CH633554A5 - Process for the preparation of novel thienamycin derivatives. - Google Patents

Description

The invention relates to a method for producing new derivatives of the antibiotic thienamycin. Preferred are white N, O and carboxyl derivatives of thienamycin.

Thienamycin (I) is described in U.S. Patent 3,950,357. Thienamycin is one of the starting products used in the process according to the invention and has the following formula:

Oh

O NH O

II II II

-C-CH2CH2NHC-NH2 -C-CH2CH2N (CH3) 2

o o

nh

-c-ch2ch2n (ch3) 3 -c-ch2ch2c-nh2

O

NH

O

-C-CH2CH2CH2NHC-CH3 -C-CH2CH2CH2N (CH3) 3

o o

-C-CH2CH2CH2N (CH3) 2 -C-CH2S-C,

^ NH

\

NH,

sch2ch2nh2

20th

/ 7 O

-n.

cooh

Another suitable starting compound is the N-acylated carboxyl derivative of thienamycin (Ia), which in the. U.S. Patent No. 634,295 filed on November 21, 1975, which is simultaneously filed, is described with internal No. 15 813 Y):

sch2ch2nr1r2

coxr

Yes

O O

II / NH II ^ NH

-C-CH2S-CH2-C -C-CH2-O-CH2C

\ NH2 nNH2

4. The method according to claim 3, characterized in that compounds are prepared in which R2 is a formyl, propionyl, butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, methylcarbamoyl, ethyl carbamoyl -, Phenylthiocarbonyl-, 3-aminopropionyl-, 4-ami-nobutyryl-, N-methylaminoacetyl-, N, N-dimethylaminoacetyl-, N, N, N-trimethyIaminoacetyl-, 3- (N, N-dimethyl-ami -nopropionyl-, 3- (N, N, N-trimethyl) -aminopropionyl-, N, N, N-triethylaminoacetyl-, pyridiniumacetyl-, guanylthioacetyl-, guanidinoacetyl-, 3-guanidinopropionyl-, N3-methylguanidi-nopropionyl-, hydroxyacetyl -, 3-hydroxypropionyl, acryl-oyl, propinoyl, malonyl, phenoxycarbonyl, amidinoacetyl, acetamidinoacetyl, amidinopropionyl, acetamidopropionyl, guanylureidoacetyl, guanylcarbamoyl, carboxymethyl-aminoacetylamino-sulfo -acetyl-, N3-dimethylaminoacetamidinopropionyl-, ureidocarb-onyl-, dimethylaminoguanylthioacetyl-, 3- (l-methyl-4-pyridi-nium) -pro pionyl-, 3- (5-aminoimidazol-l-yl) propionyl-,

Represents 3-methyl-1-imidazoliumacetyl, 3-sydnonylacetyl, o-aminomethylbenzyl or o-aminobenzoyl group.

5. The method according to claim 3, characterized in that compounds are prepared in which X is an oxygen atom and R is a hydrogen atom or a methyl, tertiary butyl, phenacyl, p-bromophenacyl, pivaloyloxymethyl, 2,2,2-tri -chloroethyl-, allyl-, 3-methyl-2-butenyl-, 2-methyl-2-propenyl-, benzyl-, benzhydryl-, p-tert-butylbenzyl-, phthalidyl-, phenyl-, 5-indanyl -, acetylthiomethyl, acetoxymethyl, propionyloxymethyl, methallyl, 3-butenyl, 4-pentenyl, 2-butene

35 wherein X, R, R1 and R2 are defined below.

The new compounds which can be prepared according to the invention have the following formula

OR

sch2ch2nr1r2

coxr

(II),

'or you can more easily by the following symbol so Th-

OR3

NR ^ 2

COXR

(iia)

are described, in which “Th” means the bicyclic nucleus of thienaymicin and the OH, amino and carboxyl groups of thienamycin, which are shown therein.

In the compounds of the formula II which can be prepared according to the invention, X denotes oxygen, sulfur or a radical of the formula NR ', in which R' = R.

60 R denotes hydrogen, an alkyl radical with 1 to 10 carbon atoms, a phenacyl radical or a nucleus-substituted phenacyl radical, where the substituent is a chlorine, bromine or fluorine atom or an alkyl radical with 1 to 6 carbon atoms, an alkoxyalkyl radical, the alkoxy part being open-chain or 65 cyclic and has 1 to 6 carbon atoms and the alkyl moiety has 1 to 6 carbon atoms, an alkanoyloxyalkyl radical with 2 to 12 carbon atoms, a halogen or perhaloalkyl radical, the halogen atom being a chlorine,

633 554

Is bromine or fluorine atom and the alkyl chain has 1 to 6 carbon atoms, an alkenyl radical with 2 to 10 carbon atoms, an alkoxycarbonylalkyl radical with 3 to 14 carbon atoms, a dialkylaminoacetoxyalkyl radical with 4 to 21 carbon atoms, an alkanoylamidoalkyl radical with 2 to 13 carbon atoms, an aralkyl radical, where the alkyl moiety has 1 to 3 carbon atoms and the aryl moiety has 6 to 10 carbon atoms, a mono- or bicyclic heteroaralkyl radical with 4 to 10 ring atoms and 1 to 6 carbon atoms in the alkyl moiety, the hetero atom (s) (a) oxygen , Sulfur or nitrogen atom (s) represents a nucleus-substituted aralkyl or heteroaralkyl radical, the substituent being a chlorine, fluorine, bromine or iodine atom or an alkyl radical having 1 to 6 carbon atoms, a mono- or bicyclic Heterocycloalkylalkylrest, wherein the heterocycle has 4 to 10 atoms and the heteroatom (s) (a) oxygen, Schwe represents fur or nitrogen atom and the alkyl portion has 1 to 6 carbon atoms, an aryl or nucleus-substituted aryl residue with 6 to 10 ring carbon atoms, the core substituent being a hydroxyl group, an alkyl residue with 1 to 6 carbon atoms, a chlorine, fluorine or Is bromine or an alkylthioalkyl radical with 2 to 12 carbon atoms, a cycloalkyl thioalkyl radical with 4 to 12 carbon atoms or an acylthioalkyl radical, the acyl component having 2 to 10 carbon atoms and the alkyl component having 1 to 6 carbon atoms,

R1 and R2 are each hydrogen or an acyl group and

R3 represents an acyl radical,

where R1, R2 and R are not all hydrogen atoms at the same time.

The new compounds of formula II can be converted into pharmacologically acceptable salts.

R1 and R2 are independently selected from the group consisting of hydrogen and acyl groups. Preferred compounds are those in which R1 is hydrogen and R2 is acyl. The term “acyl” here includes by definition the alkanoyl including derivatives and analogues thereof, such as thio analogs in which the carbonyl oxygen is replaced by sulfur, diacyl radicals in which R1 and R2 are linked together, and the sulfur and phosphorus Acyl analogs of such substituted sulfonyl, sulfinyl and sulfenyl residues and substituted P (III and V) residues, such as substituted phosphorus, phosphorus, hypophosphorous and phosphinig residues. Such acyl residues are further disclosed below.

X represents oxygen, sulfur or NR '(R' - R); and R is, among others, typically selected from the group consisting of hydrogen, common blocking groups such as trialkylsilyl, acyl and the pharmaceutically acceptable salt, ester and amide groups as are known for bicyclic β-lactam antibiotics. The preferred definitions of R are described in more detail below.

R3 is acyl as defined above. The term “acyl” here by definition includes the alkanoyl including derivatives and analogues thereof, such as the thio analogs in which the carbonyl oxygen is replaced by sulfur. Preferred acyl radicals are defined below.

There is a constant need for new antibiotics. Unfortunately, there is no static effectiveness for a given antibiotic because continued use on a broad scale of such an antibiotic selectively increases the formation of resistant strains and pathogens. In addition, the known antibiotics have the disadvantage that they are only effective against certain types of microorganisms. Therefore, research continues on new antibiotics.

It has now been found, unexpectedly, that the new compounds which can be prepared according to the invention are broad-spectrum antibiotics which are useful both for animal and human therapy and in inanimate systems.

It is therefore an object of the present invention to provide a method for producing a new class of antibiotics which contain the basic core structure of the antibiotic thienamycin, but which are characterized as its O, N and carboxyl derivatives. These antibiotics are active against a wide range of pathogens, for which both gram-positive bacteria such as S. aureus, B. subti-10 Iis and Strept. pyogenes and gram-negative bacteria such as E. boli and Salmonella are typical.

The process according to the invention for the preparation of the compounds of the formula II is characterized in that a compound of the formula oh

A

20th

/ 7th

o sch2ch2nr1r2 coxr is reacted with an acylic solvent suitable for introducing the R3 radical and, if appropriate, compounds subsequently obtained are converted into the corresponding salts.

The new compounds of the formula (II) can be prepared simply and preferably according to the following reaction scheme, which also comprises the preparation of the starting compounds of the formula IA. First, thienamycin (I) is N-acylated to give the N-acyl intermediate (1), which is then esterified to give the carboxyl derivative (2). Deriving from 2 gives II:

35

40

45

OH Th-4- NH2. COOH

OH

Th-- NR! R2 Th-- NR * R2

Th-

OR3

NRJR2 Th-COXR

50

II

L COOH 1

r OR3

NR! R2 L COOH IIa

OH

Th-

L COXR 2

OR3

nh2

COXR

IIb

Here the symbols have the meaning given above. Preferred species IIa are obtained when X is 55 oxygen and R is an easily removable blocking group. Species IIb can likewise be obtained when R1 and R2 are hydrogen or easily removable N-blocking groups by selective deblocking.

The above production method is described in greater detail below.

In a general presentation of the new compounds (II, above), the acyl radical, as represented either by R1 and R2, can be a substituted or unsubstituted aliphatic, aromatic or heterocyclic, 65 araliphatic or heterocycloaliphatic carboxylic acid radical, a substituted or unsubstituted carbamoyl residue or a carbothioic acid residue. Preferred acyl radicals R3 have the following formula

633 554

X

II

-C-R4

in which

X is an oxygen or sulfur atom and R4 is a hydrogen atom, an amino group or an alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, arylaralkyl, cycloalkyl , Heteroaryl or heteroarylkyl radical, or R3 is an acyl radical of the formula below

X

II

-C (CH2) nZR5,

in which

X is defined above,

R5 is a hydrogen atom, an amino, mercapto or hydroxyl group or an alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, Represent heteroaryl or hetero-aralkyl radical,

n is 0, 1, 2, 3 or 4 and

Z represents an oxygen, sulfur or nitrogen atom or a carbonyl group, except that when Z is an oxygen atom,

Rs does not represent a mercapto or hydroxyl group when Z is a sulfur atom,

R5 is not an amino or hydroxyl group and when Z is a nitrogen atom

R5 is not a mercapto group, or R3 is an acyl group of the formula below

X

II

-C-CHR5 I

R6

in which

X is an oxygen or sulfur atom,

R5 is defined above,

R6 is an amino, hydroxyl, azido, carbamoyl or guanidine group, an acyloxy group, a halogen atom, a sulfamino, tetrazolyl, sulfo or carboxyl group, a carbalkoxy group, a phosphono group or an alkoxy or Arylthio radical, with the proviso that both radicals R5 and R6 cannot represent hydroxyl, amino or mercapto groups, or

R3 is an acyl group of the formula below

O

II

-C (CH2) m-A- (CH2) n-Y

radical, where the alkoxy part has 1 to 6 carbon atoms and the alkyl part has 2 to 6 carbon atoms, or a cycloalkyl or cycloalkylalkyl radical, where the cycloalkyl part has 3 to 6 carbon atoms and the alkyl part has 1 to 3 carbon -5 atoms, where two R7 Residues can be linked and together with the N atom to which they are attached can form a ring with 3 to 6 atoms;

2) an amidino or substituted amidino group of the general formula

10th

-N = C-N (R7) 2

R7

where the R7 radicals are each independently a hydrogen atom, N (R8) 2, where R8 is a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, an alkyl or alkoxy radical having 1 to 6 carbon atoms, an alkoxyalkyl radical, where the alkoxy portion has 1 to 6 carbon atoms and the alkyl portion has 2 to 6 carbon atoms — when the never-20 deralkoxy-lower alkyl group is attached to a carbon atom, the alkyl part contains 1 to 6 carbon atoms - or a cycloalkyl or cycloalkylalkyl group, where the alkyl portion has 1 to 3 carbon atoms, where two radicals R7 are linked and, together with the atoms to which they are attached, can form a ring having 3 to 6 carbon atoms;

3) a guanidino or substituted guanidino group of the general formula so -NH-CN (R7) 2

II

NR7

where R7 has the meaning given in 2);

35 4) a guanyl or substituted guanyl group of the general formula

-C = NO

40

45

N (R7) 2

where R7 has the meaning given in 2); or

5) a nitrogen-containing, mono- or bicyclic heterocyclic radical with 4 to 10 nucleus or ring atoms, the heteroatom (s) being (are) oxygen and / or sulfur atom (s) apart from nitrogen .

Acyl radicals R3 of the formula below are also preferred

X

50

-C-R9

55

where m and n each represent 0 or an integer from 1 to 5,

A is an oxygen atom, NR8, where R8 is a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, a sulfur atom or a single bond and

Y represents one of the following: 60

1) an amino group or substituted amino group of the general formulas

-N (R7) 2 and -N (R7) s where the R7 radicals are each independently a water-65 atom, N (R8) 2, where R8 is a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, an alkyl or alkoxy radical having 1 to 6 carbon atoms, an alkoxyalkyl

in the

R9 is benzyl, p-hydroxybenzyl, 4-amino-4-carboxybu-tyl, methyl, cyanomethyl, 2-pentenyl, n-amyl, n-heptyl, ethyl, 3- or 4- Nitrobenzyl-, phenethyl-, ß, ß-diphenyl-ethyl-, methyldiphenylmethyl-, triphenylmethyl-, 2-methoxy-phenyl-, 2,6-dimethoxyphenyl-, 2,4,6-trimethoxyphenyl-, 3,5-dimethyl- 4-isoxazolyl-, 3-butyl-5-methyl-4-isoxazolyl-, 5-methyl-5-phenyl-4-isoxazolyl-, 3- (2-chlorophenyl) -5-methyl-4-isoxazolyl-, 3- (2,6-dichlorophenyl) -5-methyl-4-isoxazolyl-, D-4-amino-4-carboxybutyl-, D-4-N-benzoylamino-4-carboxy-n-butyl-, p-aminobenzyl -, o-Aminobenzyl-, m-Ami-nobenzyl-, p-dimethylaminobenzyl-, (3-pyridyl) -methyl-, 2-ethoxy-l-naphthyl-, 3-carboxy-2-quinoxalinyl-, 3- (2nd , 6-dichlorophenyl) -5- (2-furyl) -4-isoxazolyl-, 3-phenyl-4-isoxazolyl-, 5-methyl-3- (4-guanidinophenyl) -4-isoxazolyl-, 4-guanidi -nomethylphenyl-, 4-guanidinomethylbenzyl-, 4-guanidino-benzyl-, 4-guanidinophenyl-, 2,6-dimethoxy-4-guanidinophe-

633 554

nyl, o-sulfobenzyl, p-carboxymethylbenzyl, p-chlorobenzyl, p-methoxybenzyl, 1-naphthylmethyl, 3-isothiazolylmethyl, 4-isothiazolylmethyl, 5-isothiazolylmethyl, guanyithiomethyl, 4- Pyridylmethyl-, 5-isoxazolylmethyl-, 4-methoxy-5-iso-xazolylmethyl-, 4-methyl-5-isoxazolylmethyl-, 1-imidazolylmethyl-, 2-benzofuranylmethyl-, 2-indolylmethyl-, 2-phenylvi-nyl -, 2-phenylethynyl, 1-aminocyclohexyl, 2- or 3-thienylaminomethyl, 2- (5-nitrofuranyl) vinyl, phenyl, o-methoxyphenyl, o-chlorophenyl, o- Phenylphenyl-, p-aminomethyl-benzyl-, l- (5-cyanotriazolyl) -methyl-, difluoromethyl-, dichloromethyl-, dibromomethyl-, l- (3-methyl-imidazolyl) -methyl-, 2- or 3- (5-carboxymethylthienyl) methyl, 2- or 3- (4-carbamoylthienyl) methyl, 2- or 3- (5-methylthienyl) methyl, 2- or 3- (5-methoxythienyl) - methyl, 2- or 3- (4-chlorothienyl) methyl, 2- or 3- (5-sulfothienyl) methyl, 2- or 3- (5-carboxythienyl) methyl, 3- ( l, 2,5-thiadiazolyl) methyl, 3- (4-methoxy-l, 2,5-thiadiazolyl) methyl -, 2-furylmethyl, 2- (5-nitro uryl) methyl, 3-furylmethyl, 2-thienylmethyl, 3-thynylmethyl, tetrazolylmethyl, benzamidinomethyl or cy-clohexylamidino-methyl group means

or R3 is an acyl group of the formula below

X

II

-C (CH2) n-ZR10

where the portion -CH2) "ZR10 of the acyl radical is an allylthiomethyl, phenylthiomethyl, butylmercaptomethyl, a-chlorotromercyl methyl, phenoxymethyl, phenoxyethyl, phenoxybutyl, phenoxybenzyl, diphenoxymethyl, dimethylmethoxy methyl , Dimethylbutoxymethyl-, Dimethylphenoxymethyl-, 4-Guanidinophenoxymethyl-, 4-Pyridylthiomethyl-, p- (Carboxymethyl) -phenoxymethyl-, p- (Carboxymethyl) -phe-nylthiomethyl-, 2-Thiazolylthiomethyl-, p- (Sulfo) -phenoxy thyl-, p- (carboxymethyl) phenylthiomethyl-, 2-pyrimidinyl-thiomethyl-, phenethyl-thiomethyl-, l- (5,6,7,8-tetrahydro-naphthyl) -oxymethyl-, N-methyl-4-pyridiniumthio -, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio, amino, methylamine, dimethylamino, pyridiniummethyl, trimethylammoniummethyl, cyanomethylthiomethyl, trifluoromethylthio-methyl, 4-pyridylethyl , 4-pyridylpropyl, 4-pyridylbutyl, 3-imidazolylethyl, 3-imidazolylpropyl, 3-imidazolylbutyl,

Represents 1-pyrroloethyl, 1-pyrrolopropyl or 1-pyrrolobutyl group,

or R3 can be an acyl group of the following formula

X

II

-CCHRnR12

where the portion -CHRnR12 of the acyl residue is an a-ami-nobenzyl-, a-amino- (2-thienyl) -methyl-, a- (methylamino) -benzyl-, a-aminomethylmercaptopropyl-, a-amino-3- or - 4-chlorobenzyl-, a-amino-3- or -4-hydroxybenzyl-, a-amino-2,4-dichlorobenzyl-, a-amino-3,4-dichlorobenzyl-, D - (-) - a-hydroxybenzyl- , a-Carboxybenzyl-, a-Amino- (3-thienyl) -methyl-, D - (-) - a-Amino-3-chloro-4-hydroxy-benzyl-, a-Amino- (cyclohexyl) -methyl- , a- (5-tetrazolyl) benzyl,

2-thienylcarboxymethyl-, 3-thienylcarboxymethyl-, 2-furyl-carboxymethyl-, 3-furylcarboxymethyl-, a-sulfaminobenzyl-,

3-thienylsulfaminomethyl-, a- (N-methylsulfamino) -benzyl-, D - (-) - 2-thienylguanidinomethyl-, D - (-) - a-guanidinoben-zyl-, a-guanylureidobenzyl-, a-hydroxybenzyl-, a-azido-benzyl-, a-fluorobenzyl-, 4- (5-methoxy-l, 3-oxadiazolyl) aminomethyl, 4- (5-methoxy-l, 3-oxadiazolyl) hydroxymethyl-,

4- (5-methoxy-l, 3-sulfadiazolyl) hydroxymethyl, 4- (5-chlorothienyl) aminomethyl, 2- (5-chlorothienyl) hydroxymethyl, 2- (5-chlorothienyl) carboxymethyl -, 3- (l, 2-thiazolyl) aminomethyl, 3- (l, 2-thiazolyl) hydroxymethyl, 3- (l, 2-thiazo-lyl) carboxymethyl, 2-thiazolylaminomethyl, 2-thiazolylhy

droxymethyl, 2-thiazolylcarboxymethyl, 2-benzothienylcar-boxymethyl, 2-benzothienylhydroxymethyl, 2-benzothienyl-carboxymethyl, a-sulfobenzyl, a-phosphonobenzyl, a-diethylphosphono- or a-monoethylphosphono group 5,

or R3 denotes an acyl radical from the following group:

o nh o nh

II II II II

10-c-ch2ch2nhc-ch3 -c-ch2ch2nhc-h o nh o

II II II

-c-ch2ch2nhc-nh2 -c-ch2ch2n (ch3) 2

15

o o nh

II @ II II

-c-ch2ch2n (ch3) 3 -c-ch2ch2c-nh2

20 o nh O

II II II @

-c-ch2ch2ch2nhc-ch3 -c-ch2ch2ch2n (ch3) 3

o o

II

-c-ch2ch2ch2n (ch3) 2 -c-ch2s-c.

^ NH

\

nh,

o o

30 II XNH II ^ -NH

-c-ch2s-ch2-c -c-ch2-o-ch2c

\ nh2 xnh2

In the compounds obtained according to the invention, R2 35 is preferably a formyl, propionyl, butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, phenylthiocarbonyl, 3-aminopropionyl -, 4-aminobutyryl, N-methylaminoacetyl, N, N-dimethylaminoacetyl, N, N, N-trimethylaminoacetyl, 40 3- (N, N-dimethyl) ~ aminopropionyl, 3- (N, N, N -Trimethyl) aminopropionyl, N, N, N-triethylaminoacetyl, pyridinium acetyl, guanylthioacetyl, guanidinoacetyl, 3-guanidinopropionyl, N3-methylguanidinopropionyl, hydroxyacetyl, 3-hydroxoyloxionyl -, Propionyl-, Malonyl-, Phenoxycarbo-45 nyl-, Amidinoacetyl-, Acetamidinoacetyl-, Ainidinopropionyl-, Acetamidopropionyl-, Guanylureidoacetyl-, Guanylcarb-amoyl-, Carboxymethylaminoacetyl-, Sulfoacetylaminoacetyl-, Phosphonyl-Acino-Acetino- Ureidocarbonyl-, dimethylaminoguanylthioacetyl-, so 3- (l-methyl-4-pyridinium) propionyl-, 3- (5-aminoim idazol-l-yl) -propionyl-, 3-methyl-l-imidazoliumacetyl-, 3-Sydnonylace-tyl-, o-aminomethylbenzoyl or o-aminobenzoyl.

Acyl derivatives are preferably prepared in which X is an oxygen atom and R is a hydrogen atom or a methyl, tertiary butyl, phenacyl, p-bromophenacyl, pivaloyloxy-methyl, 2,2,2-trichloroethyl, allyl , 3-methyl-2-butenyl, 2-methyl-2-propenyl, benzyl, benzhydryl, p-tert-butyl-benzyl, phthalidyl, phenyl, 5-indanyl, acetylthiomethyl, acetoxymethyl -, Propionyloxymethyl, methallyl, 3-butenyl, 60 4-pentenyl, 2-butenyl, methoxymethyl, p-acetoxybenzyl, p-pivaloylbenzyl, p-isopropoxybenzyl, 5-indanylmethyl, dimethylaminoacetoxymethyl -, Crotonolacton-3-yl, acetamido-methyl, acetylthioethyl or pivaloylthiomethyl group.

65 An acyl class of particular interest for the acyl radicals R1 and R2 of the formula II above are those which are selected from the group consisting of customary known N-acyl blocking or protecting groups, such as carbobenzyloxy, ring

633 554

substituted carbobenzyloxy, such as o- and p-nitrocarbobenzyl-oxy, p-methoxycarbobenzyloxy, chloroacetyl, bromoacetyl, phe-nylacetyl, tert-butoxycarbonyl, trifluoroacetyl, bromethoxy-carbonyl, 9-fluorenylmethoxycarbonyl, dichloroacetyl, 2-nitro-nitro, o-nitro 2,2-trichloroethoxycarbonyl, bromo-tert-but-oxycarbonyl, phenoxyacetyl; Non-acylated protective groups such as tri-lower alkylsilyl, for example trimethylsilyl and tert-butyldimethylsilyl are also of interest.

Formulas for preferred, nitrogen-containing mono- and bicyclic heterocyclylene having 4 to 10 nucleus atoms, in which the hetero atom or the hetero atoms apart from nitrogen are selected from oxygen and sulfur. Such hererocycles are typically illustrated by the following enumeration:

-e

(R ') 2

No'

However, it goes without saying that any acyl residue can be used.

The N-acylated intermediate [1, above] is preferably prepared by treating thienamycin (I) with an acylating agent, for example an acyl halide or an acyl anhydride, such as an aliphatic, aromatic, heterocyclic, araliphatic or heterocyclic aliphatic carboxylic acid halide or anhydride. Other acylating agents which can also be used are, for example, mixed carboxylic acid anhydrides and in particular lower alkyl esters of mixed carboxylic carboxylic acid anhydrides, also carboxylic acids in the presence of a carbodiimide, such as 1,3-dicyclohexylcarbodiimide, and an activated ester of carboxy acids. such as p-nitrophenyl esters are suitable. 15 These N-acylated thienamycin starting materials are fully described in U.S. Patent Application Serial No. 634,291 dated November 21, 1975 and the Continuation-in-part U.S. Application which coexists with the present application for same applicant has been filed 20, with the internal designation 15 775YA.

The acylation reaction for the preparation of starting materials can be carried out at temperatures in the range from approximately -20 to approximately 100 ° C., but temperatures in the range from −8 ° C. to 25 ° C. are preferably used. Any solvent in which the reactants are soluble and which is essentially inert can be used, for example polar solvents such as water, alcohols and polar organic solvents, generally those such as dimethylformamide (DMF), hexamethylphosphoramide ( HMPA), acetone, disoxane, tetrahydrofuran (THF), acetonitrile, heterocyclic amines, such as pyridine, ethyl acetate, aqueous mixtures of the aforementioned, and also halogenated solvents, such as methylene chloride and chloroform. The reaction is usually carried out for a time ranging from about 5 minutes to a maximum of 3 hours, but generally a reaction time of about 0.5 to about 1 hour is sufficient. The following equation describes this process using a carboxylic acid halide; However, it goes without saying that the use of a carboxylic acid anhydride or another functional equivalent acylating agent can give the same products:

Oh

A.

ow o

.sch2ch2NH2

.cooh

Acyl halide

rSCH2ctf ^ r1 'r2'

cooh

1

In general, if an acid halide (suitable halides are chlorides, iodides or bromides) or an acid anhydride is used in the acylation reaction described above, the reaction in water or an aqueous mixture of a polar organic solvent such as acetone, dioxane, THF , DMF, acetonitrile and the like in the presence of a suitable basic acceptor such as NaHC03, MgO, NaOH, K2HP04 and the like.

When carrying out the reaction described here, it is generally not necessary to protect the 2-carboxy group or the 1 'hydroxy group; in cases where the acylating agent is extremely water sensitive, it is sometimes advantageous to carry out the acylation in a non-aqueous solvent system. Triorganosilyl (or tin) derivatives of thienamycin quickly give the tris-triorganosilyl derivatives, for example tris-trimethylsilylthienamycin Th (TMS) 3:

Th-

55

OTMS

NHTMS

COOTMS

These derivatives, which are readily soluble in organic solvents, can be easily prepared by reacting thienamycin with an excess of hexamethyldi-60 silazane and a stoichiometric amount of trimethylchlorosilane at 25 ° C. with vigorous stirring under an N2 atmosphere . The resulting NH4C1 is preferably removed by centrifugation and the solvent can be removed by evaporation to give the desired 65 silyl derivative.

In the general description of the new compounds (II, above), the radical designated by the group -COXR means, inter alia, -COOH (X is oxygen and

9 633 554

R is hydrogen) and all residues known to be effective or branched such as methyl, ethyl, tert-butyl, pentyl, decyl, pharmaceutically acceptable esters, anhydrides (R is acyl) and the like; Carbonylmethyl, including phenacyl,

Amide residues in bicyclic β-lactam antibiotics such as in p-bromophenacyl, p-tert-butylphenacyl, acetoxyacetylmethyl,

Cephalosporins and penicillins and their core ana- pivaloxyacetylmethyl, carboxymethyl and its alkyl and lie. 5 aryl esters, a-carboxy-a-isopropyl; Including aminoalkyl

Suitable residues (R) include conventional protective or 2-methylaminoethyl, 2-diethylaminoethyl, 2-acetamidoethyl,

Carboxyl blocking groups. Such blocking groups phthalimidomethyl, succinimidomethyl; Alkoxyalkyl,

are used in the preparation of species IIa, above. wherein the alkoxy part 1 to 10 and preferably 1 to 6 carbon

The term “blocking group” as used herein has atomic atoms and is branched, straight-chain or cyclic is used in the same way as it can according to io, and in which the alkyl part has 1 to 6 carbon atoms,

U.S. Patent 3,697,515, hereby incorporated by reference, such as methoxymethyl, ethoxymethyl, isopropoxymethyl, decyl

is closed, is used. Pharmaceutically acceptable, oxymethyl, ethoxypropyl, decyloxypentyl, cyclohexyloxyme

Thienamycin derivatives which can be prepared according to the invention and which are treated with thyl and the like; Alkanoyloxyalkyl, in which the alkanoyl

these hates fall are given below. Suitable oxy part is straight-chain or branched and 1 to 6 carbon

Blocking esters thus include those which have atoms in the following and the alkyl part has 1 to 6 carbon atoms, as indicated in the list which is representative of acetoxymethyl, pivaloyloxymethyl, acetoxyethyl, propionyl

but by no means an exhaustive list of the possible oxyethyl, acetoxypropyl, and the like. Haloalkyl, wherein

Represents ester groups in which X = O and R are given: halogen, chlorine, bromine, fluorine or iodine and the alkyl part

(i) R = CRaRbRc, in which at least one of Ra, Rb and Rc is straight-chain or branched and has 1 to 6 carbon atoms, is an electron donor, for example p-methoxyphenyl, 20 for example 2,2,2-trichloroethyl, trifluoroethyl, 2 -Bromopro-2,4,6-trimethylphenyl, 9-anthryl, methoxy, CH2SCH3, tetrapyl, diiodomethyl, 2-chloroethyl, 2-bromoethyl and the like; hydrofur-2-yl, tetrahydropyran-2-yl or Fur-2-yl. The remaining AJkenyï with 1 to 10 carbon atoms, which are either straight-chain Ra, Rb and Rc groups, can be hydrogen or organic or branched, for example allyl, 2-propenyl, 3-butene substitution groups. Suitable ester groups of these nyl, 4-butenyl, 4-pentenyl, 2-butenyl, 3-pentenyl, 3-methyl-3-species include p-methoxybenzyloxycarbonyl and 2,4,6-tri-25-butenyl, methallyl, 1,4 -Cyclohexadien-l-yl-methyl and derllei-methylbenzyloxycarbonyl. chen; Alkynyl of 1 to 10 carbon atoms, either

(ii) R = CRaRbRc, in which at least one of Ra, Rb and R ° - can be straight-chain or branched, for example 3-pentynyl, groups is an electron-attracting group, for example propargyl, ethynyl, 3-butyn-l-yl and the like ; Alkanoyl, benzoyl, p-nitrophenyl, 4-pyridyl, trichloromethyl, tribromo- which can be either straight-chain or branched, with 1 to 10 thyl, iodomethyl, cyanomethyl, ethoxycarbonylmethyl, aryl-30 carbon atoms, such as pivaloyl, acetyl, propionyl and the- sulphonylmethyl, 2-dimethylsulphoniummethyl, o-nitrophenyl same; Aralkyl or heteroaralkyl, wherein the alkyl is 1 to 3 or cyano. Suitable esters of this type include benzoyl carbon atoms, and hetero means that 1 to 4 hetero-methoxycarbonyl, p-nitrobenzyloxycarbonyl, 4-pyridylmethroatoms are present, selected from the group consisting of oxycarbonyl, 2,2,2-trichloroethoxycarbonyl and 2,2,2-tribromo from O, S or N, such as benzyl, benzhydryl and substituted ethoxycarbonyl. 35 benzyl, benzhydryl, for example benzyl or benzhydryl,

(ni) R = CRaRbRc, in which at least two of the Ra, Rb and substituted with 1 to 3 substituents, such as benzyl, phenoxy, Ha-Rc radicals, are hydrocarbons, such as alkyl, for example lied, lower alkyl, lower alkanoyloxy with 1 to 5 carbon atoms. Methyl or ethyl or aryl, for example phenyl and the atoms, lower alkoxy, hydroxy, nitro, blocked carboxy, residual Ra, Rb and Rc group, if present, is hydrogen or combinations thereof, for example p-chlorobenzyl. Suitable esters of this type include tert-butyl-40 o-nitrobenzyl, 3,5-dinitrobenzyl, p-methoxybenzyl, m-ben-oxycarbonyl, tert-amyloxycarbonyl, diphenylmethoxycarb- zoylbenzyl, p-tert.-butylbenzyl, m- Phenoxybenzyl, p-benzoyl-onyl and triphenylmethoxycarbonyl. benzyl, p-nitrobenzyl, 3,5-dichloro-4-hydroxybenzyl, p-meth-

(iv) R = Rd, where Rd is adamantyl, 2-benzyloxyphenyl, oxycarbonylbenzyl, p-methoxybenzhydryl, p-carboxybenzyl,

4-methylthiophenyl or tetrahydropyran-2-yl. the latter either as a free acid, as an ester or as

Silyl esters of this category of blocking groups can be present, 45,6-trimethylbenzyl, p-pivaloyloxy-45enes can be prepared in a simple manner from halosilanes, benzyl, p-tert-butoxycarbonylbenzyl, p-methylbenzyl, p-ben- or silazanes of the formula: zoyloxybenzyl, p-acetoxybenzyl, p-2-ethylhexanoylbenzoyl,

R43SiX '; R42SiX'2; R43Si.NR42; R43Si.NH.COR4; p-ethoxycarbonylbenzyl, p-benzoylthiobenzyl, p-benzamido

R43Si.NH.CO.NH.SiR43; R4NH.CO.NH4.SiR43; or benzyl, p-pivaloyloxybenzyl, m-pivaloyloxybenzyl, p-isoprop-

R4C (OSiR43); 50 oxybenzyl, p-tert-butoxybenzyl and also the cyclic ana-

HN (SiR43) 2, where X 'is halogen, chlorine or bromine, loge thereof, 2,2-dimethyl-5-coumaranmethyl, 5-indanylmethyl, and the various groups R4, which are identical or different, p-trimethylsilylbenzyl, 3,5- Bis-t-butoxy-4-hydroxybenzyl;

can be, hydrogen atoms or alkyl, for example 2-thienylmethyl, 2-furylmethyl, 3-tert-butyl-5-isothiazole-like methyl, ethyl, n-propyl, isopropyl; Aryl, for example thyl, 6-pivaloyloxy-3-pyridazinyl-ethyl, 5-phenylthio-l-tetra-phenyl; or aralkyl, for example a benzyl group. 55 zolylmethyl or the like (the term lower alkyl

More generally, pharmaceutically acceptable, new or lower alkoxy in this context are a chain with 1 carboxyl derivatives, in particular those which are derived from up to 4 carbon atoms); or phthalidyl; or Phe-reacting an N-acylated thienamycin (1) with alcohols, nylethyl, 2- (p-methylphenyl) ethyl and the arylthioalkylanalo-phenols, mercaptans, thiophenols, acylating agents and ge, aryloxyalkyl, wherein aryl is preferably a phenyl ring with the like , whereupon compounds (2, above) are obtained, which are then converted to 0 to 3 substituents, preferably 0 or 1 substituents, in the derivatives according to the invention by the R3 ortho or para position and the alkyl 1 to 6 carbon atoms -Group of the new compounds mentioned (II, above), for example (4-methoxy) -phenoxymethyl-, phenoxy-lead. methyl, (4-chloro) phenoxymethyl, (4-nitro) phenoxymethyl,

For example, esters and amides of interest are the ver (4-benzyloxy) phenoxymethyl, (4-methyl) phenoxymethyl, compounds of formula II (above) with the following group in 65 (2-methoxy) phenoxymethyl, (1- Phenoxy) ethyl, (4-amino) -the 2-position: -COXR, where X means oxygen, sulfur or NR 'phenoxymethyl, (4-methoxy) -phenylthiomethyl, (4-chloro) - (R' has the above Meaning) and R alkyl phenylthiomethyl, phenylthioethyl; Aryl, in which aryl is phenyl, having 1 to 10 carbon atoms and specifically straight-line 5-indanyl or substituted phenyl having 0 to 3 substituents,

633 554

10th

is preferably 0 to 1 substituent in the ortho or para position, for example (4-methyl) phenyl, (4-hydroxy) phenyl, (4-tert-butyl) phenyl, p-nitrophenyl, 3.5 -Dini-trophenyl or p-carboxyphenyl, the latter being present either as the free acid or as the sodium salt; Aralkenyl, wherein aryl is phenyl and alkenyl having 1 to 6 carbon atoms, such as 3-phenyl-2-propenyl; Aralkoxyalkyl, wherein aralkoxy is benzyloxy and the alkyl has 1 to 3 carbon atoms, such as benzyloxymethyl, (4-nitro) -benzyloxymethyl, (4-chloro) -benzyloxymethyl; Alkylthioalkyl, in which the alkylthio part has 1 to 10 and preferably 1 to 6 carbon atoms, but which can be branched, straight-chain or cyclic and the alkyl part has 1 to 6 carbon atoms, such as methylthioethyl, ethylthioethyl, cyclohexylthiomethyl, decylthiobutyl, methylthio-propyl, Isopropylthioethyl, methylthiobutyl and the like.

Alkoxycarbonylmethyl, dialkylaminoacetoxymethyl, and alkanoylamidomethyl, in which the alkyl groups of the last three mentioned radicals each contain 1 to 6 carbon atoms.

In addition to the esters (and thioesters) listed above, amides are also of the present invention

R '

includes, that is, where X is an -N group. Typical examples of such amides, Th-CONR'R, are those in which R 'is selected from the group consisting of hydrogen, methyl, ethyl, phenyl, p-methoxyphenyl, benzyl, carboxymethyl, methylthioethyl and heteroaryl; The grouping -COXR also includes anhydrides in which R is acyl, for example benzyloxycarbonyl, ethoxycarbonyl, benzoyl and pivaloyl.

Particularly preferred esters are those in which X is oxygen, sulfur or NR '(R' = hydrogen or lower alkyl having 1 to 6 carbon atoms) and R is aralkyl, araloxyalkyl, aralkoxyalkyl, alkylthioalkyl, halogenalkyl and alkenyl.

The most preferred compounds which can be prepared according to the invention are those in which (in relation to structure II above) X is oxygen and R is selected from the group consisting of:

Lower alkyl, lower alkenyl such as methallyl, 3-methyl-butenyl, 3-butenyl and the like; Methylthioethyl; Benzyl and substituted benzyl such as p-tert-butylbenzyl, m-phenoxybenzyl, p-pivaloyloxybenzyl, p-nitrobenzyl and the like; Piva-loyloxymethyl, 3-phthalidyl and acetoxymethyl, propionyloxy-methyl, acetylthiomethyl, pivaloylthiomethyl, allyl, 4-pentene-nyl, 2-butenyl, 3-methyl-2-butenyl, phenacyl, acetoxyacetyl-methyl, pivaloylacetamethyl, diethyl aminoethyl, methoxymethyl, p-acetoxybenzyl, p-pivaloyloxy-benzyl, p-isopropoxybenzyl-5-indanylmethyl, 5-indanyl, benzyloxymethyl, ethylthioethyl, methylthiopropyl, methoxy-carbonyloxymethyl, ethoxycarbonyloxymethyl, dimethylamino-3-yloloxymethyloxoethyloxo

The preferred N-blocking groups for the starting compound Ia are: carbobenzyloxy, ring-substituted car-bobenzyloxy, such as o- and p-nitrocarbobenzyloxy, p-methoxy-carbobenzyloxy, chloroacetyl, bromoacetyl, phenylacetyl, tert.-butoxycarbonyl, trifluoroacetyl, bromoethoxycarbonyl -renylmethoxycarbonyl, dichloroacetyl, o-nitrophenylsulfenyl, 2,2,2-trichloroethoxycarbonyl, bromine-tert-butoxycarbonyl, phenoxyacetyl; Non-acylated protecting groups such as alkylidene, e.g. benzylidene, salicylidene and the like are also of interest.

As previously indicated, the intermediate starting compounds for the preparation of 2 (above) can be prepared according to the following scheme;

10th

15

OH Th-NR'R2. COOH

1

OH Th-j- NR! R2

. COXR

2nd

where all symbols have the meaning given above.

20 In general, the transition (2—> 2) is effected according to customary prior art methods. These procedures include in particular:

25 1. Reaction of _1 with a diazoalkane such as diazomethane, phenyldiazomethane, diphenyldiazomethane and the like in a solvent such as dioxane, ethyl acetate, acetonitrile and the like at temperatures between 0 ° C and reflux for. a period of a few minutes to 2

30 hours.

2. Reaction of an alkali salt of .1 with an activated alkyl halide, such as methyl iodide, benzyl bromide or m-phenoxybenzyl bromide, p-tert-butylbenzyl bromide, pivaloyloxyme-35 thyl chloride and the like. Suitable reaction conditions include solvents such as hexamethylphosphoramide and the like, and temperatures from 0 ° C to 60 ° C for a period from a few minutes to 4 hours.

3. Reaction of! L with an alcohol such as methanol, ethanol, benzyl alcohol and the like. This reaction can be carried out in the presence of a carbodiimide condensing agent such as dicyclohexylcarbodiimide and the like. Suitable solvents at temperatures between 0 ° C and the reflux temperature and reaction times of 15 minutes to 18 hours include CHC13, CH3CH, CH2CI2 and the like.

45

so 4. Reaction of an N-acylated acid anhydride of .1, prepared by reacting the free acid 1 with an acid chloride such as ethyl chloroformai, benzyl chloroformate and the like, with an alcohol such as those listed under 3.) under the same reaction conditions as below 3.) called ge-55. The anhydride is prepared by reacting 1 and the acid chloride in a solvent such as tetrahydrofuran (THF), CH2C12 and the like at temperatures from 25 ° C to the reflux temperature for 15 minutes to 10 hours.

60

5. Reaction of labile esters of 1 or thienamycin, such as trimethylsilyl ester, dimethyl tert-butylsilyl ester or the like, with RX ', where X' is halogen, such as bromine or chlorine and R has the meaning given above, in a 65 solvents such as THF, CH2C12 and the like at temperatures between 0 ° C and reflux for 15 minutes to 16 hours. The implementation follows, for example, according to the following scheme:

Th-

OTMS

NHTMS

COOTMS 3

N-acylation Th-

ORMS NR'TMS L COOTMS

11

Esterification TH-

OTMS

NR'TMS

COOR

mild hydrolysis Th-

633 554

OH NHR1 L COOR

wherein TMS means triorganosilyl, such as trimethylsilyl, and all other symbols have the meaning given above.

The previously given schemes for the esterification are known for the related bicyclic β-lactam antibiotics and generally for organic syntheses and it should be noted here that no particular critical conditions with regard to the reaction parameters in the preparation of the N-acylated carboxyl derivatives 2 are the starting compounds for the Processes according to the invention are suitable are required.

For the ester embodiment, the most preferred R3 groups are those with a relatively low molecular weight that are hydrophilic.

The following radicals are typical and preferred for the ester embodiment: sulfo, phosphono, carbamoyl, methylsulfonyl, sufamoyl, dimethylsulfamoyl, N-methylcarbamoyl, bromoacetyl, hydroxyacetyl, aminoacetyl, dimethylaminoacetyl, triethylammoniumacetyl, guidanoacetyl, guidanacetyl, amidinoacetyl, guidanacetyl, amidinoacetyl, amidinoacetyl, guidylacetyl, amidinoacetyl, amidinoacetyl , Phosphamoyl, phosphonothioyl, thiocarbamoyl and the like.

In general, the new compounds can be obtained by a variety of known esterification reactions:

OH Th-1 NR! R2. COXR

"OR3» Th — j- NR! R2

. COXR

II

1. For the preparation of the ester embodiments, the reaction of 2 with any of the acyl groups listed above takes place in their acid form. This reaction can be carried out in the presence of a carbodiimide condensing agent such as a dicyclohexylcarbodiimide and the like. Suitable solvents include an inert solvent such as CHC13, CH2C12, DMF, HMPA, acetone, dioxane and the like at temperatures from 0 ° C to 60 ° C for a period of 15 minutes to 12 hours.

2. For the preparation of the ester embodiments, 2 is reacted with an acyl halide or an acid anhydride, the acyl groups being described above. In general, the aforementioned acylation reaction uses an acid halide (suitable halides are chlorides, iodides or bromides or acid anhydrides) and the reaction is carried out in an anhydrous organic solvent such as acetone, dioxane, methylene chloride, chloroform, DMF or the like in the presence a suitable basic acceptor such as NaHC03, MgO, triethylene, pyridine and the like, at temperatures between 0 ° C to 40 ° C for one hour to 4 hours.

Suitable acyl halides and anhydrides include: acetic anhydride, bromoacetic anhydride, propionic anhydride, benzoyl chloride, phenylacetyl chloride, azidoacetyl chloride, 2-thienylacetyl chloride, 2-, 3- and 4-nicotinyl chloride, p-nitro-benzoyl chloride, 2,6-dimethoxybenzoyl Guanidino-phenylacetyl chloride, hydrochloride, methanesulfonyl chloride, dibenzyl phosphorochloridate, dimethylthiophosphorochloridate,

2-furoylethyl carboxylic anhydride, methyl chloroformate, bis (p-10 nitrobenzyl) phosphorochloridate and the like.

3. For the preparation of the ester embodiments, 2 is reacted with a suitable substituted ketene or isocyanate, such as ketene, dimethyl ketene, methyl isocyanate, methyl isothiocyanate, chlorosulfonyl isocyanate and the like. Suitable solvents include dioxane, tetrahydrofuran, chloroform and the like at temperatures from -70 ° C to 60 ° C for a period of 15 minutes to 18 hours.

The new compounds are new valuable antibiotics that are active against various gram-positive and gramnega-20 tive bacteria and are therefore used in human and veterinary medicine. The new compounds can therefore be used as antibacterial drugs for the treatment of infections caused by gram-positive and gram-negative bacteria, for example against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Salmonella typhosa, Pseudomonas and Bacterium proteus. The new bactericides mentioned can also be used as additives for animal feed and for food preservation and as disinfectants. For example, they can be used in aqueous compositions in concentrations ranging from 0.1 to 100 parts of the antibiotic per million parts of solution to destroy or inhibit the growth of harmful bacteria on medical and dental equipment, and also as bactericides industrial applications, for example water-based paints,

in white water from paper mills to inhibit the growth of harmful bacteria.

The products obtained according to the invention can be used alone or in combination with active ingredients in a large number of pharmaceutical preparations. These antibiotics and their corresponding salts can be used in the form of capsules or as tablets, as powders or as liquid solutions or as suspensions and elixirs 45. They can be administered orally, intravenously or intramuscularly.

The compositions are preferably prepared in a form such that they are absorbed in the gastrointestinal tract. Tablets and capsules for oral administration can be in unit dosage form and can contain conventional excipients such as binders, for example syrup, acacia, gelatin, sorbose, tragacanth or polyvinylpyrrolidone; Fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbose or glycerin; Lubricants, for example magnesium stearate, talc, polyethylene glycol, silicon dioxide; Disintegrants, for example potato starch or suitable wetting agents, such as sodium lauryl sulfate. The tablets can be coated in a known manner. Oral liquid preparations can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs and the like, or can be prepared as a dry product so that they can be mixed with water or other suitable carrier material for their use can. Such liquid preparations can contain conventional additives, such as suspending agents, for example sorbose, syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible oils

633 554

12

for example, contain almond oil, fractionated coconut oil, diester, propylene glycol or ethyl alcohol; Preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid. Suppositories contain the usual substances suitable for this, for example cocoa butter or other glycerine.

Compositions for injections can be made in single doses in the form of ampoules or in containers with added preservatives for multiple doses. The compositions can be in the form of suspensions, solutions or emulsions in oil or aqueous carrier materials and they can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents. Alternatively, the active ingredients can be in the form of powder so that they can be mixed with a suitable carrier material, for example sterile, germ-free water, before use.

The compositions can also be prepared in suitable forms for absorption by the mucous membranes of the nose and the neck and bronchial tissues. A daily oral dosage contains about 15 to about 600 mg of active ingredient per kg of body weight of the patient in one or more administrations per day. A preferred daily dosage for adults is in the range of about 80 to 120 mg of active ingredient per kg of body weight.

The present compositions can be administered in various unit dosage forms, for example in solid or liquid oral dosage forms. The compositions per unit dose, whether liquid or solid, should contain about 0.1% to 99% of active material, with the preferred range being between about 10 and 60%. The compositions generally contain from about 15 mg to about 1500 mg of active ingredient; however, it is generally preferred to use a dosage amount in the range of about 250 to 1000 mg. In the case of parenteral administration, the single dose is generally the pure compound in a slightly acidified aqueous sterile solution or in the form of a soluble powder which is intended to be dissolved.

The following examples describe the invention, but without restricting it. In the examples, the compounds that can be produced according to the invention are represented by the following symbol:

Ren react at 25 ° C and then centrifuged the ammonium chloride from the suspension. The supernatant is evaporated to an oil in a stream of nitrogen, which is used for further reaction.

Example 1 O

ii

-o-c-ch.,

Th-

-NHg-O-CH 2 ~ ^ 3 ~ N ° 2

cooch rO-

NO,

15 Preparation of Ö-acetyl-N- (p-nitrobenzyloxycarbonyl) thien-amycin- (p-nitrobenzyl) ester

A solution of 50 mg of p-nitrobenzyloxycarbonyl-thien-amycin-p-nitrobenzyl ester in 0.5 ml of pyridine is mixed with 0.16 ml of acetic anhydride. The mixture is allowed to react for 3 hours at 20 room temperature and then evaporated to dryness in vacuo. The solid residue is dissolved in chloroform and the solution is chromatographed on a 20.32 × 20.32 cm (8 × 8 in.) Large 1000 [i-silica gel plate in ethyl acetate / chloroform (3: 1). The band at the Rf value of 0.55 is isolated; 25 25 mg of O-acetyl-N-p-nitrobenzyloxycarbonyl-thien-amycin-p-nitrobenzyl ester of mp 172 to 175 ° C. are obtained; NMR (CDC13) Ô, 1.41, ((d) J = 6 Hz CH3), 2.04 ((s) COCH3), 2.7 to 3.6 ((m) 4.14 (t of d) J = 3 and 9 Hz), 5.17 (s) carbamyl CH2; 5.35 (AB quartet) J = 14, Hz; 7.45 (d), 7.60 (d), 8.18 (d) J = 9 30 Hz, aromatic H; Mass spectrum M + m / e 628.

Example 2

Preparation of O-acetyl-N-azidoacetyl-thienamycin-35 benzyl ester

0

"OC-CH-r

Th

0

ti

HHCCH2U3

co2ch2

y A

45

Th-

OR3

NR ^ 2

COXR

Step A: Preparation of N-Azidoacetyl-thienamycin Sodium Salt (1) and Lithium Salt (II)

Th-

where the three functional groups are given.

Preparation 1

"OTMS NHTMS or Th (TMS) 3 COOTMS TMS = trimethylsilyl group

Production of silylated thienamycin

80 mg of thienamycin are suspended in 40 ml of tetrahydrofuran under nitrogen, the suspension is concentrated to 10 ml and 1 ml of hexamethyldisilazane and 300 μl of trimethylchlorosilane are added. The mixture is allowed to stir for 20 minutes with vigorous stirring.

Th

55

OH

KB

CO,

©

Th

©

OH

- MHCCH2N3

C02OM © M = Na, Li

(I) Dissolve 48 mg (0.18 mmol) of thienamycin in 10 ml of cold water and add the solution at 0 ° C with 147 mg (17.6 mmol) of sodium bicarbonate and 10 ml of dioxane. The solution is then mixed with 60 mg (0.50 mmol) of azidoacetyl chloride for 2 min. The reaction mixture is then stirred for 15 minutes, neutralized (up to pH 7.0) with 30% phosphoric acid and transferred to a separating funnel. The solution is extracted twice with 50 ml of ether each time. The aqueous layer is concentrated to 5 ml and placed on a UV-monitored ion exchanger containing Dowex AG-50 X 8 (sodium form).

13

633 554

abandoned pillar. The desired fractions are combined and freeze-dried; 21 mg of the product are obtained. Electrophoresis (50 V / cm, 20 min.) Of the product in phosphate buffer (pH 7.0) reveals a single bioactive band (zone) which moves 40 mm in the direction of the anode; UV X 300 nm; NMR (100 MHz, D20): Ô 1.26 (d, CH3CH); 2.92 to 3.43 (m, 3CH2 and C6-H), 4.01 (s, CH2N3) and 4.20 ppm (m, Q-HundCr-H).

(II) Dissolve 76.2 mg (0.28 mmol) of thienamycin in 10 ml of cold water and add 0.6 ml in lithium hydroxide solution and 10 ml of dioxane to the solution at 0 ° C. The reaction mixture is stirred for 1 minute, 33.6 mg (0.28 mmol) of azidoacetyl chloride are added over the course of 2 minutes, the mixture is stirred for a further minute and then neutralized with 30% phosphoric acid (up to pH 7.0). The mixture is then extracted with ether, the aqueous solution is concentrated to 5 ml and applied to an ion exchange column containing Dowex AG-50 X 8 (lithium form). The desired fractions are combined and freeze-dried; 38 mg of the product are obtained; UVa 300 nm.

Step B: Preparation of N-azidoacetyl-thienamycin-benzyl ester graphic isolated (Rf = 0.49 in ethyl acetate); IR (CHC13: 2121 (N3), 1777 (β-lactam), 1738 (OAc) and 1683 cm "1 (ester and amide); NMR (CDC13.60 MHz): 1.38 (d, CH3CH), 2, 05 (s, OAc), 3.96 (s, CH2N3), 5.20 (s, CH2C6H5) and 7.40 ppm (m, s QH5) (Ac = acetyl).

Example 3

Preparation of O-acetyl-N-azidoacetyl-thienamycin-3-methyl-2-buten-l -yl ester

10th

- O

II

o-c-ch3

Th -j- NHCCH2N3

o l c02ch2ch

'H,

XCH3

Th

OH

0

NHCCH2N3

- COoOM ©

Th

•OH

0

HHCCHoN

2 3

c02ch2

y \

(a) 3 mg of N-azidoacetyl-thienamycin lithium salt are mixed with 1 ml of hexamethylphosphoramide (HMPA) and

30 mg (0.21 mmol) of benzyl bromide were stirred. The reaction mixture is then diluted with 5 ml of ethyl acetate and washed thoroughly with water. The organic layer is separated and dried over sodium sulfate. The product (2 mg) is isolated by silica gel thin layer chromatography (Rt = 0.18 in ethyl acetate); IR (CHC13): 2125 (N3), 1777 (β-lactam) and 1687 cm "1 (ester and amide); NMR (CDC13, 100 MHz): 1.34 (d, CH3CH), 2.80 to 3, 60 (m, 3CH2 and C6-H), 3.97 (s, CH2N3), 4.21 (m, C5-H and C7-H), 5.19 and 5.34 (d, CH2C6H5), 6, 63 (m, NH) and 7.32 (m, C6H5).

(b) 30 mg (0.08 mmol) of N-azidoacetyl-thienamycin sodium salt are stirred for 30 min at room temperature with 3 ml of HMPA and 120 mg (0.70 mmol) of benzyl bromide. The product (30 mg) is isolated by the method described above.

Step C: Preparation of O-acetyl-N-azidoacetyl-thienamycin-benzyl ester

Step A: Preparation of N-Azidoacetyl-thienamycin-3-methyl-2-buten-l -yl ester

11 mg (0.029 mmol) of N-azidoacetyl-thienamycin sodium 25 salt are stirred for 30 minutes at room temperature with 1 ml of HMPA and 39 mg (0.26 mmol) of l-bromo-3-methyl-2-butene. The mixture is diluted with 10 ml of ethyl acetate and washed thoroughly with water. The desired product (10 mg) is isolated by silica gel thin layer chromatography 30 (Rf = 0.18 in ethyl acetate); IR (CHC13): 2121 (N3), 1777 (β-lactam) and 1685 cm-1 (ester and amide); MNR (CDC13,100

CH3

MHz): 1.34 (d, CH3CH), 1.73 (s, = <, 2.80 to 3.80

Th

■ oh

0

hhcch2F3

Th co2ch2

<3

-OAc

0

it ch3

35 (m, 3CH2 and C6-H), 3.98 (s, CH2N3), 4.20 (m, C7-H), 4.72 (d, CHjCH), 5.40 (t, CH2CH) and6, 70 (broad, NH); Mass spectrum (E.I.): e / m 423 (molecular ion), 405 (M + -H20), 395 (M + -N2) and 337 (M + -86).

40 Step B: Preparation of O-acetyl-N-azidoacetyl-thienamycin-3-methyl-2-buten-l -yl ester

5 mg of N-azidoacetyl-thienamycin-3-methyl-2-butten-1-yl ester are dissolved in 0.5 ml of pyridine and the solution is mixed with 0.1 ml of acetic anhydride. Then hold the mixture for 2 hours.

45 at room temperature and then evaporated to dryness. The residue is worked up by silica gel thin layer chromatography to give the desired product (rf = 0.52 in ethyl acetate); NMR (CDC13, 100 MHz): 1.38 (d, CH3CH), 1.74 / CH3

(s, =), 2.03 (s, OAc), 2.90 to 3.80 (m, 3CH2 and C6-CH3

H), 3.98 (s, CTysy, 4.20 (m, C5-H), 4.74 (d, CH2CH =) and 5.32 ppm (t, CH2CH =).

50

55

hhcch2n3

l— c02ch2

-O

Example 4

Preparation of O-acetyl-N-glycyl-thienamycin-3-methyl-2-buten-l -yl ester

O

30 mg (0.067 mmol) of N-azidoacetyl-thienamycin-benzyl ester are dissolved in 0.5 ml of pyridine and the solution is mixed with 0.2 ml of acetic anhydride. The mixture is kept at room temperature for 40 minutes. The solution is diluted with 1 ml of ethyl acetate and washed with ice water. The organic layer is separated off and dried over sodium sulfate. The desired product (10 mg) is separated by silica gel thin layer chromatography.

60

65

Th-

rOCCH3

O

II

-NHCCH2NH2

—CO, CH, CH = C

/ CH3

\

CH,

633 554

14

10 mg of O-acetyl-N-azidoacetyl-thienamycin-3-methyl-2-buten-l-yl ester are dissolved in 1 ml of ethyl acetate and the solution is poured into a hydrogenation flask which contains 10 mg of palladium (obtained from palladium oxide) and 0 Contains 5 ml of 50% methanol in ethyl acetate. The mixture is stirred for 1 hour at 25 ° C. and a hydrogen pressure of 1 atm. The catalyst is then filtered off and the product is isolated by silica gel thin layer chromatography (20% methanol / chloroform). Electrophoresis of the product in a pH 7.0 buffer (50 V / cm, 20 min.) Results in a bioactive band (zone) which migrates towards the cathode.

Preparation 2

Production of N- (o-nitrobenzyloxycarbonyl) thienamycin-p-bromophenacyl ester method migrates; UVI ^ 01 315 nm; IR (CHC13): 3300 (NH2), 1777 (β-lactam) and 1672 cm-1 (ester and amide).

Preparation 4

s Preparation of N-azidoacetyl-thienamycin-pivaloxymethyl ester [- "OH

O

II

Th + - NHCCH2N3

- CO2CH2-O-C-C- (CH3) 3 II

O

10th

15

[—O (tms)

NH (TMS) ^

1 — coo (tms)

(I)

Th ■

oh no 2

-nhc-o-ch2- / y

O O

(n)

TMS = trimethylsilyl group

24 mg of Th (TMS) 3 (I) in 0.8 ml of anhydrous tetrahydrofuran are mixed with 23 mg of o-nitrocarbobenzyloxychloride and then with 0.015 ml of triethylamine. After 30 minutes of vibration mixing at 25 ° C, the mixture is evaporated in a dry nitrogen stream to a pasty residue, which is washed three times with petroleum ether. The residue is then suspended in 1 ml of anhydrous tetrahydrofuran and 14 mg of p-bromophenacyl bromide and then 0.03 ml of triethylamine are added to the suspension. After 30 minutes of vibration mixing at 25 ° C, the mixture is evaporated to dryness at 20 ° C in a vacuum. The residue is dissolved in 2 ml of ethyl acetate and the solution is shaken for 5 minutes with 0.3 ml of pH 4 buffer. The organic layer is dried over magnesium sulfate and filtered. The filtrate is evaporated to a pasty residue and the desired product (44 mg) is isolated by preparative thin layer chromatography on silica gel eluting with ethyl acetate / CHC13 (7: 3).

Preparation 3

Preparation of N-Glycyl-thienamycin-3-methyl-2-buten-l-yl ester i-oh

O

Th -f- NHCCH2N3

y l-co2ch2ch = c

■ OHO

Th - NHCCH2NH2

CH,

11 mg (0.04 mmol) of N-azidoacetyl-thienamycin sodium salt are stirred for 30 minutes at 25 ° C. with 1 ml of HMPA and 36 mg (0.24 mmol) of chloromethyl pivalate. The mixture is then diluted with ethyl acetate and washed with water. The desired product is isolated by silica gel thin layer chromatography; (Rf = 0.18 in ethyl acetate); IR (CHC13): 2121 (N3), 1777 cm "1 (β-lactam); PMR (CDC13, 60 MHz); 1.22 (s, t-Bu), 1.32 (d, CH3CH), 3, 98 (s, CH2N3) and 5.83 ppm (dd,

co2ch2o-).

25th

Preparation 5

Preparation of N-azidoacetyl-thienamycin-2-methyl-2-pro-pen-1-yl ester

- 30 —oh

O

II

Th -1- NHCCH2N3

-co2ch2-c = ch2 I

ch3

40

20 mg (0.055 mmol) of N-azidoacetyl-thienamycin lithium salt are stirred for 30 minutes at 25 ° C. with 1 ml of HMPA and 27 mg (0.30 mmol) of 3-chloro-2-methylpropene. The mixture is diluted with ethyl acetate and washed with water. The desired product is isolated by silica gel thin layer chromatography (R £ = 0.18 in ethyl acetate); IR (CHC13): 2121 (N3), 1777 (β-lactam) and 1684 cm-1 (ester and amide).

Preparation 6

so production of N-glycyl-thienamycin esters

35

<—Co? Ch7ch = c

/ CH;

Th-

55

-CH,

VCH,

Oh

• NHCCH2NH2

O

-COOR

10 mg of N-azidoacetyl-thienamycin-3-methyl-2-buten-l-yl ester are dissolved in 1 ml of ethyl acetate and the solution is added to a hydrogenation flask which contains 10 mg of palladium (from palladium oxide) and 0.5 ml Contains 50% methanol in ethyl acetate. The mixture is stirred for 1 hour at 25 ° C. and a hydrogen pressure of 1 atm. The catalyst is then filtered off and the desired product is isolated by thin layer chromatography (Rf = 0.16 in 20% methanol / chloroform). Electrophoresis of the product in a pH 7.0 buffer (50 V / cm, 20 min.) Results in a bioactive band which is 34 mm in the

O

60

I)

II) R

65

R = -ch2-0-c-c (ch3) 3

-ch2c = ch2 I

ch,

Compounds I and II are prepared by using the reduction method of Example 20 instead of

15

633 554

N-Azidoacetyl-thienamycin-3-methyl-2-buten-l-yI-ester each uses the equivalent amount of the appropriate starting compound.

Preparation 7

Preparation of N-Benzyloxycarbonyl-thienamycin and N-Benzyloxycarbonylbenzylcarbonsäureanhydrid r — oh ho

Th

NC-O-CH,

■ COOR

rO

R =

0

A solution of 16.6 mg of thienamycin in 4 ml of 0.05 m pH 7 phosphate buffer and 2 ml of dioxane, which is in a three-necked flask equipped with a stirrer, thermometer, pH electrode and the feed line end of an automatic titration device, is placed in a CH3OH / Ice bath cooled to - 8 ° C. The pH is adjusted to 8.2 by adding 0.2N sodium hydroxide in 50% aqueous dioxane and a solution of 0.015 ml carbobenzyloxychloride in 2 ml chloroform is added. Then the mixture is stirred for 10 minutes at -6 ° C (pH 8.2), covered with ether and the pH is adjusted to 7 with in hydrochloric acid. The layers are then separated by centrifugation and the aqueous phase is extracted twice more with ether. The aqueous phase is then acidified with a layer of ethyl acetate (up to pH 2). The ethyl acetate is separated off and the aqueous layer is extracted again with ethyl acetate. The combined ethyl acetate extracts are washed with saturated sodium chloride solution, dried over magnesium sulfate and filtered. Mna stir the filtrate with water and adjust it to pH 7 with dilute sodium bicarbonate solution. Then the aqueous phase is separated off and freeze-dried. 10 mg (46%) of the sodium salt of N-benzyloxycarbonylthienamycin are obtained. The UV spectrum [X.max 303 mjx, E% 147 (e = 6290)]

gives a purity of around 80%. A 20-minute electrophoresis at 50 V / cm and a pH of 7 and subsequent bioautography on S. aureus reveals an inhibition zone at +2.5 cm.

The ether extracts of the reaction mixture contain the desired compound N-benzyloxycarbonyl-thienamycin-benzyl-carboxylic acid anhydride; UV Xmax 335 m | i.

Preparation 8

Preparation of N-benzenesulfonyl-thienamycin-2-methyl-2-pro-pen-1-yl ester stirred gnetisch. The pH is adjusted to 8.2 using an automatic burette with 2.5N NaOH and 227 (il (226 µmol)) benzenesulfonyl chloride in 500 ml of p-dioxane is added all at once, and the pH is then maintained using the automatic burette at 8.2 for 30 min and then adjust it to 7.0 with dilute aqueous phosphoric acid, then the reaction solution is concentrated to 15 ml and chromatographed on 50 cc XAD-2 resin and the column is eluted with water and then with 10% aqueous tetrahydrofuran, through which the product is washed out. The eluate obtained with the 10% aqueous tetrahydrofuran is concentrated to a third of its volume and freeze-dried. 28 mg of the product, the electrophoretic mobility ( 50V / cm, 20 min., PH 7.0.05n phosphate buffer) makes up 3.5 cm in the direction of the cathode; A.raax 303 (e = 3650) in 0.1 npH 7 phosphate buffer.

Step B: N-benzenesulfonyl-thienamycin-2-methyl-2-propen-l-yl ester

20 The desired compound is obtained by reacting N-benzenesulfonyl-thienamycin (in the form of the sodium salt) with 2-methyl-2-propen-1-yl chloride.

Preparation 9

25 Preparation of N- (N -acetimidoyl - $ - alanyl) -thienamycin-p-tert-butylbenzyl ester

Step A: N - ($ - Azidopropionyl) thienamycin

30th

Th

35

OH

NH, © -C02 ©

Th i-0H

0

NHCCH2CH2N5 -CO., © Na '

40

45

50

184 mg of thienamycin are dissolved in 30 ml of water and 0.52 g of sodium bicarbonate, 30 ml of dioxane and 163 mg of β-azidopropionyl chloride are added to the solution at 0.degree. The mixture is stirred for 15 minutes, then neutralized with 30% phosphoric acid and then extracted with ether. The aqueous layer is separated off and concentrated to 5 ml. The crude product is chromatographed on an ion exchange column (2.54 X 25.4 cm or 1X10 in.) Containing Dowex 50W X 8 (sodium form). The column is eluted with water to give 81 mg of the desired product, which shows UV absorption at X 306 nm. Electrophoresis of the product at 2 kV in 0.1 m pH 7 phosphate buffer for 2 hours results in a single bioactive zone which shifts 30 mm in the direction of the anode.

Step B: N- (β-alanyl) thienamycin

Th

■ OH

NH-

n \ /

0 N— '

-COOR

55

Th

60

■ OH

Q

-NHCCH2CH2N3 © ©

.COy «a

Th.

OH

©

■ NHCCH2CH2NH3

L-CO

0

R = -CH2C = CH2

CH,

Step A: N-benzenesulfonyl-thienamycin

52 mg (148 μmol) of thienamycin are dissolved in 25 ml of 0.1 in pH 7 phosphate buffer. The solution is cooled with ice bath cooling.

40 mg of N- (β-azidopropionyl) thienamycin (dissolved in 20 ml of water) is dried for 40 minutes at 25 ° C. and a hydrogen pressure of 1 atm in the presence of 200 mg of palladium hy-65. The solution obtained (pH 9.0) is neutralized with 30% phosphoric acid. The catalyst is then filtered off and the mixture is chromatographed on an ion exchange column containing Dowex 50W X 8 (sodium form)

633 554

16

(2.54 X 25.4 cm or 1X10 in.). The column is eluted with water, giving 20 mg of the desired product,

buffer results in a single bioactive zone which moves 10 mm in the direction of the cathode.

that shows UV absorption at X ^ 302 nm. Electrophoresis of the product for 20 min at 2 kV in 0.1 m pH 7-phosphate stage C: N- (N-acetimidoyl - $ - alanyl) -thienamycin r-OH

raw

0

©

Egg -j- NHOCH2CH2NH3 c02 ©

A solution of 125 mg of N- (β-alanyl) -thienamycin in 15 ml of water is kept at 0 ° C. and by adding 2.5N NaOH at a pH of 8.5, while keeping it within 10 min. 350 mg of O-ethyl acetate-hydrochloride were added in portions. The mixture is then stirred for 1 hour and then neutralized with 2.5N HCl. The mixture is then concentrated to 15 ml and the crude product obtained is chromatographed twice on a column containing Dowex 50W X 8 (sodium form) (2.54 X 25.4 cm or 1X10 in.). 25 mg of product are obtained, which is eluted with water. The solution is freeze-dried. Recrystallization of the product from Waser gives a crystalline solid with the following data: IR (Nujol-Mull): 1769 cm-1 (β-lactam); NMR (D20.100 MHz): 2.20 ppm (s, acetimidoyl CH3); UV X 302 nm. The 20 min. Electrophoresis of the product at 2 kV in 0.1 m pH 7-phosphate buffer results in a single bioactive zone which moves 10 mm in the direction of the cathode.

Step D: N- (N -acetimidoyl-§-alanyl) -thienamycin-p-tert-butylbenzyl ester

The desired connection is made using the appropriate starting compounds.

Example 5

Preparation of 0- (methylcarbamoyl) -N-p-nitrobenzyloxy-carbonyl-thienamycin-p-nitrobenzyl ester

0 h

[-0cnhch,

O

■ raccH2CH2ira2GN

-co "€)

CH,

15

20th

th

0

r-o-c-CH, watch1 • coor2

Th-hHHR1 "coor2

R1 = p-nitrobenzyloxycarbonyl R2 = p-nitrobenzyl

25 A solution of 50 mg N- (p-nitrobenzoxycarbonyl) thienamycin (p-nitrobenzyl) ester in 0.5 ml pyridine is mixed with 0.16 ml acetic anhydride. The mixture is allowed to react at 250 ° C. for 3 hours and then evaporated to dryness in vacuo. The solid residue is dissolved in chloroform and the solution is chromatographed on a 20.32 × 20.32 cm (8 × 8 in.) Large silica gel plate in ethyl acetate / chloroform (3: 1). The band at Rf 0.55 is isolated; 36 mg of 0-acetyl-N- (p-nitrobenzyloxycarbonyl) thienamycin (p-nitrobenzyl) ester are obtained; Mp = 172 to 175 ° C; NMR (CDCl3) ô, 35 1.41, (d, J = 6 Hz, CH3); 2.04 (s, COCH3), 2.7 to 3.6 (m), 4.14 (t of d) (J = 3 and 9 Hz), 5.17 (s, carbamyl CH2), 5.35 (AB Quartet, J = 14Hz), 7.45 (d), 7.60 (d), 8.18 (d) (J = 9Hz, aromatic H); MS: M + m / e 628.

■ »o Example 7

Preparation of N- (p-nitrobenzyloxycarbonyl) thienamycin-cin-O-methanesulfonyl-p-nitrobenzyl ester

45

Th

OSOgCHj NHR1

> - coor r1 = -coch2—:

r2 = • -ch2- £ ^ -:

■ no.

A solution of 20 mg of N- (p-nitrobenzyloxycarbonyl) thienamycin (p-nitrobenzyl) ester and 20 mg of methyl isocyanate in 5 ml of methylene dichloride is stirred at 23 ° C. for 18 hours. The solvent is then evaporated off and the residue is extracted with hexane. The residue, which is insoluble in hexane, is chromatographed on silica gel, giving essentially pure 0- (methylcarbamoyl) -N-p-nitrobenzyloxycarbonylthienamycin-p-nitrobenzyl ester.

Example 6

Preparation of O-acetyl-N- (p-nitrobenzyloxycarbonyl) thienamycin- (p-nitrobenzyl) ester

50

R1 = p-nitrobenzyloxycarbonyl

R2 = p-nitrobenzyl

A solution of 34 mg of methanesulfonyl chloride in 0.8 ml of 55 methylene dichloride is added dropwise with stirring in an ice-cold solution of 117 mg of N- (p-nitrobenzyloxycarbonyl) thienamycin-p-nitrobenzyl ester and 40 mg of triethylamine in 6 ml of anhydrous methylene dichloride with stirring . The reaction mixture is stirred for a further 90 min, then diluted with 20 ml of 60 cold methylene dichloride, washed with 10 ml of water (3 ° C), 5 ml of 0.1 m pH 3-phosphate buffer (3 ° C) and 2% aqueous Sodium bicarbonate solution (3 ° C), it dries over magnesium sulfate and filtered. Evaporation of the solvent in vacuo leaves p-nitrobenzyl-6- (l-methanesulfonyloxyethyl) -3-65 [2- (p-nitrobenzyloxycarbonylamino) ethylthio] -7-oxo-l-aza-bicyclo [3.2.0] hept-2-en-2-carboxylate (otherwise conveniently referred to as N- (p-nitrobenzyloxycarbonyl) thienamycin-0-methansulfonyl-p-nitrobenzyl ester) in the form of an oil.

17th

633 554

Example 8 spelling methods given the following invented

If instead of the compounds according to the starting materials in Examples 1-7:

Compounds used N-acyl-thienamycin carboxylic acid

esters in each case the equivalent amount of N-acylthienamycin q ^ 3

uses carboxylic acid esters, the corresponding O-, 5 N- and carboxyl-thienamycin derivatives are obtained.

Example 9

According to the in the preceding examples and in the q

10th

S

_N.

sch2ch2nr1r2

coxr

connection

1)

2)

3)

r3

-c-h

II

O

-c-nh2 II

O

-c-s-CH3

II

O

4) -C-O-CH3

II

O

5) -C-CH2Br

II

O

6) -C-CH2NH2

II

O

7) -C-CH2-N (CH3) 2

II

O

8) -C-CH2OH

II

O

9) -C-S-CH3

II s

10) -c-ch2-o-ch3 II

O

R

-CH,

* =>

-ch2-0-c-c (ch3) 3 II

O

-ch2ch2-s-ch3 -ch2-ch2-n (ch3) 2

-ch2ch = ch2

h h

-ch2-c-ch3 II

ch2

-ch2-s-c-ch3

II

O

-ch2-c-0 II

o r2 H H

-C-H

II

O

-c-o-ch3 II

0

-C-CH2Br

II

O

-c-ch2nh2 II

O

-c-ch2-n-c = nh2 II I

o ch3hc1

-c-n-ch3

ii h O

-C-CH2C1 II

O

-c-ch2ch2nh2 II

o x

o h o h o h o h o h o h o h

O H

o h

O H

11) -c-ch2n = c-nh2

II I

o h

O

"l \ H

12) -c ch2Lncnh2 h3po4

n h

H

H

-CH2-CH2-C = CH2

c-ch2-n = c-nhh2so4 o h II

Oh oh

633 554

18th

Ver R3

am-

dung

13) -c-ch2-cooh II

O

R

-ch2-o-c-o-ch3 II

O

R2

c-ch, -s:

; nh

\

nh x r1

O H

O

14) -SO3H

15) -s02nh2

16) -s02n (ch3) 2

-ch2 j ^ -q-c-chj h o

c — o-ch20 II

o o

-ch2 - <^ ^> - 0-ch (ch3) 2 c-o-ch20-nh2

O H

O H

O H

O

il

17) COCH2Hfy-0- € H3

-ch2-och2 - ^ och3 -p ^

, ONa

OCH,

O H

18) -C-CH2CC13

II

o o

II

19) C OC (CH2Br) (CH3) 2 0 = phenyl

-ch-

^) - N ° 2

0-ch2-0-c-n- (ch3) 2

-C-C = CH II

O

-S-CF,

O

H

O

H

O

Manufacture of alternative raw materials

In addition to thienamycin, it is obvious to the person skilled in the art that its various isomers, alone or as mixtures, can be used as starting materials in the preparation of the compounds according to the invention. Some of these isomers are available from natural fermentation products (see below). However, all isomers can be obtained by total synthesis (see below) in the form of a mixture of 4 diastereoisomers which have bactericidal activity and which can be separated by customary industrial processes. The 4 diastereoisomers (2 eis, 2 trans) can be separated chromatographically. The separation of a given d / 1 pair with optically active acids or bases is carried out according to customary methods. It should be noted that the absolute configuration of the first identified starting material is (I) 5R 6S 8R.

Production of thienamycin by total synthesis

SCH2CH2HH2 COOH

Step A: Preparation of 4- (2-acetoxyvinyl) azetidin-2-one

40

45

h2c = ch-ch = choc-ch3 + o = c = n-so2ci

II

O

0 0

ch = chocca,

50

n

- • R \

/

CH = CH0CCH,

S02C1

/

-NK

A solution of 1.0 ml of distilled chlorosulfonyl isocyanate (1.65 g, 11.7 mmol) in 2.5 ml of anhydrous diethyl ether is cooled in a bath of −20 ° C. under N 2.

A solution of 2.5 g L-acetoxybutadiene (22 mmol) in 2.5 ml anhydrous ether is similarly cooled under nitrogen in a -20 ° C bath.

The chlorosulfonyl isocyanate solution is added dropwise to the 60 acetoxybutadiene solution by means of a polytetrafluoroethylene tube immersed in the CSI solution and is pressurized with N2. The addition takes 10 minutes. Little or no color is discernible and the reaction is stirred at -20 ° C for 0.5 hours. The solution is clear and has a light yellow color.

A solution of 2 g of sodium sulfite and 5 g of K2HP04 in 20 ml of H20 is prepared during the aforementioned 0.5 hour reaction time and cooled on an ice bath; 20 ml ether who

19th

633 554

added to the mixture with vigorous stirring on an ice bath. At the end of the 30-minute reaction time, the reaction mixture, again using N2 pressure and a polytetrafluoroethylene tube, is transferred from the reaction flask, which is kept in a bath at −20 ° C., to the vigorously stirred hydrolysis mixture. The quick, dropwise addition is complete after 5 minutes. The hydrolysis is allowed to take place for a further 5 minutes. The hydrolysis mixture has a pH of 6-8, preferably pH 8.

The phases are separated, with a yellow-orange resin remaining in the aqueous phase. The ether phase is dried directly over MgS04. The aqueous / resin phase is extracted three times with 50 ml portions of ether, each portion being added to the starting ether / MgSO4 mixture.

The dried extracts are filtered and concentrated to 5 ml under a stream of N2; part of the product is crystallized at this stage.

A column of 10 g of Baker's silica gel, packed in ether, is made and the ether concentrate is added to the top and allowed to run through. The residue in the flask is rinsed three times with 1 ml ether, pipetted off each time and added to the column. Then it is eluted with ether.

The first 25 ml are mainly empty volumes. The next 5 10 ml fractions are collected, followed by 3 50 ml fractions and all are reduced in volume under a stream of N2. The product crystallizes from fractions 4 to 6 with traces in 3 and 7. Fractions 1 to 3 contain a yellowish, pungent-smelling material that becomes resinous when standing. Yield: 100 mg as a mixture of the ice and trans isomers.

A solution of 4- (2-acetoxyethyl) -2-azetidinone (2.24 g, 0.014 mol) in 25 ml of anhydrous methanol with a solution of sodium methylate (77 mg, 1.4 mmol ) treated in 5 ml of anhydrous methanol. After stirring for one to five hours, the solution is neutralized with glacial acetic acid. Removal of the methanol in vacuo gives the crude 4- (2-hydroxyethyl) -2-azetidinone as an oil. The product is purified chromatographically on silica gel by eluting with 10% methanol / CHCl3, giving 1.55 g of the alcohol io: mp 50 ° C .;

IR (CHC13) h 5.67; KMR (CDD13) t3.20 (broad s, 1, NH), 6.24 and 6.28 (m at t, total 3, C-4H and CH? OH respectively), 6.90 (broad s at '/ z AB pattern further splits into four by C-4H and NH, total 2, OH and C-3H respectively, 15 Jgem = 13-OHz, Jvic = 4.2 Elz, JNH = 1.6 Hz), 7.42 ('A AB pattern further splits into four by C-4H and NH, 1, C-3H,

Jgem = 13.0 Hz, Jvic = 2.2 Hz, Jnh = 1.1 Hz), 816 (m, 2, CH2CH2OH).

20 Step D: Preparation of 8-oxo-2,2-dimethyl-3-oxa-l-azabicyclo [4,2,0] octane

OH

_NH

30th

Step B: Preparation of 4- (2-acetoxyethyl) -2-acetidinone

O

OCCH-

A solution of 4- (2-acetoxyvinyl) -2-azetidinone (10.0 g,

0.065 mol) in 200 ml of ethyl acetate, containing 100 mg of a 10% palladium / C catalyst, is hydrogenated in a Parr shaker at 25 ° C. under a pressure of 2.8 kg / cm 2 over the course of 15 minutes. The mixture is filtered through a bed of Supercel and washed with additional ethyl acetate. The combined filtrate is washed in vacuo to give 4- (2-acetoxyethyl) -2-azetidinone (10.0 g) as a crystalline solid. When recrystallizing from ether, white crystals are obtained: mp 44 to 47 ° C;

IR (CHClj ^ 5.66.5.74; KMR (CDC13) t 3.44 (broad s, 1, NH), 5.82 (m, 2, CH, OCOCHQ, 6.29 (m, 1, C -4H, 6.87 (> / 2 AB pattern is further divided fourfold by C-4H and NH, 1, Jgem = 12.8Hz, J = 4.5 H Hnh = 1.9 Hz, 7.38 (V2 AB Pattern continues to be divided into four by C-4H and NH,

1, Jgem = 12.8 Hz, J = 2.3 Hz, JNH = 1.0 Hz). 7.93 and 8.02 (s on m, total 5, OCOCH3 and CH2CH2OCOCH3, respectively).

Step C: Preparation of 4- (2-hydroxyethyl) -2-azetidinone

45

50

0 ^

A solution of 4- (2-hydroxyethyl) -2-azetidinone (1.87 g, 0.016 mol) and 2,2-dimethoxypropane (1.69 g, 0.016 mol) in 25 ml of anhydrous methylene chloride is treated with boron trifluoride etherate (0.201 ml, 0.002 mol) treated at 25 ° C. The solution obtained is stirred for 10 minutes. Removal of the solvent under reduced pressure gives an oil (2.5 g). Chromatography of the crude product over a silica gel using 2: 1 ethyl acetate / benzene as eluent gives 8-oxo-2,2-dimethyl-3-oxa-l-azabi-cyclo [4,2,0] octane (1 , 59 g) as a crystalline solid. Recrystallization from ether / hexane gives a product with a melting point of 60 to 61 ° C.

IR (CHCl3) n: 5.73 (β-lactam)

KMR 6.02-6.28, m, 2H, c-4 methylene

(CDC13) t: 6.22-6.62, m, 1H, c-6 methine

6.90, dd, 1H, J7i7 = 14 Hz, J6j7 = 4.5 Hz C-7 proton ice to C-6H 7.47, dd, 1H, J7i7 = 14 Hz, J6.7 = 2 Hz • C- 7 proton trans to D-6H 7.82-8.68, m, ^ 2H, C-5 methylene 8.23, s, 3H 8.57, s, 3H

C-2 methyls

Step E: Preparation of 8-oxo-2,2-dimethyl-7a and β- (7-55 hydroxyethyl) - (3-oxa-l-azabicyclo [4,2,0] octane

A solution of 8-oxo-2,2-dimethyl-3-oxa-l-azabicyclo- [4 , 2,0] octane in anhydrous tetrahydrofuran, which has been cooled to -78 ° C

633 554

20th

is admitted. After 2 minutes, the lithium enolate obtained is treated with excess acetaldehyde. The solution is stirred at -78 ° C for 30 minutes and then poured into water. The aqueous phase is saturated with sodium chloride and extracted with ethyl acetate. The combined ethyl acetate solutions are dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give the crude product. Purification by means of chromatography on silica gel using ethyl acetate / benzene gives 8-oxo-2,2-dimethyl-7a and ß- (l-hydr-oxyethyl) -3-oxa-l-azabicyclo- [4,2, 0] octane.

Data for 8-oxo-2,2-dimethyl-7ß- (l-hydroxyethyl) -3-oxa-l-azabicyclo [4,2,0] octane:

IR (CH2Cl2) (i: 5.72 (0. (Β-lactam

KMR 5.53-6.43, m, 4H, C-4 methylene +

(CDC13) t: C-6 methine + C-9 methine

6.90, dd on broad s, 2H, J7 9 = 9 Hz J6 7 = 5.5 Hz, C-7 methine + OH 7.70-8.83, m, 2H, C-5 methylene 8.27, s, 3H 8.60, s, 3H

8.78, d, 3H, J'g 10 = 6.5 Hz, C-10 methyl

C-2 methyl

Data for 8-oxo-2,2-dimethyl-7a ~ (l-hydroxyethyl) -3-oxa-1-azabicyclo [4,2,0] octane:

IR (CHCl3) | x: 2.9 broad O-H 5.73 β-lactam 4.23-3.33, m, c-6 methine 3.33, broad s, OH

9'S'hh '} c-7 methine

2.67, dd, J = 2 Hz, 8 Hz

1.93-1.63, m, C-5 methylfen

1.63, s

KMR (acetone-d6) ô:

1.40, s

> C-2Methyl

1.23, d, -H = 6.5 Hz, C-10 methyl

Step F: Preparation of 8-oxo-2,2-dimethyl-7a (l-p-nitro-benzylcarbonyldioxyethyl) -3-oxa-l -azabicyclo [4,2,0] octane

OR l

IR 5.68 (β-lactam and carbonate), 6.19 and

(CH2C12) 2 [x: 6.54 (nitro)

KMR (CDC13): 1.67, d, 2H, ArH 2.37, d, 2H, ArH 5 4.67, s, 2H, ARCH2

4.67-5.22, m, CH3CH 5.98-6.25, m, 2H, C-4 methylene 6.25-6.62, m, 1H, C-6 methine 7.75-8.83 , m, 2H, C-5 methylene io 8.22, s. 3H, C-2 methyl

8.50-8.59, m, 5H, c-2 methyl + CH3 CH

The 7β-diastereoisomers or the 7a and 7ß-mixtures are obtained in an analogous manner.

15

Step G: Preparation of cis and trans-3- (l-p-nitrobenzylcar-bonyldioxyethyl) -4- (2-hydroxyethyl) -2azetidinone

OR

OR

R * = ^ 0CH2-0

.-no,

8-Oxo-3-oxa-2,2-dimethyl-7a (lp-nitrobenzylcarbonyl-30 dioxyethyl) -l-azabicyclo- [4.2.0] octane (1.0 g) is dissolved in 8 ml of acetic acid and Dissolved 2 ml of water and heated to 65 ° C for 1.25 hours. The acetic acid and the water are removed under reduced pressure and the residue is taken up in benzene and evaporated to give (Trans-3- (lp-nitro-35 benzylcarbonyldioxyäthyl) -4- (2-hydroxyethyl) -2-azetidinone as a mixture of Obtains diastereoisomers.

IR (CH2C12) | a: 5.67 (β-lactam), 5.72 shoulder, 6.20 and

6.57 (nitro)

KMR (CDC13): 1.73, d, 2H, H = 8.5 Hz, ArH 40 2.43, d, 2H, J = 8.5 Hz, ArH

3.63, broad s, 1H, NH 4.37-5.13, m, 1H, CH3CH 4.72, s, 2H, ArCH2 6.07-6.53, m, 1H, C-4 methine 45 6 , 23, t, 2H, J = 5.5 Hz, CH2OH

6.73-6.93, m, IH, C-3 methine 7.63-8.97, m, 3H, CH2CH2OH 8.53, d, J = 6.5 Hz, CH3CH

50 The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

OR

Under anhydrous conditions, a solution of stages D ', E', F and G 'is used as an alternative to stages D, E, F

8-0x0-2,2-dimethyl-7a- (l-hydroxyethyl) -3-oxa-l-azabicyclo- and G for the preparation of 3- (lp-nitrobenzylcarbonyldioxy- [4,2,0] -octane (60 mg, 0.302 mmol) in 0.6 ml of ether with powdered 55 ethyl) -4- (2-hydroxyethyl) -azetidinon treated potassium hydroxide (19 mg, 0.332 mmol) treated. After 15 minutes, p-nitrobenzyl chloroformate (65 mg, 0.302 mmol) is added to the reaction mixture. The mixture is stirred at 25 ° C. for a further 15 hours. The mixture is divided between In pH 7 phosphate buffer and other ether. The ether phase is washed with water and brine, dried over magnesium sulfate and filtered. Evaporation of the filtrate under reduced pressure gives 67 mg of a colorless oil. Purification by preparative thick-layer chromatography over silica gel and development with 1: 9 ethyl acetate / benzene gives 8-oxo-2,2-dimethyl-7a- (lp-nitrobenzylcarbonyldioxy-ethyl) -3-oxa-l-azabicyclo- [4 , 2,0] octane (40 mg) as a mixture of the diastereomers.

o fi

R = —COCH,

NO,

65

Levels D ', E', F and G '

Preparation of 1- (2-tetrahydropyranyl) -4- [2-tetrahydro-pyranyl) oxyethyl] -2-azetidinone

21st

633 554

A solution of 4- (2-hydroxyethyl) -2-azetidinone (62 mg, 0.539 nmol) in 0.5 ml of anhydrous p-dioxane with 2,3-dihydropyran (0.98 ml, 1.08 mmol) and p-toluenesulfonic acid monohydrate (19 mg, 0.10 mmol). The resulting solution is stirred for 60 minutes and then partitioned between 10 ml of a 0.5 M pH 7 phosphate buffer and 10 ml of ethyl acetate. The aqueous phase is extracted a second time with ethyl acetate. The combined ethyl acetate solutions are washed with salt water, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give 216 mg of the crude product. Purification by preparative thick layer chromatography and development with ethyl acetate gives 80 mg of l- (2-tetrahydropyranyl) -4- [2- (2-tetrahydropyranyl) -oxyethyl] -2-azetidinone as an oil.

io KMR 5.13-5.60, m. OCH (CDC13) t: 5.83-6.85, m, C-4H + OCH2

6.95, dd, J = 5 Hz and 15 Hz ", 7.35, dd, J = 3 Hz and 15 Hz C-3 methylene

7.62-8.95, m, CHCH2CH2CH2CH2 + 15 CHCH2CH20

Preparation of cis and trans-l- (2-tetrahydropyranyl) -3- (l-hydroxyethyl) -4- [2-tetrahydropyranyl) oxyethyl] -2-azetidinone

If you work in the for the production of 8-oxo-2,2-dimethyl-7a and ß- (l-hydroxyethyl) -3-oxa-l-azabicyclo [4,2,0] octane from 8-oxo -2,2-dimethyl-3-oxa-l-azabicyclo- [4,2,0] -octane described manner and uses l- (2-tetra-hydropyranyl) -4- [2- (2-tetrahydropyranyl) -oxyethyl ] -2-acetidi-non, you get a diastereomeric mixture of both

Cis- and Trans-l- (2-tetrahydropyranyl) -3- (l-hydroxyethyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone.

35 Preparation of cis- and trans-l- (2-tetrahydropyranyl) -3- (l-p-nitrobenzylcarbonyldioxyäthyl) -4- [2- (2-tetrahydropyranyl) -oxyethyl] -2-azetidinone

O

II

R = COCH2

- <

-no2

The procedure is the same as for the production of 8-oxo-2,2-dimethyl-7a- (lp-nitrobenzylcarbonyldioxyethyl) -8-oxa-l-azabicyclo [4,2,0] octane from 8-oxo -2,2-dimethyl-7a- (l- (l-hydroxyethyl) -3-oxa-l-azabicyclo [4,2,0] octane and uses Trans-l- (2-tetrahydropyranyl) -3 - (l-hydroxyethyl) -4- [2 (2-tetrahy-dropyranyl) -oxyethyl] -2-azetidinone, there is obtained trans-l- (2-tetrahydropyranyl) -3- (lp-nitrobenzylcarbonyldioxyethyl) -4- [ 2- (2-tetrahydropyranyl) -oxyethyl] -2-azetidinone The cis-di-stereoisomer is obtained in an analogous manner.

Preparation of cis and trans-3- (l-p-nitrobenzylcarbonyldi-oxyethyl) -4- (2-hydroxyethyl) -2-azetidinone

OR

. PH

R = COCH2-

<

.NH

no2

633 554

22

A solution of trans-l- (2-tetrahydropyranyl) -3- (lp-nitrobenzylcarbonyldioxyethyl) -4- [2-tetrahydropyranyloxyethyl] -2-azetidinone in methanol at 25 ° C is treated with a 0.1 molar equivalent of p -Toluenesulfonic acid monohydrate treated. The solution is stirred for 2 hours and then neutralized with pH 7 phosphate buffer. The product is extracted into ethyl acetate. The ethyl acetate solution is washed with salt water, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone. The cis diastereoisomer is obtained in an analogous manner.

5

Step Hr Preparation of Cis and Trans-3- (l-p-nitrobenzyl

carbonyldioxyethyl) -4- [2,2-bis- (2-hydroxyethyl) thioethyl] -2-

azetidinone

OR.

OH

/

//

NH

/ X / ^ CHO

/

.NH

<-

O

r = coch2

-Qno2

A mixture of anhydrous pyridine (0.146 ml, 1.81 mmol) and anhydrous, powdered chromium trioxide (92 mg, 0.916 mmol) in 8 ml of anhydrous acetonitrile is stirred under nitrogen at 25 ° C. for 30 minutes. 250 mg of dry supercel are added to the dark brown solution obtained, followed by a solution of trans-3- (1-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone (186 mg, 0.550 mmol) in 1 ml anhydrous acetonitrile. After stirring for one hour, the reaction mixture is filtered through a mixed packed bed of 2 g each of silica gel and magnesium sulfate.

The bed is washed repeatedly with a total of 30 ml of additional acetonitrile. The filtrate is concentrated under reduced pressure at 25 ° C to a volume of 3 ml. Thin layer chromatography (silica gel; ethyl acetate / benzene 2: 1) shows that this solution contains a product (Rf = 0.38) that is less polar than the starting material (Rf = 0.21).

The acetonitrile solution of trans-3- (lp-nitrobenzylcarbo-nyldioxyäthyl) -4- (2-oxoäthyl) -2-azetidinone, which was prepared as above, is under nitrogen at 0 ° C with 2-Mercap-toäthanol (0.386 ml , 5.5 mmol) and immediately thereafter treated with boron trifluoride etherate (0.176 ml, 1.43 mmol). After stirring for 15 minutes, this solution is divided between aqueous dipotassium hydrogen phosphate (1.5 g in 4 ml of water) and 12 ml of ethyl acetate. The aqueous phase is extracted a second time with ethyl acetate. The combined ethyl acetate solutions are washed with brine, dried over magnesium sulfate and filtered. The filtrate is reduced

Evaporated pressure to give 229 mg of an oil. The product is purified by preparative thick-layer chromatography on silica gel and developed with ethyl acetate, using 118 mg of trans-3- (l-nitrobenzylcarbonyldioxyethyl) -4-35 [2,2-bis (2-hydroxyethyl) thioethyl] -2-azetidinone as receives a colorless oil.

IR 5.75 (5.79 shoulder) β-lactam and carbonate

(CH2C12) | x: 6.20.6.55 Nitro KMR 1.70. d, J = 8.5 Hz, 2H, ArH 40 (acetone- 2.28, d. J = 8.5 Hz, 2H, ArH d6) x: 2.48-2.88, m, 1H, NH

4.63, s, ARCHz

4.63-5.12, m, CH3CH

45

50

3H

5.80, t, J = 7 Hz, CH2CH /

\ s

5.80-7.45, m, C-4H + C-3H + SCH2CH2OHj 7.63-8.33, m, 2H, CH2CH 8.53, d, J = 6.5 Hz, 3H, CH3CH

13h

The cis diastereoisomers are prepared in an analogous manner. Alternatively, er-55 are obtained in mixed diastereoisomers if a mixture of the diastereoisomers is used as the starting material.

Step I: Preparation of Trans-3- (l-p-nitrobenzylcarbonyldio-xyethyl) -4- [2,2-bis- (2-azidoethyl) thioethyl] -2-azetidinone

60

OCOOPNB

Ì

S

s ^ 0H

+ 2ch3s02c1

_NH

OCOOPNB

2Et, N NaN-,

THF DMSO

X

O ^

.NH

To a solution of 211 mg (molecular weight = 474; 0.445 mmol) of trans-3- (lp-nitrobenzylcarbonyldioxyäthyl) -4- [2,2-bis- (2-hydroxyethyl) -thioethyl] -2-azetidinone in 5 ml of tetrahydrofuran ( THF) (distilled from lithium aluminum hydride) at 0 ° C., 103 mg of mesyl chloride (molecular weight = 114; 0.904 mmol) are added to 1 ml of tetrahydrofuran and immediately thereafter 134 (xl triethylamine (molecular weight = 101; e = 0.729; 0.967 mmol) is stirred for 1 hour under N2. The triethylamine hydrochloride is filtered under N2 and washed with a few ml of additional THF. The clear colorless filtrate is concentrated under a stream of N2 and then pumped under high vacuum for 10 minutes. The dimesylate is immediately dissolved in 5 ml DMSO (distilled from CaH2 at 8 mm and stored over a 4A Linde molecular sieve) in the presence of 347 mg NaN3 (molecular weight =

23 633 554

65; 5.34 mmol). After stirring overnight and under N2, 10 ml of H20 and 20 ml of ethyl acetate (EA) are added. The layers are separated and the aqueous layer is washed three times with 10 ml EA, each organic layer being washed one to five times with 10 ml H20 and 10 ml brine. The combined ethyl acetate layers are dried over anhydrous magnesium sulfate and filtered and concentrated under a stream of N2 to give the crude diazide. Preparative thin layer chromatography on silica gel gives trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- [2,2-bis (2-azidoethyl) thioethyl] -2-azetidinone. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

i5 level J:

COoH

i 2

c (0h) o - +

i 2

c02h

2N0,

y /

c02? nb vs ^ chn2 EA / Et20 c (OH)

co2pnb

4-

OCOOPNB

/ sch2ch2n3

sck2ch2n3

Toi.

OCOOPNB

i

€

-n oh sch2ch2n3 sch2ck2m3

Y

(c02pnb) 2

- + ïî20

pnb = ch., // ^

2 \ ^ =

N02

A freshly prepared (H. Davies and M. Schwarz, JOC, 30, 1242 (1965)) solution of p-nitrophenyldiazomethane (29 mmol) in 150 ml ether is stirred with a solution of 1.0 g oxomalonic acid monohydrate (molecular weight = 136 ; 7.35 mmol) in 50 ml of ethyl acetate (EA) at 0 ° C. After 2 1/2 hours, the yellow solution is concentrated on a rotary evaporator with gentle heating to approximately half the volume, dried over anhydrous magnesium sulfate, filtered and concentrated to an oil as described above. 3.54 mg of trans-3- (lp-nitrobenzylcarbonyldioxyethyl) -4- [2,2-bis- (2-azidoethyl) -thioethyl] -2- are added to the crude p-nitrobenzyl ester in 50 ml of toluene (Toi.) azetidinone (molecular weight = 524; 6.75 mmol). The reaction mixture is heated with stirring

45

55

on an oil bath, allowing about 1 hour of the toluene to be distilled off. Toluene (dried over a 3 A Vi "" linden molecular sieve) is again added to make up the volume to 50 ml and the azeo drying process is repeated three times. The solution is then refluxed under N2 for one hour and the azeo drying process becomes a final one Repeated times and the reflux treatment is continued for another hour, concentrating the resulting solution under a stream of N2 gives crude 1 ^. The raw material is chromatographed on silica gel to give 1_. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Level K:

sch2ch2n3 sch2ch2n3

pnb).

+ soci2 Py->

THF

OCOOPNB

sch2ch2n3 'sch2ch2n3

cl /

(c02pnb) 2

OCOOPNB / '

+ 03P aq.DMF

* 0 ^

sch0ch0nq 2 2 3

sch2ch2n3

-\/H

<i

02pnb),

633 554

24th

To a solution of 2.80 g of 1 (molecular weight = 912; 3.07 mmol) in 35 ml of THF (distilled from lithium aluminum hydride) at -20 ° C, 0.3 ml of pyridine (molecular weight = 79; e = 0.982; 3, 73 mmol) (distilled from NaH and stored over a 4A Linde molecular sieve). While stirring under N2, 0.438 g of thionyl chloride (molecular weight = 119; 3.68 mmol) in 1 ml of THF are added dropwise. The reaction mixture is stirred under N2 for 5 minutes at - 20 ° C and then V2 hour at 0 ° C and finally 1 hour at 25 ° C. The pyridine hydrochloride is filtered off under N2 and washed twice with benzene (dried over a 3A Vir. "Linde molecular sieve). The combined filtrates and washing solutions are concentrated under a stream of N2, slurried in a small volume of benzene with anhydrous MgSO4, under N2 filtered and then concentrated under a stream of N2. An oil is obtained by pumping under high vacuum for V2 hour. To this freshly prepared chlorine compound is added 0.885 g of triphenylphosphine (molecular weight = 262; 3.38 mmol) in 66 ml of 9: 1 dimethylformamide (DMF) / H2Ö and then 550 mg of K2HP04 (molecular weight = 174; 3.16 mmol). The reaction mixture is stirred for 35 minutes at 25 ° C. After dilution with EA and saline, the layers are separated and the aqueous layer becomes Extracted three times with EA The combined EA layers are washed with brine 10, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of N2 i get raw 2. The material is chromatographed on silica gel, giving 2. The cis-diastereoisomers or cis-trans mixture is obtained in an analogous manner.

15

Level L:

ocoopnb

_n

S-CH2CH2N3

e

S-CH2CH2N3 + Br2 Et20 / C5H12

(Co2pnb) 2

ocoopnb?

Br 4- i

Br h

sch2cr2n3 sch2ch2n3

(co2? nb) 2

(1)

(2) kah.dkf ocoopnb

0.2 ml of Br2 (molecular weight = 160; 9 = 3.12; 3.9 mmol) are added to 7.8 ml of pentane (dried over a 4A Linde molecular sieve). To a solution of 950 mg 2 (molecular weight = 896; 1.06 mmol) in 15 ml of diethyl ether (dried over a 3A Vir "Linde molecular sieve) is added dropwise at 0 ° C. and under N2 with stirring 2.3 ml of the above 0 , 49m Br2 solution (1.13 mmol) After stirring for 10 minutes at 0 ° C. 114 | xl cyclohexene (molecular weight = 82; q = 0.81; 1.13 mmol) are added and after 5 minutes at 0 ° C. 53 mg of 57% NaH (57% of 53 mg = 30.2 mg, molecular weight 24, 1.26 mmol) in mineral oil were added to the stirred reaction mixture, followed immediately by 14 ml of ice-cold DMF (distilled from anhydrous CaSÖ4 at 40 mm and stored

55

over a 4A Linde molecular sieve). The stirring is continued under N2 at 0 ° C for 3 hours. The reaction mixture is poured onto a stirred ice-cold mixture of 2.5 ml of KH2PÖ4-40 ml of H20-75 ml of Ea. After the layers 60 have been separated, the aqueous layer is saturated with NaCl and extracted again with EA. The combined organic layers are extracted once with brine, dried over anhydrous MgSO4, filtered and concentrated under a stream of N2 and then pumped under a high vacuum to give the 65 crude! 3. Preparative thin layer chromatography on silica gel gives 13. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

Level M:

25th

633 554

OCOOPNB

/

^, SCH2CH2N3

(C02PN3) 2

■ + Br2 Xther / C5 \ 2 y

OCOOPNB

Br i

-He

HCH,

| Mii '

N

(C02PNB) 2

■ SCH2CH2N3

+ NaH

DMF

->

OCOOPNB Br

0.2 ml Br2 (molecular weight = 160; 3.9 mmol) is added to 9.16 ml pentane (dried over a 4A Linde molecular sieve). To 474 mg of 3 ^ (molecular weight = 793; 0.598 mmol) in 13 ml of diethyl ether (dried over a 3 A Vis "Linde molecular sieve) at 0 ° C. under N2, 1.52 ml of the above 0.42 m are added dropwise with stirring Br2 solution (0.63 mmol) After 15 minutes at 0 ° C., 33 mg of 57% NaH (57% of 33 mg = 18.8 mg; molecular weight = 24; 0.78 mmol) are added and immediately added 6.35 ml of ice-cold DMF (distilled from anhydrous CaS04 at 40 mm and stored over a 4A Linde molecular sieve) are added The reaction mixture is stirred for 1 hour at 0 ° C., then to a stirred ice-cold mixture of 1.6 ml of Im KH2P04-20 ml H20 and 20 ml EA poured. The layers are separated and the aqueous layer is saturated with NaCl and extracted again with further EA. The combined organic layers are washed once with salt water, dried over anhydrous magnesium sulfate and filtered. The filtrate is concentrated under a stream of N2 and then placed in a high vacuum, wob to get the raw 4. Preparative thin layer chromatography on silica gel gives 4. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

35

OCOOPNB

OCOOPNB

\

-s CH2 CH2N3

-f

N

40

45

-SCH2CH2N3

(C02PNB) 2

N>

(C02PNB) 2

to 210 mg 4 (molecular weight = 871; 0.241 mmol), dissolved in 0.5 ml DMSO (distilled from CaH2 at 8 mm and stored over a 4A Linde molecular sieve), 40 mg l, 5-diazobicyclo at 25 ° C. with stirring - [5,4,0] -undec-5-ene (distilled at 80 ° C / 2 mm) (molecular weight = 152; 0.263 mmol) in 0.7 ml of dimethyl sulfoxide (DMSO). The solution is stirred for 4 hours under N2 and then added to a stirred ice-cold mixture of 0.48 ml of KH2P04.7 ml of H20 and 10 ml of EA. After separation of the layers, the aqueous layer is extracted again with EA. The combined 60 organic layers are washed once with salt water and then dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of N2 and then placed under a high vacuum to give the crude 5. 65 is obtained by N preparative thin layer chromatography on silica gel. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Level O:

633 554

26

ocoopnb *

'f ^^ _ SCH0CH0N -, + LiI s - Col li din

0 ^ 5

(co2pnb) 2

2. 2 3

ch2ch2n3

Vsw3

4 *

c02? nb

V "- \

H C02PN3

To a solution of 187 mg 5 (molecular weight = 791; 0.236 mmol) in 2.5 ml s-collidine (distilled from powdered KOH at 30 mm pressure) 45 mg of anhydrous LiJ (dried for a few hours at 100 ° C. over P2O5 under Vacuum) (molecular weight = 134; 0.336 mmol). The reaction mixture is heated to 120 ° C. on an oil bath while stirring under N2. After a total of 25 minutes, the reaction mixture is cooled to 25 ° C., diluted with CH2C12 and transferred to a round bottom flask in order to concentrate under a stream of N2 and under high vacuum. The residue is divided between 10 ml EA and 1.8 ml in 10 ml H2O in KH2P04 and then the aqueous layer is extracted two more times with EA. The combined organic layers are extracted with salt water, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of nitrogen to give crude 6. Preparative thin layer chromatography on silica gel gives 6. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

15

0

ocopnb

DMSO

0 ^

-N.

sch2ch2n3

c02pn3

25th

35

To a solution of the mixed diastereoisomers 6 (34 mg; molecular weight = 612; 0.055 mmol) in 0.2 ml DMSO (distilled from CaH2 at 8 mm and stored over a 4A Linde molecular sieve), 9.5 μl. 1 liter are added with stirring , 5-diazobicyclo- [5,4,0] -undec-5-ene (distilled at - 80 ° C / 2 mm) (molecular weight = 152; q = 1; 0.0625 mmol). The solution is stirred under nitrogen for 15 minutes and diluted to a total volume of 1 ml with CHC13 and added immediately to 2 to 10000 silica gel plates. The band of the product shows 7 as a mixture of cis and trans diastereoisomers.

Level Q:

OCOqPNB

-N-

sch2ch2n3

OH

A

-c02pnb

H2 / 2 3 8 kg / cïn

/

y \

rsch2ch., nh2

-n-

.co2h

In the presence of 61 mg Pt02 61 mg 7 (molecular 50 deacetylation of N-acetylthienamycin weight = 612; 0.1 mmol) in 6 ml of dioxane, 6 ml of THF, 3 ml of a 1% (weight / volume) suspension of fruit-

H20 4 hours at a pressure of 2.8 kg / cm2 with water lawn soil is produced by hydrogenating 1 g of the lawn earth in cloth. The reaction mixture is passed through a Celite-Fil 100 ml sterile phosphate buffer saline solution, the phosphate being filtered and washed with 2 ml of 0, in pH 7 phosphate buffer. phatbuffer saline has the following composition: after concentration in vacuo to cloud point 55

the aqueous mixture is extracted with ethyl acetate. The phosphate buffer saline aqueous layer is concentrated to a small volume of NaCl 8.8 g and added to a column of 100 g of XAD-2 resin. For elu-phosphate buffer, pH 7.5, 10 ml with H20 and discarding the initial fractions, distilled water 1000 ml, the fractions containing the product, freeze-dried 60 phosphate buffer, pH 7.5

where 8 is a mixture of cis and trans diastereoisomes

in KH2P04 are mixed with 84 ml in K2HP04.

the pH of the phosphate buffer is adjusted by adding a small amount. The procedure described below for the en amount of either Im KH2P04 or Im K2HP04 to 7.5 zymatic N-deacylation of thienamycin is applicable for 65.

All isomers of thienamycin, especially for the pronounced aliquots of this 1% stock earth suspension, are N-acetyl isomers 890A and 890A3, which are subsequently prepared for the preparation of LOx, 100X and 100X diluent.

are written. mentions.

27th

633 554

1 ml portions of the stock suspension or 1 ml portions of the lOx, lOOx and lOOOx dilutions are added to 2 ml portions of sterile, 1.0% agar solutions at 48 ° C. The mixture is quickly poured over the surface of sterile petri dishes 85 mm in diameter containing 20 ml of medium A. Medium A has the following composition:

Medium A

KH2P04 3.0 g

K2HP04 7.0 g

MgS04 0.1 g distilled H20 1000 ml

N-acetylethanol

amine solution 8.5 ml

+ N-acetylethanolamine solution

N-Acetylethanolamine is diluted 10X in H20 and membrane sterilized. This solution is added after the autoclave treatment.

For solid media: add 20 g agar.

The petri dishes are incubated at 28 ° C for 18 days. A well-isolated colony is picked up and spread on a Petri dish containing Medium B. Medium B has the following composition:

Medium B

Tomato paste 40 g ground wheat flour 15 g dest. Water 1000 ml pH: adjusted to pH 6 using NaOH For solid media: add 20 g agar.

A single culture is selected and grown on a medium B agar culture at 28 ° C for 2 days. Part of the growth of this culture is spread onto the surface of 6 cultures made from Medium B. These cultures are incubated at 28 ° C for 2 days. This culture was identified as Protaminobacter ruber and designated MB-3528 in the culture collection by Merck & Co., Ine, Rahway, N.J., USA and a sample was deposited with the Agricultural Research Service, U.S. Department of Agriculture, Accession No. NRRL B-8143.

Part of the growth of Protaminobacter ruber MB-3528 agar culture is used to inoculate a 250 ml Erlen-Meyer flask containing 50 ml of medium C. Medium c has the following composition:

Medium C

Dextrose 20 g

Pharmamedia * 8 g

Soaked corn liquid (wet basis) 5 g distilled water 1000 ml pH: adjusted to 7 with NaOH or HCl N-acetylethanolamine solution + 8.5 ml

+ N-acetylethanolamine solution N-acetylethanolamine is diluted 10X in H20 and membrane-sterilized. This solution is added after the autoclave treatment.

The flask is shaken at 28 ° C and 220 rpm on a vibrator for 4 days. A 25 ml portion from the flask is centrifuged at 8000 rpm for 15 minutes. The supernatant is removed and the cells on the surface of the solid medium are scraped off and added to a 0.5 ml 0.05 M potassium phosphate buffer, pH 7.4. The suspension obtained is subjected to ultrasound treatment using a Branson Instruments Model LS-75 Sonifier with a V2 inch probe at setting 4 with four 15 second intervals while the suspension is cooled in ice water during and between interruptions. A 10- ^ 1-5 portion of the sound-treated product is mixed with 25 (xl solution containing 840 (xg / ml N-acetylthienamycin in 10 mmol potassium phosphate buffer, pH 7) and incubated overnight at 28 ° C. Controls, which the antibiotic and containing buffers alone and sonicated cells and buffers without antibiotics were also made After overnight incubation at 28 ° C., 2- [iI amounts are placed on cellulose-coated thin-layer chromatography plates which were developed in ethanol: H20.70 : 30. After air drying, the thin layer chromatography plate is placed on a plate infected with 15 Staphylococcus aureus ATCC 6538P for 5 minutes.

The infected plates were produced as follows: The overnight growth of the organism Staphylococcus aureus ATCC 6538P used for the infection in meat broth 20 plus 0.2% yeast extract is diluted to a suspension with meat broth plus 0.2% He-fe extract, which has a 60% transmission at a wavelength of 660nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g / 1 Difco yeast extract at 37 to 48 ° C to obtain a composition containing 33.2 ml of the suspension per liter of agar. 40 ml of this suspension are placed on petri dishes,

22.5 cm X 22.5 cm, cast and these plates are cooled and kept at 4 ° C until used (5 days maximum).

30 The thin layer chromatography plate is removed and the plate containing the organism is incubated overnight at 35 ° C. In addition to the unreacted bioactive N-acetylthienamycin with a point at Rf 0.7-0.89, a bioactive point was observed at Rf 0.44-0.47, which is attributed to 35 thienamycin. Control incubation mixtures of the antibiotic plus buffer, sonicated cells plus buffer and antibiotic plus buffer, to which sonicated cells had been added shortly before thin layer chromatography application, showed no bioactive material at 40 rf 0.44-0.47.

Production of 890Aj, a special isomer

SCH2CK2NHCCK3 COOH

50

A tube of a freeze-dried culture of Strep-tomyces falvogriseus MA-4434 (NRRL 8139) is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis salt of the following composition 55:

Davis salt

Sodium citrate 0.5 g

K2HP04 7.0 g

KH2P04 3.0 g

60 (NH4) 2S04 1.0 g

MgS04 • 7H20 0.1g distilled water 1000 ml

This suspension is used to incubate four 65 cultures of Medium A with the following composition: Medium A

Glycerin 20.0 g primary yeast 5.0 g

633 554

28

Fish meal 15.0 g distilled water 1000 ml

Agar 20.0 g pH: adjusted to 7.2 using NaOH

The inoculated cultures are incubated at 27-28 ° C for one week and then stored at 4-6 ° C until use. An 'A portion of the growth of three cultures is used to inoculate 9 stoppered Erlenmeyer flasks containing 50 ml of Medium B of the following composition:

Medium B

Yeast autolysate (+ ardamine) 10.0 g

Glucose 10.0 g

Phosphate buffer ++ 2.0 ml

MgS04-7H20 50 mg distilled water 1000 ml pH: adjusted to 6.5 using HCl or NaOH

+ Ardamine: Yeast Products Corporation

++ phosphate buffer solution kh2po4

Na2HP04 distilled water

91.0 g 95.0 g 1000 ml

The flasks containing the germs are shaken for 1 day at 27-28 ° C. on a vibrator at 220 rpm. The flasks and contents are kept at 4 ° C for 1 day.

33 2-1 Erlenmeyer flasks, each containing 250 ml of medium C, are inoculated with 8 ml per flask of growth from the flask containing the germs. Medium C has the following composition:

Medium C

Tomato paste 20.0 g primary yeast - 10.0 g

Dextrin (Amidex) 20.0 g

CoCl2 • 6H20 5.0 mg distilled water 1000 ml pH: adjusted to 7.2-7.4 by NaOH After the inoculation, the production flasks are at

Incubate 24 ° C with shaking on a shaker operating at 212 rpm for 4 days. The flasks are then emptied and the contents checked for activity using standard Salmonella gallinarum MB 1287 and Vibrio percolans ATCC 8461 plates using 1.25 cm discs immersed in the centrifuged broth samples. If necessary, the samples are diluted with 0.02m phosphate buffer, pH 7.0. The results are given below:

Time of harvest

(Harvest Age hours) h 96

pH 7.2

Salmonella gallinarum (mm zone) 29.5

Vibrio percolans Vio thinner (mm zone) 31

890 test units 40

71 of the entire fermentation broth are cooled to 3 ° C. and centrifuged in 200 ml portions at 9000 rpm for 15 minutes.

7 ml of 0.1 m neutral EDTA are added to the combined supernatant liquids and the entire sample is placed on a Dowex-1 X 2 (Cl ~), 50-100 mesh column with a bed dimension of 5.1 X 25 cm and a running speed of 40 ml / min. The column is washed with 500 ml of deionized water, containing 5 ml in Tris-HCl buffer, pH 7.0, and

25 jxmoles of neutral EDTA, washed. The antibiotic is eluted with 1 liter of deionized water containing 50 g of sodium chloride, and the column is then washed with 300 ml of deionized water. 100 ml fractions are collected starting from the first appearance of salt at the column exit. The bioactivity is present in fractions 1 to 10 with a peak in fraction 2. Fractions 2 to 5, which contain 17% of the activity used, are combined.

The pooled fractions are concentrated to 110 ml io by means of a rotary evaporator under reduced pressure and the pH is adjusted to 5.8 by adding 4.2 ml in HCl. The concentrate thus adjusted is placed on a column of XAD-2 with a bed dimension of 3.8 × 50 cm, which had been washed beforehand with 3160% 15 aqueous acetone (volume / volume) and then with 31 deionized water, 3 1 of 5% (weight / volume) sodium chloride and 1125% (weight / volume) sodium chloride in deionized water. The concentrate used is drained to the level of the bed. The antibiotic 20 is eluted with deionized water at a flow rate of 15 ml / min. 22 fractions of 100 ml each are collected, calculated from the first application of the sample. The bioactivity appears in fractions 6 to 22 with a peak in fractions 9 and 10. 25 fractions 9 to 20 are combined for further processing. Similar fractions are pooled and the pooled fractions are concentrated to 70 ml using a rotary evaporator under reduced pressure.

The concentrate is placed on a Dowex-1 X 4 (Cl ~) 400 Maso see column with a bed dimension of 2.2 X 27 cm. The column is washed with 50 ml deionized water and the antibiotic eluted with 210.70m NaCl + 0.005m NH4C1 4-0.0001m NH3 in deionized water at a flow rate of 2 ml / min. 9.5 ml fractions are collected.

The main peak of the antibiotic is eluted in fractions 142 to 163. Fractions 146 to 157 are combined for further processing.

The combined fractions 146-157 are concentrated to 40 3 ml by evaporation under reduced pressure on a rotary evaporator and the concentrate is brought to pH 6.5 by adding 5 ml in NH3. The concentrate is placed on a column (2.2 X 70 cm) of a Bio-Gel P-2 (200 to 400 mesh), which had been washed beforehand with 20 ml of 45 5M NaCl and 500 ml of deionized water. After the concentrate had run down to bed level, 2 rinses with 1 ml of deionized water each were carried out and again run down to bed level. The column is then eluted with deionized water at a flow rate of 0.6 ml / min. 2.9 ml fractions are collected.

The main peak of the antibiotic is eluted in fractions 63 to 70. Fractions 64 and 65 are pooled for further processing.

55 The combined fractions 64 and 65 are concentrated on a thin layer evaporator under reduced pressure to 2 ml and then frozen in an envelope and lyophilized for 8 hours in a 14 ml screw-capped vessel, giving 2.25 mg of the essentially 60 pure antibiotic 890A! receives. The N-acetyl group is cleaved off as described previously, giving the free base:

65

OK

I.

/ \

_ÏL

sch2ch2tse2

cooh

0 <

29

633 554

Production of 890A3, a special isomer

OH

Cr

-n

SCH, CH0NHCCH, i | p 2 2 3

iLcc

JOOH

A tube of a lyophilized culture of Streptomyces flavogriseus MA-4434 (NRRL 8139) is opened aseptically and the contents are suspended in a tube containing 0.8 ml of a sterile Davis saline solution of the following composition:

Davis salt

Sodium citrate 0.5 g

K2HP04 7.0 g

KH2P04 3.0 g

(NH4) 2S04 1.0 g

MgS04-7H20 0.1g distilled water 1000 ml

This suspension is used to inoculate 4 cultures of medium A with the following composition:

Medium A

Glycerin 20.0 g primary yeast 5.0 g

Fish meal 15.0 g distilled water 1000 ml

Agar 20.0 g pH: adjusted to 7.2 under NaOH. The inoculated cultures are incubated at 27-28 ° C for 1 week and then stored at 4-6 ° C until use (no longer than 21 days).

A V3 portion of the growth from 4 cultures is used to inoculate 12 plugged 250 ml Erlenmeyer flasks containing 50 ml Medium B of the following composition:

Medium B

Yeast autolysate (+ ardamine) 10.0 g

Glucose 10.0 g

Phosphate buffer ++ 2.0 ml

MgS04 • 7H20 50 mg distilled H20 1000 ml pH: adjusted to 6.5 under HCl or NaOH + Ardamine: Yeast Products Corporation

++ phosphate buffer solution kh2po4

Na2HP04 distilled water

91.0 g 95.0 g 1000 ml

The bottle with the germs is shaken for 1 day at 27-28 ° C on a vibrating machine at 220 rpm. The bottle and its contents are kept stationary at 4 ° C for 1 day.

44 2-1 Erlenmeyer flasks, each containing 200 ml of medium C, are mixed with 8 ml per flask of growth from the inoculated bottles. Medium C has the following composition:

Medium C

Tomato paste 20.0 g primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl2 • 6H20 5.0 mg distilled water 1000 ml pH: adjusted to 7.2-7.4 with NaOH After the inoculation, the production flasks are operated at 24 ° C. for 4 days and 5 hours on a shaking machine which is operated at 212 rpm is shaken. The flasks are then emptied and checked for activity using

Standard Salmonella gallinarum MB1287 and Vibro percolans ATCC 8461 sample plates using 1.25 cm Pro-disks immersed in the centrifuged samples of the broth. The samples are diluted with 0.2m Phos-5 phate buffer, pH 7.0, if necessary. The results are given below:

Time of harvest (Harvest

Age hours) h 101

pH 7.2

io Salmonella gallinarum (mm zone) 35

Vibrio percolans' / io dilution (mm zone) 34

890 sample units 121

A total of 7 l of the total of 15 broth are obtained from this fermentation and these are cooled to 3 ° C. and centrifuged in 200 ml portions at 9000 rpm for 15 minutes each. Become the combined overarching solutions

1.7 ml of 0.1m neutral EDTA are added and the mixture is kept at 3 ° C.

20 The above fermentation is repeated under identical conditions with the exception that the 44 2-1 Erlen-meyer flasks are inoculated with 7 ml per bottle of growth from the bottles containing the cultures; the pH and the test results obtained are given below: 25 Harvest Agehours h 101

pH 7.3

Salmonella gallinarum (mm zone) 38

Vibrio percolans Vio thinner 30 (mm zone) 39

890 sample units 92.8

A total of 7.41 of the entire broth obtained from this fermentation are cooled to 3 ° C. and centrifuged for 35 minutes each in 200 ml portions at 9000 rpm. One gives to the combined protruding solutions

1.8 ml 0.1m neutral EDTA.

The supernatant from the centrifuged broths as obtained from the above two fermentations in this example is combined to give a total volume of 13 liters.

The combined supernatant solutions are passed through a column of Dowex-1 X 2 (Cl ~), 50-100 meshes with a bed dimension of 4.7 cm X 50 cm and a flow rate 45 of 60 ml / min. The column is washed with 11 deionized water and the antibiotic is eluted with 5 l of a 5% (weight / volume) NaCl solution containing 0.01 M Tris-HCl buffer, pH 7.0, and 25 (iMol EDTA. 220 ml fractions are collected at a flow rate of 50 ml / min and the fractions are checked against Salmonella gallinarum MB 1287 plates.

Antibiotic activity is present in fractions 3 to 26 with a peak in fractions 5 and 6. Fractions 5 to 9 are combined for further processing. The pH of the 55 pooled fractions is 10.8 and the pooled fractions contain 24% of the initial bioactivity as measured against Salmonella gallinarum MB 1287 plates.

The combined fractions 5 to 9 are concentrated to 150 ml by means of a rotary evaporator under reduced pressure 60 and placed on a column (4.9 cm X 47 cm) of XAD-2, which had been washed beforehand with 5 160% (volume / Volume) of aqueous acetone and then with 5 1 of deionized water and 5 150 g / 1 NaCl in deionized water. The sample is applied in 20 ml portions with the column soaked to 65 bed level each time. When application is complete, three 20 ml portions of deionized water are added and each time allowed to flow to bed level. The sample is eluted with deionized water

633 554

30th

a flow rate of 20 ml / min. All measures using the XAD column are carried out at room temperature (24 ° C) and the eluted fractions are quickly cooled in an ice bath immediately after collection. Fractions from 95 to 230 ml are collected.

Antibiotic activity appears in fractions 2 to 21, as measured from a sample of Salmonella gallinarum MB 1287 plates, with a peak in fractions 5 to 7 (which make up 510 to 895 ml of eluted volume, calculated from the first application of the deionized Water). Fractions 6 to 21 are pooled.

The pooled fractions 6 to 21 are concentrated to 68 ml on a rotary evaporator under reduced pressure and then diluted to 112 ml by adding deionized water. The concentrate is placed on a column (2.2 cm X 21 cm) of Dowex-1 X 4 (Cl ~) 400 mesh. The column is washed with 20 ml deionized water and the antibiotic is eluted with 2 10.07m NaCl + 0.005m NH4C1 + 0.001m NH3 in deionized water at a flow rate of 1.6 ml / min. Fractions of 8 ml each are collected. Fractions 157-179 are the most active and are pooled.

These combined fractions are concentrated on a rotary evaporator under reduced pressure to 7 ml, the pH is adjusted to 7.5 by adding 20 [xl in NaOH. The solution is further concentrated to 5 ml and placed on a column (2.2 X 75 cm) of Bio-Gel P-2,200 to 400 mesh. The sample is washed in the column bed with two washes of 1 ml of deionized water each and eluted with deionized water at a flow rate of 0.6 ml / min. Ten fractions of 3.3 ml and then 60 fractions of 2.65 ml and ten fractions of 2.0 ml are collected.

The fractions are adjusted to the pH range between 7.5 and 8.0 by adding 1.5 to 2.5 (il 0.1m NaOH. The fractions 62, 63, 64 and 65 are frozen and individually in 14- ml glass tubes lyophilized and stored at - 20 ° C under vacuum.

For the isolation of the antibiotic 890A3 and 890A! the advantage of the relatively higher resistance of the antibiotic 890A3 against degradation by penicillinase is used in the following way:

Bio-Gel fraction 63 is combined with fractions 61, 66 and 67 from the Bio-Gel column. To these four combined fractions, 0.2 ml in Tris-HCl buffer, pH, 7.5, and 0.2 ml

Penicillinase (Difco «Bacto-Penase») given. After 113 minutes at 23 ° C, another 0.2 ml of penicillinase are added. After a further 7 hours at room temperature, a further 0.2 ml of penicillinase are added, and after a further 2 hours at room temperature the reaction mixture is cooled on an ice bath. The finished reaction mixture is diluted to 15 ml by adding 5 ml of deionized water.

The reaction mixture is absorbed on a Dowex-1X4 (CI ~) 400 mesh column, bed dimension 10 2.15 X 40 cm. The antibiotic 890A3 is eluted with 0.07m NaCl + 0.005m NH4CI + 0.0001m NH3 in deionized water at a flow rate of 3 ml / min. Fractions of 13.8 ml are collected in each case. Fractions 187 to 200 are pooled.

15 The combined fractions are concentrated to 4 ml using a rotary evaporator under reduced pressure and the concentrate is added to a column (2.2 X 70 cm) Bio-Gel P-2, 200-400 meshes. The antibiotic 890A3 is eluted with deionized water at a flow rate 20 of 0.6 ml / min. 30 fractions of 3.3 ml and then 50 fractions of 2.65 ml are collected.

Fractions 66 to 70 are combined and the combined samples are concentrated to 1.5 ml under reduced pressure using a rotary evaporator and the concentrate is frozen and freeze-dried to give 5.4 mg of a solid which contains the antibiotic 890A3 and the rest Contains salt. The N-acetyl group is split off, as previously described, so that the free base is obtained:

30th

35

OH

'ì cooh

The antibiotic thienamycin and its preparation are described in DT-OS 25 52 638. It is available from breeders of the microorganism Streptomyces cattleya (MA-4297), a culture of which is in the culture collection of the Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, US Department of Agriculture, Peoria, Illinois, V.St.A. deposited 45 and is designated with NRRL 8057.

C.

Claims (2)

633 554 2 PATENT CLAIMS 1. Process for the preparation of new compounds of the formula X II -C-R4 OR (II), sch2ch21tr1r2 coxr wherein X is oxygen, sulfur or a radical of the formula NR ', where R' is R, R is a hydrogen atom, an alkyl radical having 1 to 10 carbon atoms, a phenacyl radical or nucleus-substituted phenacyl radical, where the substituent is a chlorine, bromine or fluorine atom or an alkyl radical with 1 to 6 carbon atoms, an alkoxyalkyl radical, the alkoxy part being open-chain or cyclic and 1 to 6 carbon atoms and the alkyl portion has 1 to 6 carbon atoms, an alkanoyloxyalkyl radical with 2 to 12 carbon atoms, a halogen or perhaloalkyl radical, the halogen atom being a chlorine, bromine or fluorine atom and the alkyl chain having 1 to 6 carbon atoms, an alkenyl radical with 2 to 10 carbon atoms, an alkoxycarbonylalkyl radical with 3 to 14 carbon atoms, a dialkylaminoacetoxyalkyl radical with 4 to 21 carbon atoms fatomen, an alkynoylamidoalkyl radical with 2 to 13 carbon atoms, an aralkyl radical, where the alkyl portion has 1 to 3 carbon atoms and the aryl portion has 6 to 10 carbon atoms, a mono- or bicyclic heteroaralkyl radical with 4 to 10 ring atoms and 1 to 6 carbon atoms in the alkyl portion, where that or the hetero atom (s) represents an oxygen, sulfur or nitrogen atom (s), a nucleus-substituted aralkyl or heteroaralkyl radical, the substituent being a chlorine, fluorine, bromine or iodine atom or an alkyl radical having 1 to 6 carbon atoms is a mono- or bicyclic heterocycloalkylalkyl radical, the hererocycle having 4 to 10 atoms and the heteroatom (s) representing (representing) oxygen, sulfur or nitrogen atom (s) and the alkyl moiety has 1 to 6 carbon atoms, an aryl radical or nucleus-substituted aryl radical with 6 to 10 ring carbon atoms, the nucleus substituent being a hydroxyl group, an alkyl radical with 1 to 6 carbon atoms, a chlorine, fluorine or bromine atom or an alkylthioalkyl radical with 2 to 12 carbon atoms, a cycloalkylthioalkyl radical with 4 to 12 carbon atoms or an acylthioalkyl radical, the acyl component having 2 to 10 carbon atoms and the alkyl component 1 to 6 carbon atoms, R1 and R2 each represent a hydrogen atom or an acyl radical and R3 an acyl radical, where R1, R2 and R are not all hydrogen atoms at the same time, and their pharmacologically tolerable salts, characterized in that a compound of the formula. where j X is an oxygen or sulfur atom and R4 is a hydrogen atom, an amino group or an alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, arylaralkyl -, Cycloalkyl-, 0 heteroaryl or heteroaralkyl radical, or R3 is an acyl radical of the following formula X II 1S -C (CH2) nZR5, where X is an oxygen or sulfur atom, R5 is a hydrogen atom, an amino, mercapto or Represent hydroxyl group or an alkylamino, dialkylamino, alkyl, 20 alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alinyl, aryl, aralkyl, cycloalkyl, heteroaryl or heteroaralkyl radical, n 0.1 , 2, 3 or 4 and Z represents an oxygen, sulfur or nitrogen atom or a carbonyl group, except that if Z is an oxygen atom, R5 does not represent a mercapto or hydroxyl group, if Z is a sulfur atom, R5 does not represent an amino group. or hydroxyl group, and when 30 Z is a nitrogen atom, R5 is not a mercapto group , or R3 is an acyl group of the formula below 35 X II -C-CHR5 I R6 40 where X is an oxygen or sulfur atom, R5 is defined above, R6 is an amino, hydroxyl, azido, carbamoyl or guanidine group , an acyloxy radical, a halogen atom, a sulf-45 amino, tetrazolyl, sulfo or carboxyl group, a carb alkoxy radical, a phosphonograppe or an alkoxy or arylthio radical, with the proviso that not both radicals R5 and R6 are hydroxyl- , Amino or mercapto groups, or so R3 is an acyl radical of the formula below O -C (CH2) ra-A- (CH2) nY 55 oh SS O _N_ sch2ch2nr1r2 coxr (IA) with an acyl suitable for introducing the radical R3 - Reacting agent and then optionally converting the compounds obtained into the corresponding salts. 2. The method according to claim 1, characterized in that compounds are prepared in which R3 is an acyl radical of the formula below wherein m and n each 0 or an integer from 1 to 5, A is an oxygen atom, NR8, where R8 is a hydrogen atom or a Is an alkyl radical having 1 to 6 carbon atoms, a sulfur atom or a single bond and Y represents one of the following radicals: 1) an amino group or substituted amino group of the general formulas 65 -N (R7) 2 and -N (R7) 3 where the R7 radicals are each independently a hydrogen atom, N (R8) 2, where R8 is a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, an alkyl or 3,633,554 Alkoxy radical having 1 to 6 carbon atoms, an alkoxyalkyl lyl, 5-methyl-3- (4-guanidinophenyl) -4-isoxazolyl, 4-guanidi radical, the alkoxy part having 1 to 6 carbon atoms and the nomethylphenyl, 4- Guanidinomethylbenzyl-, 4-guanidino-alkyl portion have 2 to 6 carbon atoms, or a Cy- benzyl-, 4-guanidinophenyl-, 2,6-dimethoxy-4-guanidinophe-cloalkyl or cycloalkylalkyl radical, the cycloalkyl portion 3 nyl-, o- Sulfobenzyl, p-carboxymethylbenzyl, p-chlorobenzyl, up to 6 carbon atoms and the alkyl portion have 1 to 3 carbon atoms, 5 p-methoxybenzyl, 1-naphthylmethyl, 3-isothiazolylmethyl, atoms, where two R7- Residues link 4-isothiazolylmethyl, 5-isothiazolylmethyl, guanylthiome and together with the N atom to which they are bound thyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4-methoxy-5-isos, a ring can form with 3 to 6 atoms; xazolylmethyl-, 4-methyl-5-isoxazolylmethyl-, 1-imidazolylme- 2) an amidino or substituted amidino group of the all-, 2-benzofuranylmethyl, 2-indolyimethyl, 2-phenylvi-general formula io nyl, 2-phenylethynyl, 1-aminocyclohexyl, 2- or 3-thienyl - aminomethyl, 2- (5-nitrofuranyl) vinyl, phenyl, o-methoxy - N = C-N (R) 2 phenyl, o-chlorophenyl, o-phenylphenyl, p-aminomethyl I benzyl-, l- (5-cyanotriazolyl) methyl, difluoromethyl, dichloro- R7 methyl, dibromomethyl, 1- (3-methylimidazolyl) methyl, 2- ,. ... . ,. i5 or 3- (5-carboxymethylthienyl) methyl, 2- or 3- (4-carb- where the radicals R7 are each independently a water amoylthienyl) .methyl.> 2. or 3- (5-methylthienyl) methyl-, 2- substance atom, N (R8) 2, where R is a hydrogen atom or an Al- or 3_ (5_MethoXythienyl) methyl, 2- or 3- (4-chlorothie ky rest with 1 to ó carbon atoms is an alkyl or nyl) _me \ hyl-, 2- or 3- (5-sulfothienyl) methyl, 2- or 3- (5- Alkoxy radical having 1 to 6 carbon atoms, an alkoxyalkyl-carboxythienyi) methyl-, 3- (l, 2,5-thiadiazolyl) -methyl-, 3- (4- rest, the alkoxy part having 1 to 6 carbon atoms and the M methoxy_1; 2,5-thiadiazolyl) methyl, 2-furylmethyl, 2- (5-Ni) Alkyl content have 2 to 6 carbon atoms - if the Netrot £ 1) _methyK 3-Furylmethyl-, 2-Thienylmethyl-, 3-Thie- the alkoxy-lower alkyl radical bonded to a carbon atom nylmethyl tetrazolylmethyl-, benzamidinomethyl- or cy- the alkyl portion contains 1 to 6 carbon atoms - or clohexylamidinomethyl group, A cycloalkyl or cycloalkylalkyl radical, where the alkylane R3 is an acyl radical of the formula below, in part has 1 to 3 carbon atoms, where two are 25 Residues R7 must be linked and together with the atoms to X which they are bonded can form a ring with 3 to 6 carbon atoms; _q (CH2) n-ZR10 3) a guanidino or substituted guanidino group of the " general formula 30 where the fraction - (CH2) nZR10 of the acyl radical is an allylthiome 7 thyl, phenylthiomethyl, butyl mercaptomethyl, a-chlorocro n tylmercaptomethyl, phenoxymethyl, phenoxyethyl, phenoxy "? butyl, phenoxybenzyl, diphenoxymethyl, dimethylmethoxy ^ methyl, dimethylbutoxymethyl, dimethylphenoxymethyl, where R7 has the meaning given in 2); 35 4-guanidinophenoxymethyl-, 4-pyridylthiomethyl-, p- (carb- 4) a guanyl or substituted guanyl group of the general oxymethyl) -phenoxymethyl-, p- (carboxymethyl) -phenylthio-my formula methyl-, 2- (thiazolylthiomethyl-, p- (sulfo) -phenoxymethyl-, P _ vrR7 p- (carboxymethyl) phenylthiomethyl-, 2-pyrimidinylthiome- _ Ethyl, phenethylthiomethyl, 1- (5,6,7,8-tetrahydronaphthyl) - 40 oxymethyl, N-methyl-4-pyridiniumthio, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio, amino, methylamino, where R7 has the meaning given in 2); or dimethylamino, pyridiniummethyl, trimethylammoniumme 5) a nitrogen-containing, mono- or bicyclic hetero-, cyanomethylthomethyl, trifluoromethylthiomethyl, cyclic radical with 4 to 10 nucleus or ring atoms, the 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 3 -Imidazo (the) heteroatom (s) is (are) oxygen (and) oxygen and / 45 lylethyl, 3-imidazolylpropyl, 3-imidazolylbulyl, 1-pyrrolo or sulfur atom (s). represents ethyl, 1-pyrrolopropyl or 1-pyrrolobutyl group, 3. The method according to claim 1, characterized in that the R3 is an acyl radical of the formula below that compounds are prepared in which R1 is a hydrogen atom X and R3 is an acyl group of the formula below 11 50 -CCHRnR12 X II where the portion -CHR1 JR12 of the acyl radical is an a-amino-C-R9 benzyl-, a-amino- (2-thienyl) -methyl-, a- (methylamino) - benzyl-, a-aminomethylmercaptopropyl-, a-amino-3- or in the 55 -4-chlorobenzyl-, a-amino-3- or -4-hydroxybenzyl-, R9 is a benzyl, p-hydroxybenzyl, 4-amino-4-carboxybu-a-amino-2,4-dichlorobenzyl, a-amino-3,4-dichlorobenzyl, tyl, methyl, cyanomethyl, 2 -Pentenyl-, n-amyl-, n-heptyl-, D - (-) - a-hydroxybenzyl-, a-carboxybenzyl-, a-amino- (3-ethyl-, 3- or 4-nitrobenzyl-, phenethyl- , ß, ß-diphenyl-thienyl) -methyl-, D - (-) - a-amino-3-chloro-4-hydroxy-benzyl-, ethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxy-a-amino- (cyclohexyl) -methyl-, a- (5-tetrazolyl) -benzyl-, phenyl-, 2,6-dimethoxyphenyl-, 2,4,6 -Trimethoxyphenyl-, 3,5-60 2-thienylcarboxymethyl-, 3-thienylcarboxymethyl-, 2-furyl-dimethyl-4-isoxazolyl-, 3-butyl-5-methyl-4-isoxazolyl-, 5-Me-carboxymethyl-, 3-furylcarboxymethyl-, a-sulfaminobenzyl-, thyl-4-phenyl-4-isoxazolyl-, 3- (2-chlorophenyl) -5-methyl-4- 3-thienylsulfaminomethyl-, a- (N-methylsulfamino) benzyl- , isoxazolyl-, 3- (2,6-dichlorophenyl) -5-methyl-4-isoxazolyl-, D - (-) - 2-thienylguanidino-methyl-, D - (-) - a-guanidinoben- D-4-amino-4-carboxybutyl-, D-4-N-benzoylamino-4-cyl-, a-guanylureidobenzyl-, a-hydroxybenzyl-, a-azido- carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl, m-amino 65 benzyl, a-fluorobenzyl, 4- (5-methoxy-l, 3-oxadiazolyl) ami nobenzyl, p -Dimethylaminobenzyl-, (3-pyridyl) -methyl-, nomethyl-, 4- (5-methoxy-l, 3-oxadiazolyl) -hydroxymethyl-, 2-ethoxy-l-naphthyl-, 3-carboxy-2-quinoxalinyl-, 3- (2,6- 4- (5-methoxy-l, 3-sulfadiazolyl) hydroxymethyl-, 4- (5-chloro-dichloro -phenyl) -5- (2-furyl) -4-isoxazolyl-, 3-phenyl-4-isoxazothienyl) -aminomethyl-, 2- (5-chlorothienyl) hydroxymethyl-, N (R7) 2 633 554 2- (5-chlorothienyl) carboxymethyl-, 3- (1,2-thiazolyl) aminomethyl-, 3- (1,2-thiazolyl) -hydroxymethyl-, 3- (1,2-thiazolyl) -carboxymethyl-, 2-thiazolylaminomethyl-, 2-thiazolyIhy-droxymethyl-, 2-thiazolylcarboxymethyl-, 2-benzothienylcar-boxymethyl-, 2-benzothienylhydroxymethyl-, 2-benzothienyl-carboxymethyl-, a-sulfobenzyl-, a-phosphonobyl represents a-diethylphosphono or a-monoethylphosphono group, or R3 represents an acyl radical from the following group: o nh o nh II II II II -c-ch2ch2nhc-ch3 -c-ch2ch2nhc-h nyl-, methoxymethyl-, p-acetoxybenzyl-, p-pivaloylbenzyl-, p-isopropoxybenzyl-, 5-indanylmethyl-, dimethylaminoacet-oxymethyl-, crotonolacton-3-yl Acetamidomethyl, acetyl-thioethyl or pivaloylthiomethyl group mean.