 ml (7.92 mmol) of N,N-diisopropylethylamine was added and the solution of 3.3 g (7.67 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2- (N-ethanesulfonyl-N-methylhydrazono) methyl-4-mercaptopyrrolidine obtained in EXAMPLE 33-4 in 50 ml of anhydrous acetonitrile was added dropwise to the mixture solution. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature for 48 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and methylenechloride was added to the residue to extract the product. The extract was treated, as discribed in EXAMPLE 8-1, to obtain 5.35g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform:acetone=30:1) and purified to give 3.42 g (57.5%) of the mixtures of (E) and (Z) of (4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 -[ 1- (p-nitrobenzyloxycarbonyl) -2- (N-ethanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylate.

NMR(CDCl₃) δ:

1.30(m,9H), 2.10(m,1H), 2.70(m,1H), 3.10(m,1H), 3.50(dd,1H,J=4.4Hz,J=l1Hz), 3.80(m,1H), 4.05(m,1H), 4.12(m,2H), 4.70(m,1H), 5.10-5.50(m,4H), 7.05(m,1H), 7.45(d,2H,J=8.1Hz), 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 morpholinepropanesulfonic acid buffer (pH 7.0) was added 0.70g (0.90 mmol) of

(4R,5S, 6S ,8R, 2' S, 4' S) -p-nitrobenzyl -3 -[ 1- (p-nitrobenzyloxycarbonyl) -2- (N-ethanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6- (1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylic acid obtained in EXAMPLE 17-1 and stirred to dissolve. 0.280 g of 10% of the palladium/carbon catalyst was added and stirred at room temperature for 7 hours under an atmospheric pressure 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 was concentraed under reduced pressure at 20°C, then the extracted impurity was removed by filtration. The clarified filtrate was treated, as is 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-ethanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

NMR(DMSO-d₆) δ:

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

EXAMPLE 18

( 2S,4S) -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 stirred to dissolve, 1.92 g (18.12 mmol) of sodium bicarbonate was added dropwise 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 -butyldimethylsilyloxypyrrolidine in 360 ml of methylalcohol was added dropwise to the reaction mixture solution. After the addition, the reaction mixture solution was stirred overnight. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the solvent, 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=5:1) and purified to 7.45 g (66.9%) of (E)- (2S,4R) -1-(p-nitrobenzyloxycarbonyl)-2-methoxyimino-4-tert-butyldimethylsilyloxypyrr ol idine and 1.7 g (15%) of (Z)-(2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-methoxyimino-4-tert-butyldimethylsilyloxypyrrolidine. NMR(Acetone-d₆)

(1) E form: δ:

0.03(s,6H), 0.85(s,9H), 2.10(m,2H), 3.50(m,2H), 3.75(s,3H), 4.55(m,2H), 5.22(m,2H), 7.38(d,1H), 7.65(d,2H), 8.21(d,2H)

(2) Z form: δ:

0.03(s,6H), 0.85(s,9H), 2.10(m,2H), 3.65(m,2H), 3.85(S,3H), 4.60(m,2H), 5.30(dd,2H), 7.38(d,1H), 7.65(d,2H), 8.21(d,2H)

EXAMPLE 18-2 To 100 ml of tetrahydrofuran was added 4.85 g ( 11.09 mmo l ) o f ( E ) - ( 2 S , 4R ) - 1 - ( p -nitrobenzyloxycarbonyl) -2-methoxyimino-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 18-1 and stirred at room temperature to dissolve. Then 13.2 ml (13.2 mmol) 1 M tetrabutylammonium fluoride(tetrahydrofuran solution) was added dropwise and stirred at room temperature for 30 minutes. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the solvent, and the residue was dissolved in ethylacetate and washed successively with water and sodium 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 chloroform: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-d₆) δ:

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

4.23(m,1H), 4.43(m,1H), 7.39(d,1H), 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 ) - 1 - ( 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 solution was concentrated under reduced pressure to evaporate the solvent, and the residue was extracted with ethylacetate and washed successively with water and sodium 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 residue. The residue was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1.5:1) and purified to give 3.96 g (94.9%) of (E)-(2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-methoxyimino-4-methanesulfonyloxypyrrolidine.

NMR(Acetone -d₆) δ:

2.39-2.64(m,2H), 3.19(s,3H), 3.74(m,3H), 3.75-4.05(m,2H), 4.62(m,1H), 5.25(m,3H), 7.41(d,1H), 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) was dissolved 3.31 g (8.68 mmol) of (E)-(2S,4R)-1-(p-nitrobenzyloxycarbonyl ) -2-methoxyimino-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 18-3. After 16.2 g (14.2 mmol) of potassium thioacetate was added and was refluxed for 3 hours, the reaction solution was concentrated under reduced pressure and the residue was extracted with ethylacetate. The organic layer was washed with water and sodium chloride, and the separated organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 3.51 g of the residue. The residue was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1.5:1) and purified to give 2.4 g ( 72 . 5% , E : Z = 3 : 2 ) of ( 2S , 4S ) - 1 - ( p-nitrobenzyloxycarbonyl) -2-methoxyimino-4-acethylthiopyrrolidine.

NMR(Acetone -d₆) δ:

2.32(s,3H), 2.52-2.82(m,2H), 3.14(m,1H), 3.75(s,3Hx3/5), 3.81(s,3Hx2/5), 3.94-4.10(m,2H), 4.40(m,1H), 5.22(m,2H), 6.82(dd,1HX2/5), 7.41(d,1HX3/5), 7.62(d,2H), 8.21(d,2H)

EXAMPLE 18-5

In 70ml of methyl alcohol was dissolved 2.4 g (6.29 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-methoxyimino-4-acetylpyrrolidine obtained in EXAMPLE 18-4. After the reaction solution was 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 solution was adjusted to pH 4.5 using saturated citric acid and concentrated under reduced pressure to evaporate the organic solvent. To the residue was added ethylacetate to extract the product from the solution, washed with water and saturated sodium chloride, separated the organic solvent and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated to give 3.0 g of crude material. This crude material was 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-4-mercaptopyrrolidine.

NMR(Acetone-d₆) δ:

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

EXAMPLE 19

(2S,4S)-1-(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-butyIdimethylsilyloxypyrrolidine was added in 400 ml of methyl alcohol and dissolved (the reaction solution B). After solution B was added to the solution B, the reaction mixture solution was stirred at room temperature for 20 hours, the precipitate was filtered, and the filtrate was concentrated under reduced pressure. 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-hydroxyimino-4-tert-butyldimethylsilyloxypyrrolidine.

NMR(Acetone -d₆) δ:

0.10(d,6H), 0.89(m,9H), 2.15-2.40(m,1H), 2.82(m,1H), 3.52(m,2H), 4.55(m,2H), 5.23(m,2H), 6.78(m,1Hx1/3), 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)-1-(p-nitrobenzyloxycarbonyl)-2-hydroxyimino-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 19-1 and stirred to dissolve. Then 15.31 g (110.8 mmol) of potassium carbonate and 4.91 ml (44.32 mmol) of ethylbromoacetate were added and refluxed at room temperature for 3 days . After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the solvent, the residue was dissolved in water and ethylacetate, and the reaction solution was adjusted to pH 5.5 using saturated citric acid solution. Then the excess ethylacetate was added to the reaction solution and shaken, the mixture solution was washed successively with water and sodium chloride, the organic layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 13.5 g of the crude material. This crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=2.5:1) and purified to 8.83 g (78.2%) of (2S,4R)-1-(p- nitrobenzyloxycarbonyl)-2-ethoxycarbonyl methoxyimino-4-tert-butyldimethylsilyloxypyrrolidine.

NMR(Acetone-d₆) δ:

0.03(d,6H), 0.83(s,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,1Hx1/3), 7.41(d,1Hx2/3), 7.50(m,2H), 8.20(d,2H)

EXAMPLE 19-3

To 120 ml of tetrahydrofuran was added 8.08 g (15.86 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-ethoxycarbonylmethoxyimino - 4 - te rt -butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 19-2 and stirred to dissolve. Then 19.8 ml (19.8 mmol) of 1 M- tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwise and stirred at room temperature for 30 minutes. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the solvent, and the residue was extracted with ethylacetate and dried over anhydrous magnesium sulfate. In the reaction solution were added dropwise 5.27 ml (39.65 mmol) of triethylamine and 1.85 ml (23.9 mmol) of methanesulfonyl chloride, and after the removal of the ice bath, stirred at room temperature for 1 hour. After the reaction, the mixture solution was concentrated under reduced pressure to evaporate the solvent, and the residue was extracted with ethylacetate. The extract was washed successively with water and saturated sodium chloride, and the organic layer was dried over anhydrous magnesium sulfate, filtered and then concentrated under reduced pressure to give 8.4 g of the crude material. This crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate =1:1.5). After filtration, the filtrate was concentrated under reduced pressure and the residue was dissolved in 160 ml of methylene chloride, and cooled to 0°C using an ice bath and purified to 5.84 g (77.8%) of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-ethoxycarbonylmethoxyimino-4-methanesulfonyloxypyrrolidine.

NMR(Acetone-d₆) 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,1H×1/3), 7.58(d,1Hx2/3), 7.63(d,2H), 8.21(d,2H)

EXAMPLE 19-4

In 300 ml of the solution of acetonitrile was dissolved 3.31 g (8.68 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-ethoxycarbonylmethoxyimino-4-methanesulfonylpyrrolidine obtained in EXAMPLE 19-3. After 5.64 g (49.4 mmol) of potassium thioacetate was added and refluxed for 3 hours, the reaction solution was concentrated under reduced pressure and the residue was extracted with ethylacetate. The organic layer was washed with water and sodium chloride and the separated organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 3.51 g of the residue. The residue was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1.5:1) and purified to 4.16 g (72.8%) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-ethoxycarbonylmethoxyimino-4-acethylthiopyrrolidine.

NMR(Acetone -d₆) δ:

1.10(m,3H), 2.01(m,1H), 2.30(s,3H), 2.70(m,1H), 3.20-3.62(m,2H), 3.92-4.22(m,4H), 4.50-4.62(m,2H), 5.25(s,2H), 6.92(dd,1Hx1/3), 7.54(d,1HX2/3), 7.64(d,2H), 8.23(d,2H)

EXAMPLE 19-5

In 50 ml of ethyl alcohol was dissolved 4.16 g (9.18 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-ethoxycarbonyl methoxyimino-4-acetylthiopyrrolidine obtained in EXAMPLE 19-4. After the reaction solution was cooled to 0°C using the ice bath, 6 ml of 2 N sodium hydroxide aqueous solution was added dropwise 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 pressure to evaporate the organic solvent. The residue was extracted with ethylacetate, and the organic layer washed with water and saturated sodium chloride, and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give 5.78 g of the residue. The residue 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-d₆) δ:

1.12(m,3H), 2.03(m,1H), 2.65(m,1H), 3.25- 3.75(m,2H) , 3.95-4.25(m,2H), 4.43-4.70(m,2H) ,

5.30(s,2H), 6.98(dd,1Hx1/3), 7.51(d,1Hx2/3), 7.65(d,2H), 8.21(d,2H)

EXAMPLE 20

(2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N,N-dimethylaminocarbonylmethoxyimino)-4-mercaptopyrrolidine

EXAMPLE 20-1

1.85 g (3.63 mmol) of (2S,4R)-1(p-nitrobenzyloxycarbonyl)-2-ethoxycarbonylmethoxyimino-4-tert-butyldimethylsilyloxypyrrolidine was added to 30 ml of acetone and stirred to dissolve. At room temperature, 2.8 ml of 2 N Sodium hydroxide aqueous solution was added dropwise and stirred for 3 hours, and at the same temperature the pH of the solution was adjusted to pH 2.0 using saturated citric acid. The layer was washed several times with water and saturated sodium chloride after separation, the organic layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated under reduced pressure. 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(CDCl₃) δ:

0.03(d,6H), 0.81(s,9H), 2.10(m,2H), 3.50(m,2H), 4.38(m,1H), 4.58(m,3H), 5.20(m,2H), 6.80(m,1Hx1/3), 7.40(d,1Hx2/3), 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 -butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 20-1 and cooled to 0°C using an ice bath. Then, 1.56 ml (11.19 mmol) of triethylamine and 0.93 ml (10.35 mmol) of ethylchloroformate was added dropwise and stirred at the same temperature for 30 minutes. 0.93 ml (10.3 mmol) of 50% of the dimethylamine aqueous solution was added dropwise, 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 extracted with ethylacetate. The organic layer was washed successively with water and saturated aqueous sodium chloride. After separation, the organic layer was dried over anhydrous 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-butyldimethylsilyloxypyrrolidine. This crude material was used at the next reaction without purification. NMR(CDCl₃) δ:

0.03(s,6H), 0.80(s,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, 1Hx1/3), 7.50(d,1Hx2/3), 8.19(d,2H)

EXAMPLE 20-3

To 70 ml of tetrahydrofuran was added 4.3 g (8.46 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N,N- dimethylaminocarbonyl methoxyimino)-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 20-2 and added dropwise 10.5 ml (10.5 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran solution) which was stirred at room temperature for 30 minutes. After the reaction, the reaction mixture was concentrated under reduced pressure and extracted with ethylacetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give 3.29 g of the crude material. The crude material was dissolved in 70 ml of methylenechloride. After the temperature of the reaction solution 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 dropwise, 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 pressure and extracted with ethylacetate. The orgainc layer was washed successively with water and saturated aqueous sodium chloride. After separation, the organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to give 5.6 g of the residue. The residue was subjected to the column chromatography on sil ica gel ( eluted with chlorof orm: methylalcohol=20 : 1 ) and puri f ied to 3 . 4 g ( 85% ) of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N,N-dimethylaminocarbonyl methoxyimino ) -4 -methanesulfonyloxypyrrolidine. NMR(Acetone-d₆) δ :

1.80-2.20(m,2H), 2.80-3.05(d,6H), 3.09(s,3H), 3.50-3.70(m,2H), 4.50-4.80(m,3H), 5.10(m,1H), 5.20(m,2H), 6.90(dd,1Hx1/3), 7.50(d,1Hx2/3), 7.65(d,2H), 8.21(d,2H) EXAMPLE 20-4

In 100 ml of acetonitrile was dissolved 1.55 g

(32.84 mmol) of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2- (N,N-dimethylaminocarbonylmethoxyimino ) -4 -methanesulfonyloxypyrrolidine obtained in EXAMPLE 20-3. After 1.1 g (96.3 mmol) of potassium thioacetate was added and refluxed for 4 hours, as discribed in EXAMPLE

19-4, and was carried out to give 1.2 g of crude material. This crude material solution was concentrated under reduced pressure to give 3.51 g of the residue. The residue 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-dimethylaminocarbonylmethoxyimino)-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ :

2.05(m,2H), 2.12(s,3H), 2.90(d,6H), 3.55(m,1H), 3.95(m,2H), 4.50-4.80(m,3H), 5.19(s,2H), 6.80(d,1Hx1/3), 7.50(d,1Hx8/3), 8.18(d,2H)

EXAMPLE 20-5

in 30 ml of methyl alcohol was dissolved 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 using an ice bath. 1.5 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 solution was adjusted to pH 4.2 using saturated citric acid. The reaction mixture was concentrated under reduced pressure, as discribed in EXAMPLE 18-5, and was carried out to give 0.82 g of crude material. This crude material was subjected 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(CDCl₃) δ :

2.0(m,2H), 2.40-2.60(m,2H), 2.82(s,6H), 3.32(m,1H), 3.95(m,2H), 4.44(m,1H), 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 solution of 6.6 g (13.7 mmol) of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(carboxymethoxyimino)-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 20-1 in 150 ml of N,N-dimethylformamide was 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 was stirred for a day, concentrated under reduced pressure by a high vacuum pump, and then extracted with ethylacetate. After the extracted solution was washed with water and saturated aqueous sodium chloride, the organic layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 6.04 g (73.3%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(p-methoxybenzyloxycarbonyl methoxyimino)-4-tert-butyldimethylsilyloxypyrrolidine

NMR(CDCl₃) δ:

0.03(d,6H), 0.80(s,9H), 2.00(m,2H), 3.50(m,2H), 3.80(s,3H), 4.40(m,1H), 4.60(m,3H), 5.10(m,2H), 5.20(m,2H), 6.83(d,1Hx7/3), 7.23(d,2H), 7.50(m,1HX8/3), 8.19(d,2H)

EXAMPLE 21-2

(2S,4R) -1- (p-nitrobenzyloxycarbonyl) -2- (p-methoxybenzyloxycarbonylmethoxyimino) - 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(CDCl₃) δ:

1.80(t,1H), 2.02(m,2H), 2.39-2.90(m,1H), 3.30- 3.70(m,2H), 3.78(s,3H), 4.00(m,1H), 4.42-4.64(m,2H) , 4.70-5.05(m,1H), 5.15(m,4H), 7.00(d,1Hx2/3), 7.56(d,1Hx1/3), 8.22(d,4H)

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

EXAMPLE 22-1

4.29 g (8.91 mmol) of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-carboxymethoxyimino-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 20-1 was dissolved in 100 ml of tetrahydrofuran and cooled to 0°C using 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 successively with water and saturated aqueous sodium chloride, the organic layer was dried over anhydrous magnesium sulfate, Filtered and then the filtrate was concentrated under reduced pressure to give 6.-7 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 3.25 g (76%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-aminocarbonylmethoxyimino-4-tert-butyldimethylsilyloxypyrrolidine.

NMR(CDCl₃) δ:

0.03(d,6H), 0.83(s,9H), 1.70-2.15(m,2H), 3.46(m,2H), 4.35-4.65(m,4H), 5.20(m,2H), 6.60(d,1Hx1/3), 7.49(d,1Hx8/3), 4.35-8.20(d,2H) EXAMPLE 22-2

In 80 ml of tetrahydrofuran was dissolved 2.52 g (5.26 mmol) of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-aminocarbonyl methoxyimino- 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 dropwise and stirred at room temperature for 30 minutes. After the reaction, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethylacetate and dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue was dissolved 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 was added dropwise, removed from the ice bath, and stirred at room temperature for 1 hour. After the reaction, the mixture solution was concentrated under reduced pressure, extracted with ethylacetate and washed successively with water and brine. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate and filtrate, and then the filtrate was concentrated to give 2.8 g of the residue. The residue was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=25:1) and purified to 1.56 g (66.9%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-am 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(CDCl₃) δ:

2.01-2.80(m,2H), 3.03(s,3H), 3.70(m,1H), 4.01(m,1H), 4.42-4.70(m,3H), 5.20(m,3H), 6.62(d,1Hx1/3), 7.49(d,1H×8/3), 8.20(d,2H)

EXAMPLE 22-3

1.28 g (2.89 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-aminocarbonylmethoxyimino-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 22-2 was dissolved in 100 ml of acetonitrile. Then, 0.825 g (7.23 mmol) of potassium thioacetate was added and refluxed for 4 hours. After the reaction, the mixture solution was concentrated under reduced pressure, and the residue was treated, as discribed in EXAMPLE 19-4, to obtain 2.2 g of the crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=25:1) and purified to 1.0 g (81.7%) of ( 2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-aminocarbonylmethoxyimino-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ:

2.03(m,1H), 2.28(s,3H), 2.60(m,1H), 3.34(m,1H), 3.98(m,2H), 4.43(m,H), 5.19(s,2H), 6.01-6.43(m,2H), 6.70(d,1Hx1/3), 7.50(d,1Hx8/3), 8.19(d,2H)

EXAMPLE 22-4

In the mixture solvent of 50 ml of methylalcohol and 10ml of tetrahydrofuran was dissolved 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 sodium hydroxide was added dropwise and stirred for 3 minutes. The pH of the solution was adjusted to pH 3.5 using a saturated citric acid solusion. The reaction solution was concentrated under reduced pressure, the residue was extracted with ethylacetate, and then treated, as discribed in EXAMPLE 19-1, to give 0.95 g of crude material. The crude material was subjected to the column chromatography on silica 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(CDCl₃) 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,1H), 5.12(s,2H), 5.85- 6.80(m,2H), 6.80(d,1Hx1/3), 7.42(d,1Hx8/3), 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 using an ice bath was added 5.31 g (11.03 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-carboxymethoxyimino-4-tert-butyldimethylsilyloxypyrrolidine and stirred to dissolve. 2 ml(14.36 mmol) of triethylamine and 1.27 ml (13.28 mol) of ethyl chloroformate was added and the reaction mixture solution stirred at the same temperature for 30 minutes. Then, 1.07 ml (14.3 mmol) of 40% methylamine aqueous solution added and stirred for 1 hour without an ice bath. After the reaction, the mixture solution was concentrated under reduced pressure, and then extracted with ethylacetate. The extract was treated as discribed in EXAMPLE 20-2, to give 5.3 g (97.2% ) 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 -methylaminocarbonylme thoxyimino ) - 4 - tert -butyldimethylsilyloxypyrrolidine. The product was used for the next reaction without purification.

NMR(CDCl₃) δ:

0.03(s,6H), 0.80(s,9H), 2.05(m,2H), 2.92(d,3H), 3.48(m,2H), 4.29-4.72(m,4H), 5.21(m,2H), 6.80(m,1Hx1/3), 7.51(m,1Hx8/3), 8.20(d,2H)

EXAMPLE 23-2

(2S,4R) -1- (p-nitrobenzyloxycarbonyl)-2-(N-methylaminocarbonylme thoxyimino ) - 4 - tert -butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 23-1 was treated, as discribed in EXAMPLE 20, to give ( 2S , 4R) - 1 - ( p-nitrobenzyloxycarbonyl ) -2 - (N-methylaminocarbonylmethoxyimino)-4-mercaptopyrrolidine.

NMR(CDCl₃) δ:

2.10(m,1H), 2.55(m,1H), 2.60(d,3H), 3.20- 3.70(m,2H), 4.05(m,1H), 4.42(d,2H), 4.50-4.80(m,1H), 5.32(d,2H), 7.01-7.50(m,1H), 7.70(d,2H), 8.22(d,2H) EXAMPLE 24

(2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-hydroxyimino4-mercaptopyrrolidine

EXAMPLE 24-1

In 200 ml of methylenechloride, cooled to 0°C using an ice bath, was dissolved 19.9 g (61.36 mmol) of (E)-1-(p-nitrobenzyloxycarbonyl)-4-hydroxy-L-prolinemethyl ester. 11.12 ml (79.78 mmol) of triethyl amine and 4.99 ml (64.45 mmol) of methanesulfonylchloride was added and stirred at room temperature for 3 hours after removing the ice bath. After the reaction, 200 ml of water was added, shaken vigorously, and the water layer separated from the organic layer. To the water layer was added 250 ml of methylenechloride which was shaken vigorously to extract the product. The organic layer was combined with the previous organic layer and washed successively with water and brine. The organic layer was separated again, dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 24.45 g (99%) of crude material of (E)-1-(p-nitrobenzyloxycarbonyl)-4-methanesulfonyl-L-prolinemethyl ester. This material was used for the next reaction without purification.

NMR(CDCl₃) δ:

2.02(m,1H), 2.35(m,1H), 3.01(s,3H), 3.50-3.70(m,2H), 3.60(s,3H), 4.10(m,1H), 5.20(s,2H), 5.22(m,1H), 7.43(d,2H), 8.15(d,2H)

EXAMPLE 24-2

After 2.16 g (19.46 mmol) of anhydrous calcium chloride was added to 30 ml of anhydrous ethylalcohol, stirred and dispersed the mixture solution was cooled to 0°C using an ice bath and 1.07 g (28.28 mmol) sodium borohydride (solution A) was added. After stirring at the same temperature, the solution of 2.85 g (7.08mmol) of (E)-1-(p-nitrobenzyloxycarbonyl)-4-methanesulfonyl-L-prolinemethyl ester obtained in EXAMPLE 24-1 in 11 ml of anhydrous ethylalcohol was added dropwise to solution A. After removal of the ice bath, the reaction mixture solution was added and stirred at room temperature for 3 hours, saturated citric acid solution was added to remove the excess sodium borohydride, and the mixture was concentrated under reduced pressure. The residue was extracted with ethylacetate and washed successively with water and brine. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtred and then the filtrate was concentrated to give 2.59 g (7.7%) of nearly pure material of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-hydroxymethyl-4-methanesulfonyloxypyrrolidine. This material was used for the next - reaction without purification.

NMR(CDCl₃) δ:

2.03(m,1H), 2.32(m,1H), 3.02(s,3H), 3.50-4.20(m,5H), 5.19(s,2H), 5.21(m,1H), 7.22(d,2H), 8.15(d,2H)

EXAMPLE 24-3

In 10 ml of anhydrous methylenechloride, cooled to -10°C using a dry ice bath, was dissolved 298 μl (3.49 mmol) of oxalylchloride, the reaction mixture solution cooled to -78°C. then, 454 μl (6.40 mmol) of anhydrous dimethylsulfoxide was added dropwise, stirred at the same temperature for 10 minutes, and the solution of 1.09 g (2.91 mmol) of (2S,4R)-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 was stirred for 1 hour, concentrated under reduced pressure, and the residue was extracted with ethylacetate and washed successively with water and brine. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtred and then the filtrate was concentrated to give 1.3 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=40:1) and purified to 0.95 g (87.7%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-formyl-4-methanesulfonyloxypyrrolidine.

NMR(CDCl₃) δ:

2.10-2.60(m,2H), 3.01(d,3H), 3.60-4.01(m,2H), 4.45(m,1H), 5.19(m,1H), 5.22(s,2H), 7.43(d,2H), 8.15(d,2H), 9.50(d,1H)

EXAMPLE 24-4

After 86.8 ml (1.25 mmol) of sodium bicarbonate and 164 mg (1.55 mmol) of hydroxylamine hydrochlorate salt was added to 10 ml of water, the solution of 442 mg (1.19 mmol of ( 2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-formyl-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 24-3 in 10 ml of ethylalcohol was added and then stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure and the residue was extracted with ethylacetate. The organic layer was washed with water and brine and the separated organic layer was dried over anhydrous magnesium sulfate and filtred. 640 mg of the obtained crude material was concentrated under reduced pressure to give 350mg of the residue. The residue was subjected to the column chromatography on silica gel (eluted with chloroform: methylalcohol=25:1) and purified to 350 mg (75.9%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-hydroxyimino-4-methanesulfonyloxy pyrrolidine.

NMR(CDCl₃) δ:

2.01-2.90(m,2H), 3.00(d,3H), 3.65(m,1H), 3.95(m,1H), 4.63(m,1H), 5.20(m,3H), 6.80(d,1Hx1/3), 7.15(d,1Hx2/3), 7.50(d,2H), 8.15(d,2H)

EXAMPLE 24-5

In 10 ml of acetonitrile was dissolved 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 potassium thio acetate was added and refluxed at room temperature for 2 hours, it was concentrated under reduced pressure to evaporate the organic solvent. The residue was extracted with ethylacetate, washed with water and brine, separated from the organic solvent and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated to give 118 mg of crude material. This crude material was subjected 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-hydroxyimino-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ:

1.80(m,1H), 2.60(m,1H), 3.30(m,1H), 4.0(m,2H), 4.5-5.0(s,1H), 5.20(s,2H), 6.75, 7.30(d,dd,1H), 7.60(d,2H), 8.20(d,2H), 10.8-11.0(m,1H)

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,4S)-1-(p-nitrobenzyloxycarbonyl)-2-hydroxyimino-4-acetylthiopyrrolidine obtained in EXAMPLE 24-5 was added and stirred to dissolve 0.32 ml of 2 N sodium hydroxide solution at the same temperature for 3 minutes, and the pH of the solution was adjusted to pH 3.5 using saturated citric acid. The reaction solution was concentrated under reduced pressure and the residue was extracted with ethylacetate. The organic layer was washed successively with water and brine and the separated organic layer was dried over anhydrous magnesium sulfate and filtrend. The filtrate was concentrated under reduced pressure to give 101 mg of the crude material. This crude material was subjected 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(CDCl₃) δ: 2.01(m,1H), 2.30(d,2H), 2.71(m,2H), 3.21- 3.59(m,2H), 4.01(m,1H), 4.91-5.01(m,1H), 5.31(s,2H), 7.71(d,2H), 8.20(d,2H), 10.01(m,1H)

EXAMPLE 25

(2S,4S) -1-(p-nitrobenzyloxycarbonyl)-2-(N-methaneεulfonyl -N-methylhydrazono )methy1- 4 -mercaptopyrrolidine

EXAMPLE 25-1

After 8.16 g (19.97 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl ) - 2 - f ormyl - 4 - tert -butyldimethylsilyloxypyrrolidine was added to 200 ml of methylenechloride, 1.5 ml (28.20 mmol) of methylhydrazine was added and stirred for 2 hours.

After the reaction, the mixture solution was washed with 200 ml of water and the organic layer was dried over anhydrous sodium sulfate, filtred, and then the filtrate was concentrated under reduced pressure 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-butyldimethylsilyloxypyrrolidine.

NMR(CDCl₃) δ:

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

2.75(s,3H), 3.5(m,2H), 4.39(br,s,1H), 4.59(m,1H), 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-(N-methylhydrazono)methyl-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 25-1. And then 27 ml (27.0 mmol) of 1 M tetrabutylammonium fluoride( tetrahydrofuran solution) was added dropwise and stirred for 30 minutes. After the ice bath was removed, the reaction mixture was stirred at room temperature for 1 hour, and concentrated under reduced pressure, and 200 ml of methylenechloride was added to the residue and washed with water and brine. After the separation of the organic layer, it was dried over anhydrous sodium sulfate, filtred, and then the filtrate was concentrated to give 7.5 g of crude material. This crude material was 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(CDCl₃) δ:

2.10(rα,1H), 2.56(m,1H), 2.73(s,3H), 3.58(m,2H), 4.44(brs,1H), 4.62(m,1H), 5.19(m,2H), 7.49(d,2H), 8.18(d,2H)

EXAMPLE 25-3

To 100 ml of methylenechloride was 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 methanesulfonylchloride was added and stirred at room temperature for 6 hours. After the reaction, the mixture solution was washed with water and dried over anhydrous sodium sulfate, filtered and the filtrate concentrated under reduced, pressure 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,4R)-1-(p-nitrobenzyloxycarbonyl) -2-(N-methanesulfonyl-N-methylhydrazono)methyl-4-methanesulfonyloxypyrrolidine. NMR(CDCl₃) δ:

2.40(m,2H), 2.83(s,3H), 2.94(s,3H), 3.04(m,3H), 3.63(m,1H), 3.89(m,1H), 4.70(m,2H), 5.19(s,2H), 5.21(m,1H), 7.40(d,2H), 8.16(d,2H)

EXAMPLE 25-4

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

After 1.67 g (4.62 mmol) of potassium thioacetate added and was refluxed for 4 hours, the reaction mixture was concentrated under reduced pressure, and methylenechloride was added dissolved, and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to give 7.7 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1:1.5) and purified to 3.0 g (62.6%) of (2S,4S)-1- (p-nitrobenzyloxycarbonyl) -2-(N-methanesulfonyl -N-methylhydrazono (methyl- 4 -acetylthiopyrrolidine.

NMR(CDCl₃) δ:

2.10(m,1H), 2.64(m,1H), 2.98(s,3H), 3.16(s,3H), 3.42(m,1H), 4.04(m,1H), 4.62(m,2H), 5.23(s,2H),

7.10(m,1H), 7.52(d,2H), 8.11(d,2H)

EXAMPLE 25-5

In 80 ml of methylalcohol, cooled to 0°C using an ice bath, was added 1.5 g (3.27 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono)methyl-4-acetylthiopyrrolidine obtained in EXAMPLE 25-4, and stirred to dissolve, 3.3 ml of 2 N sodium hydroxide was added dropwise and stirred for 2 minutes. Then, at the same temperature, the pH of the reaction solution was adjusted to pH 4.0 using saturated citric acid and concentrated under reduced pressure to evaporate the organic solvent. In the residue methylenechloride was dissolved and the extrate was washed with water. The organic layer was dried over anhydrous sodium sulfate. After the filtration, the filtrate was concentrated under reduced pressure, and the produced crystal was dried in a vacuum to give 1.0 g (73.4%) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl -N-methylhydrazono (methyl- 4 -mercaptopyrrolidine.

NMR(CDCl₃) δ:

2.05(m,1H), 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)-1-(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 hours. After the reaction, the organic layer obtained by washing with water was dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 6.8 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1:1) 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 -butyldimethylsilyloxypyrrolidine.

NMR(CDCl₃) δ:

0.03(d,6H), 0.83(s,9H), 2.19(m,2H), 3.52(m,2H), 4.42(m,1H), 4.63(m,1H), 5.19(m,2H), 6.72(m,1H), 7.01-7.30(m,2H) , 7.31-7.60(m,3H), 7.90-8.20(m,2H) ,

9.20(brs,1H)

EXAMPLE 26-2

To 100 ml of tetrahydrofuran, cooled to 0°C using an ice bath, was 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 -butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 26- 1 and stirred at room temperature to dissolve. Then, 13.01 ml (13.0 mmol) of 1 M tetrabutylammonium fluoride( tetrahydrofuran solution) was added dropwise and stirred for 30 minutes. After the removal of the ice bath, the mixture solution was stirred at room temperature for 1 hour. The mixture solution was concentrated under reduced pressure, 100 ml of methylenechloride was added, stirred to dissolve and treated, as discribed EXAMPLE 25-2, to give 7.1 g of crude material. The crude material was subjected to the column chromatography on silica 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(CDCl₃) δ:

2.20(m,2H), 3.64(m,2H), 4.39(m,1H), 4.75(m,1H), 5.20(m,2H), 6.72(m,1H), 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 dissolve. Then 2.9 ml (20.9 mmol) of triethyamine and 0.9 ml (11.63 mmol) of methanesulfonylchloride was added and stirred at room temperature for 1 hour. After the reaction, the mixture solution was 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 -methanesulfonyloxypyrrolidine.

NMR(CDCl₃) δ:

2.35-2.80(m,2H) , 3.75(m,1H) , 4.02(m,1H) , 4.80(m,1H), 5.22(m,3H), 6.80(m,1H), 7.05(m,2H), 7.40- 7.60(m,3H), 7.90-8.42(m,4H)

EXAMPLE 26-4

To the solution of 100 ml of anhydrous acetonitrile and 5 ml of dimethylformamide was added 3.9 g (8.4 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-N-(2-py r i d i ny 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 potassium thioacetate was added and refluxed for 7 hours, the reaction solution was concentrated under reduced pressure and methylenechloride was added to dissolve, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 5.3 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 2.87 g (76.9%) of (2S.4S)-1-(p-ni t r o b en z y l o xy c a r b on y l ) - 2 - N - ( 2 -pyridinylhydrazono)methyl-4-acethylthiopyrrolidine. NMR(CDCl₃) δ:

2.05(m,1H), 2.32(s,3H), 3.41(m,1H), 4.05(m,2H), 4.62(m,1H), 5.20(m,2H), 6.74(m,1H), 7.12(m,2H), 7.38- 7.62(m,3H), 7.89-8.23(m,3H), 8.98(m,1H)

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-ni t r o b en z y l o xy c a r b ony 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 stirred 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 pressure to evaporate the organic solvent. The residue was dissolved in methylenechloride, washed with water, and the organic solvent separated and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give 4.3 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with chloroform: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(CDCl₃) 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(m,3H), 7.80-8.40(m,4H)

EXAMPLE 27

(2S,4S)-1-(p-nitrobenzyloxycarbonyl) -2-N-(p-methoxybenzyloxycarbonylphenylhydrazono(methyl-4-mercaptopyrrolidine

EXAMPLE 27-1

After 5.16 g (12.63 mmol) of (2S,4R)-1(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 stirred at room temperature for 15 hours. After the reaction, the mixture solution was washed with 100 ml of water, the pH of the organic layer was adjusted to pH 5.0 using saturated citric acid. After the mixture solution was washed with water and saturated sodium chloride, the organic layer was dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure. Until the crystal was produced, the mixture solution was dried, and dried in vacuum to give 5.0 g (73.0%) of (2S,4R)-1-(p-ni t r oben z y l o xy c a r b ony l ) - 2 - N - ( 4 -carboxyphenylhydrazono(methyl -4 -tert-butyldimethylsilyloxy pyrrolidine.

NMR(CDCl₃) δ:

0.03(d,6H), 0.82(s,9H), 2.01(m,2H), 3.21(m,2H), 4.39(m,1H), 4.45(m,1H), 5.05(m,2H), 6.75(d,2H), 7.30(m,2H), 7.70(m,3H), 7.98(d,1H), 9.10(d,1H)

EXAMPLE 27-2

To 30 ml of dimethylf ormamide was 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 -butyldimethylsilyloxy pyrrolidine obtained in EXAMPLE 27-1 and stirred to dissolve. Then 2.54 g (18.4 mmol) of 1 M potassium carbonate and 1.729 g ( 11.04mmol ) of p-methoxybenzylchloride was added and raised the temperature to 50°C. At this temperature the mixture solution was stirred for 4 hours, 150 ml of methylene chloride was added and shaken vigorously, and the mixture solution was washed with water (100 ml × 3). After separation of the organic layer, it was dried over anhydrous sodium sulfate 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(CDCl₃) δ:

0.03(d,6H), 0.82(s,9H), 2.01-2.40(m,2H), 3.50(m,2H), 3.80(s,3H), 4.43(m,1H), 4.64(m,1H), 5.20(m,4H), 6.90(dd,4H), 7.40(dd,4H), 7.90(m,2H), 8.20(m,1H)

EXAMPLE 27-3

The ester compound obtained in EXAMPLE 27-2 was treated, as discribed in EXAMPLE 25, to give the desired product of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-N-(4-methoxybenzyloxyearbonylphenylhydrazono(methyl-4-mercaptopyrrolidine.

NMR(CDCl₃) δ:

1.95-2.10(m,2H), 3.20-3.80(m,2H), 3.80(s,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-nitrobenzyloxycarbonyl)-N-methylhydrazono]methyl-4-mercaptopyrrolidine

EXAMPLE 28-1

After 8.7 g (19.93 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-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 hours. The reaction mixture was washed with water and the organic layer was dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure. The residue was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1:1) 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(CDCl₃) 6:

0.03(d,6H), 0.82(s,9H), 2.07(m,2H), 3.11(d,3H), 3.52(m,2H), 4.40(m,1H), 4.75(m,1H), 5.10-5.40(m,4H), 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-nitrobenzyloxycarbonyl)-2-[N-(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 - butyldimethylsilyloxypyrrolidine 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 stirred at room temperature for 1 hour. After the reaction, the mixture solution was concentrated under reduced pressure, and methylenechloride was added to the residue. The reaction mixture solution was 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-nitrobenzyloxycarbonyl ) -2 - [N- (p-nitorbenzyloxycarbonyl)-N-methylhydrazono]methyl-4-hydroxypyrrolidine.

NMR(CDCl₃) δ:

2.20(m,2H), 3.29(d,3H), 3.60(m,2H), 4.50(m,1H), 4.80(m,1H), 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- [N-(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 was added and stirred at room temperature for 1 hour. After the reaction, the mixture solution was treated, as described in EXAMPLE 25-3, to give 2.7 g of crude material. The crude material was dissolved in 100 ml of anhydrous acetonitrile and 0.732g(6.43 mmol) of potassium thioacetate was added and refluxed for 5 hours. After the reaction, the mixture solution was concentrated under reduced pressure, 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=1:2) and purified to 1.8 g (70.3%) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-[N-(p-nitorbenzyloxycarbonyl)-N-methylhydrazono]methyl-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ:

2.10(m,1H), 2.30(s,3H), 2.62(m,1H), 3.20(s,3H), 3.40(m,1H), 4.03(m,2H), 4.63(m,1H), 5.20(m,2H), 5.30(s,2H), 7.01(m,1H), 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, was 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 was adjusted to pH 4.0 and concentrated under reduced pressure. The residue was dissolved in 100 ml of methylenechloride, washed with water and the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and the produced crystal was dried in a vacuum to give 1.51 g (90.7%) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-[N-(p-nitorbenzyloxycarbonyl)-N-methylhydrazono]methyl-4-mercaptopyrrolidine.

NMR(CDCl₃) 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)-1-(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 was 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 was added and stirred for 3 hours. After the reaction, the mixture solution was washed with water and the organic layer was dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 3.2 g of the residue. 150 ml of tetrahydrofuran, cooled to 0°C using an ice bath, was added to the residue, and 12.14 ml (12.14 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwise and stirred for 40 minutes. After removal of the ice bath, the mixture solution was stirred at room temperature for 1.5 hours, washed with water and saturated sodium chloride, and the organic layer was drier over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give 6.8 g of crude material.

The crude material was 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-nitrobenzyloxycarbonyl)-2-(N,N-dimethylhydrazono)methyl-4-hydroxypyrrolidine.

NMR(CDCl₃) δ:

1.92-2.60(m,2H), 2.70(s,6H), 3.58(m,2H), 4.42(br,s,1H) , 4.62(m,1H) , 5.19(m,2H) , 7.42(d,2H,J=8.6HZ), 7,17(d,2H,J=8.5Hz)

EXAMPLE 29-2

(2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N,N-dimethylhydrazono)methyl-4-hydroxypyrrolidine obtained in EXAMPLE 29-1 was treated, as described in EXAMPLE 26, with a result of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2- (N,N-dimethylhydrazono)methyl-4-hydroxypyrrolidine. NMR(CDCl₃) δ:

2.01(m,1H), 2.61(m,1H), 2.75(s,6H), 3.40(m,2H), 4.50(m,1H), 5.20(m,3H), 7.40(d,2H, J=8.7Hz), 8.19(m,2H) EXAMPLE 30

(2S,4S)-1-(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 -butyldimethylsilyloxypyrrolidine was added to 150 ml of methylenechloride and stirred at room temperature, 4.3ml(30.8 mmol) of triethylamine and 2.09g(16.8 mmol) of diethylhydrazine hydrochloride salt were added and stirred for 4 hours. After the reaction, the mixture solution was concentrated under reduced pressure, and then extracted with ethylacetate. After the extracted solution was washed several times with water and saturated aqueous sodium chloride, the organic layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 7.2 g of the residue. The residue was subjected to the column chromatography on silica gel (eluted with n-hexane :ethylacetate=2:1) 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 -butyldimethylsilyloxypyrrolidine.

NMR(CDCl₃) δ:

0.03(s,6H), 0.80(s,9H), 1.01(t,6H), 1.85-2.15(2,

2H), 3.05(q,4H), 3.23(m,2H), 4.40(m,1H), 4.55(m,1H), 5.18(m,2H), 7.44(d,2H,J=8.9Hz), 8.15(d.2H,J=8.1Hz)

EXAMPLE 30-2

(2S,4R)-1-(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 -butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 30-1 was treated with the same separation of EXAMPLE 26 to give the desired product (2S,4S) -1- (p-nitrobenzyloxycarbonyl)-2-(N,N-diethylhydrazono)methyl- 4-mercaptopyrrolidine.

NMR(CDCl₃) δ:

1.00(m,6H), 2.01(m,1H), 2.50(m,2H), 3.08(q,4H), 3.20-3.60(m,2H), 4.50(m,1H), 5.20(m,1H), 7.44(m,2H), 8.17(m,2H)

EXAMPLE 31

(2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-[N-methyl-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 was 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 was added dropwise and refluxed for 6 hours. After the reaction, the mixture solution was concentrated under reduced pressure, and then extracted with ethylacetate. After the extracted solution was washed. with water and saturated sodium chloride, the organic layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 8.4 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=5:1) and purified to 7.35 g (92.2%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-[N-methyl-N- (N',N'-dimethylsulfamoyl) hydrazono]methyl-4-tert-butyldimethylsilyloxypyrrolidine. NMR(CDCl₃) δ:

0.02(d,6H), 0.82(5,9H), 2.02(m,1H), 2.85(s.3H), 2.90(5,6H), 3.01(m,1H), 3.41(m,2H), 4.40(m,1H), 4.60(m,1H), 5.20(m,2H), 7.44(d,2H,J=8.7Hz), 8.20(d,2H,J=8.6Hz)

EXAMPLE 31-2

To 120 ml of tetrahydrofuran was added 7.3 g (13.43 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-[N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazono]methyl-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 31-1, stirred to dissolve, and cooled to 0°C using an ice bath. Then 16 ml (16.0 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwise and stirred for 20 minutes. After removal of the ice bath, the mixture solution was stirred for 30 minutes and concentrated under reduced pressure, and the residue was dissolved in ethylacetate and washed successively with water and saturated aqueous sodium chloride. After the separation of the organic layer, the organic, layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was 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 stirred 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 anhydrous sodium sulfate, filtered, and then the filtrate was concentrated to give 5.7 g of crude material. The crude material was subjected to the column chromatography on silica gel(eluted with chloroform:methanol=300:1) and purified to 4.2 g (61.6%) of (2S,4R) -1-(p-nitrobenzyloxycarbonyl) -2-[N-methyl-N-(N' ,N'-dimethylsulfamoyl)hydrazono]methyl-4-methanesulfonyloxy hydroxyhydroxyoxypyrrolidine.

NMR(CDCl₃) δ:

2.30(m,2H), 2.84(s,6H), 3.02(s,3H), 3.13(s,3H), 3.60-4.01(m,2H), 4.75(m,1H) , 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 anhydrous acetonitrile was added 2.0 g (3.94 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-[N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazono]methyl-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 31-2 and stirred to dissolve. Then, 1.37 g (12.0 mmol) of potassium thioacetate was added and refluxed for 3 hours. After the reaction, the mixture solution was concentrated under reduced pressure, and the residue was dissolved in ethylacetate and washed successively with water and saturated aqueous sodium chloride. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 2.5 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=50:1) and purified to 1.39 g (72.4%) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-[N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazono]methyl-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ:

2.10(m,1H), 2.30(S,3H), 2.60(m,1H), 2.90(s,6H), 3.12(s,3H), 3.40(m,H), 4.00(m,2H), 4.60(m,1H), 5.20(br s,2H), 7.48(d,2H, J=8.3Hz), 8.20(d,2H,J=8.7Hz)

EXAMPLE 31-4

To the mixed solvent of 50 ml of methylalcohol and 5 ml of tetrahydrofuran was added 1.39 g (2.85 mmol) of (2S,4S)-1-(p-nitrobenzyloxyearbonyl)-2-fN-methyl-N-(N',N'-dimethylsulfamoyl) hydrazono]methyl-4-acetylthiopyrrolidine obtained in EXAMPLE 31-3 was stirred to dissolve. Then 3.0 ml of 2 N sodium hydroxide was added dropwise and stirred for 3 minutes. After the reaction, the pH of the solution was adjusted to pH 4.2 using saturated citric acid, and the reaction mixture was treated, as described in EXAMPLE 19-5, to give 1.1 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1:1.5) and purified to 0.69 g (54.3%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-[N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazono)methyl-4-mercaptopyrrolidine.

NMR(CDCl₃) δ:

2.05(m,2H), 2.50-2.79(m,2H), 2.83(s,3H), 2.93(s,3H), 3.10(s,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 -butyldimethylsilyloxypyrrolidine was added to 100 ml of methylenechloride at room temperature. And 1.92 ml (36.09 mmol) of methylhydrazine and 2 g of anhydrous magnesium sulfate was added and refluxed for 5 hours.

After the reaction, the mixture solution was cooled, filtered, and then the filtrate was concentrated under reduced pressure to give 8.47 g of the residue. The residue was 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,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methylhydrazono(methyl-4-tert-butyldimethylsilyloxypyrrolidine.

NMR(CDCl₃) δ:

0.02(s,6H) , 0.82(s,9H) , 1.52( s,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,1H), 4.60(m,1H), 5.03-5.30(m,2H), 7.43(d,2H,J=8.9Hz), 8.19(d,2H,J=8.4Hz)

EXAMPLE 32-2

To 80 ml of methylenechloride was added 6.8 g

(15.09 mmol) of (2S,4R)-1-(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 - butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 32- 1, stirred to dissolve, 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 minutes. After the reaction, the reaction mixture solution was washed with satusatedcitric acid and saturated aqueous sodium chloride. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and 100 ml of tetrahydrofuran was added to the residue and stirred to dissolve. Then, 18 ml (18 mmol) of 1 M-tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwise and stirred at room temperature for 30 minutes. After the reaction, the mixture solution was concentrated under reduced pressure, the residue was dissolved in ethylacetate and washed successively with water and saturated aqueous sodium chloride. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 6.4 g of the residue. The residue was 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-methanesulfonyl-N-methylhydrazono)methyl-4-hydroxypyrrolidine.

NMR(CDCl₃) δ:

1.95(5,1.8H), 1.98(m,1,2H), 2.10-2, 64(m,2H), 2.74- 2.76(s,s,3H), 2.79-2.83(s,s,3H), 3.50-3.80(m,2H), 4.50(s,1H), 4.63(m,1H), 5.10(m,2H), 7.44(d,2H,J=8.7Hz), 8.18(dd,2H,J=8.6Hz,J=8.6Hz)

EXAMPLE 32-3 To 70 ml of methylenechloride was added 4.5 g (10.86 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono(methyl-4-hydroxypyrrolidine obtained in EXAMPLE 32-2 and stirred to dissolve. The reaction mixture solution was cooled to 0°C using an ice bath, and 2.23 ml (16.0 mmol) of triethyamine and 0.95 ml (12.3 mmol) of methanesulfonylchloride was added and stirred at the same temperature for 30 minutes. After the reaction, the organic layer was washed with water, and washed successively with citric acid solution and sodium chloride solution. 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-methanesulfonyl-N-methylhydrazono(methyl-4-methanesulfonyloxypyrrolidine.

This material itself was used for next reaction. NMR(CDCl₃) δ:

1.99(s.1.6H), 2.05(s.l.4H), 2.20(m,1H), 2.60(m,1H), 2.78-2.79(s,s,3H), 2.80-2.87(s,s,3H), 3.73(m,1H), 4.01(m,1H), 4.68(t,1H), 5.05-9.32(m,3H), 7.42(d,2H,J=8.2Hz), 8.20(dd,2H,J=7.2,8.5Hz)

EXAMPLE 32-4

To 80 ml of acetonitrile was added 4.9 g (9.95 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono(methyl-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 32-3. After 2.8 g (24.52 mmol) of potassium thioacetate was added and refluxed for 5 hours, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethylacetate. The organic layer was washed with water and saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 5.1 g of the crude material. The residue was 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(methyl-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ:

1.99-2.02(s,s,3H), 2.30(s,3H), 2.70(m,1H), 2.80-3.01(m,6H), 2.90(m,1H), 3.30(m,1H), 3.95(m,1H), 4.16(m,1H), 4.52(t,3H), 5.12(m,2H), 7.43(d,2H,J=8.3Hz) 8.18(d,2H,J=5.4Hz),

EXAMPLE 32-5

To 60ml of methyl alcohol was added 3.21 g (6.79 mmol) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono(methyl-4-acetylpyrrolidine obtained in EXAMPLE 32-4 and stirred to dissolve. After the reaction solution was cooled using the ice bath, 5 ml of 2 N sodium hydroxide aqueous solution was added dropwise and stirred for 4 minutes. 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 residue ethylacetate was added, washed successively with water and saturated aqueous sodium chloride, and dried over anhydrous magnesium sulfate. After the filtration, the filtrate was concentrated to give 3.7 g of crude material. This crude material was subjected to the column chromatography on silica gel (eluted with n-hexane:ethylacetate=1:3) and purified to

2.31 g (79%) of ( 2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-methanesulfonyl-N-methylhydrazono)methyl- 4-mercaptopyrrolidine.

NMR(CDCl₃) δ:

1.75(d,1H), 2.01(s,1.5H), 2.03(s,1.5H), 2.70(m,2H),

2.80-2.90(m,6H), 3.30(m,1H), 3.70(m,1H), 4.10(m,1H), 4.50(m,1H), 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 -butyldimethylsilyloxypyrrolidine was added to 120 ml of methylenechloride and stirred to dissolve. 2.4 ml (45.11 mmol) of methylhydrazine and 3 g of anhydrous magnesium sulfate was added, stirred at room temperature for 1.5 hour, and cooled to 0°C using an ice bath. 8.4 ml (60.22 mmol) of triethylamine and 4 ml (35.3 mmol) of ethanesulfonylchloride was added, and stirred for 2 hours, washed successively with water and saturated aqueous sodium chloride and the organic layer was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give 15.8 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chloroform:methylalcohol=30:1) and purified to 14.7 g (92.7%) of (2S,4R)-1- (p-nitrobenzyloxycarbonyl)-2- (N-ethanesulfonyl-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 -butyldimethylsilyloxypyrrolidine.

NMR(CDCl₃) δ:

0.03(d,6H), 1.83(s,9H), 1.13(t,3H), 2.10(m,2H), 3.10(m,5H), 3.50(m,2H), 4.40(m,1H), 4.70(m,1H), 5.10(m,2H), 7.48(d,2H,J=8.5Hz), 8.20(d,2H,J=8.6Hz) EXAMPLE 33-2

To 160 ml of tetrahydrofuran was added 14.7 g (27.80 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-ethanesulfonyl-N-methylhydrazono(methyl-4-tert-butyldimethylsilyloxypyrrolidine obtained in EXAMPLE 33- 1 and stirred to dissolve. Then, 37 ml (3.7 mmol) of 1 M tetrabutylammonium fluoride (tetrahydrofuran solution) was added dropwise and stirred at room temperature for 3 hours. After the reaction, the mixture solution was concentrated under reduced pressure, and the residue was dissolved in methylenechloride and washed successively with water and saturated aqueous sodium chloride. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure 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 was added dropwise, and stirred at 0°C for 1 hour. The reaction mixture solution was washed successively with citric acid solution and saturated aqueous sodium chloride. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was 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-ethanesulfonyl-N-methylhydrazono)methyl-4-methanesulfonyloxypyrrolidine. NMR(CDCl₃) 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,1H), 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 was added 12.6 g (11.07 mmol) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-ethanesulfonyl-N-methylhydrazono)methyl-4-methanesulfonyloxypyrrolidine obtained in EXAMPLE 33-2 and stirred to dissolve. Then, 4.73 g (41.4 mmol) of potassium thioacetate was added and refluxed for 5 hours. After the reaction mixture solution was cooled to room temperature, the mixture solution was concentrated under reduced pressure to evaporate the solvent, and the residue was dissolved in 300 ml of methylenechloride and washed successively with water and saturated aqueous sodium chloride. After the separation of the organic layer, it was dried over anhydrous magnesium sulfate, filtered, and then the filtrate was concentrated to give 9.8 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 8.2 g (62.9%) of (2S,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-ethanesulfonyl-N-methylhydrazono)methyl-4-acetylthiopyrrolidine.

NMR(CDCl₃) δ:

1.30(m,3H), 2.10(m,1H), 2.30(s,3H), 2.60(m,14), 3.10(m,5H), 4.05(m,2H), 4.65(m,1H), 5.10(s,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,4R)-1-(p-nitrobenzyloxycarbonyl)-2-(N-ethanesulfonyl-N-methylhydrazono)methyl-4-acetylthiopyrrolidine obtained in EXAMPLE 33-3, stirred to dissolve, and cooled to 0°C using an ice bath. After 11.7 ml of 2 N sodium hydroxide solution was added and stirred for 3 minutes, the pH of the solution was adjusted to pH 3.7 using citric acid solution. The reaction solution was concentrated under reduced pressure and 200 ml of ethylacetate was added, washed successively with water and saturated aqueous sodium chloride, and the separated organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 5.7 g of crude material. The crude material was subjected to the column chromatography on silica gel (eluted with chlorof orm:methylacohol =20:1) and purified to 4.1 g (60.8%) of (2S,4S)-1-(p-nitrobenzyloxycarbonyl)-2-(N-ethanesulfonyl-N-methylhydrazono) methyl-4 -mercaptopyrrlidine.

NMR(CDCl₃) δ:

1.14(m,3H), 2.10(m,2H), 3.01(s,3H), 3.10(m,5H), 3.50(m,3H), 3.70(m,1H), 4.65(m,1H), 5.10(m,2H),

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

Claims

WHAT IS CLAIMED IS:

1. A compound of the formula (I)

or a pharmaceutically acceptable salt or ester thereof;

wherein R₁ is selected from the group consisting of a hydrogen atom or a methyl group, R₂ is selected from the group consisting of a hydrogen atom, a metal or a nonmetal salt group, or a carboxy protecting group, R₃ is selected from the group consisting of a hydrogen atom or an imino protecting group, and R₄ is selected from the group consisting of a hydrogen atom, a lower alkyl group, a hydroxy group, a cyano group, or a halogen atom, R₅ is selected from the group consisting of a hydroxy group, a lower alkoxy group, a protected amino group, unprotected amino group, or one of the following formula (1)-(4);

wherein R₆ and R₇ are each selected from the group consisting of a hydrogen atom or a lower alkyl group, R₆ is selected from the group consisting of a hydroxy group, a cyano group, a halogen atom, a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, a protected or unprotected amino group, or the following general formula,

wherein R₉ is selected from the group consisting of a lower alkyl sulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄)alkylsulfonyl or an N,N-lower dialkylsulfamoyl group, n is an integer of 0 to 4

wherein R₆ is selected from the group consisting of a hydrogen atom or a lower alkyl group, n is an integer of 0 to 4

wherein R₆ and R₇ are each selected from the group consisting of a hydrogen atom or a lower alkyl group, n is an integer of 0 to 4

(4

wherein R₆ is selected from the group consisting of a hydrogen atom or a lower alkyl group, R₁₀ is selected from the group consisting of a lower alkyl group, a lower alkyl sulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄)alkylsulfonyl group, an N- (lower)alkylsulfamaoyl group, an N,N- (lower)dialkylsulfamoyl group, a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms which may be optionally substituted with appropriate substituent, or the following general formula,

wherein R₁₁ is selected from the group consisting of

, a

, a halogen atom, a hydroxy group, or a cyano group, R₆ and R₇ are each selected from the group consisting of a hydrogen or a lower alkyl group.

2. The compound according to claim 1, wherein R1 is a methyl group, R₂ is a hydrogen atom.

3. The compound according to claim 1, wherein each of R₁ and R₂ is a hydrogen atom.

4. The compound according to claim 1, wherein R₁ is a methyl group, each of R₂ and R₃ is a hydrogen atom.

5. The compound according to claim 1, wherein each of R₁, R₂ and R₃ is a hydrogen atom.

6. The compound according to claim 4, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of a hydroxy group or a lower alkoxy group.

7. The compound according to claim 4, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of a carbamoyl group or a lower alkoxy carbamoyl group.

8. The compound according to claim 4, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group, or a lower alkylsulfamoyl amino group.

9. The compound according to claim 4, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of an imidazolylamino group, a pyrimidinylamino group or a pyridinylamino group.

10. The compound according to claim 4, wherein R₄ is selected from the group consisting of a methyl group, R₅ is a hydroxy group or a lower alkoxy group.

11. The compound according to claim 4, wherein R₄ is selected from the group consisting of a methyl group, R₅ is a carbamoyl group or a lower alkylcarbamoyl group.

12. The compound according to claim 4, wherein R₄ is selected from the group consisting of a methyl group, R₅ is an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group, or a lower sulfamoyl amino group.

13. The compound according to claim 4, wherein R₄ is a methyl group, R₅ is selected from the group consisting of an imidazolylamino group, a pyrimidinylamino group, or a pyridinylamino group.

14. The compound according to claim 4, wherein R₄ is a hydroxy group, R₅ is selected from the group consisting of a carbamoyl group or a lower alkylcarbamoyl group.

15. The compound according to claim 4, wherein R₄ is a hydroxy group, R₅ is selected from the group consisting of an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group, or a lower alkylsulfamoyl group.

16. The compound according to claim 4, wherein R₄ is a hydroxy group, R₅ is selected from the group consisting of an imidazolylamino group, a pyrimidinylamino group, or a pyridinylamino group.

17. The compound according to claim 4, wherein R₄ is a halogen atom, R₅ is selected from the group consisting of a carbamoyl group or a lower alkylcarbamoyl.

18. The compound according to claim 4, wherein R₄ is a halogen atom, R₅ is selected from the group consisting of an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group, a lower alkylsulfamoylamino group.

19. The compound according to claim 4, wherein R₄ is a halogen atom, R₅ is selected from the group consisting of an imidazolylamino group, a pyrimidinylamino group, or a pyridinylamino group.

20. The compound according to claim 5, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of a hydroxy group or a lower alkoxy group.

21. The compound according to claim 5, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of a carbamoyl group or a lower carbamoyl group.

22. The compound according to claim 5, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group or a lower alkylsulfamoylamino group.

23. The compound according to claim 5, wherein R₄ is a hydrogen atom, R₅ is selected from the group consisting of an imidazolylamino group, or a pyrimidinylamino group.

24. The compound according to claim 5, wherein R₄ is a hydroxy group, R₅ is selected from the group consisting of a carbamoyl group, or a lower alkylcarbamoyl group.

25. The compound according to claim 5, wherein R₄ is a hydroxy group, R₅ is selected from the group consisting of an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group, a lower alkylsulfamoyl group.

26. The compound according to claim 5, wherein R₄ is a hydroxy group, R₅ is selected from the group consisting of an imidazolylamino group, a pyrimidinylamino group, or a pyridinylamino group.

27. The compound according to claim 5, wherein R₄ is a halogen atom, R₅ is selected from the group consisting of a carbamoyl group, or a lower alkylcarbamoyl group.

28. The compound according to claim 5, wherein R₄ is a halogen atom, R₅ is selected from the group consisting of an amino group, a lower alkyl amino group, a lower alkanesulfonylamino group, or a lower alkylsulfamoylamino group.

29. The compound according to claim 5, wherein R₄ is a halogen atom, R₅ is selected from the group consisting of an imidazolyl amino group, a pyrimidinylamino group, or a pyridinylamino group.

30. The compound according to claim 1, wherein the three dimensional structure is (5R,6S,8R,2'S,4'S) or (4R,5S,6S,8R,2'S,4'S).

31. The compound according to claim 1, wherein the compound i s ( 4R, 5S, 6S, 8R, 2 'S , 4 ' S ) - 3 - ( 2 - methoxyiminopyrrolidin-4-yl -thio) -4-methyl-6- (1 - hydroxyethyl)-1-azabicyclo[ 3,2,0] -hept-2-ene-7-one-2- carboxylic acid.

32. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-(N,N-dimethylaminocarbonyl methoxyimino)pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0)-hept-2-ene-7-one-2-carboxylic acid.

33. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-(2-aminocarbonylmethoxyiminopyrrolidin-4-yl-thio)-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

34. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-(N-methylaminocarbonyl methoxyimino(pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

35. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-(N-methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]- hept-2-ene-7-one-2-carboxylic acid.

36. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-(N- methanesufonylhydrazonomethyl) pyrrolidin-4-yl-thio]-4- methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2- ene-7-one-2-carboxylic acid.

37. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)- 3-[2-(2- imidazolylhydrazonomethyl) pyrrolidin-4-yl-thio]-4- methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

38. The compound according to claim 1, wherein the compound is (4R, 5S,6S,8R,2'S,4'S)-3-[2-(2-pyrimidinylhydrazonomethyl) pyrrolidin-4-yl-thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

39. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-(N-methyl-N- (N',N'-dimethylsulfamoyl(hydrazonomethyl]pyrrolidin-4-yl- thio] -4 -methyl-6- (1 -hydroxyethyl) -1 -azabicycl o [3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

40. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-[N-(N',N'-dimethylsulfamoyl) hydrazonomethyl]pyrrolidin-4-yl- thio]-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]- hept-2-ene-7-one-2-carboxylic acid.

41. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-f2-(N-methyl-N- methylsulfamoyl) hydrazonomethyl]pyrrolidin-4-yl-thio]- 4-methyl-6-(1-hydroxyethyl)-1-azabicycl o [3,2,0]-hept-2- ene-7-one-2-carboxylic acid.

42. The compound according to claim 1, wherein the compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-(1-N- methanesulfonyl-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio]-4-methyl-6-(1-hydroxyethyl)-1-azabi cycl o [3,2,0]- hept-2-ene-7-one-2-carboxylic acid.

43. The compound accordinc to claim 1, wherein tne compound is (4R,5S,6S,8R,2'S,4'S)-3-[2-N-(N',N'- dimethylsulfamoyl)-N-methylhydrazonomethyl)pyrrolidin-4-yl- thio] -4 -methyl- 6- ( 1 -hydroxyethyl ) -1 -azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

44. The compound according to claim 1, wherein the compound is (5R,6S,8R,2'S,4'S)-3-[2-N-methanesulfonyl-N-methyl hydrazonomethyl)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

45. The compound according to claim 1, wherein the compound is ( 5R,6S,8R,2'S,4'S)-3-[2-(N-methanesulfonylhydrazonomethyl)pyrrolidin-4-yl-thio]-6- (1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

46. The compound according to claim 1, wherein the compound is (5R,6S,8R,2'S,4'S)-3-[2-(N-methyl-N-(N',N'-dimethylsulfamoyl)hydrazonomethyl)pyrrolidin-4-yl-thio]- 6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one- 2-carboxylic acid.

47. The compound according to claim 1, wherein the compound is (5R,6S,8R,2'S,4'S)-3-[2-[N-(N',N'- dimethylsulfamoyl) hydrazonomethyl]pyrrolidin-4-yl- thio]-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene- 7-one-2-carboxylic acid.

48. The compound according to claim 1, wherein the compound is ( 5R ,6S, 8R ,2 'S , 4 'S ) - 3- (2 - methoxyiminopyrrolidin-4-yl-thio)-6-(1-hydroxyethyl)-1- azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

49. The compound according to claim 1, wherein the compound is (5R,6S,8R,2'S,4'S)-3-(2-aminocarbonyl methoxyiminopyrrolidin-4-yl-thio)-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

50. The compound according to claim 1, wherein the compound is ( 5R, 6S, 8R, 2 'S , 4' S ) - 3 -[ 2 - ( N-methylaminocarbonyl methoxyimino)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclo[3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

51. The compound according to claim 1, wherein the compound is ( 5R, 6S, 8R, 2'S ,4' S) - 3- [ 2- (N,N-dimethylaminocarbonyl methoxyimino)pyrrolidin-4-yl-thio]-6-(1-hydroxyethyl)-1-azabicyclof3,2,0]-hept-2-ene-7-one-2-carboxylic acid.

52. A process for preparing a compound of the following formula (IV)

wherein R₁, R₂, R₃, R₄, R₅ and R₁₄ are the same as defined below; or a pharmaceutically acceptable salt and ester thereof, comprising the steps of;

reacting a compound of the formula (II)

wherein R₁ is selected from the group consisting of a hydrogen atom or a methyl group, R₂ is selected from the group consisting of a hydrogen atom, a metal or an non metal salt group, or a carboxy protecting group, R₁₄ is selected from the group consisting of a hydrogen atom or a hydroxy protecting group, or a reactive derivative thereof,

with a compound of the formula (III),

wherein R₃ is selected from the group consisting of a hydrogen atom, an imino protecting group, R₄ is selected from the group consisting of a hydrogen atom or a lower alkyl group, a hydroxy group, a cyano group, or a halogen group, R₅ is selected from the group consisting of a hydroxy group, a lower alkoxy group, a protected or unprotected amino group, or one of the following formula (1)-(4);

wherein R₆ and R₇ are each selected from the group consisting of a hydrogen atom, a lower alkyl group, R₈ is selected from the group consisting of a hydroxy group, a cyano group, a halogen atom, a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, a protected or unprotected amino group, or the following general formula,

wherein R₉ is selected from the group consisting of a lower alkylsulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄) alkylsulfonyl or an N,N-lower dialkylsulfamoyl group, n is an integer of 0 to 4

wherein R₆ is selected from the group consisting of a hydrogen atom or a lower alkyl group, R₁₀ is selected from the group consisting of a lower alkyl group, a lower alkyl sulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄) alkylsulfonyl group, an N- (lower) alkylsulfamoyl group, an N,N- (lower)dialkylsulfamoyl group or a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, or the following general formula

wherein R₁₁ is selected from the group consisting of

, a

, a halogen atom, a hydroxy group, or a cyano group, R₆ and R₇ are each selected from the group consisting of a hydrogen or a lower alkyl group; and

removing or introducing any protecting group, if necessary.

53. An antibacterial agent comprising an antibacterial effective amount of a compound of the formula (I), a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable vehicle or adjuvant wherein formula (I) contains:.

wherein R₁ is selected from the group consisting of a hydrogen atom or a methyl group, R2 is selected from the group consisting of a hydrogen atom, a metal or a nonmetal salt group, or a carboxy protecting group, R₃ is selected from the group consisting of a hydrogen atom or an imino protecting group, and R₄ is selected from the group consisting of a hydrogen atom, a lower alkyl group, a hydroxy group, a cyano group, or a halogen atom, R₅ is seiected from the group consisting of a hydroxy group, a lower alkoxy group, a protected or unprotected amino group, or one of the following formula ( 1 ) - ( 4 ) ;

wherein R₆ and R₇ are each selected from the group consisting of a hydrogen atom or a lower alkyl group, R₈ is a hydroxy group, a cyano group, a halogen atom, a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, a protected or unprotected amino group, or the following general formula,

wherein R₆ is selected from the group consisting of a hydrogen atom or a lower alkyl group, R₁₀ is selected from the group consisting of a lower alkyl group, a lower alkyl sulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄) alkylsulfonyl group, an N- (lower)alkylsulfamoyl group, an N,N- (lower)dialkylsulfamoyl group, a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, or the following general formula,

wherein R₁₁ is selected from the group consisting _of

, a , a halogen atom, a hydroxy group,

or a cyano group, R₆ and R₇ are each selected from the group consisting of a hydrogen or a lower alkyl group.

54. A process for preparing a compound of the formula (III)

wherein R₆ and R₇ are each selected from the group consisting of a hydrogen atom, a lower alkyl group, R₈ is selected from the group consisting of a hydroxy group, a cyano group, a halogen atom, a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, appropriate substituent, a protected or unprotected amino group, or the following general formula,

wherein R₉ is selected from the group consisting of a lower alkylsulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄) alkylsulfonyl or an N,N-lower alkylsulfamoyl group, n is an integer of 0 to 4

wherein R₆ and R- are each selected from the group consisting of a hydrogen atom or a lower alkyl group, n is an integer of 0 to 4

wherein R₆ is selected from the group consisting of a hydrogen atom or a lower alkyl group, R₁₀ is selected from the group consisting of a lower alkyl group, a lower alkyl sulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄) alkylsulfonyl group, an N- (lower)alkylsulfamoyl group, an N,N- (lower)dialkylsulfamoyl group or a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms, the following general formula

wherein R₁₁ is selected from the group consisting of

, a , a halogen atom, a hydroxy group,

or a cyano group, R₆ and R₇ are each se-lected from the group consisting of a hydrogen or a lower alkyl group, comprising the steps of;

reacting a compound of the formula (V),

wherein R₃, R₄, R₅ are the same as defined above, R₁₅ is R₁₆SO₂ wherein R₁₆ is a lower alkyl group of C₁-C₄ with a compound of the following formula (VI),

wherein R₁₆ is the same as defined above, M is an alkali metal, an alkali earth metal to produce a compound of the following formula (VII)

treating the resulting product with alkali solution.

55. A compound of the formula (VIII)

where R₃ is selected from the group consisting of a hydrogen atom, an imino protecting group, R₄ is selected from the group consisting of a hydrogen atom or a lower alkyl group, a hydroxy group, a cyano group, or a halogen group, R₁₇ is selected from the group consisting of a hydrogen atom or R₁₆

where R₁₆ is a lower alkyl group of C₁-C₄, R₅ is selected from the group consisting of a hydroxy group, a lower alkoxy group, a protected or unprotected amino group, or one of the following formula (1)-(4);

wherein R₆ is selected from the group consisting of a hydrogen atom or a lower alkyl group, R₁₀ is a lower alkyl group, a lower alkyl sulfonyl group, a halo lower alkylsulfonyl group, a phenyl (C₁-C₄) alkylsulfonyl group, an N-(lower)alkylsulfamoyl group, an N,N- (lower)dialkylsulfamoyl group or a heterocyclic group of 5 or 6 membered ring containing 1 to 4 heteroatoms or the following general formula

wherein R₁₁ is selected from the group consisting of

, a , a halogen atom, a hydroxy group,

56. A method of treating bacterial infection comprising administering the compound recited in claim

1 to an animal suffering from a bacterial infection.

57. A method as recited in claim 56 wherein the compound is administered by a method selected from the gruop consisting of oral, topical, intravenous or parenteral.

58. A methods as recited in claim 56 wherein the composition is administered in a dose of 5 to 50 mg/kg of body weight/day.

59. A methods as recited in claim 56 wherein the composition is administered in a dose to 5 to 25 mg/kg of body weight/day.