NO790956L - ANALOGICAL PROCEDURES FOR THE PREPARATION OF CEPHALOSPORINE ANALOG OR PHARMACEUTICAL ACCEPTABLE SALTS THEREOF - Google Patents

Description

Analogy process for the preparation of cephalosporin analogues or pharmaceutically acceptable salts thereof.

The present invention relates to the production of cephalosporin analogues, and more specifically 7-acylamino-1-larba-3-cephem-4-carboxylic acids with the general formula:

where

X means an acyl group with the formula X^CO, where X"^ means one of the following five groups:

(l) a cyanomethyl group or a group of the formula:

where B means an unsaturated, six-membered, carbocyclic group selected from cyclohexenyl, cyclohexadienyl and phenyl or a five- or six-membered, heterocyclic group, A"<*>" means hydrogen, hydroxy, alkoxy with 1-4 carbon atoms, halogen, nitro, amino, aminomethyl, methylsulfonamido or acyloxy with 1-4 carbon atoms, n is an integer from 0-5, and A means hydrogen, amino, hydroxy, carboxyl or sulfoxyl,

(2) a group with the general formula:

where B, A 1 and n have the meaning given above, and A^ 3 and A^ are the same or different and each means hydrogen, alkyl of 1-4 carbon atoms, a group of the general formula -CO-A^, where A^ means alkyl of 1-4 carbon atoms, or a group with the general formula: where A^ and A^ are the same or different and each of them means hydrogen, alkyl of 1-4 carbon atoms or alkali metal, 3 4 or and A together with the nitrogen atom to which they are attached, form a group with the general formula:

O O f

where A and A are the same or different and each means hydrogen or alkyl with 1-4 carbon atoms, or a group with the general formula:

where kQ J means hydrogen, alkyl with 1-4 carbon atoms, methyl-sulfonyl or furfurylideneimino, and A en » means hydrogen or

alkyl with 1-4 carbon atoms,

(3) a group with the general formula:

where B, A"1" and n have the meaning given above, and A"^ means a substituted aryl group or a mono-, bis- or tricyclic heterocyclic group,

(4) a group with the general formula:

where B, A"*" and n have the above meaning, and A"<1>""<*>" means hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon atoms , cycloalkyl with 3-6 carbon atoms or aryl, where the groups are optionally substituted with suitable substituents selected from carboxyl, cyan, halogen, carbamoyl and alkoxycarbonyl with 1-4 carbon atoms, or (5) a group with the general formula:

where B"<*>"means trifluoromethyl, cyanomethyl or a group of the formula (A<1->)^—B-, where B, A<1>and n have the meaning given above, and Z means oxygen or sulphur,

R<1> represents hydrogen or a carboxylic acid protecting group selected from alkyl with 1-5 carbon atoms, halogenated alkyl with 1-5 carbon atoms, arylmethyl with 7-20 carbon atoms, which may be substituted on the phenyl ring, substituted silyl and a group, which is easily removed enzymatic or non-enzymatic in vivo, and

R 2 means hydrogen, alkyl with 1-5 carbon atoms or acyloxy with 1-5 carbon atoms, or pharmaceutically acceptable salts thereof.

The carbacefem nomenclature refers to the Journal of the American Chemical Society volume 96, 7584, 1974. Until now, only those with a substituted methyl group in the 3-position, such as (-)-l-carba-cephalotin, have been known as carbacefem compounds with the formula: and some have been reported to have antibacterial activity (the above reference and J.Med.Chem., 20. 551, (1977)). As a result of various investigations to provide hitherto unknown analogues of cephalosporin with strong antibacterial activity, the inventors in this case have previously made the invention-related cephalosporin analogue with the general formula (II) below, i.e. 4- or 5-substituted carbacefem ( the numbering system in formula (II) below is used in the following); pg salts thereof (Japanese patent application no. 34696/78).

"wherein X1 means amino, azido or phthalylimino, R<1> means hydro-

gen, halogen, hydroxy, lower alkoxy, aryloxy, aralkoxy, acyloxy, alkylsulfonyloxy, arylsulfonyloxy, lower alkylthio, arylthio, aralkylthio, lower alkylsulfinyl, arylsulfinyl, aral-+ 45 alkylsulfinyl, a sulfonium group of general formula -S R R ,

4 5

where R and R are the same or different and each means a lower alkyl, aryl or aralkyl group, lower alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a quaternary ammonium group of the general formula N+R^R R®, where R^, R^ and R Q are the same or different and each represents a lower alkyl, aryl or aralkyl group, arylseleno or

2 1

arylseleninyl, where R means the same as R or lower alkyl, which is optionally substituted with one or more halogen atoms, azido, nitrile or an amino group with the general

9 10 9 10

formula N R R„ , where R and R are the same or., different and each means hydrogen, lower alkyl, aryl or aralkyl, and R means hydrogen or an optional alkyl, aryl, aralkyl or silyl group, and salts thereof.

As a result of further investigations, it has now been shown that hitherto unknown cephalosporin analogues with unexpectedly strong antibacterial activity can be obtained by acylation of compounds of the above-mentioned formula (II),

1 12

where X is NH0, R is H, R is H, lower alkyl or lower

"5 1

acyloxy, and R is R .

These hitherto unknown compounds with general formula (i) are prepared according to the invention by a cephalosporin analogue with the general formula:

1 2 where R and R have the meaning stated above, or a functionally equivalent compound (hereinafter referred to as the 7-amino compound) is acylated with a carboxylic acid of the general formula: or a reactive derivative thereof, where X 2 means one of the following five groups: 1') cyanomethyl or a group with the general formula:

where B and n have the above meaning, A. means hydrogen, hydroxy, protected hydroxy, alkoxy with 1-4 carbon atoms, halogen, nitro, protected amino, protected aminomethyl, methylsulfonamido or acyloxy with 1-4 carbon atoms, and A 2' means hydrogen, protected amino, hydroxy, carbonyl or protected sulfoxyl,

2') a group with the general formula:

1' 3 4

where B, A , n, kr and A have the meaning stated above,

3') a group with the general formula:

where B, A"^, n and A^ have the above meaning,

4') a group with the general formula:

11 12 where B, A and n have the above meaning, and A means hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon atoms, cycloalkyl with 3-6 carbon atoms, or aryl, where the groups are optionally substituted with suitable substituents selected from protected carboxyl, cyano, halogen, carbamoyl and alkoxycarbonyl with 1-3 carbon atoms, or

5') a group with the general formula:

where B means trifluoromethyl, cyanomethyl or a group of the formula (A11^ B-, where B, A<11> and n have the meaning given above, and Z means oxygen or sulphur,

after which, if necessary, the protecting group in the X CO- and/or -COO^ group is removed in a conventional manner, and, if desired, the compound formed is converted to a pharmaceutically acceptable salt thereof.

With respect to B, examples of the 5- or 6-membered, heterocyclic group can be mentioned as furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, isothiazolyl', -isoxazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidi- nyl, pyrazinyl, pyridazinyl, triazinyl and 5,6-dihydro-1,4-dithiin-2-yl.

With respect to A", examples of the mono-, bi- or tricyclic heterocyclic group can be mentioned those with the following formulas:

which may have substituents such as those represented by

a\ on the rings.

With regard to R"*", examples of halogenated alkyl can be mentioned as chloromethyl, 2,2,2-trichloroethyl and 2,2,2-trifluoroethyl, as examples of optionally substituted arylmethyl with 7-20 carbon atoms are mentioned benzyl, diphenylmethyl and triphenyl-methyl, which may have a methoxy group, nitro group, etc. on the phenyl ring, as examples of substituted xylyl are mentioned tri-methylxylyl and triphenylxylyl, and as examples of a group that is easily removed enzymatically or non-enzymatically in vivo,

a group with the general formula is mentioned

where R means hydrogen or alkyl with 1-6 carbon atoms,

and R^" means alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms or phenyl.

In the case that R"*" in the general formula (I) is hydrogen, or that X is an acyl group with a free carboxyl group or amino group, the compounds of the general formula (I) may be salts of pharmaceutically acceptable inorganic or organic bases or acids.

The compounds of formula (I) include all the stereoisomers in the 6-, 7- and 4-positions and mixtures thereof. Among the stereoisomers, those having cis configuration at the 6 and 7 positions have higher antibacterial activities than trans isomers, so that the cis isomers are more useful as antibiotics. The group =N0R in the acyl group has the following two geometrical isomers, i.e. syn and anti: As the syn isomer is better than the anti isomer with regard to antibacterial activity, the syn isomers are more useful as antibiotics. In general, it is known that the thiazolyl group

shows reversible conversion to the thiazolinyl shown below

group, and both are usually treated as identical. In the present context, both isomers are represented; at the thiazolyl group

The compounds of formula (I) are prepared by acylation of the compounds (II') corresponding to formula<r>(II),

1 2

where X is NH9, R is H, R is H, alkyl with 1-5 carbon atoms

3 1

or acyloxy with 1-5 carbon atoms, and R is. R , according to reaction scheme I.

React. ion. tbsp. jam in

1 2

where R , R and X have the meaning given above.

The acylation reaction (condensation) is carried out according to a conventional acylation method used in penicillin and cephalosporin chemistry.

The compounds of formula (I) (hereinafter referred to as compound (I)) are thus prepared by condensation of a compound of formula (II') (hereinafter referred to as compound (II<1>)), a salt thereof or a thus functionally equivalent compound (in the following summary referred to as the "7-amino compound") and a carboxylic acid of the formula:

or reactive derivative thereof, if necessary followed by

removal of the protecting group in the X 2 - or -CO OR 1 group by conventional means. In formula (III), X 2 CO corresponds to X 1CO,

i.e. X in the compound of formula (I), and X o in formula (III) means the above-defined group X"*" or a suitably protected X~<*>" group in the event that X"*" contains groups which are susceptible to acylation, such as hydroxy, amino, carboxyl or mercapto.

As protective groups for amino, hydroxy, etc. in the X 2 group, those used in cephalosporin and penicillin chemistry, and those shown below, are used.

As salts of compound (II<1>) can e.g. mention is made of inorganic salts such as hydrochloride, sulphate, carbonate and phosphate, and organic salts such as formate, trifluoroacetate and malate. Furthermore, examples may be mentioned of the sodium, potassium, calcium and ammonium salts and the salts with organic amines etc. of the carboxylic acids of formula (II'), where R"'" is hydrogen. The salts are produced in a conventional manner.

As compounds which are functionally equivalent to compound (II'), e.g. mention is made of 7-monosilyl or 7-disilylamino derivatives of compound (II')•

As reactive derivatives of the carboxylic acid with the general formula (III), X<2>COOH, e.g. mention is made of 1) an acid halide, 2) an acid anhydride, 3) a mixed acid anhydride, 4) an active ester, 5) an active thioester and 6) an acid azide.

Condensation (acylation) reactions using the above derivatives are explained in more detail below:

(1) Method_under_renewal_of_it

A 7-amino compound and an acid halide are subjected to condensation in an inactive solvent, preferably in the presence of a proton acceptor. The acid halide is prepared in a conventional manner.

As a proton acceptor, e.g. inorganic and organic bases, preferably sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxy, triethylamine, N-methylmorpholine

and pyridine.

Any inactive solvent that does not affect the reaction can be used as a solvent, preferably water, an ether, such as tetrahydrofuran and dimethoxyethane, an ester, such as ethyl acetate, an amide, such as dimethylacet-

amide and hexamethylphosphoric acid triamide, or a sulfoxide such as dimethyl sulfoxide, alone or in combination.

The reaction is carried out at a temperature between

-40 and +40°C, preferably between 0°C and room temperature.

(2) Method_using-of an--acid anhydride

A 7-amino compound and an acid anhydride are subjected to condensation in an inactive solvent.

The acid anhydride is prepared in a conventional manner.

Any solvent that does not affect the reaction can be used as a solvent, preferably the same solvents as in the above-mentioned method. The temperature range for this reaction is the same as for the above-mentioned method.

The typical method using carbodiimide such as dicyclohexylcarbodiimide is exemplified as the acid anhydride method.

(3) Me<52>^§_<y>^<ir>_§^<Y>§S§£l§§<_>§Y_<®J>_<5i>§S^Jacid anhydride

A 7-amino compound and a mixed acid anhydride are subjected to condensation in an inactive solvent.

The mixed acid anhydride prepares in a conventional manner, e.g. by reacting the corresponding carboxylic acid, X 2C00H, and a chloroformate ester, such as ethyl chloroformate or isobutyl chloroformate, in the presence of a base.

Any inactive solvent that does not affect the reaction can be used as a solvent, usually an anhydrous solvent or a mixture of water and an anhydrous solvent, preferably the same solvent as in the above-mentioned methods.

The temperature range for this reaction is the same as for the above-mentioned methods.

(4) Method_under_anyendelse_ay_an active ester

A 7-amino compound and an active ester are subjected to condensation in an inactive solvent. Solvents and reaction temperatures are the same as in methods 1-3 stated above.

As the active ester can e.g. mention is made of a phenyl ester such as p-nitrophenyl ester and trichlorophenyl ester, a methyl ester with an electronegative group such as cyanomethyl ester, and an N-oxydiacyl imide ester such as N-hydroxysuccin imide ester.

The active ester is prepared in a conventional manner by reacting a corresponding carboxylic acid and a hydroxy compound in the presence of a dehydrating condensation reagent, such as dicyclohexylcarbodiimide.

(5) Method_under_use_of_an_active_t

This method is carried out in the same way as method 4.

As the most preferred thioester can be mentioned p-nitrothiophenyl ester which is prepared by a mixed-acid anhydride method or dicyclohexylcarbodiimide method.

(6) Method_under_renewal_ay_e^

A 7-amino compound and an acid azide are subjected to condensation in an inactive solvent in the same way as in the above-mentioned methods 1-5.

The acid azide is produced by reacting the hydrazide of a corresponding carboxylic acid with nitric acid at a temperature of between -20 and 0°C.

In the event that the above-mentioned reactive derivatives of the carboxylic acid contain such groups as amino, hydroxy, carboxy or mercapto, which are susceptible to acylation, these groups are preferably protected with a suitable protecting group in a conventional manner for the condensation reaction with the amino compounds.

As suitable protecting groups, those used in the synthesis of penicillins and cephalosporins can be mentioned.

As examples of the amino protecting group, mention may preferably be made of t-butyloxycarbonyl (Boe), benzyloxycarbonyl (Cbz), trichloroethyloxycarbonyl, trityl, formyl, chloroacetyl, trialkylsilyl, proton, (3-diketone and (3-ketoester). As an example of a compound protected with a proton can be mentioned the compound with the formula:

As an example of a compound protected with a (3-ketoester, mention may be made of the compound with the formula:

As examples of the hydroxy protecting group

mention may be made of benzyl, benzyloxycarbonyl, trityl, tetrahydropyranyl and t-butyl. As the reactivity (the nucleophilic activity) of most amino groups is higher than that of the hydroxy group, the protection of the hydroxy group is not absolutely necessary, depending on the acylation method used.

As examples of the carboxy protecting group

can e.g. mention is made of t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl and benzhydryl.

As examples of the mercapto protecting group

mention may be made of benzyl, trityl, benzyloxycarbonyl and p-nitrobenzyl.

The removal of the above-mentioned protecting groups is carried out in a conventional manner in the synthesis of penicillins and cephalosporins.

The following can be mentioned as examples of methods for removing amino-protecting groups: Boe is removed by a method using an acid such as formic acid, acetic acid, trifluoroacetic acid and hydrochloric acid.

Cbz is removed by catalytic reduction or one-method using hydrogen bromide/acetic acid. Trityl is removed by a catalytic reduction or a method using an acid such as trifluoroacetic acid.

Formyl is removed by hydrolysis using an acid or alkali.

Chloroacetyl is removed by a method using thiourea.

Trialkylsilyl is removed by hydrolysis.

A proton is removed by neutralization.

The additive with P-diketone or (3-ketoester) is removed by acid hydrolysis.

The following can be mentioned as examples of methods for removing hydroxy-protecting groups: Benzyl is removed by catalytic reduction or a method using hydrogen fluoride.

Cbz is removed by catalytic reduction or a method using hydrogen bromide/acetic acid.

Trityl is removed by catalytic reduction or a method using trifluoroacetic acid.

Tetrahydropyranyl is removed by acid hydrolysis.

t-butyl is removed by a method using an acid such as trifluoroacetic acid, hydrobromide/acetic acid and hydrochloric acid.

As examples of methods for the removal of. carboxy protecting groups can be mentioned as follows: t-butyl is removed by a method using an acid such as trifluoroacetic acid.

Benzyl or p-nitrobenzyl is removed by catalytic reduction or a method using a Lewis acid such as AlCl₂.

Benzhydryl or p-methoxybenzyl is removed by catalytic reduction or a method using hydrobromide/acetic acid, hydrochloric acid/methanol, trifluoroacetic acid, etc.

The following can be mentioned as examples of methods for removing mercapto-protecting groups: Benzyl is removed by a method using hydrogen fluoride, etc.

Cbz is removed by a method using hydro-

gen bromide/acetic acid, trifluoroacetic acid, etc.

p-nitrobenzyl is removed by catalytic reduction.

The above-mentioned deprotection can very well be carried out simultaneously with the conversion of the R^ group to hydrogen, i.e. deesterification.

If desired, the acylation reaction is preferably promoted by silylation of the starting compound (II) with a silylating agent such as trimethylchlorosilane/base, hexamethyldisilazane and N,0-bis-trimethylsilylacetamide, to solubilize the starting material in organic solvents and activate the amino group .

The starting compounds of the general formula (II') can be prepared by a method described in Japanese patent application No. 34,696/78. A couple of examples are illustrated in the following reference reaction schemes (I') and

(II').

In the reference reaction schemes (I') and (II<1>), the compounds 6, 7, 6', 7', 11 and 12 are examples of the starting compounds with the general formula (II<1>). The production of the starting compounds is described in the subsequent reference examples.

Compound (I') corresponding to the general formula (I), where R"^ is H, can be prepared by removing the carboxyl protecting group according to reaction scheme (II).

1

where R is a carboxylic acid protecting group, and R and X have the above meaning.

The de-esterification reaction is carried out by a conventional method used in the synthesis of penicillins and cephalosporins.

As examples of the reaction which converts -COOR"*" to -COOH, mention may be made of 1) catalytic reduction, 2) azidolysis, 3) cleavage reaction with a Lewis acid, 4) hydrolysis, 5) reduction using reducing agents and 6 ) a method using an esterase. Each method is explained in more detail below.

1) Catalytic reduction. John

The group COOR"<1>" is converted to the group COOH in the presence of a catalyst in a hydrogen atmosphere in an inactive solvent. Any solvent which does not affect the reaction can be used as a solvent, preferably ethanol, water, tetrahydrofuran, dioxane, ethyl acetate, acetic acid or mixtures thereof. Examples of the catalyst can be mentioned palladium-carbon, platinum oxide, palladium-calcium carbonate and Raney nickel. The reaction is usually carried out at a pressure of 1 - 50 atm. and a temperature of 0.0-100°C, preferably at atmospheric pressure and room temperature.

This method is preferably used in cases where. R 1 is benzyl, p-nitrobenzyl, diphenylmethyl, p-methoxybenzyl, etc.

2) Acidolysis

The group COOR"'" is converted to the group COOH with an acid in an inactive solvent. Examples of the acid include hydrochloric acid, p-toluenesulfonic acid and trifluoroacetic acid. Any solvent which does not affect the reaction can be used as a solvent, preferably ethyl acetate, benzene, ethanol, acetic acid, dioxane, methylene chloride, chloroform or mixtures thereof.

The reaction is carried out at a temperature between -15 and +50°C, preferably 0 - 25°C, over a period of from 10 minutes to 5 hours, preferably from 30 minutes to 3 hours.

This method is preferably used in cases where R"^" is t-butyl, trityl, etc.

3) Cleavage using a Lewis acid

The group COOR"'' is converted to the group COOH by cleavage in the presence of a Lewis acid in an inactive solvent. Any solvent which does not affect the reaction can be used as a solvent, preferably a mixture of a nitroalkane such as nitromethane, and a haloalkane such as methylene chloride. Examples of the Lewis acid include aluminum chloride, boron trifluoride, titanium tetrachloride and tin tetrachloride. The acid is used in an amount of 1.0 - 1.5 molar equivalents in relation to compound (I<1>). The reaction is preferably carried out in the presence of of a carbonium cation absorbing agent, such as anisole.The reaction is carried out at a temperature of 0 - 50°C, preferably at room temperature, for a period of 1 - 10 hours.

This method is preferably used in cases where R"<*>" is p-nitrobenzyl etc.

4) Hydrolysis

The group COOR<1> is converted to the group COOH by hydrolysis in the presence of an acid or alkali in an inactive solvent. Examples of the acid include p-toluenesulfonic acid, hydrochloric acid and acetic acid. Any solvent which does not affect the reaction can be used as a solvent, preferably 2% aqueous methanol, N,N-dimethylformamide or acetic acid/water/tetrahydrofuran. The reaction is carried out at a temperature of 0 - 50°C, preferably 15 - 25°C, over a period of from 10 minutes to 2 hours.

This method using acids is used

preferably in cases where R<1> is t-butyldimethylsilyl.

Calcium carbonate is preferably used as the base

in an amount of 1 - 6 molar equivalents in relation to compound (II"). The solvent can be used.. any solvent which does not affect the reaction, preferably tetrahydrofuran/water, dioxane/water or acetone/water. The reaction is usually carried out at a temperature of 0 - 30°C

in increments of 30 minutes to 24 hours.

This method using a base is preferably used in cases where R1 is methyl, ethyl, etc.

5) 'Reduction using reducing agents (different from catalytic reduction)

The group COOR<1> is converted to the group COOH by reduction in an inactive solvent. Zinc/acid can be mentioned as an example of the reducing agent. As a solvent, e.g. acetone, water, dioxane, tetrahydrofuran, ethanol, acetonitrile, N,N-dimethylformamide, acetic acid or mixtures thereof. Examples of the acid include hydrochloric acid, formic acid and acetic acid. The reaction is carried out at a temperature of

0 - 100°C, preferably 0 - 40°C, over a period of 1 - 10 hours.

The amount of zinc used for the reaction is usually 1 - 10 molar equivalents.

If suitable reagents are used, the protecting group in group X can be removed simultaneously with the deesterification.

The compounds of the general formula (I) produced by the method according to the invention, in which R1

is H, as mentioned in the example 2 below, have such excellent antibacterial activities against gram-positive and gram-negative bacteria that they are expected to be useful as anti-infectives or antibacterial agents against these bacteria. The compounds of formula (I), where R<1> is different from H, can also be used as starting materials for the preparation of compounds of formula (I), where R<1> is H.

The compounds produced according to the invention can be used in pharmaceutical preparations containing compound (I) or a pharmaceutically acceptable salt thereof as active ingredient in connection with a pharmaceutical carrier or a pharmaceutical diluent. The compounds can be administered parenterally (intramuscular, intraperitoneal, intra-venous or subcutaneous), oral or rectal and. can be put together in dosage forms suitable for each mode of administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or . emulsions. Examples of non-aqueous solvents or media are propylene glycol, polyethylene glycol, vegetable oils, such as olive oils, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain additives such as preservatives, wetting agents, emulsifiers and dispersants. They can be sterilized, e.g. by filtering through a bacteria-retaining filter, by incorporating sterilizing agents into the preparations, by irradiating the preparations or by heating the preparations. They can also be prepared in the form of sterile, solid mixtures, which can be dissolved in sterile water or another sterile injectable medium immediately for use.

Agents for oral administration may be in a form suitable for absorption in the gastrointestinal tract. Tablets and capsules for oral administration may be in unit dosage form and may contain conventional excipients such as binders, e.g. syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone, fillers, e.g. lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine, glitter agents, e.g. magnesium stearate, talc, polyethylene glycol or silicon dioxide, disintegrants, e.g. potato starch, or an acceptable wetting agent such as sodium lauryl sulfate. The tablets can be coated according to well-known methods in the art. Oral, liquid preparations can be in the form of aqueous or oil-based suspensions, solutions, emulsions, syrups or can be available as a solid product for mixing with water or another suitable medium for use. Such liquid preparations may contain conventional additives such as suspending agents, e.g. sorbitol syrup, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose and aluminum stearate gel, emulsifiers, e.g. lecithin or sorbitan mono-oleate, non-aqueous media which may include edible oils, e.g. almond oil, coconut oil, propylene glycol or ethylene glycol, and preservatives, e.g. methyl or propyl p-hydroxybenzoate or sorbic acid...

Means for rectal administration are preferably suppositories, which, in addition to the active substance, may contain excipients such as coconut oil or a suppository wax.

The content of active ingredient in the preparations can be varied, but it is necessary that the amount of the active ingredient is such that a suitable dosage form is obtained. The chosen dosage depends on the desired therapeutic effect, on the method of administration and on the duration of the treatment. Dosages of 5 - 350 mg/kg body weight are usually administered daily to mammals to achieve an antibiotic effect.

As examples of pharmaceutically acceptable salts

of compound (I) can be mentioned inorganic salts, such as hydrochloride, sulphate, carbonate and phosphate, and organic salts such as formate and malate. Furthermore, examples can be mentioned of sodium; potassium, calcium and ammonium salts and salts with organic amines of the carboxylic acids of general formula (i), in which R<1> is hydrogen. The salts are produced conventionally

manner.

The following examples serve to further illustrate the invention...

Example 1

Preparation of (-)-cis-2-carboxy-7-(2-(thiophen-2-yl)acetyl-amino7-1-azabicyclo/2J-,2,0_7oct-2-en-8-one with the formula: 88 mg (0.489 mthpl) of the amino compound prepared in reference example 8 is dissolved in 2 ml of deionized water and 1 ml of acetone and 84 mg of aqueous sodium bicarbonate solution are added. 78 mg of 2-thienylacetyl chloride dissolved in 0.5 ml of acetone is added dropwise to the mixture under ice cooling. The mixture is then stirred for 30 minutes and washed with ethyl acetate. The aqueous layer obtained is adjusted to pH 2.0 with hydrochloric acid. The resulting white suspension is extracted 3 times with 5 ml of ethyl acetate. The ethyl acetate layer is washed with saturated sodium chloride solution, washed with anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow oily product. The product is fed onto a column packed with 4.0 g of silica gel ("Wako-gel C-200", the same silica gel used in the following examples and reference examples) and eluted with chloroform. The syrup obtained is treated with chloroform/ethanol to obtain 30 mg of crystals. The crystals are identified as the desired compound on the basis of the following properties. Dividend 20.1%.

Melting point: 181 - 183°C

IR(KBr)V£m~s: 1775, 1690, 1650, 1615

NMR (CT>30D)£(ppm): 7.16-6.88 (3H, m), 6.18 (1H, t), 3.16 (1H, d, J = 5Hz), 3.80 ( 1H, m), 3.37 (2H, s), 2.5-1.30 (4H, m) Example 2

The antibacterial activities of the (<i>)-cis-2-carboxy-7-[5-(thiophen-2-yl)acetylamino7-1-azabicyclo/Zj., 2,07oct-2-en- 8-on is shown below. The regular agar dilution method is used at pH 7.0.

Example 3

Preparation of (-)-cis-7-(2-(2-tritylamino-4-thiazolyl)-2-anti-methoxyiminoacetamido7-2-t-butyloxycarbonyl-1-azabicyclo/5,2,07oct-2-ene-8 -on:

Method a)

75 mg (0.307 mmol) of (-)-cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo/4\ 2,07oct-2-en-8-one, prepared as in ..reference example 9, and 135 .9 mg (0.307 mmol) of 2-(2-trityl-amino-4-thiazolyl)-2-anti-methoxyiminoacetic acid is dissolved in 2 ml of anhydrous methylene chloride, and 69.6 mg (0.307 mmol) of dicyclohexylcarbodiimide dissolved in 1 ml is added anhydrous methylene chloride under stirring and cooling. The mixture is stirred for 3 hours and allowed to react at a temperature of 10°C overnight.

The reaction mixture is washed with 1% aqueous phosphate, saturated sodium bicarbonate solution and saturated sodium chloride solution. The washed solution is dried with magnesium sulfate and concentrated under reduced pressure to obtain 2.57 mg of crude product. The product is purified by flash chromatography using 12 g of silica gel and a solvent consisting of n-hexane and ethyl acetate in the volume ratio 1:1 to obtain 73 mg (35.9%) of the desired compound as a pale yellow glassy substance.

-1

IR(KBr) Y™ks: 1780, 1725, l695(sh), 1690, 1635

NMR(CDCl 3 ) in (ppm): 8.48 (d, 1H, J=6, 4Hz), 7.25 (s, 15H),

6.30 (t, 1H, J=3, 0Hz), 5.35 (t, 1H, J=6, 4Hz), 4.05 (s, 3H), 2.5-1.6 (m, 4H ), 1.52 (s, 9H)

Method b)

524.9 mg (1.18 mmol) of 2-(2-tritylamino-4-thiazolyl)-,2-anti-methoxyiminoacetic acid is dissolved in 10 ml of concentrated tetrahydrofuran. To the solution are added 1.18 ml (1.18 mmol) IN-1N-methylmorpholine/tetrahydrofuran and 1.18 ml (1.18 mmol) IN-" isobutyl chloroformate/tetrahydrofuran at a temperature of

-30°C, and<:>the mixture is stirred for 40 minutes. A solution of 235 mg (0.987 mmol) (-)^cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo/5.,2,07oct-2-en-8-one in 5 ml of anhydrous methylene chloride is added dropwise to the above mixture. The mixture is allowed to react for 30 minutes and is further stirred at 0°C for 2 hours. 10 ml of ethyl acetate is added to the reaction mixture and the mixture is washed with water, saturated sodium bicarbonate solution and saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 865 mg of crude product.

By silica gel chromatography using 40 g of silica gel according to method a) 580 mg (87.5%) is obtained

of the desired connection. The IR and NMR spectra of the product agree with the spectra of the compound prepared by method a).

Example 4 Preparation of (-)-cis-7-^2-(2-amino-4-thiazolyl)-2-anti-methoxyimino-acetamidoZ-1-azabicyclo/fr,2,07oct-2-en-8-one - 2- carboxylic acid:

500 mg (0.754 mmol) (-)-cis-7-(2-(2-tritylamino-4-thiazolyl)-2-anti-methoxyimino-acetamido7-2-t-butyloxycarbonyl-1-azabicyclo/4,2,07oct -2-en-8-one, prepared in example 3, is dissolved in a mixture of 5 ml of trifluoroacetic acid, 2.5

ml of anhydrous methylene chloride and 2.5 ml of anisole. The solution is left at 0°C for 3 hours and 40 minutes and then concentrated under reduced pressure. Add 5 ml of 50% aqueous acetic acid to the concentrate. The mixture is stirred at room temperature for 3 hours and concentrated under reduced pressure. The concentrate is thoroughly triturated with ether and filtered to obtain 244 mg of crude product. The product is purified by flash chromatography with 10 ml "Diaion HP-10" and a solvent consisting of methanol and water in a volume ratio of 2:5 to obtain 90 mg (32.7%) of a pale yellow powder.

IR(KBr)V^ks: 1760, 1670, 1630

NMR(CD₂OD) c£ (ppm): 7.47 (s, 1H), 640 (m, 1H), 5.51 (d, 1H, .

J=5, 0Hz), 4.05 (s, 3H), 4.3-3.7 (m, 1H), 2.6-1.1 (m, 4H) Example 5

Preparation of (-)-cis-7-(TR)-2-phenyl-2-t-butyloxycarbonyl-aminoacetamido7-2-t-butyloxycarbonyl-1-azabicyclo[4] 2,07oct-2-en-8-one:

Method a)

81 mg (0.34 mmol) (-)-cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo/3-, 2,07oct-2-en-8-one and 94.0 mg (0, 34 mmol) (R)-N-t-butyloxycarbonylphenylglycine dissolved in 2 ml of anhydrous methylene chloride. A solution of 77 mg (0.34 mmol) of dicyclohexylcarbodiimide in 1 ml of anhydrous methylene chloride is added to the solution while cooling with ice and sodium chloride. The mixture is allowed to react while cooling with ice for 2 hours and 2 drops of acetic acid are added. The mixture is stirred for 20 minutes and suction filtered. The filter cake is washed with 20 ml of ethyl acetate. The filtrate and the washing solutions are combined and 20 ml of ether is added. The mixture is washed with 1%

in aqueous phosphoric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 187 mg of crude product. The product is purified by silica gel chromatography with 9 g of silica gel and a solvent consisting of n-hexane and ethyl acetate in the volume ratio 1:1 to obtain 104 mg

(64.9%) of the desired compound as a colorless glassy substance.

IR(KBr) Y™ks": 1770, 1750, 1720, 1630

NMR(CDCl 3 ) δ (ppm).: 7.32 (s, 5H), 6.31 (m, 1H), 5.90 (m, 1H), 2.50-1.70 (m, 4H), 1.50 (s, 9H), 1.40 (s, 9H)

Method b)

297.3 mg (1.18 mmol) of (R)-N-t-butyloxycarbonyl-phenylglycine are dissolved in 5 ml of anhydrous tetrahydrofuran, and 1.18 ml (1.18 mmol) of IN N-methylmorpholine/tetrahydrofuran and 1.18 ml are added (1.18 mmol) 1N isobutyl chloroformate/tetrahydrofuran at -30°C. The mixture is stirred for 30 minutes and

; ^subsequently added! 234 mg. (0.983 mmol) (-)-cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo^Zt-,2,Q7oct-2-en-8-one dissolved in 5 ml of anhydrous methylene chloride. The mixture is allowed to react at

-30°C for 45 minutes and at 0°C for 4 hours and 15 minutes. The reaction mixture is diluted with 15 ml of methylene chloride and washed successively with water, IN hydrochloric acid, water and saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and concentrated to obtain 588 mg of crude acyl compound. Purification by silica gel chromatography with 28 g of silica gel is carried out according to method a) to obtain 322 mg (69.4%) of the desired compound as a colorless, glassy substance. of the product. IR and NMR spectra agree with the spectra of the compound prepared by method a).

Example 6

Preparation of (-)-cis-7-(TR)-2-phenyl-2-aminoacetamido7-1-azabicyclo^4", 2,07oct-2-en-8-one-2-carboxylic acid:

280 mg (0.59 mmol) (<±>)-cis-7-ZtR)-2-phenyl-2-tert-butyloxycarbonylaminoacetamido7-2-t-butyloxycarbonyl-r-azabicyclo/5-,2,07oct-2-ene- 8-one, prepared in example 5, is dissolved in 2.5 ml of anhydrous methylene chloride and 2.5 ml of anisole, and 5.0 ml of trifluoroacetic acid is added under ice cooling. The mixture is allowed to stand for 4 hours and 50 minutes under ice cooling, after which it is concentrated. 10 ml of ether is added to the residue, and the mixture is stirred at room temperature for 1 hour to form a precipitate. The precipitate is collected by filtration, whereby 202 mg (70.9%) of the desired compound is obtained as a pale

yellow powder.

IR(KBr)Ym|ks: 1765, 1680, 1630

NMR(D 2 O with DSS as an international standard) & (ppm): 7.51 (d, 5H), 6.31 (m, 1H), 5.19 (s, 1H), 4.95 (d, 1H ),

3.8-3.5 (m, 1H), 2.6-'2.9 (m, 4H) -

Separation of the diastereoisomers of (-)-cis-7-(TR)-2-phenyl-2-aminoacetamido7-1-azabicyclo/Zj., 2,Q7oct-2-en-8-one-carboxylic acid: The compound prepared above (50 mg) is dissolved in 150 ml of water, and the information is subjected to high pressure liquid chromatography using "Bondapak C-18" as a carrier and a solvent consisting of 7% methanol in 0.2N potassium hydrogen phosphate solution, eight times. The isolation of 2 fractions is checked by a spectroscopic analysis at a wavelength of 25.4 nm. After removal of methanol under reduced pressure, each fraction is lyophilized. The dried material is dissolved in water and adsorbed on a soyle packed with 20 ml "Diaion-HP-10". The swab is washed with 200 ml of water and eluted with 20% ethanol. The fractions which are positive for the ninhydride sample are collected and lyophilized, whereby 14.0 mg of the A-isomer and 24.6 mg of the B-isomer are obtained as white powders consisting of potassium salt.

A: most polar fraction

/<a>7o<2>° (H20, C=0.5): -74.2° -1

IR(KBr)Y^ks: 1750'1690'1640

PMR(D20) & (ppm): 7.51 (5H, s), 6.15 (1H, 5, J=3, 9Hz), 5.20 (1H, d, J=4, 9Hz), 5, 19 (1H, s), 3.88 (1H, octet, J=8, 3, 7, 4.9 Hz), 2.41-1.41 (4H, m)

B: least polar fraction

</a7j>f° (<H>20, c=0.5): +57.2°

IRCKBr)^™^: 1760, 1690, 1640

PMR(D20)/(ppm): 7.51 (5H, s), 6.08 (1H, t, J=4, 2Hz), 5.41 (1H, d, J=4, 9Hz), 3, 83 (1H, octet, J=8.6, 3.7, 4.9 Hz), 2.28-1.01 (4H, m)

When the structure-activity relationship of the cephalosporins is taken into account, the least polar isomer is assigned, which

has dextro ^a/^ value and stronger antimicrobial activity than the most polar isomer as shown in the table below, absolute 6(R)7(S) configuration.

Example 7

Preparation of (±)-cis-7-(CR)-2-phenyl-2-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-acetamido7-2-t-butyloxycarbonyl-1-azabicyclo/4 ", 2, Ojokt-2-en-8-on:

in

Method a)

68 mg (0.286 mmol) (<i>)-cis-7-amino-2-t-butyloxy-carbonyl-1-azabicyclo[5-, 2.07oct-2-en-8-one and 100.4 mg (0.286 mmol).(R)-2-phenyl-2-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)acetic acid is dissolved in 2 ml of anhydrous methylene chloride and

70 mg (0.315 mmol) of dicyclohexylcarbodiimide dissolved in 1 ml of anhydrous methylene chloride are added under ice-cooling. The mixture is stirred for 6 hours and stirred further at a temperature of 10°C overnight. The reaction mixture is filtered and the filter cake is washed with methylene chloride. The filtrate and the washing solutions are combined and washed successively with 1% phosphoric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and concentrated to obtain 189 mg of crude acyl compound. The product is purified by "silica gel chromatography using 9 g of silica gel and a solvent consisting of h-hexane and ethyl acetate in the volume ratio 1:2 to obtain 43 mg of the most polar isomer, 20 mg of the least polar isomer and 11.1 mg of a mixture of those two

"isomers. The total yield is 54.1%."

The most polar isomer:

IR(CHC1,)Yc n : 1780, 1720, 1695 (sh), 1685

max

NMR(CDCl 3 ) S(ppm): 7.73 (d, 1H!, J=7, OHz), 7.37 fs, 5H), 6.25 (m, 1H), 5.7-5.0 ( m, 2H), 4.3-3.0 (m, 7H), 2.6-0.7 ,(m, 4H),

1.50 (s, 9H), 1.20 (h, 3H)

The least polar isomer:

.orr,"1

IR; (CHCl3)Y^<m>ks: 1780, 1695, 1685, 1620

NMR(CDCl 3 ) δ (ppm): 7.77 (d, 1H, J=8, OHz), 7.30 (m, 5H), 6.21 .(m, 1H), 5.67-5.33 (m, 2H), 4.5-3.2 (m, 7H), 3.5-1.0 (m, 4H), 1.50 (s, 9H), 1.20 (t~ 3H)

Method b)

Dissolve 428.5 mg (1.13 mmol) of (R)-2-phenyl-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)acetic acid in 10 ml of concentrated tetrahydrofuran and add 1.25 ml (1.25 mmol) 1N N-methylmorpholine/tetrahydrofuran and 1.25 ml (1.25 mmol) 1N isobutyl chloroformate/tetrahydrofuran at a temperature of -30°C. The mixture is stirred for 30 minutes and 235 mg (1.13 mmol) is added

(-)-cis-7-amino-2-t-butyroxycarbonyl-1-azabicykiso/4,2,Q7oct-2-en-8-one dissolved in 5 ml of anhydrous methylene chloride. The mixture

allowed to react for 1 hour and stirred at a temperature of 10°C

overnight. The reaction mixture is diluted with 20 ml of ethyl acetate and washed successively with water, 0.1N hydrochloric acid, saturated sodium bicarbonate and water. The washed solution is dried with anhydrous sodium sulfate and concentrated to obtain 570 mg of crude acyl compound. The product is purified and fractionated according to method a), with the exception that 27 g of silicon dioxide gel is used to obtain 73 mg of the most polar isomer and 61 mg of the least polar isomer (total yield 65.4%). The IR and NMR spectra of the isomers agree with the spectra of the isomers primarily in method a)

Example 8

Preparation of (-)-cis-7-(1R)-2-phenyl-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-acetamido7-1-azabicyclo/4\ 2,07oct-2-ene- 8-one-2-carboxylic acid:

103 mg (0.248 mmol) (<±>)-Cis-7-ZlR)-2-phenyl-2-(4-ethyl-2, 3-dioxo-1-piperiazinylcarbonylamino)-acetamido7-2-t-butyloxycarbonyl-1 -azabicyclo/Zi-, 2,07oct-2-en-8-one (the least polar isomer) prepared in Example 7, is dissolved in a mixture of 5 ml of trifluoroacetic acid, 5 ml of methylene chloride and

-.2 drops of anisole. The mixture is allowed to react at 0°C for 2 hours and is concentrated under reduced pressure. Add to the concentrate

benzene is used, and the mixture is concentrated again to obtain an oily product. Ether is added to the product and the mixture is stirred at room temperature to form a yellow precipitate. By filtration, 104 mg of crude product is collected as a yellow powder. The crude product is dissolved in ethyl acetate and extracted with 5 ml saturated sodium bicarbonate solution 3

times. The extracts are washed with ethyl acetate. The washed solution is adjusted to pH 2.5 with 0.5N hydrochloric acid under ice-cooling and extracted with 5 ml of ethyl acetate 3 times. The extract is washed with saturated sodium chloride solution, dried with magnesium sulfate and concentrated under reduced pressure to obtain 41 mg (46.0%) of a pale yellow powder.

about" 1

IR(KBr) v/max: 1175'1"720»l685'" 1620 (sh)

NMR(CD 3 OD);_J(ppm): 7.31 (s, 5H), 6.33 (t, 1H, J=4.0Hz), 5.40 (m, 2H), 4.30-3.1 (m, 7H), 2.40-0.7 (m, 4H), 1.27 (t, 3H)

Based on the strong antimicrobial activity shown in the table below, this compound is assigned the absolute 6(R)7(S) configuration.

Example 9 Preparation of (±)-cis-7|3-/TR)-2-phenyl-2-t-butyloxycarbonyl-aminoacetamido7-4(3-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4\2 ,07Oct-2-A-8-Wed:

132 mg (0.53 mmol) (R)-N-t-butyloxycarbonylphenyl-glycine. is dissolved in 5 ml anhydrous tetrahydrofuran and 0.53 ml (0.53 mmol) IN N-methylmorpholine and 0.53 ml (0.53 mmol) IN isobutyl chloroformate are added at a temperature of 0°C. The mixture is stirred for 15 minutes and then 0.07 ml (0.5 mmol) triethylamine and 1.44 mg (0.5 mmol) hydrochloride (-)-cis-7P-amino-4|3-methyl-2-t are added -butyloxycarbonyl-1-azabicyclo/Zj., 2,07-oct-2-en-8-one, prepared in reference example 12.. The mixture is stirred at a temperature of 0 o C for 1 hour and then at a

temperature of 5..- 10°C overnight.

The reaction mixture is diluted with 10 ml of ethyl acetate and washed successively with 10% citric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and concentrated to obtain a crude acyl compound.. The product

purified by flash chromatography using 30 g of silica gel and a solvent consisting of n-hexane and

ethyl acetate in the volume ratio 3:1. 150 mg (61.7%) of the desired compound is obtained as a powder.

-1

IR(KBr)Y^ks<:>3290, 1780, 1720, 1685, 1665

NMR(CDCl 3 )<£(ppm): 7.34 (5H, s), 6.60, 6.49 (respectively 1H and d, J=7), 6.11 (respectively 1H and d, J=2Hz) , 5.66, 5.60 (respectively 1H and\d, J=7Hz), 5.18 (2H, m), 3.86 (1H, m), 2.46 (1H, m), 1.75 (2H/<->br), 1.51 (9H, s), 1.42 (9H, s), 1.15, 0.98

(respectively 3H and d, J=7.5Hz)

Example 10

Preparation of the trifluoroacetate of (-)-cis-7(3-/TR)-2-phenyl-2-aminoacetamido74(3-methyl-2-carboxy-1-azabicyclo/4\ 2,Q7oct-2-en-8- on:

100 mg (0.21 mmol) (-)-eis-7P-((R)-2-phenyl-t-butyl-oxycarbonyl-aminoacetamido7-4(3-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4\2, 07<->kt-2-en-8-one, prepared in example 9, is dissolved in 1 ml of anhydrous methylene chloride, and 1 ml of trifluoroacetic acid is added while decoupling in an ice bath. The mixture is kept at a temperature of 0 - 5° C for 3.5 hours with occasional shaking. The reaction mixture is concentrated under reduced pressure. The concentrate is triturated with 5 mL of anhydrous diethyl ether and the ether layer is removed by decantation. The treatment is repeated 3 times and the resulting cake is dried under reduced pressure to obtain 70 mg (75 %) of the desired compound as powder.

-1

IR(KBr)V^<m>k9: 3450 (sh), 3230 (sh), 3060, 2960 - 2800, 1976,

1695 (sh), 1681 (sh), 1673

NMR(DMSO-dg) δ (ppm): 9.29 (1H, t, J=8Hz), 7.49 (5H, s), 6.11,

6.04 (respectively 1H and d, J=2Hz), 5.30 (1H, m), 4.97 (1H, d, v J=4Hz), 3.82 (1H, br), 2.44 ( br, partially overlapping with the signal .to DMS0-d6), 1.8.2 (2H, br), 1.14, 0.91' (respectively 3H and d, J=5.7Hz)

Example 11

Preparation of (-)-cis-7|3-j/rR-)-2-phenyl-2-t-butyloxycarbonyl-acetamido7-4a-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4\ 2,07- oct-2-en-8-on:

The same method as in example 9 is used, with the exception that 144 mg is used as starting compound

(*)-cis-7P-amino-4oc-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,Q7oct-2-en-8-one, prepared in reference example 11. As a result, 140 mg (57 .6%) of the desired compound.

.nm"1

IR(KBr)V^ks. 3330>1972 (sh))1782>1730, 1692, 1675

NMR(CDCl 3 ) é (ppm): 7.34 (5H, s), 6.73, 6.60 (1H and d, J=7Hz, respectively), 6.28 (1H, t, J=6Hz), 5 .60 (1H, m), 5.43-5.18 (2H, m), 3.82 (1H, m), 2.55 (1H, m), 1.69' (2H, m), 1 .51 (9H, s), 1.41 (9H, ,s~), 1.09, 103 (respectively 3H and d, J=7Hz) Example 12

Preparation of (-)-cis-7|3-(1R)-2-phenyl-2-aminoacetamido7-4oc-methyl-2-carboxy-1-azabicyclo/4,2,07oct-2-en-8-one:

The same method as "in example 10" is used, with the exception that "80 mg (-)-cis-7(3-/TR)-2-phenyl-2-t-butyloxycarbonyl-aminoacetamido7-4oc is used as starting compound" -methyl-2-t-butyloxycarbonyl-1-azabicyclo[alpha], 2,07oct-2-en-8-one;, prepared in Example 11. As a result, 73 mg (100%) of the desired compound is obtained. IR(KBr)Y^kg: 3430, 3200, 3060, 2960-2650, 1780 (sh), 1770, 1695 (sh), 1680" NMR(DMSO-dg) S (ppm): 9.36 (1H, d , J=8Hz), 7.47 .(5H, s), 6.28 (1H, d, J=6Hz), 5.40 (1H, m), 4.98 (1H, m), 3.70 (1H, br), 2.45 (br, partially overlapping with the signal for DMSO-dg), 1.80 (2H, m), 1.06, 0.95 (3H and d, respectively, J=7.5Hz) 30 mg of the above compound is dissolved in 100 µl of water, and the solution is subjected to high pressure liquid chromatography using "Bondapak C-18" as a carrier and a solvent consisting of 7% methanol in 0.2N potassium hydrogen phosphate solution, 5 times . The isolation of the two fractions is checked by means of a spectroscopic analysis at a wavelength of 254 nm. After removal of methanol under reduced pressure, each fraction is lyophilized. The used material is dissolved in water and adsorbed on a swab packed with 20 ml of "Diaion HP-10". The swab is washed with 200 ml of water and eluted with 20% ethanol. The eluate is taken up in 20 ml fractions. The fractions which are positive for ninhydria dprbven, is collected and lyophilized to obtain 8.9 mg (yield 29%) of the C-isomer, which is eluted at the earliest by high-pressure liquid chromatography, and 11.4 mg (yield 38%) of the D-isomer, which is eluted later. These are the potassium salts of the desired compound. The specific rotations of the isomers are as follows:

no.O

C-isomer"38.4° (H 2 O, C = 0.5)

in Peace

D-isomer /α7^ +4.23 (H2O, C = 0.52)

The D-isomer, which has stronger antimicrobial activity, than the diastereomixture (\-)-form7 as shown in the table below, is assigned the absolute 6(R)7(S) configuration.

D-Isomef:

' 'orr,"1

IR(KBr)Y^ks:3420, 1766; 1695, 1633

PMR(D 2 O ) /(ppm): 7.51 (5H, s), 6.10 (1H, d, J=5,D, 5.47 (1H,

•d, J=4.7), 5.19 (1H, s), 3.89 (1H, m), 2.45 (1H, m), 1.44-1.04 (2H, m), 1.00 (3H, d, J=7.4)

Example 13 - --

Preparation of (-)-cis-7|3-/-2-(2-tritylamino-4-thiazolyl-2-methoxyiminoacetamido7-4a-methyl-2-t-butyloxycarbonyl-1-azabicyclo/Zr, 2.07Oct-2 -en-8-on:

Method A

88 mg (0.35 mmol) (<±>)-cis-7p-amino-4a-methyl-2-t-butyloxycarbonylf-1-azabicyclo^4, 2,Q7oct-2-en-8-one is obtained

in 1.5 ml of anhydrous methylene chloride and 155 mg (0.35 mmol) of 2-(2-tritylamino-4-thiazolyl)-2-anti-methoxyimino-acetic acid are added. Furthermore, 1.5 ml of anhydrous dioxane is added to make the mixture more homogeneous. The mixture is then added with 80 mg (0.39 mmol) of dicyclohexylcarbodiimide dissolved in 1 ml

dioxane, and the resulting mixture is stirred at a temperature of 5-10°C overnight. The resulting white precipitate is filtered off and 10 ml of ethyl acetate and 5 ml of ether are added to the filtrate. The mixture is washed successively with 5 ml of cold 1% phosphoric acid three times and with saturated sodium bicarbonate solution. The washed solution is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 290 mg of crude product as a semi-solid. The crude product is filtered on a sponge packed with 27 g of silica gel and eluted with a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1. The eluate is concentrated under reduced pressure to obtain 170 mg (72%) of the desired compound. Method B

Dissolve 243.9 mg (0.05 mmol) of 2-(2-tritylamino-4-thiazolyl)-2-anti-methoxyiminoacetic acid in 5 ml of anhydrous tetrahydrofuran] and add 0.55 ml (0.55 mmol) IN N-methylmorpholine. 0.55 ml (0.55 mmol) IN isobutyl chloroformate/tetrahydrofuran is added dropwise to the mixture at a temperature of 0°C with stirring, and the mixture is stirred for a further 15 minutes. 0.11 ml (0.5 mmol) of triethylamine is added to the mixture followed by the addition of 144 mg (0.5 mmol) of (-)-cis-7(3-amino-4a-methyl-2-t-butyloxycarbonyl) hydrochloride -l-

azabicyclo/^,2,07oct-2-en-8-one. The mixture is stirred at a temperature of 5 - 10°C overnight and concentrated under reduced pressure. 10 ml of ethyl acetate is added to the concentrate and the resulting mixture is washed successively with 5% hydrochloric acid, saturated sodium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The product is applied to a sponge packed with 25 g of silicon dioxide gel and eluted with a mixture of n-hexane and ethyl acetate in a volume ratio of 5:3. The eluate is concentrated under reduced pressure to obtain 250 mg (74.0%) of the desired compound. The physical properties of the compound agree with the compound produced by method A.

Example 14

■Preparation of (-)^-cis-7P-(2-(2-amino-4-thiazolyl)-2-anti-methoxyiminoacetamido7-4a-methyl-2-carboxyl-1-azabicyclo-[%, 2,Q7oct- 2-on-8-on:

70 mg (0.10s mmol)'(-)-eis-7p-(2-(2-tritylamino-4-thiazolyl)-2-anti-methoxyimino-acetamido7-4a-methyl-2-t-butyl-poxycarbonyl- 1-azabicyclo/7r, 2, Q7oct-2-en-8-one, prepared in Example 13, is dissolved in 0.5 ml of anhydrous methylene chloride and 0.1 ml of anisole. The mixture is cooled to 0°C followed by the addition of 0.5 ml! trifluoroacetic acid. The resulting mixture is placed in an ice bath for 3.5 hours.

The reaction mixture is concentrated under reduced pressure. The concentrate is triturated with 5 ml of anhydrous diethyl ether and filtered to obtain a white powder. The powder is dissolved in 2 ml of 50% acetic acid. The reading is left at room temperature for 2.5 hours and then at 5 - 10°C overnight. The solution is further allowed to stand at room temperature (25°C) for 6 hours and is then concentrated under reduced pressure to obtain a glassy product. The glassy product is thoroughly triturated with ether and filtered. The filtrate is dried to obtain 20 mg (51%) of the desired compound.

■ -1

IR(KB<r>)v^ks<:>3480, 3300, 1770, 1680, 1635

NMR(DMSO-dg) & (ppm): 9.17 (1H, d, J=8Hz), 7.50 (1H, s), 7.24 (2H,.m), 6.31 (1H, d , J=6Hz), 5.52 (1H, m), 4.00 (3H, s), 2.65 (br, partially overlapping with the signal for DMSO-dg), 1.06

(3H, d.J=7.5Hz)

Example 15

Preparation of (i)-cis-7|3-(2-(2-tritylamino-4-thiazolyl)-2-methoxyiminoacetamido7-4p-methyl-2-t-butyloxycarbonyl-1-azabicyclo/Zi-, 2.07oct- 2-on-8-on:

The same method as in example 13 is used, with the exception that 202 mg (0.7 mmol) (-)-cis-7P-amino-4P-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4 is used as starting compound ", 2, Q7oct-2-en-8-one. As a result, 251 mg (53%) of the desired compound is obtained.

Melting point: 201.0 - 202.0°C

IR(KBr)V^ks: 3225, 1780 (sh), 1760, 1725, 1668, 1635

NMR (CDCl 3 ^CD 3 OD) Hppm): 7.35 (1/2H, s), 7.30 (15H, .s), 6.%

(1/2H, s), 6.14 (1H, d, J=2Hz), 5.38 (1H, d-d, J=5Hz), 4.09 (3/2H, s), 4.01 (3 /2H, s), 3.96 (1H, m), 2.45 (1H, m), 2.05 (2H, m),,1.53 (9/2H, s), 1.52 (9 /2H, s), 1.16 (3H, d-d,

J=7.5Hz)

Example 16

Preparation of (-)-cis-7|3-(2-(2-amino-4-thiazolyl)-2-methoxy-iminoacetamido7-|4(3-methyl-2-carboxy-1-azabicyclo/4\ 2, 07Oct-2-A-8-Wed:

The same method as in example 14 is used, with the exception that 70 mg of (-)-cis-7P-(2-(2-tritylamino-4-thiazolyl)-2-methoxyiminoacetamido7-4(3-methyl- 2-t-Butyloxycarbonyl-1-azabicyclo/Zi-,2,Q7oct-2-en-8-one, prepared in Example 15. As a result, 22 mg (56%) of the desired compound is obtained.

IR(KBr)V^ks: 3460, 3280, 1780 (sh), 1770, 1670, 1630

NMR(DMS0-d6) <f (ppm): 9.24 (1/2H, d, J=8Hz), 9.17 (1/2H, d, J=8Hz), 7.50 (1/2H, s), 7.25 (2H, m), 6.78 (1/2H, s), 6.10 (1H,: d, J=2Hz), 5.47 (1H, m), 4.00 ( 3/2H, s), 3.85 (3/2H, s), 2.60 (br, partially overlapping with the signal for DMS0-dg), 1.91 (2H, m), 1.12 (3H, d-d ,.J=7.5Hz)

. Example 17 Preparation of (-)-cis-7P-(2-(2-chloroacetylamino-4-thiazolyl)-2-syn-methoxyiminoacetamido7-4a-methyl-2-carboxy-1-azabicyclo-phe, 2,07oct-2 -en-8-on:

"'., '";• 172 mg (0.62 mmol) of (2-chloroacetylamino-4-thiazolyl)-2-syn-methoxyiminoacetic acid is suspended in 3.6 ml of anhydrous dichloromethane and 68.9 mg (0.68 mmolLtriethylamine to make the solution homogeneous.While cooling in an ice/

, sodium chloride bath, 129 mg (0.62 mmol) of phosphorus pentachloride are added to the mixture with stirring and the resulting mixture with stirring•and the resulting mixture is stirred for 1.5 hours. 13.8 ml of n-hexane are added to the mixture and the supernatant is removed by decantation. 1.3 ml of anhydrous tetrahydrofuran is added to the residue to obtain an acid chloride solution.

On the other hand, 160 mg (0.52 mmol) of trifluoroacetate (-)-cis-7(3-amino-4a-methyl-2-carboxy-1;-azabicyclo[4,2,Q]oct-2-ene-8 -one, prepared in Reference Example 13, in 1 ml of 50% tetrahydrofuran/water, and 209 mg (2.06 mmol) of triethylamine are added. The mixture is added to the acid chloride solution under ice cooling and stirring. After stirring at the same temperature for 1.5 hours, the mixture is adjusted to pH 4-5 with 1N hydrochloric acid and extracted with 10 ml of ethyl acetate. 3 times. The ethyl acetate extracts are washed with sodium chloride solution, dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 147 mg. (52.1%) of the desired connection.

IR(KBr)Y°mks: 17.65, 1680

PMR(DMS0-d5) é (ppm): 7.40 (1H, s), 6.32 (1H, d, J=5.2Hz), 5.53 (1H, m), 4.35 (2H, s), 3.90 (3H, s), 2.50 (1H, m), 1.90-1.27 (2H, m), 1.10 (3H, d, J=7.5Hz)

Example 18

Preparation of (<i>)-cis-7P-(2-(2-amino-4-thiazolyl)-2-syn-methoxyiminoacetamido7-4a-methyl-1-azabicyclo/4\ 2,Q7oct-2-ene-8 -one-2-carboxylic acid

147 mg (0.321 mmol) of the chloroacetyl compound prepared in Example 17 is dissolved in 0.5 ml of dimethylsulfoxide and 2.5 ml of dimethylformamide, and 47 mg (0.64 mmol) of thiourea is added at room temperature with stirring. The mixture is stirred for 14 hours. After adding ether, the supernatant is removed by decantation and the residue is dissolved in a small amount of dimethyl sulphoxide. The solution is adsorbed on a swab packed with 10 ml "Diaion HP-10". The rinse is washed with 240 ml of water and eluted with a solvent consisting of methanol and water in a volume ratio of 1:10 to 1:2. The eluates are collected and the methanol is removed under reduced pressure. The residue is again adsorbed on a sponge packed with 10 ml of "Diaion HP-10" and the sponge is washed with 500 ml of water. The elution is carried out with a solvent consisting of methanol and water in the volume ratio 1: 1. The eluates are collected and concentrated under reduced pressure to obtain 50.2 mg (41.1%) of the desired compound.

IR(KBr)y£mks: 1760, 1680, 1655

PMR(DMS0-d6) S (ppm): 9.27 (1H, d, J=9.0Hz), 7.15 (2H, br), 6.75. (1H, s), 6.31 (1H, d, J=4.2Hz), 5.58 (1H, br), 3.85

(3H, s), 2.60 (1H, m), 1.67 (2H, br), 1.08 (3H, d, J=8Hz)

Example 19

Preparation of (-)-cis-7-(2-(2-chloroacetylamino-4-thiazolyl)-2-syn-methoxyiminoacetamido7-1-azabicyclo[5,2,07oct-2-en-8-one-2-carboxylic acid] :

54.2 mg] (0.195 mmol) of 2-chloroacetylamino-4-thiazolyl-2-syn-methoxyiminoacetic acid (syn-type) is suspended in 0.98 ml of anhydrous methylene chloride and 23.48 mg (0.195 mmol) of triethylamine is added. 40.8 mg (0.195 mmol) of phosphorus pentachloride is added to the reaction mixture under ice-cooling. After stirring for 20 minutes, 3.92 ml of hexane are added, and the supernatant is removed by decantation. The residue is dissolved in 1.96 ml of tetrahydrofuran to obtain an acid chloride solution. On the other hand, 45.9 mg (0.155 mmol) of trifluoroacetate (-)-cis-7-amino-2-carboxy-1-azabicyclo/4", 2,07-oct-2-en-8-one, prepared in Reference Example 8, in 2 ml of 50% tetrahydrofuran/water, and 47.4 mg (0.469 mmol) of triethylamine are added. To this solution is added the above acid chloride solution under ice-cooling and the mixture is stirred for 2 hours. The mixture is adjusted to pH 2, 0 with 10% hydrochloric acid and extracted 3 times with ethyl acetate. The extracts were washed with saturated sodium chloride solution. The washed solution was dried with anhydrous sodium sulfate and concentrated to obtain 80 mg of the desired compound as a pale yellow powder.

orr,"1

IR(KBr)Y£mks: 1760, 1710, 1660

PMR(DMS0-d6) & (ppm): 16.64 (1H, br), 9.39 (1H, d, J=8.8Hz), 7.47 (1H, s), 6.31 (1H, t), 5.51 (1H, d-d, J=5.5, 8.8Hz), 4.38 (2H, s), 3.89 (3H, s), 2.54-0.8 (4H, m)

Example 20

Preparation of (-)-cis-7-(2-(2-amino-4-thiazolyl)-2-syn-methoxyacetamido7-1-azabicyclo/4", 2.07<->kt-2-en-8- on-2-carboxylic acid:

80 mg of the compound prepared in example 19 is dissolved in 0.96 ml of dimethylacetamide. 27.5 mg of thiourea is added to the solution at room temperature with stirring, and mix-N

no stirring for 14 hours. After adding ether, the supernatant is removed by decantation to obtain a red oily residue. The residue is purified by chromatography using "Diaion HP-10" to obtain 19.2 mg of the desired compound

about" 1

IR(KBr)v^ks: 1760'1670'16''30

PMR(DMSO-dg) é (ppm): 9.26 (1H, d, J=8.6Hz), 7.11 (2H, br), 6.75 (1H, s), 6.30 (1H, t), 5.47 (1H, d-d, J=5!.4 8.8 Hz), 3.8.9 (3H, s), 2.5-1.0 (4H, m)

Example 21

Preparation of (-)-cis-7(3-(2-thienylacetamido)-4oc-acetoxy-1-azabicyclo/4,2,07oct-2-ene-8-2-carboxylic acid:

126 mg of (i)-cis-7(3-amino-4oc-acetoxy-1-azabicyclo/4,2,07oct-2-en-8-one-2-carboxylic acid, prepared in reference example 14, is dissolved in 3.0 ml of dioxane and 4.0 ml of water. The solution is cooled in an ice/sodium chloride bath. To the solution is added 105 mg of sodium bicarbonate and also 84 mg of 2-thienylacetyl chloride dissolved in 1 ml of dioxane. The mixture

toss for 1 hour. The reaction mixture is then adjusted to pH 2.0 with 1N hydrochloric acid and extracted 3 times with ethyl acetate. The extracts are combined and washed with saturated sodium chloride solution. The washed solution is dried with anhydrous sodium sulfate and filtered. The filtrate is concentrated and the concentrate is fed onto a funnel saturated with 20 g of silicon dioxide gel. The elution is carried out with a mixture of chloroform and ethanol in the volume ratio 20:1. The fractions containing the desired compound are combined and concentrated to dryness, whereby 89.1 mg of the desired compound is obtained as a pale yellow powder. Dividend 47%. The properties of the compound are listed below.

orr,"1

IR(KBr)V™ks: 1-780, 1745, 1660

NMR(CDC13+ GD30D) in (ppm): 7.27-6.93 (3H, m), 6.39 (1H, d, J=5.4Hz), 5.43 (1H, d, J=4, 9Hz), 5.40 (1H, m), 3.79 (2H, s), 2.10-1.26 (2H, m), 2.06 (3H, s)

Antibacterial activities of the compounds prepared in Examples 4, 6, 8, 10, 12, 14, 16, 18, 20 and 21 are determined by the heart infusion agar dilution method (pH 2.7). The results are shown in the following table. Cefazolin is used as a reference. Example 22 Preparation of (-)-cis-7-phenylacetamido-1-azabicyclo/^,2,0j-oct-2-en-8-one-2-carboxylic acid

50.1 mg (0.28 mmol) (-)-cis-7-amino-1-azabicyclo-/Zt, 2,07oct-2-en-8-one-2-carboxylic acid, prepared as in reference example 16 (or 8), is dissolved in 1 ml of water and 2 ml of acetone, and 55.5 mg of sodium bicarbonate is added. To the mixture is added 0.3 ml of 1N phenylacetyl chloride/methylene chloride solution at a temperature of -20°C and the mixture is stirred at the same temperature for 20 minutes. After the temperature has been raised to room temperature, the reaction mixture is stirred for 2 hours. The mixture is then adjusted to pH 1.5 with 10% hydrochloric acid under ice-cooling and extracted 3 times with 5 ml of ethyl acetate. The extract is washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crystals are recovered by filtration and washed with a small amount of ethyl acetate, whereby 39.7 mg (67.8%) of the desired compound is obtained. The properties of the compound are listed below. Melting point: 192.0 - 193.0°C.

-1

IR(KBr)V^ks: 1770'1690'1650'1615

NMR(CD 3 OD) /(ppm): 7.22 (5H, s), 6.40 (1H, 5, J=4.0), 5.33 (1H, d, J=5i0), 3.57 ( 2H, s), 2.5-1.5 (9H, m)

Example 25

Preparation of (-)-cis-7(3-phenylacetamide-4oc-methyl-1-azabicyclo/5-,2,Q7oct-2-en-8-one-2-carboxylic acid

399 mg (1.28 mmol) trifluoroacetate of (-)-7|3-amino-4a-methyl-1-azabicyclo/5,2,07oct-2-en-8-one-2-carboxylic acid, prepared as in reference example 18 (or 13), dissolve in 6.4 ml of water and 12.8 ml of acetone, and 432 mg (5.14 mmol) of sodium bicarbonate are added. The Blaricling is stirred under ice-cooling. After adding Tav' 258.5 mg (5.14 mmol) phenylacetyl-

chloride, the mixture is allowed to react under ice-cooling for 3 hours. The reaction mixture is adjusted to pH 3 with 0.1N hydrochloric acid and extracted 5 times with 10 ml of ethyl acetate. The extract is washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated under reduced pressure. Ether is added to the concentrate and the crystals formed are recovered by filtration. Thus, 242.7 mg of the desired compound are obtained.

The aqueous layer obtained by the above-mentioned ethyl acetate extraction is extracted again 5 times with 10 ml of ethyl acetate. The above-described crystallization process is repeated, whereby 27.6 mg of the desired compound is obtained. Saturated sodium chloride solution is added to the remaining water layers and the mixture is extracted 5 times with 10 ml of ethyl acetate. The above-mentioned crystallization process is repeated, whereby 21.3 mg of the desired compound is obtained. The total amount of the desired compound is 293.6 mg. Yield 72.6%. The properties of the compound are listed below.

orr,"1

IR(KBr)Ymax: 1775'1698'1655

NMR(CD 3 OD) δ (ppm): 7.31 (5H, s), 6.48 (1H, d, J=5.0), 5.42 (1H, d, J=5.2), 3, 85 (1H, m), 3.60 (2H, s), 2.62 (1H, m), 1.60 (2H, m), 1.10 (3H, d, J=7.5)

Example 24

Preparation of (-)-cis-7-cyanoacetamido-2-t-butyloxycarbonyl-r-azabicyclo/2+, 2,07oct-2-en-8-one

42.5 mg (0.5 mmol) cyanoacetic acid and 57.5 mg (0.5 mmol) N-hydroxysuccinimide are dissolved in 2.5 ml dried dioxane, and 103.3 mg (0.5 mmol) N,N-dicyclohexylcarbodiimide is added. The mixture is stirred at room temperature for 1 hour. The resulting crystals are removed by filtration and the filtrate is concentrated

under reduced pressure. 5 ml of dried dichloromethane is added to the residue obtained and insoluble material is removed by filtration. three ring to obtain a solution of the succinimide ester of cyanoacetic acid.

On the other hand, 123 mg (0.45 mmol) of (-)-cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo-2,Q7oct-2-en-8-one (in the subsequent calculated as the amino compound) and 0.077 ml of triethylamine in 5 ml of dried dichloromethane. To this solution is added the above solution of the succinimide ester of cyanoacetic acid. The mixture is stirred at room temperature for 4 hours and then concentrated under reduced pressure. The oily product thus obtained is subjected to silicon dioxide gel bubble chromatography using a mixture of chloroform and methanol in the volume ratio 15:1. The fractions containing the desired compound are combined and concentrated under reduced pressure to obtain 112 mg of the desired compound. Yield 73.4%.

IRCNaCiyQ^: 3310, 2265, 1765, 1720, 1680

NMR(CDCl3)<f (ppm): 7.90 (1H), 6.32 (1H, m), 5.35 (1H, m),

3.75 (1H, m), 2.03 (6H, m), 1.52 (9H, s)

Example 25

Preparation of (<i>)-cis-7-[2-(2-furyl)-2-syn-methoxyimino-acetamido7-2-t-butyloxycarbonyl-1-azabicyclo([5-, 2,07oct-2-ene -8-Mon

The same method as in example 24 is repeated with the exception that instead of cyanoacetic acid, 158 mg (0.934 mmol) of 2-(2-furyl)-2-methoxyiminoacetic acid is used, and that 247 mg (0.9 mmol) of the amino compound is used . As a result

1313 mg of the desired compound is obtained.

Yield: 89.3%

-1

IR(NaCl)V°<m>ks: 3310, 1765, 1725, 1715, 1685, 1655

NMR(CDCl 3 ) "f (ppm): 7.65 (1H, m), 7.52 (1H, m), 6.73 (1H, m), 6.46 (1H, m), 6.29 ( 1H, m), 5.40 (1H, m), 4.02 (3H, s), 3.78 (1H, m), 2.19 (4H, m), 1.50 (9H, s)

Example 26

Preparation of (-)-cis-7-cyanomethylthioacetamido-2-t-butyloxycarbonyl-1-azabiecyclo/4,2,Q7oct-2-en-8-one:

The same method as in example 24 is repeated, with the exception that instead of cyanoacetic acid, 65.6 mg (0.5 mmol) of cyanomethylthioacetic acid is used. As a result, 136.5 mg of the desired compound is obtained. Yield 86.3%.

IR(NaCl)V£mks: 3315, 2250, 1760, 1725, 1715., 1675, 1655, 1650

NMR(CDCl 3 ) δ (ppm): 7.90 (1H, m), 6.32 (1H, m), 5.38 (1H, m), 3.72 (5H, m), 2.18 (4H , m), 1.52 (9H, s)

Example 27

Preparation of (-)-cis-7-(2-(2-furyl)-2-hydroxy-iminoacet-amido72-t-butyloxycarbonyl-1-azabicyclo/4", 2,07oct-2-en-8-one:

The same method as in example 24 is repeated with the exception that instead of cyanoacetic acid, 156 mg (1 mmol) of 2-(2-furyl)-2-hydroxyiminoacetic acid is used, and that 137 mg (0.5 mmol) of the amino compound is used. As a result, 115 mg of the desired compound is obtained. Yield: 61.3%.

IR(NaCl)V^ksr7 3290, 1770, 1720, 1690, 1680, 1655

NMR(CDCl 3 ) ^(ppm): 7.51 (4H, m), 6.49 (1H, m), 6.28 (1H, m), 5.46 (1H, m), 3.88 (1H , m), 2.18 (4H, m), 1.49 (9H, s) Example 28

Preparation of (-)-cis-7-(2-(2-thienyl)-2-hydroxyiminoacet-amido7-2-t-butyloxycarbonyl-1-a^abicyclo/Zr, 2,07oct-2-en-8-one:

The same method as in example 24 is repeated with the exception that instead of cyanoacetic acid, 85.6 mg (0.5 mmol) of 2-(2-thienyl)-2-hydroxyiminoacetic acid is used, and that 137 mg (0.5 mmol ) of the amino compound. As a result, 103 mg of the desired compound are obtained. Yield: 52.6%.

, -1

IR(KBr)V^ks: 1760, 1720, 1675, 1655, 1650

NMR(CDCl 3 ) δ (ppm): 8.02 (1H, m), 7.22 (4H, m), 6.18 (1H, m), 5.37 (1H, m), 3.78 (1H , m), 2.09 (4H, m), 1.46 (9H,'s) Example 29

Preparation of (<i>)-cis-7-(1-tetrazolyl)acetamido-2-t-butyloxycarbonyl-1-azabicyclo/4,2,07oct-2-en-8-one:

79 mg (0.55 mmol) of (1-terazolyl)acetic acid and 137 mg (0.5 mmol) of the amino compound are dissolved in 5 ml of N,N-dimethylformamide and 113 mg (0.55 mmol) of N,N-dicyclohexylcarbodiimide are added. The mixture is stirred at room temperature for 2 hours and then 50 ml IN sodium bicarbonate solution is added. The resulting crystals are removed by filtration and the filtrate is extracted with 50 ml of ethyl acetate twice. The ethyl acetate layers are combined, dried with magnesium sulfate and concentrated under reduced pressure. The oily product thus obtained is purified by silica gel chromatography to obtain 157 mg of the desired compound. Yield: 90.1% -1

IR(NaCl)V^ks: 3290, 1765, 1725, 1715, 1685, 1680, 1670, 1635

NMR(CDCl 3 ) S (ppm): 8.94 (1H, s), 8.24 (1H, m), 6.29 (1H, m), 5.61 (3H, m), 3.77 (1H , m), 2.16 (4H, m), 1.49 (1H, s) Example 30

Preparation of (-)-cis-7-(1-tetrazolyl)acetamido-2-t-butyloxycarbonyl-1-azabicyclo/4", 2,07oct-2-en-8-one:

The same method as in example 29 is repeated with the exception that 79 mg (0.55 mmol) of (2-tetrazolyl)acetic acid is used instead of (1-tetrazolyl)acetic acid. As a result is achieved

142 mg" of the desired compound. Yield 8; 1.4%.

about" 1

IR(rNaCl)max: 3300'1760'1725'1705'1690

NMR(CDCl 3 ) Å (ppm): 8.60 (1H, m), 8.52 (1H, s), 6.29 (1H, m), 5.47 (2H, s), 5.35 (1H , m), 3.72 (1H, m), 2.15 (4H, m), 1750 (9H, s)

Example 31

Preparation of (-)-cis-7-(4-pyridyl)thioacetamido-2-t-butyloxycarbonyl-1-azabicyclo[5-,2,07oct-2-en-8-one:

The same method as in example 29 is repeated with the exception that instead of (1-tetrazolyl)acetic acid, 181 mg (0.725 mmol) of hydrogen bromide of 4-pyridylthioacetic acid is used. As a result, 148.6 mg of the desired compound is obtained. Yield: 76.3%.

IR(NaCl)V£mk^: 3290, 1775, 1725, 1715, 1690

NMR(CDCl 3 )£ (ppm): 8.38 (2H, m), 8.24 (1H, m), 7.25 (2H, m), 6.25 (1H, m), 5.35 (1H , m), 3.78 (2H, s), 3.78 (1H, m), 2.13 (4H, m), 1.52 (9H, s)

Example 32

Preparation of (-)-cis-7-(2-tetrazolyl)acetamido-2-carboxyl-azabicyclo/4,2,07oct-2-en-8-one: 3 ml of denatured trifluoroacetic acid is added to 150 mg (0.431 mmol) ( - )-cis-7-v(,2'-tetra2olyl)acetamido=2-t-butyloxycarbonyl-1-azabicyclo^4\ 2,07oct-2-en-8-one (hereinafter referred to as the ester compound), prepared in example 30. The solution is stirred for 10 minutes and then concentrated under reduced pressure at room temperature. 10 ml of ether is added to the resulting highly viscous substance. The mixture is stirred for 20 minutes and then filtered to obtain a solid product. The product is dissolved in 2 ml of methanol and the mixture is left in an ice bath for 1 hour. The resulting crystals are filtered off and dried, whereby 11.5 mg of the desired compound is obtained. Yield: 87.3%.

-1

IR(KBr)Y£<m>ks: 3340, 1765, 1720, 1685, 1635, 1555

Example 53

Preparation of (-)-cis-7-(1-tetrazolyl)-acetamido-2-carboxyl-azabicyclo[5-,2,Q7oct-2-en-8-one:

The same method as in example 32 is repeated, with the exception that 70 mg (0.201 mmol) (-)-cis-7-(1-tetrazolyl)acetamido-2-t-butyloxycarbonyl-1-azabicyclo/4 is used instead of the ester compound, 2,07oct-2-en-8-one, prepared in Example 29. As a result, 53.6 mg of the desired compound is obtained. Yield: 91.2%.

IR(KBr)V^kg: 3280, 1770, 1725, 1675, 1635, 1565

Example 54

Preparation of (-)-cis-7-(4-pyridyl)thioacetamido-2-carboxy-1-azabicyclo/5.,2,07oct-2-en-8-one:

The same method as in example 32 is repeated, with the exception that instead of the ester compound, 105 mg (0.27 mmol) (-)-cis-7-(4-pyridyl)thioacetamido-2-t-butyloxycarbonyl-1-azabicyclo/ ^., 2,07oct-2-en-8-one, prepared in Example 31. As a result, 70 mg of the desired compound is obtained. Yield: 78.0%.

,-1

IR(KBr)V^ks: 1760, 1708, 1680, 1630

Example 35

Preparation of (-)-cis-7-cyanacetamido-2-carboxy-1-azabi-cylo/4,2,07oct-2-en-8-one:

The same method as in example 32 is repeated with the exception that instead of the ester compound, 112 mg (0.367 mmol) (<i>)-cis-7-cyanacetamido-2-t-butyloxycarbonyl-1-azabicyclo/^., 2.07oct -2-en-8-one, prepared in Example 24.

As a result, 81 mg of the desired compound is obtained. Yield: 88.6%.

orrT1

IR(KBrW™ks: 2270, 1755, 1708, 1690, 1655, I63O

Example 56

Preparation of (-)-cis-7-(2-(2-furyl)-2-syn-methoxyimino-acetamido7-2-carboxy-1-azabicyclo/4', 2,Q7oct-2-en-8-one:

The same method as in example 32 is repeated with the exception that instead of the ester compound, 131 mg (0.336 mmol) of (-)-cis-7-(2-(2-furyl)-2-methoxyiminoacet-amido7-2-t-butyloxycarbonyl) is used -1-azabicyclo/5-,2,07oct-2-en-8-one, prepared in example 25. As a result, 78 mg of the desired compound are obtained. Yield: 69.6%.

IR(KBr)Y^k<s:>1760, 1690, 1660, 1640, 1620

Example 37

Preparation of (-)-cis-7-cyanomethylthioacetamido-2-carboxyl-azabicyclo/4,2,07oct-2-en-8-one:

The same method as in example 32 is repeated with the exception that instead of the ester compound 136 mg (0.387 mmol) (-)-cis-7-cyamethyl-thioacetamido-2-t-butyloxycarbonyl-1-azabicyclo/5-, 2.07oct -2-en-8-one, prepared in Example 26. As a result, 100 mg of the desired compound is obtained. Dividend.78.0%.

IR(KBrW£mks: 2250, 1755, 1730, 1665, 1635

Example 58

Preparation of (-)-cis-7-(2-(2-furyl)-2-hydroxyiminoacet-amido7-2-carboxy-1-azabicyclo/5., 2,07oct-2-en-8-one:

The same method as in example 32 is repeated, with the exception that instead of the ester compound 115 mg (0.3 mmol) of (-)-cis-7-(2-(2-furyl)-2-hydroxyiminoacetamido7-2-t-butyloxycarbonyl) is used -1-azabicyclo/4\ 2,07oct-2-en-8-one, prepared in Example 27. As a result, 92 mg of the desired compound is obtained. Yield 69%.

orr,"1

,- IR(KBr)Vmax: 1760'1720'1680'1650'1535

Example 59

Preparation of (<i>)-cis-7-(2-(2-thienyl)-2-hydroxyimino-acetamido7-2-carboxy-1-azabicyclo[4,2,07oct-2-en-8-one]:

The same method as in example 32 is repeated, with the exception that 103 mg (0.263 mmol) (<i>)-cis-7-(2-(2-thienyl)-2-hydroxyiminoacet-amido7-2-t is used instead of the ester compound -butyloxycarbonyl-1-azabicyclo/5,2,07oct-2-en-8-one, prepared in Example 28. As a result, 71.6 mg of the desired compound are obtained. Yield: 81.2%.

-1

IR(KBr)Y^ks<:>1760, 1690, 1665, I63O

Example 40

Preparation of (-)-cis-7-(2-(5,6-dihydro-1,4-dithiinyl)-acetamido7-2-carboxy-1-a:zabicyclo-4', 2,Q7oct-2-ene-8 -on:

176 mg (1 mmol) of 2-(5,6-dihydroxy-1,4-dithianyl)-acetic acid and 115 mg (1 mmol) of N-hydroxysuccinimide are dissolved in 6 ml of dried dioxane, and 206 mg (1 mmol) of N ,N-dicyclohexylcarbodiimide is added. Bian.cL:i.ngen is stirred at room temperature for 2 hours. The resulting crystals are removed by filtration and the filtrate is concentrated. The residue is dissolved in 6 ml of dried dichloromethane.

On the other hand, 109 mg of hydrochloride of (i)-cis-7-amino-2-carboxy-l-azabicyclo/5,2,Q7oct-2-en-8-one, prepared by the same method as in Reference Example 16, is dissolved and 21 ml of triethylamine in 6 ml of dried dichloromethane. To this solution is added the above-mentioned solution and the mixture is stirred at room temperature for 10 hours, after which it is concentrated under reduced pressure. 10 ml of water is added to the residue and insoluble material is removed by filtration. The filtrate is adjusted to pH 2.5 with 2N hydrochloric acid. After cooling, the solution is filtered and the filtered material is dried to obtain 120 mg of the desired compound. Yield: 70.5%.

-1

IR(KBr<K>/£<m>ks: 3275, 1765, 1690, 1648, 1630

Example 41

Preparation of (-)-cis-7-^XR)-2-phenyl-2-hydroxyacetamido7-2-carboxy-1-azabicyclo-[4",2,07oct-2-en-8-one:

The same method as in example 40 is repeated with the exception that instead of 2-(5,6-dihydroxy-1,4-dithianyl)acetic acid, 152 mg of D-mandelic acid is used. As a result, 142.5 mg of the desired compound is obtained. Yield: 90.1%.

about"1 IR(KBr)Vmax: 3325' 1770' 1715' l690' l655' 1635

Example 42

Preparation of (-)-cis-7-(2-phenyl-2-carboxy-acetamido)-2-carboxy-1-azabicyclo/4", 2,07oct-2-en-8-one

90 mg (0.5 mmol) of phenylmalonic acid are dissolved in a mixture of 5 ml of dried ether and 61 mg (0.51 mmol) of thionyl chloride. A drop of N,N-dimethylformamide is added to the heating mixture and the mixture is heated under reflux for 2 hours. The mixture is then concentrated under reduced pressure. 5 ml of dried benzene is added to the residue and the mixture is concentrated again. The residue is dissolved in dry ether and a solution is added to the solution, in which 103 mg (0.47 mmol) hydrochloride of (-)-cis-7-amino-2-carboxy-1-azabicyclo/Z)., 2.07Oct-2 -en-8-one, prepared by the same method as in reference example 16,

is dissolved in 2 ml of 0.5N sodium bicarbonate solution and 1 ml of ether under ice-cooling. The mixture is stirred at room temperature for 1 hour and then adjusted to pH 2 with IN hydrochloric acid. The mixture is extracted with 20 ml of ethyl acetate 5 times. The extract is evaporated to dryness and the solid obtained is completely suspended in 5 ml of ether. The suspension is filtered and dried to obtain 100 mg of the desired compound. Yield: 60.1%.

IR(KBr)V™ks: 1755, 1720, 1700, 1660, 1630

Example 43

Preparation of triethylamine salt of (-)-cis-7-(2-phenyl-2-sulfo-acetamido)-2-carboxy-1-azabicyclo/4",2,Q7oct-2-en-8-one:

109 mg (0.5 mmol) hydrochloride of (-)-cis-7-amino-2-carboxy-1-azabicyclo/£,2,Q7oct-2-en-8-one and 101 mg of triethylamine are dissolved in 6 ml of dried dichloromethane. 105 mg (0.5 mmol) of oc-triethylammonium sulph or enylacetic acid-ethylcarbonic anhydride is added to the solution under ice-cooling. The mixture is stirred for 1.5 hours, after which the solvent is removed by distillation. The rest is revealed. in 5 ml of water and the solution is adjusted to pH 1.5 with IN hydrochloric acid. The solution is then extracted with 10 ml of n-butanol twice, the n-butanol layers are combined and concentrated under reduced pressure. The solid obtained is washed with 2 ml of dichloromethane and dried to obtain 196 mg of the desired compound. Yield: 81.3%.

IR(KB) £mks: 3275, 1770, 1725, 1682, 1628

Antibacterial activities of the compounds prepared in Examples 32, 33, 34, 36, 38, 41 and 42 are determined by the heart infusion agar/dilution method (pH 7.2). The results are shown in the table below.

1: Staphylococcus aureus 209-p 2: Staphylococcus aureus Smith 3: Staphylococcus epidermidis 4: Escherichia coli NIHJC-2

5: Escherichia coli Juhl 6: Klebsiella pneumoniae 8045

7: Klebsiella pneumoniae 8045

8: Serratia marcescens T-26

9: Serratia marcescens T-55

10: Proteus mirabilis 1287

11: Proteus vulgaris 6897

12: Proteus morganii KY4298

13: Proteus courtship KY4289

14: Pseudomonas aeruginosa 145 15: Pseudomonas putida F264

m: The compound prepared in example 32 n: " 33 o: " "<»>34 p:""ii36q: »»<»>38 r: " " " 41 s: " " " 42 t: Cefazolin

Example 44

Preparation of 7|3-(TR)-2-p-hydroxyphenyl-2-aminoacetamido7-4a-methyl-1-azabicyclo/4\2,07oct-2-en-8-one-2-carboxylic acid:

The same method as in example 12 is used, with the exception that 40 mg of (i)-cis-7(3-/TR)-2-p-hydroxyphenyl-2-t-butyloxycarbonyl-aminoacetamido7-4a- is used as starting compound methyl-2-t-butyloxycarbonyl-1-azabicyclo-2,07oct-2-en-8-one, prepared by the same method as in example 9. As a result, 29.3 mg (85%) of the least polar isomer of the desired connection.

laj<-,1-0>:+12.8° IM phosphate buffer (pH 7.0), c=0.51

-1

IR(KBr)Y^mks: 3420, 3260, 1760, 1685

PMR(D2O)cf(ppm): 7.35 (2H, d, J=8.0), 6.96 (2H, d, J=8.0), 6.10 (1H, d, J=5 ,l), 5.45 (1H, d,; J=4.9), 5.11 (1H, s),

3.92 (1H, m), 2.45 (1H, m), 1.50-1.08 (2H, m), 1.01 (3H, d, J=7,D

Based on the strong antimicrobial activity shown in the table below, this compound is assigned the absolute 6(R)7(S) configuration.

Example 45

Preparation of 7β-[1R)-2-phenyl-2-(4-ethyl-2,3-dioxo-1-piperiazinylcarbonylamino)acetamido7-4oc-methyl-azabicyclo/4", 2,07oct-2-en-8- on-2-carboxylic acid:

/

The same method as in example 8 is used, with the exception that 55 mg (<i>)-cis-7(3-/lR)-2-phenyl-2-(4-ethyl-2,3- dioxo-1-piperazinylcarbonyl-amino)acetamido7-4oc-methyl-2-t-butyloxycarbonyl-1-azabicyclo-IJ\, 2,07oct-2-en-8-one (the least polar isomer), prepared

by the same method as in Example 7. As a result, 25.7 mg (72%) of the desired compound is obtained as a white powder.

/α705: -36.3° (MeOH, c = 1.0)

IR(KBr)V^k\ 3450, 3300, 1775, 1720, 1683, 1630

PMR(CD30D) <$(ppm): 9.90 (1H, d, J=6.4), 7.80 (5H, br), 6.39 (1H, d, J=4.9), 5 .46 (2H, d, J=4.9), 4.00 (2H, m), 3.85-

3.40 (6H, m), 2.48 (1H, br), 1.20 (5H, m), 1.04 (3H, d, J=7.4) Based on the strong antimicrobial activity as shown in the table below assigns this compound the absolute 6(R)7(S) configuration.

Example 46

Preparation of 7(3-/TR)-2-p-hydroxyphenyl-2-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)acetamido7-4oc-methyl-1-azabicyclo/2+, 2.07oct- 2-en-8-one-2-carboxylic acid:

The same method as in example 8 is used, with the exception that 57 mg of (<i>)-cis-7|3-/TR)-2-p-hydroxyphenyl-2-(4-ethyl-2,3 -dioxo-1-piperazinyl-carbonylamino)acetamido'7-4a-methyl-2-t-butyloxycarbonyl-1-azabicyclo/z;., 2,Q7oct-2-en-8-one, prepared by the same method as in example 7. As a result, 40 mg (78%) of the desired compound is obtained as a white powder.

/«7^<5>: -7.1° (MeOH, c = 1.0)

IR(KBrW£mk : 5430, 330, 1773, 1721, 1685, 1620 PRM(CD3Q1XPPm): 7.28 (2H, d, J=8.5), 6.78 (2H, d, J=8.5 ), 6.00 (1H, d, J=5,D, 5.37 (1H, s), 5.36 (1H, d, 'J=4.4),

4.00 (2H, m), 3.85-3.35 (6H, m), 2.40 (1H,. br), 1.37-1.05 (5H, m), 1.01 (3H , d, J=6.8)

Based on the strong antimicrobial activity shown in the table below, this compound is assigned the absolute 6(R)7(S) configuration.

Example 47

Preparation of 7(3-(TR)-2-p-hydroxyphenyl-2-aminoacetamido7-1-azabicyclo/4^,2,07oct-2-en-8-one-2-carboxylic acid:

The same method as in example 6 is used, with the exception that 78 mg of (<i>)-cis-7-/TR)-2-p-hydroxyphenyl-2-t-butyloxycarbonyl-amino-acetamido7-2 is used as starting material -t-butyloxycarbonyl-1-azabicyclo[4] 2,Q7oct-2-en-8-one, prepared by the same method as in Example 5. As a result, 29.5 mg (80%) of an isomer of the desired compound is obtained .

/a7^<5>°: +107.5°

IR(KBr)V^ks: 3450'3290'3090'1700 (sh)'1685'1640

PMR(D2O)cf (ppm): 7.36 (2H, d, J=8.8), 6.95 (2H, d, J=8.8), 6.06 (1H, t, J=3 .9), 5.40 (1H, d, J=4.6), 5.12 (1H, s),

3.84 (1H, m), 2.22 (2H, m), 1.62 (1H, m), 1.12 (1H, m)

Based on the strong antimicrobial activity shown in the table below, this compound is assigned the absolute 6(R)7(S) configuration.

Antibacterial activities of the compounds prepared in Examples 44, 45, 46 and 47 are determined by the heart infusion agar dilution method (pH 7.2). The results are shown in the following table.

The following reference examples illustrate the production of the output connections used in the examples.

Reference example 1

Preparation of (-)-cis-2-t-butyloxycarbonyl-7-azido-1-azabicyclo[5-,2,Q7oct-2-en-8-one with the formula:

The above compound is prepared by the following methods 1 and 2. In the following, cis and trans refer to the stereochemistry at the 3- or 4-position in the 2-azatidinone ring or at the 6- or 7-position in l-azabicyclo/4, The 2.07 octane ring.

1) Preparation of 2-(4-(3-butenyl)-3-azido-2-oxoazetidin-1-yl7-2-diethylphosphonoacetate-5-butyl ester

447 mg (1.78 mmol) of t-butyl-α-aminodiethylphosphonoacetate (the compound has the following properties: an oily product;

-1

IR(pure)V™kg: 3400, 1735-1745, 1020-1060; NMR(CDCl 3 ) S (ppm):

4.20 (d-q, 4H), 3.83 (d, 1H, J=20Hz), 1.76 (br, 2H), 1.50 (s, 9H), 1.35 (t, 6H); mass spectrum (m/e): 268 (M<+>)) is dissolved in 25 ml of anhydrous ether, and 164 mg (1.96 mmol) of 4-penten-1-al is added. The solution is stirred at room temperature for 1 hour and then 200 mg of molecular sieve ("4A, if the same molecular sieve is used in the following) and 150 mg of anhydrous magnesium sulfate are added to the solution. The mixture is stirred for 1 hour.

The reaction mixture is subjected to suction filtration and the filtrate is concentrated under reduced pressure to obtain a pale yellow oily product. Anhydrous benzene is added to the product and the mixture is concentrated under reduced pressure to obtain a pale yellow oily product. The presence of a Schiff base in the product is confirmed by nuclear magnetic resonance spectrum. The product is dissolved in 12.5 ml cyclohexane and 12.5 ml anhydrous benzene and 0.369 (2.66 mmol) triethylamine and 200 molecular sieve "4A" are added. 319 mg (2.66 mmol) of azidoacetyl chloride dissolved in 12.5 ml of cyclohexane are added dropwise to the mixture with stirring at room temperature for 1.5 hours. The reaction mixture is stirred for a further 30 minutes and diluted with 10 ml of benzene. reaction solution-

one is washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate solution, deionized water and saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain a brown oily product identified as a crude product of the desired compound. The oily product is lined on a packed spool

with 45 g of silica gel. The elution is carried out with a "mixture of n-hexane and ethyl acetate in the volume ratio 1:2 to obtain two types of isomers. The properties of the isomers are given below and they are identified as the isomers at the 3- and 4-position, i.e. 345 mg of the cis-isomer and 58 mg of trans-isomer.The total yield is 54.2%.

Cis isomer:

om"1 IR(CHCl3)>/^ks: 2120, 1775, 1770 (sh), 1750, 1740 (sh),

1645,

NMR(CDCl 3 ) S (ppm): 6.13-6.33 (1H, m), 4.93-5.17 (2H, m), 4.50-4.93 (2H, m), 3, 80-4.40 (5H, m), 1.93-2.17 (4H, m), 1.50 (9H, s), 1.33 (6H, t)

Trans isomer:

-1

IR(CHC13)V °<m>ks: 2120, 1780, 1755, 1750 (sh), 1650

NMR'(CDCl 3 ) δ (ppm): 5.43-6.20 (1H, m), 4.80-5.30 (2H, ,,m) , 3.75-4.75 (7H, m) , 2.0-2.50 (4H, m), 1.50 (9H, d), 1.17

(6H, m)

2) Preparation of (-)-cis-2-t-butyloxycarbonyl-7-azido-1-azabicyclo/4v2,07oct-2-en-8-one: — — ~v— — ———————— — 298 mg (0.716 mmol) of cis-2-^4-(3-butenyl)-3-azido-2-oxoazetidin-1-yl7-2-diethylphosphonoacetate-t-butyl ester, prepared above, is dissolved in 8.5 ml of dioxane and 2.5 ml of deionized water and 30 mg of osmium tetroxide are added. The solution is stirred for 30 minutes. To the black reaction solution, 496 mg (2.32 mmol) of powdered sodium periodide are added over the course of 20 minutes. After stirring for 1.5 hours, the reaction solution is extracted with 50 ml of ether three times. The ether extracts are combined and washed with saturated sodium chloride solution. The resulting solution is dried<*>with anhydrous sodium sulfate and concentrated under reduced pressure to obtain ... a dark brown oily product. The product is applied to a swab packed with 5 g of silicon dioxide gel and eluted with a solvent consisting of benzene and ethyl acetate in a volume ratio of 1:2.

xThe fractions which are positive for the 2,4-dinitrophenylhydrazine reaction are collected and concentrated to obtain 235 mg of a

oily product, which is the cis isomer of the aldehyde compound. The oily product is dissolved in 15 ml of anhydrous acetonitrile. To the solution is added 27.1 mg (0.563 mmol) of 50% sodium hydride in a stream of nitrogen under stirring at room temperature. After stirring for 20 minutes, the reaction mixture is poured into 20 ml of 2% aqueous acetic acid, and the solution is extracted with 50 ml of ether four times. The ether extracts are combined and washed with saturated sodium chloride solution. The resulting solution is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 180 mg of an oily product identified as a crude product of the desired cis compound.

The oily product is applied to a sponge packed with 5 g of silicon dioxide gel and eluted with a solvent consisting of n-hexane and ethyl acetate in a volume ratio of 3.5:1. This gives 91 mg of white crystals of the desired compound. Dividend 51%. The properties of the compound are as follows: Melting point: 64.5 - 65.5°C

-1

IR(CHCl3)V™ks: 2130, 1790, 1730, 1640

NMR(GiDCl3)J (ppm)': 6.30 (1H, t, J=4Hz), 4.93 (1H, d, J=5Hz), 3.80 (1H, q), 1.6-2 .6 (4H, m), 1.52 (9H, s)

Reference example 2

Preparation of (-)-trans-2-t-butyloxycarbonyl-7-azido-1-azebicyclo[,2,Q]oct-2-en-8-one of the formula:

767 mg (1.84 mmol) trans-2-(4-(3-butenyl)-3-azido-2-oxoazetidin-1-yl7-2-diethylphosphonoacetate-t-butyl ester, prepared as in reference example 1 - 1), is dissolved in 22 ml of dioxane and 6.5 ml of deionized water, and 100 mg of osmium tetroxide is added.

The mixture is stirred for 30 minutes. To the black reaction mixture, 1.5 g (7.04 mmol) of powdered sodium periodate is added over a period of 30 minutes. After stirring for 1 hour, the reaction mixture is extracted three times with 150 ml of ether. The ether extracts are combined and dried with anhydrous sodium sulfate. solution is concentrated under reduced pressure to obtain an oily product. The oily product is applied to a sponge packed with 20 g of silica gel and eluted with a solvent consisting of benzene and ethyl acetate in the volume ratio 1:2. This gives 561 mg of oily product from the fractions which are positive for the 2,4-dinitrophenylhydrazine reaction. The product is the trans compound of the :aldehyde compound. The product is dissolved in 6 mL of anhydrous acetonitrile and 61.4 mg (2.56 mmol) of 50% sodium hydride is added. The mixture is heated to a temperature of 50 °C and allowed to react for 10 minutes. The reaction mixture is poured into 6 ml of 2% aqueous acetic acid and extracted four times with 50 ml of ether. The ether extracts combined ined and dried with anhydrous sodium sulfate.

The resulting solution is concentrated under reduced pressure to obtain an oily product. The oily product is applied to a sponge packed with 20 g of silicon dioxide gel and eluted with a solvent consisting of n-hexane and ethyl acetate in a volume ratio of 3.5:1. This gives 218 mg of white crystals of the desired compound. The compound identified as the trans compound is the desired compound. The properties of the compound are as follows: Melting point: 80.5 - 81.5°C

about" 1

IR(CHCl3)Y^<m>ks: 2110, 1780, 1720, 1635

NMR(CDCl 3 ) / (ppm):- 6.27 (1H, t), ~4.28 (1H, d, J=2Hz), 3.53 (1H, q), 2.0-2.6 ( 4H,m), 1.63 (9H,s)

Reference example 5

Preparation of (-)-cis-2-carboxy-7-azido-1-azabicyclo-2,Q7oct-2-en-8-one of the formula:

55 mg (0.224 mmol) (-)-cis-2-t-butyloxycarbonyl-7- azido-1-azabicyclo/5-,2,07oct-2-en-8-one, prepared in Reference Example 1, is dissolved in 2 ml of trifluoroacetic acid and dissolved the mixture is left at room temperature for 10 minutes. The solution is concentrated under reduced pressure. Benzene is added to the concentrate and the resulting solution is concentrated under reduced pressure to give 51 mg of a yellow semi-solid. The properties of the substance are as follows, and it is identified as the desired carboxylic acid. Yield: 100%.

about"1'<

IR(CHC13)V max: 2120'1770 (sh)'1760'1715»1635

NMR(CD 3 OD) δ (ppm): 6.48 (1H, 5, J=4Hz), 5.10 (1H, d, J=5Hz),

.3.83 (1H, q), l.,l-2.5 (4H, m)

Reference example 4

Preparation of (<*>)-cis-2-t-butyloxycarbonyl-4-bromo-7-azido-1-azabicyclo/4,2,07oct-2-en-8-one of the formula:

in

50 mg (0.203 mmol) (<±>)-cis-2-t-butyloxycarbonyl-7-azido-1-azabicyclo[5-,2,07oct-2-en-8-one, prepared in reference example 1, is dissolved in 2 ml of anhydrous chloroform and 36.0 mg (0.202 mmol) of N-bromosuccinimide and a catalytic amount of azobis-isobutyronitrile are added. The mixture is heated under reflux and stirring for 30 minutes and then diluted with 5 ml of chloroform. The diluted solution is washed with 3 ml of water and 3 ml of saturated sodium chloride solution and dried with anhydrous

sodium sulfate. The resulting solution is concentrated under reduced pressure to obtain 53 mg of oily product.

The product is applied to a sponge packed with 4.0 g of silicon dioxide gel and eluted with a solvent consisting of n-hexane and ethyl acetate in a volume ratio of 3.5:1. This gives 23 mg of oily product. The product is identified as the desired cis compound based on the following properties. Yield: 33%.

-1

IR(CHC13W ™k£J: 2120, 1790, 1730, 1620

NMR(CDCl3),V(ppm): 6.33 (1H, d, J=6Hz), 5.07 (1H, d, J=5Hz),

4.93 (1H, m), 4.50-3.90 (1H, m), 2.50-1.72 (2H, m), 1.52

(9H, s)

Reference Example 5 Preparation of (-)-trans^-t-butyloxycarbonyl-^-bromo^-azido-1-azabicyclo/^,2,07oct-2-en-8-one of the formula

100 mg (0.407 mmol) (<i>)-trans-2-t-butyloxycarbonyl-7-azido-1-azabicyclo/Zi<->,2,07oct-2-en-é-one, prepared in reference example 2, is dissolved in 5 ml of anhydrous carbon tetrachloride and 72.4 mg of N-bromosuccinimide is added. The mixture is heated under reflux and. stir for 30 minutes. Then 10 ml of methylene chloride are added to the reaction mixture and the resulting

mixture is washed with 5 ml of deionized water and 5 ml of saturated sodium chloride solution. The resulting solution is dried with water

free sodium sulfate and concentrated under reduced pressure to obtain 102 mg of oily product. The oily product is applied to a sponge packed with 5 g of silicon dioxide gel and eluted with a solvent consisting of n-hexane and ethyl acetate in the volume ratio (3.5:1).

24 mg of oily product is obtained. The product is identified as the desired trans compound based on the following properties. Dividend 18.1%.

IR(CHCl3)V^ks: 2130, 1790, 1730, 1620

NMR(CDCl 3 ) cT(ppin): 6.23 (1H, d, J=6Hz), 5.93 (1H, m), 4.37 (1H, d), 4.00 (1H, m), 2 .93-1.93 (2H, m), 1.50 (9H, s).

Reference example. 6

Preparation of (<i>)-cis-2-t-butyloxycarbonyl-4a-acetoxy-7(3-azido-1-azabicyclo[4] 2,07oct-2-en-8-one of the formula:

75. mg (0.219 mmol) (<±>)-cis-2-t-butyloxycarbonyl-4-•bromo-7-azido-1-azabicyclo/4:,2,07oct-2-en-8-one, prepared as in reference example 4, dissolve in 2 ml of acetic acid. While protecting the reaction system from light, 39.4 mg is added

(0.241 mmol) of blue acetate to the solution and the mixture is stirred for 2 hours and 20 minutes. The reaction mixture is filtered and concentrated under reduced pressure to obtain an acetoxy crude product of the desired compound. The product is fed onto a sponge packed with 3.5 g of silica gel and eluted with a solvent consisting of n-hexane and ethyl acetate in a volume ratio of 3.5:1 to obtain 51 mg of oily product. The product is identified as the desired cisr compound based on the following properties. Yield: 72.1%.

- 1

■IR('CHC13)V max: 2130'1790'1750'1730 (sh)'1635

NMR(CDCl 3 ) <S'(ppm): 6.21 (1H, d, J=5Hz), 5.42 (1H, m), 5.01 (1H, d, J=5Hz), 3.95 ( 1H, m), 2.02 (3H, s), 2.6-1.7 (2H, m), 1.53 (9H, s)

Reference example 7

Preparation of (<i>)-cis-2-t-butyloxycarbonyl-4-methyl-7-azido-1-azabicyclo/4,2,07oct-2-en-8-one of the formula:

The above compound is prepared by the following methods 1 and 2.

1) Preparation of 2-(4-(2-methyl-3-butenyl)-3-azido-2-oxo-azetidin-1-yl7-2-diethylphosphonoacetate tert-butyl ester

2.13 g" (8 mmol) of t-butyl-α-aminodiethylphosphonoacetate are dissolved in 80 ml of anhydrous ether and 902 mg (9.2 mmol) of 3-methyl-4-pentenal are added with stirring. The mixture is stirred at

room temperature for 1 hour and 900 ml molecular sieve, ("4A") and 700 mg

■■_ magnesium sulphate is added. After stirring for 1.5 hours, the reaction mixture is subjected to suction filtration. The filtrate is concentrated to obtain a pale yellow oily product. 30 ml of anhydrous benzene is added to the product and the resulting solution is concentrated again to obtain 2.82 g of oily product. The presence of a Schiff base in production

tet is confirmed by NMR spectrum. The oily product is dissolved in 56 ml of dried cyclohexane and 56 ml of anhydrous benzene and 900.

mg of molecular sieve ("4A") and 1.67 ml (12 mmol) of triethylamine are added. To the mixture is added dropwise 1.43 g (12 mmol) of azidoacetyl chloride dissolved in 56 ml of dried cyclohexane in the volume of

1.5 hours at room temperature while stirring. The mixture is stirred for a further 30 minutes, after which 30 ml of benzene is added.

The mixture is transferred to a separatory funnel >and washed with 30 ml

each of 10% citric acid, saturated sodium chloride solution, sat

sodium bicarbonate solution and saturated sodium chloride solution in this order. The resulting solution is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2.8 g of oily product. The presence of a mixture of two main isomers in the product is confirmed by thin layer chromatography (silica gel, n-hexane/ethyl acetate (1:1)). The product is applied to a swab packed with 300 g of silica gel and eluted with a solvent

reagent consisting of a mixture of n-hexane and ethyl acetate in the volume ratio 1:1 to obtain 380 g (yield 11.0%) of the least polar isomer of the desired compound, 570 mg (yield 16.7%) of the most polar isomers of it

desired compound and 201 mg (yield 5.8%) of a mixture of the two isomers.

The properties of each isomer are described below.

From this data, the most polar isomer is identified as the cis isomer of the desired compound.

The least polar isomer:

IR(CHCl3)V<£>£<k>s": 2110, 1770, 1745

NMR(CT>ei3) é (ppm): 5.40-6.10 (1H, m), 5.27-4.90 (2.5H, m), 4.68 (0.5H, d), 4.23 (6H, m), 2.60-1.77 (3H, m), 1.53 (9H, s), 1.37 (6H, t, J=7.0Hz), 1.10 ( 3H, d, J=6.0Hz)

The most polar isomer (cis compound):

-1

IR(CHCl3)V£<m>ks: 2110, 1765, 1745

NMR(CDCl 3 )& (ppm): 5.45-6.13 (1H, m), 4.83-5.20 (2.5H, m), 4.67 (0.5H, d), 3, 97-4.45 (6H, m), 1.77-2.55 (3H, m), 1.50

(9H, s), 1.33 (6H, t), 1.08 (3H, d)

2) Preparation of (^~]j — cis-2-t-butyloxycarbonyl-4-methyl-7- azido-1-azabicyclo/4,2,07oct-2-en-8-one with the formula:

240 mg (0.56 mmol) tert-butyl ester of (-)-cis-2-(4-(2-methyl-3-butenyl)-3-azido-2-oxoazetidin-1-yl7-2-diethyl-phosphonoacetate , prepared in reference example 7-1), is dissolved in 6.6 ml of dioxane and 2 ml of deionized water. 20 mg of osmium tetroxide is added and the mixture is stirred for 10 minutes. To the black reaction solution, 390 mg (1.82 mmol) of powdered sodium periodate are added in small portions over the course of 30 minutes.

After stirring for 40 minutes, the reaction solution is extracted 3 times with 30 ml of ether and the extracts are combined. The combined extracts are washed with saturated sodium chloride solution, washed with anhydrous sodium chloride and concentrated to obtain 230 mg of oily product. The oily product is applied to a sponge packed with 6 g of silicon dioxide gel and eluted with a solvent consisting of benzene and ethyl acetate in a volume ratio of 1:2. The fractions positive for the 2,4-dinitrophenylhydrazine reaction are combined and concentrated to obtain 185 mg of oily product, which is the cis compound of the aldehyde compound. The product is immediately dissolved in 8 ml of anhydrous acetonitrile and 21.6 mg (0.45 mmol) of 50% sodium hydride is added to the solution in a stream of nitrogen under stirring at room temperature. After stirring for 30 minutes, the reaction solution is poured into 15 ml of 2% aqueous acetic acid and the mixed solution is extracted twice with 20 ml of ether. The ether layers are washed with saturated sodium chloride solution, washed with anhydrous sodium chloride and concentrated under reduced pressure to obtain an oily product. The product is identified as a crude product of the desired cis compound. The oily product is applied to a sponge packed with 20 g of silica gel and eluted with a solvent consisting of

n-hexane and ethyl acetate in the volume ratio 3.5:1. This gives 70 mg of the desired compound as a colorless, oily substance

product which crystallizes on standing. The product's properties are described below. Yield: 48.1%.

, -1

IR(KBr)\;°mks: 2110, 1784, 1715, 1623

NMR(CDCl 3 ) δ(ppm): 6.30 (4/5H, d, J=5.1Hz), 6.10 (1/5H, d,

J=2.7Hz), 4.98 (4/5H, d, J=5.0Hz), 4.89 (1/5H, d, J=5.0Hz), 3.60-3.90 (1H , m), 2.65 (1H, m), 1.70-1.80 (2H, m), 1.51 (9H, s), 1.20 (3/5H, d, J=8.0Hz ), 1.13 (12/5H, d, J=8.0Hz)

The crystals obtained are identified as a mixture of the 4α-methyl isomer and the 4(3-methyl isomer in a ratio of about 4:1 based on the above-mentioned NMR data.

Reference example 8

Preparation of (-)-cis-2-carboxy-7-amino-1-azabicyclo/4,2,07oct-2-en-8-one: 91 mg of (i)-cis-2=carboxy-7-az' Ido-1-azabicyclo/4, 2,07oct-r2-en-8-one, prepared as in reference example 3, is dissolved in 6.5 ml of ethanol and 26 mg of 10% palladium carbon is added. The mixture is stirred at room temperature and atmospheric pressure in a stream of hydrogen for 2 hours. The mixture is then filtered to remove the catalyst and the [filtrate" is concentrated under reduced pressure. The concentrate is redissolved in 10 ml of methanol and 26 mg of 10% palladium-carbon is added. The mixture is subjected to catalytic reduction at room temperature and atmospheric pressure for 3 hours and 50 minutes and then filtered using a filter aid, "Hyflo Super Cel". The filtrate is concentrated under reduced pressure to obtain 88 mg (100%) of a semi-solid product. The product is identified as the desired amino compound on the basis of the following data.

-1

IR(KBr)V™ks: 3450, 2950, 1770, 1650

Reference example 9

Preparation of (-)-cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo/4,2,07oct-2-en-8-one:

178 mg (0.67 mmol) of (-)-cis-7-azido-2-t-butyloxycarbonyl-1-azabicyclo/4,2,Q7oct-2-en-8-one, prepared in reference example 1, is dissolved in 10 ml of ethanol and 25 mg of 10% paiiadium carbon (catalyst) are added. The mixture is stirred at room temperature in a stream of hydrogen for 50 minutes. The reaction solution is filtered to remove the catalyst and the filtrate is concentrated under reduced pressure to give the desired compound as a yellow oily product.

Amount of product: 159, mg

Yield: '100%

'■-room'-1

IR(CHCl 3 )V™ks: 1775, 1725, 1640

NMR(CDCl 3 ) cC (ppm): 6.27 (m, 1H), 4.50 (m, 1H), 4.2-3.1

(m, 3H), 2.6-1.7 (m, 4H), 1.5 (s, 9H)

In the case that the catalytic reduction is carried out in ethanol containing 1 equivalent of hydrochloric acid, the hydrochloride of the desired compound is obtained.

Reference example 10

Preparation of (-)-cis^-t-butyloxycarbonyl^-methyl^-azido-1-azabicyclo/^-, 2,07oct-2-en-8-one of the formula

The above compound is prepared as follows

procedures l) and 2).

1) Preparation of the t-butyl ester of 2-[£-(2-methyl-3-butenyl)-3-azido-2-oxoazetidin-1-yl7-2-diethylphosphonoacetate:

2.13 g (8 mmol) of t-butyl-oc-aminodiethylphosphonoacetate are dissolved in 80 ml of absolute ether and 902 mg (9.2 mmol) of 3-methyl-4-pentenal are added with stirring. The mixture is stirred at room temperature for 1 hour and then 900 mg molecular sieve ("4A") and 700 mg magnesium sulfate are added. The mixture is stirred for 1.5 hours and then subjected to suction filtration. The filtrate is concentrated to obtain a pale yellow oily product. 30 ml of anhydrous benzene and the mixture are added to the product

~ ' is concentrated again to obtain 2.82 g of oily product.

The presence of a Schiff base is confirmed by NMR spectrum. The oily product is dissolved in 56 ml of dried. cyclohexane and 56 ml of anhydrous benzene and 900 mg of molecular sieve ("4A") and 1.67 ml (12 mmol) of triethylamine are added. 1.43 g (12 mmol) of azidoacetyl chloride dissolved in 56 ml of dried cyclohexane are then added dropwise to the mixture with stirring at room temperature over a period of 1.5 hours, and the mixture is stirred for 30 minutes. The reaction mixture is transferred to a separatory funnel together with 30 ml of benzene. The benzene layer is washed with 30 ml each of 10% citric acid, saturated sodium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The resulting solution is washed with anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2.8 g of oily product. The presence of two isomers in the product is detected by thin layer chromatography (silica gel, n-hekai/ethyl acetate (1:1)). The product is fed onto a sponge packed with 300 g of silicon dioxide gel and eluted with a mixture of n-hexane and ethyl acetate in a volume ratio of 1:1. This gives 380 mg (yield 11.0%) of the least polar isomer of the desired compound, 570 mg (yield 16.7%) of the most polar isomer and 201 mg (yield 5.8%) of a mixture of the two isomers.

The properties of each isomer are listed below. The most polar isomer is identified as the cis isomer of the desired compound.

The least polar isomer (trans-isomer):

!R(CHCl3)\/™kg: 2110, 1770, 1745

NMR (CDC130 ^ (ppm): 5.40-6.10 (1H, m), 5.27-4.90 (2.5H, m), 4.68 (0.5.H, d)r , 4.23 (6H, m), 2.60-1.77 (3H, m), , 1.53

(9H, s), 1.37 (6H, t, J=7.0Hz), 1.10 (3H, d, J=6.0Hz)

The most polar isomer (cis-isomer):

IR(CHCl 3 ) V ™ kJ: 2110, 1765, 1745 NMR(CDC 3 ) J (ppm): 5.45-6^.3 (1H, m), 4.83-5.20 (2.5H, m) , 4.67 (0.5H, d), 3.97-4.45 (6H, m), 1.77-2.55 (3H, m), 1.50 (9H, s), 1.33 (6H, t), 1.08 (3H, d) 2) Preparation of; (-)-cis-7-azido-2-t-butylboxycarbonyl-4-methyl-7azido-1-azabicyclo/4,2,07oct-2-en-8-one: 240 mg (0.56 mmol) t- butyl ester of (-)-cis-2-(4-(2-methyl-3-butenyl)-3-azido-2-oxoazetidin-1-yl7-2-diethylphosphono-acetic acid, prepared in reference example 10-1), dissolves in 6.6 ml of dioxane and 2^11 of deionized water and 20 mg of osmium tetroxide are added. The mixture is stirred for 10 minutes and 390 mg (1.82 mmol) of powdered sodium periodate is added to the black reaction mixture in small portions over 30 minutes. After stirring for 40 minutes, the reaction solution is extracted 3 times with 30 ml of ether. The ether layers are combined, washed with saturated sodium chloride solution, washed with anhydrous sodium sulfate and concentrated to obtain 230 mg of oily product. The oily product is applied to a sponge packed with 6 g of silicon dioxide gel and eluted with a solvent consisting of benzene and ethyl acetate in a volume ratio of 1:2. The fractions positive for the 2,4-dinitrophenylhydrazine reaction are combined and concentrated to give 185 mg of oily product, the aldehyde compound of the desired compound. The product is immediately dissolved in 8 ml of anhydrous acetonitrile and 21.6 mg (0.45 mmol) of 50% sodium hydride is added at room temperature under stirring in a stream of nitrogen. After stirring for 50 minutes, the reaction mixture is poured into 15 ml of 2% aqueous acetic acid, and the mixture is extracted twice with 20 ml of ether. The ether layers are washed with saturated sodium chloride solution, washed with anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily product. The product is fed onto a sponge packed with 20 g of silicon dioxide gel and eluted with a solvent consisting of n-hexane and ethyl acetate in a volume ratio of 3.5:1. 70 mg of the desired product are obtained as a colorless oily product which crystallizes on standing. Dividend 48.1%. The properties of the product are as follows: IR(KB;r)N/°mks: 2110, 1784, 1715, 1623 NMR(CDC13) / (ppm): 6.30 (4/5H, d, J=5.1Hz ), 6.10 (1/5H, d, J=2.7Hz), 4.98 (4/5H, d, J=5.0Hz), 4.89 (1/5H, d, J=5, 0Hz), 3.60-3.90 (1H, m), 2.65 (1H, m), 1.70-1.80 (2H, m), 1.51' (9H, s), 1, 20~(3/5H, d, J=8.0Hz), 1.13 (12/5H, d, J=8.0Hz) On the basis of these NMR data, the obtained crystals are identified as a mixture of 4a-methyl the -isomer and the 48-.i methyl isomer in a ratio of about 4:1. The compounds can be separated by silica gel chromatography using a solvent consisting of n-hexane and ethyl acetate in the volume ratio 3:1. The most polar isomer corresponding to the 46-CH^ isomer, i.e. (-)-cis-76-azido-46-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,Q7oct-2-ene- 8-on, and the :least polar isomer corresponds to the 4a-CH3 isomer. They have the following. respective properties:

IN

4 B-CH^ isomer:

Melting point: 84.0 - 86.5°C

IR(KBr)V™k<s>"<:>2135, 1783, 1715, 1622

NMR(CDCl 5 ) $ (ppm): 6.13 (1H, d, J=2.7Hz), 4.90 (1H, d, J=5.0-Hz), 3.93-3.73 (1H , m), 2.53 (1H, m), 2.16-1.75 (2H, m), 1.53 (9H, s), 1.20 (3H, d, J=6.0Hz)

4 a-CH-j-isomer:

3

Melting point 82.0 - 84.0°C

-1

IR(KBr)V °mks: 2120, 1790, 1721, 1630

NMR(CDCl 3 ) Å(ppm): 6.33 (1H, d, J=5.0Hz), 5.00 (1H, d, J=5.5Hz), 3.89-3.68 (1H, m ), 2.66 (1H, m), 1.82-1.57 (2H,

m), 1.53 (9H, s), 1.12 (3H, d, J=7.0Hz)

Reference example 11 1

Preparation of (<i>)-cis-78-amino-4oc-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,07oct-2-en-8-one:

255 mg (0.67 mmol) (-)-cis-76-azido-4a-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,07oct-2-en-8-one, prepared in reference example 10 -2) as a least polar isomer, is dissolved in 10 ml of ethanol, and 100 mg of 10% pallaclium carbon is added. The mixture is subjected to catalytic hydrogenation for 1.5 hours after which it is filtered to remove the catalyst. The catalyst is washed with methanol. The filtrate and washing solutions are combined and concentrated under reduced pressure to obtain a pale yellow oily product. The product is dissolved in 8 ml of ethyl acetate and the solution is extracted five times with 3 ml of 10% citric acid. The aqueous layer is set

to pH 6 - 7 with potassium carbonate to obtain a white suspension. The suspension is extracted twice with 5 ml of ethyl acetate and washed with saturated sodium chloride solution. The washed extract is dried with anhydrous sodium sulfate and evaporated to obtain 177 mg (76.6%) of oily product.

IR(CHCl3)v/^<kg>: 3400, 1770, 1720, 1630

NMR(CDCl 3 ) ^(ppm): 6.23 (1H, d, J=5.0Hz), 4.53 (1H, d, J=5.8Hz), 3.93-3.47 (1H, m ), 2.56 (1H, m), 1.92 (2H, br), 1.80-1.60 (2H, m), 1.50 (9H, s~), 1.31 (sH, d , J=7.0Hz)

In the case that the catalytic hydrogenation is carried out in the presence of an equivalent of hydrochloric acid, the hydrochloride of the desired compound is obtained.

Reference example 12

Preparation of (-)-cis-7B-amino-4B-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,07oct-2-en-8-one:

655 mg (2.35 mmol)^(i)-cis-7B-azido-4B-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,Q7oct-2-en-8-one, prepared in reference example 10-2) as a most polar isomer, is dissolved in 6 ml of ethanol and 0.79 ml (2.37 mmol) of 3N hydrochloric acid is added. The mixture is subjected to hydrogenation with 200 mg of 10% palladium-carbon for 70 minutes. Methanol is added to the resulting mixture to dissolve the precipitated salt of the desired compound. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude product. The product is thoroughly triturated with ether and filtered. The filtered substance is used to obtain 512 mg (75.4%)

of the hydrochloride of the desired compound.

Melting point: 216 - 221°C (decomp.)

-1

IR(KBr)^^ks<:>3430,2590, 1780, 1762, 1712, 1630

Reference example 15

Preparation of the trifluoroacetate of (-)-cis-7B-amino-4oc-methyl-2-carboxy-1-azabicyclo/4,2,Q7oct-2-en-8-one:

196 mg (0.78 mmol) (-)-cis-7B-amino-4a-methyl-2-t-butyloxycarbonyl-1-azabicyclo/4,2,Q7oct-2-en-8-one, prepared in reference example 11 , is dissolved in 4.2 ml of anhydrous dichloromethane and 1.8 ml of trifluoroacetic acid is added at room temperature with stirring. After 1.5 hours, the mixture is concentrated under reduced pressure. The concentrate is subjected to azeotropic distillation with anhydrous benzene to obtain an oily product. The product is triturated with ether and filtered to obtain 167 mg (69.3%) powder of the desired compound.

-1

IR(KBr)y ^ks:

PMR(D20, with DSS as internal standard)^ (ppm): 6.77 (1H, d, J=5.8Hz), 5.00 (1H, d, J=5.6Hz), 4.10 (1H , m), 2.83 (1H, m), 1.86 (2H, m), 1.15 (3H, d, J=8.0Hz)

Reference example 14

Preparation of (-)-cis-7B-amino-4a-acetoxy-1-azabicyclo/5,2,Q7oct-2-en-8-one-2-carboxylic acid: l) Preparation of (-)-cis-7B- azido-4a-acetoxy-1-azabicyclo/4,2,07oct-2-en-8-one-2-carboxylic acid: 179 mg (-)-cis-7(3-azido-4a-acetoxy-2-t- Butyloxycarbonyl-lazabicyclo/4,2,07oct-2-en-8-one, prepared as in Reference Example 6, is dissolved in 3 ml of methylene chloride and 3 ml of trifluoroacetic acid.The solution is left at room temperature for 2 hours, after which it is concentrated to obtain 145 mg of the desired compound as a yellow powder. Yield: 100%. The properties of the compound are listed below. -1 IRtCHCl^Vmåks12130,1790, 1715, 1445 2) Preparation of (-)-cis-7|3-amino-4a-acetoxy -l-azabi cyclo/4,2,07oct-2-en-8-one-carboxylic acid; f

145 mg (<i>)-cis-7|3-azido-4a-acetoxy-1-azabicyclo-2,07oct-2-en-8-one-2-carboxylic acid, prepared as in reference example 14-1), is dissolved in 14 ml of ethanol and 40 mg of 10% palladium-carbon are added. The mixture is subjected to catalytic hydrogenation at atmospheric pressure with stirring for 1 hour. The reaction mixture is filtered and the filtrate is concentrated to obtain 126 mg of the desired compound.

Reference example 15

Preparation of (-)-cis-76-azido-2-t-butoxycarbonyl-4a-hydroxy-1-azabicyclo/4,2,07oct-2-en-8-one with the formula:

200 mg of (-)-cis-2-t-butoxycarbonyl-7-azido-1-azabicyclo/4,2,07oct-2-en-8-one, prepared as in reference example 1, is dissolved in 8.8 ml of carbon tetrachloride , and 134.9 mg of N-bromosuccinimide and a catalytic amount of a,oe'-azobisisobutyronitrile are added. The mixture is heated under reflux for 30 minutes. After cooling, the reaction mixture is diluted with 5 ml of chloroform and washed with 3 ml of water and 3 ml of saturated sodium chloride solution. The washed solution is washed with anhydrous sodium sulfate and filtered. The filtrate is concentrated to obtain an oily bromine compound which is the same oily product as in example 4. The product is immediately dissolved in 10 ml of acetone, and 50 mg of sodium bicarbonate and 50 µl of water are added. The mixture is stirred at room temperature for 10 minutes. The reaction mixture is filtered and concentrated to obtain a crude product. The product is applied to a sponge packed with 20 g of silicon dioxide gel and eluted with a mixture of 1-hexane and ethyl acetate in a volume ratio of 2:1. The eluates are concentrated to obtain 86.4 mg of the desired compound as pale yellow crystals. Dividend 40.7%. The crystals properties are listed below.

Melting point: 100.0 - 101.0°C

orrT1

IR(CHCl3)y ™ks: 2130, 1790, 1635, 1630

NMR(CDCl 3 ) δ (ppm): 6.30 (1H, d, J=5Hz), 5.03 (1H, d, J=5.2), 4.47 (1H, m), 3.93 ( 1H, m), 3.20 (1H, br), 2.1-1.8 (2H, m), 1.55 (9H, s)

Reference example 16

Preparation of (-)-cis-7-amino-1-azabicyclo/4,2,07oct-2-en-8-one-2-carboxylic acid:

300 mg of (<i>)-cis-7-amino-2-t-butyloxycarbonyl-1-azabicyclo/4,2,Q7oct-2-en-8-one, prepared as in Reference Example 9, is dissolved in 3 ml of methylene chloride and 3.0 ml of trifluoroacetic acid is added. The mixture is left at room temperature for 1 hour and 20 minutes. The reaction mixture is concentrated and benzene is added to the "residue". The solution is concentrated again to give 250 mg of trifluoroacetate of the desired compound as a yellow powder. The product's properties are listed below.

IR(KBr)V™ks: 1780, 1680, 1630

The above trifluoroacetate is dissolved in 2 ml of water and the solution is adjusted to pH 7.0 with saturated sodium bicarbonate solution to form crystals. Thus, 129 mg of the desired compound are recovered by filtration. The product's properties are consistent with the product

in reference example 8.

Reference example 17

Preparation of (T)-cis-7B-azido-2-carboxy-4a-methyl-1-azabicyclo/Zr, 2,Q7oct-2-en-8-one:

To 238 mg (0.703 mmol) (<±>)-cis-7P-azido-2-t-butyl-oxycarbonate-4ct-methyl-1-azabicyclo/4,2,Q7oct-2-en-8-one, prepared as in reference example 10, 4 ml of trifluoroacetic acid is added, and the mixture is allowed to stand at room temperature for 10 minutes. The reaction mixture is concentrated at 25°C under reduced pressure. The concentrate is extracted twice with 5 ml of dried benzene. 255 mg of the oily product obtained is then dissolved in 5 ml of ethyl acetate. The solution is extracted with 2 ml of 10% potassium carbonate solution twice, and the aqueous layer is adjusted to a pH of approx. 3 with 0.5N HCl. The solution is extracted with 5 ml of ethyl acetate twice, and the combined extract is dried over anhydrous sodium sulfate. The solvent is distilled off under reduced pressure to obtaining 166 mg of the desired compound as an oily product, which crystallizes on standing. Yield 83.8%. The product's properties are listed below.

Melting point: 121.5 - 123.0°C.

IR(CHCl3)Y£™ks~: 2110, 1769, 1750, 1716, 1630

NMR(CD 3 OD) δ (ppm): 6.47 (1H, d, J=5.6Hz), 5.22 (1H, d, J=5.0), 4.2-3.7 (1H, m ), 2.3-2.9 (1H, br), 1.11 (3H, d, J=7.2)

Reference example 18

Preparation of the trifluoroacetate of (<i>)-cis-76-amino-4a-methyl-1-azabicyclo/4,2,07oct-2-en-8-one-2-carboxylic acid:

200 mg of (-)-cis-76-azido-2-t-butyloxycarbonyl-4a-methyl-1-azabicyclo/4,2,07oct-2-en-8-one is dissolved in 2 ml of water and 2 ml of ethanol, and 75 mg of 10% palladium-carbon is added.

The mixture is stirred in a stream of hydrogen gas at atmospheric pressure. After 20 hours, the reaction mixture is suction filtered. Add 2 ml of trifluoroacetic acid to the filter cake.

After removal of the catalyst by filtration, the filtrate is concentrated under reduced pressure followed by the addition of 10 ml of dried ether. The crystals formed are recovered by filtration whereby 120 mg of the desired compound is obtained. Dividend 43%. The properties of the compound are consistent with the product in Reference Example 13.

Claims (1)

Analogy method for the preparation of cephalosporin analogues with the general formula: where X means an acyl group of the general formula X"*"C0, where X"^ means one of the following forward groups: (1) a cyanomethyl group or a group of the general formula where B means an unsaturated, six-membered, carbocyclic group selected from cyclohexenyl, cyclohexadienyl and phenyl or a five- or six-membered, heterocyclic group, A"*" means hydrogen, hydroxy, alkoxy with 1-4 carbon atoms, halogen, nitro, amino, aminomethyl , methylsulfonamido or acyloxy with 1-4 carbon atoms, n is an integer from 0 to 5, A 2 means hydrogen, amino, hydroxy, carboxyl or sulfoxyl, (2) a group with the general formula: 1 3 where B, A and n have the meaning given above, and A and/ , A are the same or different and each individually means hydrogen, alkyl with 1-4 carbon atoms, a group with the general formula -CO-A < 3> , where A <3> means alkyl with 1-4 carbon atoms, or a group with the general formula: 6 7 where A and A are the same or different and each means hydrogen, alkyl with 1-4 carbon atoms or alkali metal, 3 4 or A. and A together with the nitrogen atom to which they are attached form a group with the general formula: 8 8' where A and A are the same or different and each means hydrogen or alkyl with 1-4 carbon atoms, or a group with the general formula: 0 A9' -N^ Ay where A 9 means hydrogen, alkyl with 1-4 carbon atoms, methyl sulfonyl or furfuryldenimino, and A 9' means hydrogen or alkyl of 1-4 carbon atoms, (3) a group of the general formula where B, A and n have the meaning given above, and A"^ means a substituted aryl group or a mono-, bis- or tricyclic heterocyclic group, (4) a group of the general formula: where B, A <1> and n have the meaning given above, and A^ means hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon atoms, cyclo-, alkyl with 3-6 carbon atoms or aryl, where the groups are optionally substituted with suitable substituents selected from carboxyl, cyano, halogen, carbamoyl and alkoxycarbonyl with 1-4 carbon atoms, or (5) a group with the general formula: where B"*" means trifluoromethyl, cyanomethyl or a group of the formula (A^-^j—B-, where B, A"<*>" and n have the meaning given above, and Z means oxygen or sulphur, R"*" means hydrogen or a carboxylic acid protecting group selected from alkyl of 1-5 carbon atoms, halogenated alkyl of 1-5 carbon atoms, arylmethyl of 7-20 carbon atoms, which may be substituted on the phenyl ring, substituted silyl and a group that is easily removed enzymatically or non-enzymatically in vivo, and R 2 means hydrogen, alkyl with 1-5 carbon atoms or acyloxy with 1-5 carbon atoms, or pharmaceutically acceptable salts thereof, characterized in that a cephalosporin analogue of the general formula: 1 2 where R and R have the above meaning, or a functionally equivalent compound (hereinafter referred to as the 7-amino compound) is acylated with a carboxylic acid with the general formula: or a reactive derivative thereof, where X 2 means one of the following five groups:1 <1> ) cyanomethyl or a group with the general formula: where B and n have the above meaning, A 1 » means hydrogen, hydroxy, "protected hydroxy, 1-4 carbon atom alkoxy, halogen, nitro, protected amino, protected aminomethyl, methylsulfonamido or 1-4 carbon atom acyloxy, and A 2' means hydrogen, protected amino, hydroxy, carbonyl or protected sulfoxyl, (2') a group with the general formula: 1' 3 4 where B, A , n, A v 3 and A have the meaning stated above, (3') a group with the general formula: where B, A <11> , n and A <10> have the above meaning, (4') a group with the general formula: 11 12 where B, A and n have the above meaning, and A means hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon atoms, cycloalkyl with 3-6 carbon atoms or aryl, where the groups are optionally substituted with suitable substituents chosen from protected carboxyl, cyano, halogen, carbamoyl and alkoxycarbonyl with 1-4 carbon atoms, or (5 <1> ) a group with the general formula: where B" ^ means trifluoromethyl, cyanomethyl or a group of the formula (A"*""1"-}^—B-, where B, A"^ and n have the meaning given above, and Z means oxygen or sulphur, after which, if necessary, the protecting group in the X CO- and/or -COOR"'" group is removed in a conventional manner, and, if desired, the compound formed is converted to a pharmaceutically acceptable salt thereof.