DE2724560A1 - O DERIVATIVES OF THIENAMYCINE - Google Patents

Description

Patent attorneys 2 7 2 A 5 6

Dr.-Ing. Walter AbitZ

Dr. Dieter F. M ο rf Dipl.-riiys. M. Gritschneder 8 Munich 8b, Pienzenauwstr. 28

May 31, 1977 15821IA

MERCK & C0. ₃ INC. Rahway, New Jersey 07065, V.St.A.

O-derivatives of thienamycin

809851/0039

The invention relates to O-derivatives (esters and ethers of the secondary alcohol group) of the antibiotic thienamycin, which has the following structure:

SCH ₂ CE ₂ NH ₂ COOH

These derivatives are useful antibiotics. The invention also relates to processes for production of these derivatives, pharmaceutical preparations containing these derivatives and methods of treatment in which such derivatives and preparations are administered when an antibiotic effect is indicated.

The invention relates to certain O-derivatives of the new Antibiotic thienamycin. The term "O-derivatives" means such derivatives as esters and ethers of the secondary alcoholic function of thienamycin. Such derivatives are useful antibiotics.

The invention further relates to processes for the preparation of these compounds, containing these compounds pharmaceutical preparations and methods of treatment using these compounds and preparations administered when antibiotic action is indicated.

Thienamycin can serve as a starting material for the preparation of the compounds according to the invention. It will produced according to the method described in DT-OS 2 552 638 using Streptomyces Cattleya (NRRL 8057 of the ARS Culture Collection, Peoria). It is known that thienamycin has the following structural formula

809851/0039

15821IA

OH

A / \

.COOH

owns.

The inventive O-derivatives of thienamycin can be represented by the following general structural formula II

OR

-N.

.COOM

II

or zv / more useful by the symbol II

Th--

-OR

NH ₂
L COOM

in which "Th" means the bicyclic nucleus of thienamycin and the OH, amino and carboxyl groups des thienamycins are shown, M is a hydrogen, a salt cation from the group of alkali, alkaline earth metals or represents an amine salt and R 1) represents an acyl group (in general the group OR is referred to as an ester) or 2) R denotes an alkyl, aryl, aralkyl or similar group (so that the group OR generally denotes an ether will). According to its definition, the term “acyl” also includes the alkanoyl groups, including theirs Derivatives and analogs, such as thio analogs, in which the carbon

- 2 -

809851/0039

nyl oxygen is replaced by sulfur, as well as sulfur and phosphoracyl analogs such as substituted sulfonyl, sulfinyl and sulfenyl groups and substituted P (III and V) groups such as substituted phosphorous acid, phosphoric acid, phosphonous acid and phosphonic acid groups. These acyl groups according to the invention are described below explained in more detail as well as the groups from 2) above, which are also included in the present invention.

There is an ongoing need for new antibiotics. Unfortunately there is no static or constant effectiveness Effectiveness of a given antibiotic as a result of continued frequent use of any such antibiotic resistant strains of the pathogens are formed. The known antibiotics still have the disadvantage that they are only active against certain types of microorganisms. Therefore, the search for new antibiotics continues.

Surprisingly, it has now been found that the invention Compounds of broad spectrum antibiotics are those used in animal therapy and in human therapy and are useful in inanimate systems.

The present invention is therefore based on the object of creating a new class of antibiotics, which have the basic core structure of the antibiotic thienamycin, but which are referred to as its O-derivatives can. These antibiotics are effective against a wide range of pathogens that are both gram positive Bacteria such as S. aureus, S. pyogenes and B. subtilis as well as gram-negative bacteria such as E. coli, Proteus morganii and Klebsiella. According to the invention, chemical processes for the production of such antibiotics are also intended and their non-toxic pharmaceutically acceptable

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15821IA ^ 272Λ560

Salts, pharmaceutical preparations containing these antibiotics and methods of treatment in which such Antibiotics and preparations administered when an antibiotic effect is indicated, provided will.

In the general representation of the compounds according to the invention (II above), the acyl group represented by R can be used will be, inter alia, substituted or unsubstituted aliphatic, aromatic or heterocyclic, araliphatic or heterocycloaliphatic carboxylic acid groups, a substituted or unsubstituted carbamoyl group or a carbothionic acid group. A group of acyl groups can be represented by the general formula

If

-C-R "

are represented, in which X is 0 or S and R "hydrogen, Amino, substituted amino such as alkyl- and dialkylamino, wherein the alkyl group comprises 1 to about 6 carbon atoms, substituted or unsubstituted, straight or branched chain alkyl wherein the alkyl group has 1 to about 6 carbon atoms. Mercapto such as alkylthio, typically with 1 to 6 carbon atoms, arylthio, typically with 6 to 10 carbon atoms, Hydroxy such as alkoxy, typically with 1 to 6 carbon atoms, aryloxy, typically with 6 to 10 carbon atoms, alkenyl or alkynyl groups, typically with 2 to 6 carbon atoms, aryl such as phenyl, Aralkyl such as benzyl, cycloalkyl, typically of 3 to carbon atoms, or a heteroaryl or heteroaralkyl group (mono- and bicyclic) in which the alkyl moiety typically contains 1 to 3 carbon atoms and the heterocyclic ring typically i | up to 10 carbon atoms contains and the heteroatom or the heteroatoms

-U-

809851/0039

are selected from O, N and S, where the above-mentioned groups can be unsubstituted or substituted by groups such as OH, SH, SR (where R is lower alkyl or aryl such as phenyl), alkyl or alkoxy groups with 1 to about 6 carbon atoms, Halo such as Cl, Br, P and I, cyano, carboxy, sulfamino, carbamoyl, sulfonyl, azido, amino, substituted amino such as alkylamino including quaternary ammonium in which the alkyl group contains 1 to 6 carbon atoms, haloalkyl such as trifluoromethyl, carboxyalkyl, carbamoylalkyl, N -substituted carbamoylalkyl in which the alkyl moiety of the aforementioned four groups contains 1 to about 6 carbon atoms, amidino, guanidino, N-substituted guanidino, guanidino lower alkyl, etc. Examples of such acyl groups which may be mentioned are those in which R "is benzyl , p-Hydroxybenzyl, Ij-amino-I-carboxybutyl, methyl, cyanomethyl, 2-pentenyl, β, β-diphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl, 2,6-D imethoxyphenyl, 2, ^ l, 6-trimethoxyphenyl, 3,5-dimethyl-4-isoxazolyl, 3-butyl-5-methyl- ⁱ -isoxazolyl, 5-methyl-3-phenyl-4-isoxazolyl, 3- (2 -Chlorophenyl) -5-methyl-4-isoxazolyl, 3- (2,6-dichlorophenyl) -5-methyl- ⁱ l-isoxazolyl, D-4-amino-4-carboxybutyl, D-il-N-benzoylamino- ^ -carboxy-nbutyl, p-aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, (3-pyridyl) -methyl, 2-ethoxy-1-naphthyl, 3-carboxy-2-quinoxalinyl, 3 ~ (2, 6-dichlorophenyl) -5- (2-furyl) -4-isoxazolyl, 3-phenyl- ² l-isoxazolyl, 5-methyl-3- ( ¹ * -guanidinophenyl) -4-isoxazolyl, 4-guanidinomethylphenyl, 4-guanidinomethylbenzyl , ^ I-guanidino.benzyl, 4-guanidinophenyl, 2,6-dimethoxy - ^ - guanidino, o-sulfobenzyl, p-carboxymethylbenzyl, p-carbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl, p-methoxybenzyl, 1-naphthylmethyl, 3-isothiazolylmethyl, ^ -isothiazolylmethyl, 5-isothiazolylmethyl, guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4-methoxy-5-isoxazolylmethyl, 4-methyl-5-isoxazolidazolylmethyl, 1-im hyl, 2-benzofuranylmethyl, 2-indolylmethyl ^ 2-phenyl-

809851/0039

vinyl, 2-phenylethynyl, 1-aminocyclohexyl, 2- and 3-thienylaminomethyl, 2- (5-nitrofuranyl) vinyl, phenyl, o-methoxyphenyl, o-chlorophenyl, o-phenylphenyl, p-aminomethylbenzyl, 1- ( 5-cyanotrizolyl) methyl, difluoromethyl, dichloromethyl, dibromomethyl, 1- (3-methyliraidazolyl) methyl, 2- or 3- (5-carboxymethylthienyl) methyl, 2- or 3- ( ¹ * -carbamoylthienyl) methyl, 2- or 3 ~ (5-methylthienyl) -methyl, 2- or 3 - (- methoxythienyl) -methyl, 2- or 3 ~ ( ¹ * -chlorothienyl) -methyl, 2- or 3- (5-sulfothienyl) - methyl, 2- or 3- (5-carboxythienyl) methyl, 3- (1,2,5-thiadiazolyl) methyl, 3- (i »-Methoxy-1,2,5-thiadiazolyl) methyl, 2- Furylmethyl, 2- (5-nitrofuryl) methyl, 3-furylmethyl, 2-thienylmethyl, 3-thienylmethyl, tetrazolylmethyl, benzamidinomethyl and cyclohexylamidinomethyl,

The acyl group can also be a group of the formula:

41,

-C (CE ₂ J _n ZR "

where X is O or S and η is O to 4, Z is oxygen, Is sulfur, carbonyl or nitrogen, and R ″ has the definition given above.

Exemplary representatives of the substituent

which may be mentioned are Allylthiomethyl, phenylthiomethyl, Butylmercaptomethyl, ot-Chlorcrotylmercaptomethyl, phenoxymethyl, phenoxyethyl, phenoxybutyl, phenoxybenzyl, Diphenoxymethyl, Dimethylmethoxymethyl, Dimethylbutoxymethyl, dimethylphenoxymethyl, il-Guanidinophenoxymethyl, 4-pyridylthiomethyl, p- (carboxymethyl) phenoxymethyl _y> p -iCarboxymethyD-phenylthiomethyl, 2-thiazolylthiomethyl ^

809851/0039

p- (sulfo) -phenoxymethyl, ρ- (carboxymethy1) -phenylthiomethyl, 2-pyrimidinylthiomethyl, phenethylthiomethyl, 1- (5,6,7 »8-tetrahydronaphthyl) oxomethyl, N-Methy1-4-pyridylthio, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio, Amino, methylamino, dimethylamino, pyridiniummethyl, trimethylammoniummethyl, Cyanomethylthiomethyl, trifluoromethylthiomethyl, 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 3-imidazolylethyl, 3-imidazolylpropyl, 3-imidazolylbutyl, 1-pyrroloethyl, 1-pyrrolopropyl and 1-pyrrolobutyl.

Alternatively, the acyl group can be a group of the formula:

-C-CHR "
R " ¹

where R "has the definition given above, and R" ' means a group such as amino, hydroxy, azido, carbamoyl, guanidino, amidino, acyloxy, halo such as Cl, F, Br, I, sulfamino, Tetrazolyl, SuIfo, Carboxy, Carbalkoxy, Phosphono etc.

Exemplary members of the substituent

-CHR "
R " ¹

which can be mentioned are a-aminobenzyl, X-amino- (2-thienyl) -methyl, ! X- (methylamino) -benzyl, iX-aminomethylmercaptopropyl, (X-amino-3- or 4-chlorobenzyl, iX -amino-3- or 4-hydroxybenzyl, (X-amino-2,4-dichlorobenzyl, 0 (-amino-3 ^ 4-dichlorobenzyl, D {-) - Ä ~ hydroxybenzyl, Ä-carboxybenzyl, (X-amino- (3-thienyl) -methyl-D - (-) - fX-amino-3-chloro-4-hydroxybenzyl, Qt-amino-) cyclohexyl) -methyl, * - (5-tetrazolyl) -benzyl,

809851/0039

2-thienyl-carboxymethyl, 3-thienyl-carboxymethyl, 2-furyl-carboxymethyl, 3-furyl-carboxymethyl, «-Sulfaminobenzyl, 3-thienyl-sulfaminomethyl, ^ - (N-methylsulfamino) -benzyl, D - (-) - 2- Thieny 1-guanidinomethyl, D - (-) - (λ-guanidinobenzyI, (X-guanylureidobenzyl, Ok-hydroxybenzyl, (X-azidobenzyl, (X- fluorobenzyl, 4- (5-methoxy-1,3-oxa-diazolyl) -aminomethyl, 4- (5-methoxy-1,3-oxadiazolyl) -hydroxymethyl, 4- (5-methoxy-1,3-sulfadiazolyl) -hydroxymethyl, 4 - (5-chlorothienyl) -aminomethyl, 2- (5- chlorothienyl) -hydroxymethyl, 2- (5-chlorothienyl) carboxymethyl, 3- (l, 2-thiazolyl) aminomethyl ₁ 3 ~ (1,2-thiazolyl) -hydroxymethyl, 3- (1,2-thiazolyl) -carboxymethyl , 2- (1, U-thiazolyl) -aminomethyl, 2- (1,4-thiazolyl) -hydroxymethyl, 2- (1,4-thiazolyl) -carboxymethyl, 2-benzothienylaminomethyl, 2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl, c <-Sulfobenzyl, tx-phosphonobenzyl, (X-diethylphosphono and oc-monoethylphosphono.

Other acyl groups of interest in this class are, if X = oxygen, are:

-CCHIT IT

wherein R and R have the definitions given above. R ^ can be hydrogen, halo such as chlorine, fluorine, bromine, iodine, Amino, guanidino, phosphono, hydroxy, tetrazolyl, carb-

oxy, SuIfο or SuIfamino mean, and R means phenyl, substituted phenyl, a mono- or bicyclic heterocyclyl containing one or more oxygen, nitrogen or contains sulfur atoms in the ring, such as furyl, quinoxalyl, thienyl, quinolyl, quinazolyl, thiazolyl ^ Isothiazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl and similar substituted heterocycles, phenylthio, phenyloxy, Lower alkyl having 1 to 6 carbon atoms, heterocyclic or substituted heterocyclic thio groups or cyano. The substituents in the parts of the molecule R and

809851/0039

15821IA 20 ζ 7 2 Λ 56 O

R can be halo, carboxymethyl, guanidino, guanidinomethyl, carboxamidomethyl, aminoethyl, nitro, methoxy or methyl. If R is selected from the group: hydrogen, hydroxy, amino or carboxy, and R is selected from the group: phenyl or a 5- or 6-membered heterocyclic ring with 1 or 2 sulfur, oxygen or nitrogen heteroatoms such as tetrazolyl, thienyl , furyl and phenyl, the following acyl groups examples: phenylacetyl, 3-bromophenylacetyl, p-Aminomethylphenylacetyl, ¹ J-Carboxymethylphenylacetyl, 4-Carboxyamidomethylphenylacetyl, 2-furylacetyl, 5-nitro-2-furylacetyl, 3-Purylacetyl, 2-thienylacetyl, 5-chloro-2-thienylacetyl, 5 ~ methoxy-2-thienylacetyl, Λ-guanidino-2-thienylacetyl, 3-thienylacetyl, 2- (4-methylthienyl) acetyl, 3-isothiazolylacetyl, 4-methoxy-3-isothiazolylacetyl, 4-isothiazolylacetyl, 3 ~ methyl-4-isothiazolylacetyl, 5-isothiazolylacetyl, 3 ~ chloro-5-isothiazolylacetyl, 3-methyl-1,2,5-oxadiazolylacetyl, 1,2,5-thiadiazolyl-4-acetyl, 3 ~ Methyl-1,2,5-thiadiazolylacetyl, 3-chloro-1,2,5-thiadiazolylacetyl, 3-methoxy-1,2,5-thiadiazolylacetyl, phenylthioacetyl, 4-pyri dylthioacetyl, cyanoacetyl, 1-tetrazolylacetyl, 5t -fluorophenylacetyl, D-phenylglycyl, ^ -hydroxy-D-phenylglycyl, 2-thienylglycyl, 3-thienylglycyl, phenylmalonyl, thienylmalonyl, 3-chlorophenyl-3-omalonyl, 3-chlorophenylmalonyl , cx-Amino-cyclohexadienylacetyl, C \ - sulfaminophenylacetyl, iX-hydroxyphenylacetyl, C \ -Tetrazolylphenylacetyl and Ov-SuIfophenylacetyl.

The acyl substituent can also be selected from sulfur (1) and phosphorus (2) groups:

(0) m (X) η

-SY -PY ^f

(b) n Y "

809851/0039

where in (1) m and η integers of 0 or 1 and Y = 0 ν) μ, -N (R ") ₂ and R" mean, where M ^ is selected from hydrogen-alkali metal cations and organic bases, and R " has the definition given above, for example alkyl, alkenyl, aryl and heteroaryl, and in which in (2) X = O or S, η = 0 or 1 and Y ¹ and Y "are selected from the group 0 ® M ®, - N (R ") ₂ , R" and ZR ", in which all symbols have the definitions given above, for example R" and ZR "can be, for example: alkyl, alkenyl, aryl, heteroaryloxy, and where Y ¹ and Y" including the R "Molecular parts can be bonded together to form cyclic esters, ester-amide and amide functions. Examples of (1) are 0- (methylsulphonyl) -thienamycin, 0- (o-nitrophenylsulphonyl) -thienamycin, O-Cp-chlorophenylsulphinylJ-thienamycin, O- (o-Nitrophenylsulphenyl) thienamycin, O-sulphamoylthienamycin, O-dimethylsulphamoylthienamycin and thienamycin-O-sulphonic acid sodium salt. Examples of (2) are 0- (dimethoxyphosphino) -thienamycin, O- (dibenzyloxyphosphino) -thienamycin, O- (dihydroxyphosphino) -thienamycin disodium salt, 0- (dimethoxyphosphinyl) -thienamycin, 0- (dimethoxyphosphino) -thienamycin, 0- (dimethoxyphosphino-thienothioosphyl) and O- (dihydroxyphosphinyl) thienamycin disodium salt.

One acyl class of particular interest is the class comprising diacyl groups which is selected from the group of known blocking agents. or masking groups such as carbobenzyloxy, ring-substituted carbobenzyloxy such as o- and p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy, chloroacetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl, trifluoroacetyl, bromoethoxycarbonyl, 2,2-fluorophenyl, -oxsylmethoxycarbonyl, 9-fluoroacetyl, bromoethoxycarbonyl, trifluoroacetyl, tri-ethoxylphenyl, 9-fluoro-phenyl, tri-ethoxycarbonyl, tri-fluoro-phenyl-2-trichloro-phenyl-phenyl-2-dichloro-phenylphenloro-and p-nitrocarbobenzyloxy and p-nitrocarbobenzyloxy , bromo-t-butoxycarbonyl, phenoxyacetyl \ Nichtacylschutzgruppen such as Triniedrigalkylsilyl, trimethylsilyl and t-butyldimethylsilyl are also of interest.

- 10 809851/0039

Ά 272456Ü

The following groups according to the definition given above for acyl are preferred: formyl, propionyl, butylryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, phenylthiocarbonyl, 3-aminopropionyl, 4-aminobutyryl, N-methylaminoacetyl -Dimethylaminoacetyl, N, N, N-trimethylaminoacetyl, 3- (N, N-dimethyl) -aminopropionyl, 3- (N, N, N-trimethyl) -aminopropionyl, N, N, N-triethylaminoacetyl, pyridinium acetyl, guanylthioacetyl, guanidinoacetyl , 3-Guanidinopropionyl, N -Methylguanidinopropionyl, hydroxyacetyl, 3-hydroxypropionyl, acryloyl, propynoyl, malonyl, phenoxycarbonyl, Amidinoacetyl, Acetamidinoacetyl, Amidinopropionyl, Acetamidinopropionyl, Guanylureidoacetyl, Guanylcarbamoyl, Carboxymethylaminoacetyl, Sulfoacetylaminoacetyl, SuIfo, phosphono, Phosphonoacetylaminoacetyl, N -Dimethylaminoacetamidinopropionyl, ureidocarbonyl , Dimethylaminoguanylthioacetylj 3 ~ (1-methyl- ⁱ l-pyridinium) propionyl, 3- (5-aminoimidazol-1-yl) propio nyl, 3-methyl-l-imidazoliumacetyl, 3-sydnonylacetyl, o-aminomethylbenzoyl, o-aminobenzoyl,

Il f \ | f JDCH ₃ f

-P (OCH ₃ ) _{2 /} -P (OCH ₃ J ₂ , -P ^ _Na , 4 [N (CH ₃ ) ₂ I.

-PN (CH ₃ J ₂ , -P [N (CH J] 4-N (CH,),

-PN (CH ₃ J ₂ , -P [N (CH J] -P

ONa

A particularly preferred class of acyl groups are terminally substituted acyl groups in which the substituent a basic group such as substituted and unsubstituted amino, amidino, guanidino, guanyl and nitrogen f-containing mono- and bicyclic heterocycles (aromatic and non-aromatic), wherein the hetero atom or

- 11 809851/0039

15821IA

the atoms in addition to nitrogen are selected from oxygen and sulfur, is. Such preferred sub substituted acyl groups can be represented by the following formula

in which m and η are integers selected from 0 to 5, A is 0, NR '(R ^{1 is} hydrogen or lower alkyl having 1 to 6 carbon atoms), S or A is a single bond and Y is selected from the following group will:

1. Amino or substituted amino :

-N (R) ₂ and -N (R) ₃

wherein the meanings for R are independently selected from: hydrogen, N (R ') ₂ ( ^RI is hydrogen or lower alkyl having 1 to 6 carbon atoms), lower alkyl and lower alkoxyl. having 1 to 6 carbon atoms, lower alkoxy-lower alkyl, in which the alkoxy moiety contains 1 to 6 carbon atoms and the alkyl moiety contains 2 to 6 carbon atoms, cycloalkyl and cycloalkylalkyl, in which the cycloalkyl moiety contains 3
contains from 1 to 6 carbon atoms, and the alkyl moiety contains from 1 to 3 carbon atoms, and wherein two groups R can be bonded together with the N atom to which they are attached to form a ring of 3 to 6 atoms .

- 12 -

809851/0039

2. Amidino and substituted Ami dino:

-N = C-N (R) -

ι *

wherein the meaning for R is independently selected from the group: hydrogen, N (R ¹ ) ₂ (R ^f is hydrogen or lower alkyl with 1 to 6 carbon atoms), lower alkyl and lower alkoxyl with 1 to 6 carbon atoms, lower alkoxy lower alkyl, in which the alkoxyl moiety is 1 to 6 carbon atoms and the alkyl moiety contains 2 to 6 carbon atoms (if the lower alkoxyl lower alkyl group is bonded to the carbon atom, then the alkyl moiety contains 1 to 6 carbon atoms), cycloalkyl and cycloalkylalkyl, wherein the alkyl moiety contains 1 to 3 carbon atoms, and where two groups R together with the atoms to which they are attached can form a ring of 3 to 6 carbon atoms.

3. Guanidino and substituted guanidino :

-UH-CN (R) ₉

NO
where R has the definition given under 2. above.

JJ. Guanyl and substituted guanyl : _

N (R) ₂

where R has the definition given under 2. above.

5. Nitrogen-containing mono- and bicyclic heterocycles (aromatic and non-aromatic) with H to 10 ring atoms, in which the heteroatom or the heteroatoms in addition to the nitrogen are selected from oxygen and sulfur. Examples of such heterocycles are given in the following list of groups (R ¹ means H or lower alkyl having 1 to 6 carbon atoms):

- 13 -

809851/0039

15821IA

N (R ¹ )

-ο

N -R N /

-Ο ·

8098 5 1/0039

^{15821IA %} 2 72 A 5.6

The following specific acyl groups fall into this class are also examples and are preferred.

-CCH ₂ CH ₂ NhJ-CH

-CCH ₂ CH ₂ NHi-H

3 NH -CCH ₂ CH ₂ NHC-NH ₂

Il

-CCH ₂ CH ₂ N (CH ₃ ) ₂

-CCH ₂ CH ₂ N

-CCH ₂ CH ₂ C-NH ₂

-CCH ₂ CH ₂ CH ₂ NHC-Ch ₃ O

Ii e

-CCH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ O

Il

-CCH ₂ CH ₂ CH ₂ N (CH ₃ )

f p

-CCH ₀ SC

² \

809851/0039

- 15 -

, NH NH.,

-CCH ₂ S-CH ₂ -C

-I

CH ₂ -O-CH ₂ C

NH ₂

It should be noted, however, that any acyl group can be used in the present invention, and is encompassed by the present invention.

In the so-called fither embodiments according to the invention (2.), see II above, R is selected from the acyl groups listed above, in which the carbonyl moiety

0 X

t? Il

-C- or in general -C-

is omitted or removed. R is thus selected from the following groups, in which all symbols have those previously have given definitions:

-R " - (CH-) ZR" -CER "

zn,

-CHR ³ R ⁴ - (CH,) -A- (CE,) -Y

^ m / η

- 16 -

809851/0039

For the ether embodiments (2.) as well as for the ester embodiments (1.) The most preferred groups (structure II above) are those that are relatively low molecular weight and are hydrophilic. In the case of the ether embodiments (2.) the following groups are special preferred and can be given as examples: methoxymethyl, Hydroxymethyl, methoxyethyl, dimethylaminomethyl, dimethylaminoethyl, methylthioethyl, amidinoethyl, guanidinoethyl and similar groups. For the ester versions (1.) the following groups are examples and preferred: sulfo, phosphono, carbamoyl, methylsulphonyl, sulfamoyl, Dimethylsulfomoyl, N-methylcarbamoyl, bromoacetyl, hydroxyacetyl, Aminoacetyl, dimethylaminoacetyl, methoxyacetyl, guanylacetyl, Guanylthioacetyl, phosphamoyl, phosphonothioyl, thiocarbamoyl and like groups.

In general, the antibiotics of the invention prepared by using a fully protected or masked or blocked Thi ^ namycinkern (III) and then split off the protective group:

OR j-OR

SCH ^ CHoNR ^ R "" Th -NR ¹ R Th- -NH

COOR

COOR

^L C00H

III Ilia II

1 2
where R and R denote the usual N-protecting groups (norma-

12 3

Usually R or R denotes hydrogen) and R ^ denotes an means known carboxyl protecting group. The preparation of the N- and carboxyl-masked and protected species, respectively III is explained in more detail below and in the examples

1 2 purifies. The only requirement for the protecting groups R _f R and R (or the group XR attached to the carbonyl carbon atom, where X is oxygen or sulfur)

- 17 -

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is that they do not interfere with the desired reaction and that they are ultimately removable to form the compound of For mel II . The term "protecting group" or "blocking group" is used in the present application in the same manner as in US Pat. No. 3,697,515.

The preferred N-protective groups for the starting materials III are: carbobenzyloxy, ring-substituted carbobenzyloxy, such as o- and p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy, chloroacetyl, bromoacetyl, phenylacetyl, t- butoxoxycarbonyl, trifluoroacetycarbonyl, bromoethycarbonyloxycarbonyl, dichloroacetycarbonyl, bromoethycarbonyloxycarbonyl , o-nitrophenylsulfenyl, 2,2,2-trichloroethoxycarbonyl, bromo-t-butoxycarbonyl, phenoxyacetyl; Non-acyl protecting groups such as alkylidene, e.g. benzylidene, salicylidene and similar groups are also of interest.

Suitable protecting groups R for the starting material III are those that are selected from the following list, the list only showing examples of possible ester groups are included and where X is oxygen and R ^ has the following meanings:

(i) R = CR ^a RR °, in which at least one of the substituents R, R and R is an electron donor, for example p-methoxyphenyl, 2,4,6-trimethylphenyl, 9-anthryl, methoxy, CH ₅ SCH ₇ ,, tetrahydrofuran -2-yl, tetrahydropyran-2-yl or fur-

ABC

2-yl. The remaining R, R and R groups can be hydrogen or organic substituent groups. Suitable ester groups of this type include p-methoxybenzyloxycarbonyl and 2, ¹ 1,6-trimethylbenzyloxycarbonyl,

(ii) R -CR ^a RR ^c , wherein at least one of the substituents R ^a , R and R ^{c is} an electron-withdrawing group, z, B, benzoyl, p-nitrophenyl, 4-pyridyl, trichloromethyl, tribromomethyl, iodomethyl, cyanomethyl, ethoxycarbonylmethyl ,

- ■ 18 -809851 / 0039

Arylsulphonylmethyl, 2-dimethylsulphoniumethyl, o-nitrophenyl or cyano. Suitable esters of this type are benzoylmethoxycarbonyl, p-nitrobenzyloxycarbonyl, li-pyridylmeth oxycarbonyl, 2,2,2-trichloroethoxycarbonyl and 2,2,2-tribromoethoxycarbonyl.

(iii) R = CR ^a RR ^c , in which at least two of the substituents R ^a , R and R ^{c are} hydrocarbons such as alkyl, for example methyl or ethyl, or aryl, for example phenyl, and the remaining R, R and R groups , if present, denotes hydrogen. Suitable esters of this type include t-butyloxycarbonyl, t-amyloxycarbonyl, diphenylmethoxycarbonyl and triphenylmethoxycarbonyl.

(iv) R-R where R is adamantyl, 2-benzyloxyphenyl, 4-methylthiophenyl or is tetrahydropyran-2-yl.

Of this category of protecting groups, silyl esters can suitably be selected from a halosilane or a silazane of the formula rVsiX ¹ ; R ^ _n SiX '; rVSi .NR ⁴ _O ; R ^, Si .NH. ^14th

VsiX; R _n SiX; rVSi .NR _O ; R,

H J U c-n έ & ä ^d -> H H

R ..Si.NH.CO.NH.SiR ,; R NH.CO.NR .SiR \ or R C (OSiR,)

■> a ■> ■> J

HN (SiR,) ₂ are prepared, where X ^{1 is} a halogen such as chlorine or bromine and the various groups R, which can be the same or different, hydrogen atoms or alkyl groups, for example methyl, ethyl, n-propyl, isopropyl, aryl, for example Phenyl, or aralkyl, for example benzyl groups, mean.

In this context it should be noted that preferred R- "protecting groups" include subgroups of the groups defined above, such as aralkyl, haloalkyl, alkanoyloxyalkyl, alkoxyalkyl, alkenyl, substituted alkyl, or aralkoxyalkyl, and also alkylsilyl, the alkyl groups having up to 10 carbon atoms contain. For example, suitable R ^ "protective groups" are benzyl, phenacyl, p-nitrobenzyl, methoxymethyl, trichloroethyl, trimethylsilyl ^ tributyl _z i _nn

- 19 809851/0039

p-methoxybenzyl, benzhydryl. These protecting groups are preferred as they are generally used as easily removable protecting groups in the cephalosporin and penicillin fields are valid.

Generally, the N-, carboxyl-protected reaction intermediate Species III prepared by first N-acylating and then de-

1 2 transferred privately. However, the protective reaction can result in R / R and R at the same time with a joint reaction, if this allows the identity of the selected protection group. The protective groups are preferably split off in one step single stage according to methods known per se. Often

1 2 fig, however, it depends on the identity of R / R and

R · ^ prefers to split off the protective groups in separate stages.

The introduction of the protecting groups can be carried out using the free thienamycin, I, as the starting material using, or by using its protected derivative such as a tris-silylatthienamycin:

Th-

r-OTMS

-NHTMS -CO ₂ TMS

where, for reasons of convenience, the previously introduced symbol for thienamycin is used and TMS Triorganosi-IyI such as trimethylsilyl means. For example, the preparation of such species is detailed below Example 21 described.

12th

Preferred N-protecting groups R and R are those in which R

ρ
Hydrogen and R a substituted or unsubstituted carbobenzyloxy group}

- 20 -

809851/0039

15821IA

3 ^

(R ') n

means where η = 0 to 2 (n = 0, R ¹ = hydrogen) and R 'means lower alkoxy or nitro. Another particularly preferred N-protecting group is bromo-t-butoxycarbonyl.

The preferred carboxyl protecting group R is benzyl or substituted benzyl groups such as p-nitrobenzyl:

where η = 0-2 (n = 0, R '= H) and R' is lower alkoxyl or means nitro.

The preferred starting material III can thus be represented as follows:

raw

Th. YEAR "

III

(R ') n

R ² = -CO-CH.

■> ⁹ S ^H3

R ² = COC-CH ₀ Br
CH, ²

or

-21-

809851/0039

15821IA # 27 2 A 56 O

In general, III is prepared by treating thienamycin in water or a mixture of water and a polar organic solvent such as dioxane in the presence of a base with an acyl halide such as carbobenzyloxy chloride. The resulting N-acyl-thienamycin derivative is isolated by methods known per se. The carboxyl protecting group is expediently introduced in the second stage. In this case, the N-protected thienamycin is treated with an activated halide such as benzyl bromide in a solvent such as hexamethylphosphoramide or the like at a temperature of 0 ⁰ C to 60 ° C for several minutes to hours h. The resulting N-protected and carboxyl-protected species III is then converted into the intermediate species IHa by the method described below. The protective group or groups are split off from the intermediate product species IHa by methods known per se, including hydrolysis and hydrogenation, to form the compound II according to the invention. If preferred N- and carboxyl protecting groups are used, the preferred method for splitting off the protecting groups is hydrogenation. In the hydrogenation, the intermediate species IHa is hydrogenated in a solvent such as a lower alkanol in the presence of a hydrogenation catalyst such as Pt, Pd or their oxides.

In making II, the N-protected species (1) can be made first:

-OH

Th_.NH ₂

_COOH

-OH

_COOH

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809851/0039

In general, the N-protected species (I), above,

12 f *

worm R and R are H or acyl, by treating Thienamycin (I) with an acylating agent, for example an acyl halide or acyl anhydride such as an aliphatic, aromatic, heterocyclic, araliphatic or heterocyclic-aliphatic carboxylic acid halide or anhydride. Other acylating agents can also be used, for example mixed ones Carboxylic acid anhydrides and especially lower alkyl esters of mixed carboxylic acid-carbonic acid anhydrides. Farther can be used carbocyclic acids in the presence of carbodiimide such as 1,3-dicyclohexylcarbodiimide, and activated esters of a carboxylic acid such as p-nitrophenyl ester. The acylation reaction can take place at a temperature can be carried out in the range from about -20 ° C to about 100 ° C. Preferably it is at a temperature in the range from -8 ° C to 25 ° C. You can use any solvent in which the reactants are soluble and essentially inert, use, for example, polar solvents such as water, alcohols, and polar organic solvents in general such as dimethylformamide (DHF), hexamethylphosphoramide (HMPA), acetone, dioxane, tetrahydrofuran (THF), acetonitrile, heterocyclic amines such as pyridine, ethyl acetate, aqueous mixtures of the above, as well as halogenated solvents such as methylene chloride and chloroform. The implementation is carried out for a period of about 5 minutes to a maximum of 3 hours. In general, one reaction time is sufficient from about 0.5 to 1 hour. In the following equation, this method is illustrated using a Carboxylic acid halide explained. However, one can also use a carboxylic acid anhydride or any other functional use equivalent acylating agent and obtain similar products.

- 23 -

809851/0039

15821IA

26th

-SCH ₂ CH ₂ NH ₂

OH

-COOH

Acyl halide υ

/ ^ X-SCH-CH-NR ¹ R ²

.N-

COOH

Generally, when in the above acylation reaction an acid halide (suitable halides are chlorine, iodine or bromine compounds) or an anhydride are used the reaction in water or an aqueous mixture of a polar organic solvent such as acetone, dioxane, THF, DMF, acetonitrile or similar solvents in the presence of a suitable acceptor base such as NaHCO ,, MgO, NaOH, KpHPOjj or similar bases carried out.

If the acylating agent is extremely sensitive to water, it is sometimes advantageous to carry out the acylation in a non-aqueous solvent. Triorganosilyl (or Tin) derivatives of thienamycin are suitable for this purpose. The silylation of thienamycin is rapid and the tris-triorganosilyl derivative is obtained, for example tris-trimethylsilylthienamycin, Th (TMS).

Th

I-OTMS

NHTMS -COOTMS

Such derivatives work easily in organic solvents Soluble are made expedient by treating thienamycin with an excess of hexamethyldisilazane and a stoichiometric amount of trimethylchlorosilane at 25 ° C with vigorous stirring under ^ atmosphere. The resulting NH ^ Cl is removed by centrifugation and the solvent is removed by evaporation, wherein the desired silyl derivative is obtained.

809851/0039

15821IA Ί><ο 27 2 A 56 O

In general, the intermediate III used as starting material from the N-protected species I is after produced according to the following scheme:

ΟΗ

-NK R> Th - \ - l

COOH

ΓΟΗ

-COOR

wherein all symbols have the definition given above. In general, the conversion occurs 1> III after

methods known per se. These procedures are for example:

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 a temperature from 0 C to the reflux temperature for a few minutes to 2 hours.

2. Reaction of an alkali metal salt of I ^ with an activated alkyl halide such as methyl iodide, benzyl bromide, or m-phenoxybenzyl bromide, pt-butylbenzyl bromide, pivaloyloxymethyl chloride and the like. Suitable reaction conditions include solvents such as hexamethylphosphoramide and the like at a temperature of from 0 ^° C. to 60 ° C. for a few minutes to 4 hours.

3. Reaction of 1 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 or the like. Suitable solvents are at a temperature of 0 ⁰ C to reflux temperature and a time of 15 minutes to 18 hours CHCl ,, CH, CH, CH ₂ Cl ₂ and the like.

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809851/0039

15821IA } l 272A560

¹ I. Reaction of an N-acylated acid anhydride of 1, which was prepared by reacting the free acid with an acid chloride such as ethyl chloroformate, benzyl chloroformate and the like, with an alcohol, as indicated above under 3, under the same reaction conditions as they listed above at 3-. The anhydride is prepared by reacting III and the acid chloride in a solvent such as tetrahydrofuran (THF) CH ₂ Cl ₃ and the like at a temperature from 25 ° C to reflux temperature for 15 minutes to 10 hours.

5. Reaction of the labile esters of thienamycin (I) such as the trimethylsilyl ester, dimethyl-t-butylsilyl ester or the like with RX *, where X 'is halogen such as chlorine or bromine and R is as defined above, in a solvent such as THF, CH ₂ Cl ₂ and the like at a temperature from 0 C to reflux temperature for 15 minutes to 16 hours, for example according to the following scheme:

-OTMS

Th - NHTMS

N-acylation

L-COOTMS

-OTiIS

Ver

2 esterification

-NR TMS * ■.

L-COOTMS

OTMS 2

Th -NO

lCOOR

TMS 3

mil rip

"OH Th _L NHR ²

-COOR ³

wherein TMS is triorganosilyl such as trimethylsilyl and all other symbols have the definition given above.

The aforementioned esterification schemes are good in the related β-lactam bicyclic antibiotic area known and common in organic synthesis. For the reaction parameters in the production of the invention

- 26 -

809851/0039

15821IA U 272-560

according to N-acyl and carboxylthienamycin (starting materials III) there are no unusual restrictions.

In general, the compounds according to the invention are used after a large number of esterification or etherification reactions known per se (III> purple) made that

with the secondary alcoholic group of thienamycin in its protected form III. The process for splitting off the protective groups (purple - ) II) with the preparation of the compounds according to the invention has been described in more detail above. These procedures (III ) purple) are for example:

1. For the preparation of the ether compounds according to the invention the acid-catalyzed reaction of III with a diazoalkane such as diazomethane, phenyldiazomethane, diphenyldiazomethane and the like in an inert solvent such as dioxane, tetrahydrofuran (THF), halohydrocarbons such as CHpCIp, ethyl acetate and the like in the presence a catalytic amount of a strong acid or Lewis acid such as toluenesulfonic acid, trifluoroacetic acid, fluoboric acid, Boron trifluoride and the like at a temperature of -78 C to 25 C for a period of several minutes up to 2 hours.

2. For the preparation of ether compounds according to the invention, the reaction of III with an alkylating agent such as active halides, e.g. methyl iodide, benzyl bromide, m-phenoxybenzyl bromide and the like, alkyl sulphonates such as dimethyl sulfate, diethyl sulfate, methyl fluorosulfonate and the like in the presence of a strong base capable of forming the alcoholate anion of III. Suitable bases are the alkali and alkaline earth metal oxides and the hydrous oxides, alkali metal alkoxides like potassium tertiary butoxide, tertiary amines like tri-

- 27 809851/0039

158211A ™

ethylamine, alkali metal alkyls and aryls such as phenyllithium, and alkali metal amides such as sodium amide. Suitable solvents are, for example, inert anhydrous solvent such as t-butanol, dimethylformamide (DMF), THF, hexamethylphosphoramide (HMPA) dioxane and the like at a temperature of -78 ⁰ C to 25 ° C and a time of a few minutes to 4 hours.

3. For the preparation of ester compounds according to the invention, the reaction of III with any of the above-mentioned acyl groups in their acid form. This reaction can be carried out in the presence of a carbodiimide condensing agent such as dicyclohexylcarbodiimide or the like. Suitable solvents are any inert solvent such as CHCl,., CHpCl _2, DMF, HMPA, acetone, dioxane and the like at a temperature of 0 C to 6O ⁰ C for 15 minutes to 12 hours.

4. For the preparation of ester compounds according to the invention the reaction of III with an acyl halide or an acid anhydride, the acyl moiety being the above has the meaning described. Generally, when an acid halide is used in the acylation reaction described above (suitable halides are chlorides, iodides or bromides or if an acid anhydride is used). the Reaction in an anhydrous organic solvent such as acetone, dioxane, methylene chloride, chloroform, or DMF Similar solvents in the presence of a suitable acceptor base such as NaHCO- ,, MgO, triethylene, pyridine and the like at a temper;

Carried out for 4 hours.

borrowed at a temperature of 0 ° C to 40 ⁰ C during 1 to

Suitable acyl halides and anhydrides are, for example: acetic anhydride, bromoacetic anhydride ^ propionic anhydride, Benzoyl chloride, phenylacetyl chloride, azidoacetyl

- 28 -

809851/0039

Chloride, 2-thienylacetyl chloride, 2-, 3- and 4-nicotinyl chloride, p-nitrobenzoyl chloride, 2,6-dimethoxybenzoyl chloride, il-guanidinophenylacetyl chloride, hydrochloride methanesulf onyl chloride, dibenzyl phosphorus chloride, dimethyl thiophosphorochloridate, 2-furoylethyl, 2-furoylethyl carbonic anhydride, Methyl chloroformate, bis (p-nitrobenzyl) phosphorochloridate and the like.

5 · For the preparation of ester compounds according to the invention, the reaction of III with a suitable substituted ketene or isocyanate such as ketone, dimethyl ketone, methyl isocyanate, methyl isothiocyanate, chlorosulfonyl isocyanate and the like. Suitable solvents include dioxane, tetrahydrofuran, chloroform and the like at a temperature of -7O ⁰ C to 6O ⁰ C for 15 minutes to 15 hours.

The compounds (II) according to the invention form a wide variety of pharmacologically acceptable salts with inorganic salts and organic bases. These are, for example, the metal salts, which are derived from alkali metal or alkaline earth metal hydroxides, Derive carbonates or bicarbonates, and the salts that are different from primary, secondary or tertiary Amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylenediamines, Ν, Ν-diaralkyl-lower alkylenediamines, Aralkylamines, amino-substituted lower alkanols, N, N-di-lower alkylamino-substituted lower: alkanols, Amino, polyamino and guanidino substituted lower alkanoic acids and nitrogen containing heterocyclic Derive amines. Examples are the salts derived from sodium hydroxide, sodium carbonate, Sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, Trimethylamine, triethylamine, piperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, Morphine, benzylamine, ethylenediamine, Ν, Ν'-dibenzylethylenediamine, Diethanolamine, piperazine, dimethylaminoethanol,

- 29 809851/0039

2-amino-2-methyl-1-propanol, theophylline, N-methylglucamine. and similar. Acid addition salts with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, toluene-p-sulphonic acid and methanesulphonic acid can also be used.

The salts can be monosalts like the monosodium salt, obtained by treating one equivalent of sodium hydroxide with one equivalent of the product (II), or they can be mixed disalts obtained by treating one equivalent of a monosalt with an equivalent of a different one Base receives. Alternatively, the salts can be obtained by treating one equivalent of a base with a divalent one Cation such as calcium hydroxide can be produced with an equivalent of the product (II). Furthermore are subject of the invention mixed salts and esters such as those obtained by treating the product (II) with one equivalent of sodium hydroxide and then get lactic acid with one equivalent. The salts according to the invention are pharmacologically acceptable, non-toxic derivatives that act as the active ingredient can be used in suitable pharmaceutical forms in unit dose. You can continue with others Medicines are combined to obtain preparations with a wide range of activities. In addition, you can the salts according to the invention and also the corresponding esters and amide derivatives / intermediates in the preparation of carboxylic acid products represented by Formula II above can be used.

The new thienamycin derivatives according to the invention are valuable antimicrobial compounds that act against various gram-positive and gram-negative pathogens such as Bacillus subsilis, Salmonella schottmuelleri and Proteus vulgaris are active. The free acid and especially hers Salts such as the amine and metal salts, especially the alkali metal and alkaline earth metal salts, are useful

- 30 809851/0039

tericides and can be used for the removal of sensitive pathogens from dental and medical facilities, for the separation of microorganisms and used for therapeutic use in humans and animals. For this latter purpose, pharmacologically acceptable salts with inorganic and organic bases such as them are used are known to the person skilled in the art and are used in the administration of penicillin and cephalosporins. For example, one can use salts such as alkali metal and alkaline earth metal salts and primary, secondary and tertiary Use amine salts for this purpose. These salts can be mixed with pharmaceutically acceptable liquid and solid carriers forming suitable unit dosage forms such as pills, tablets, capsules, suppositories, syrups, elixirs and the like are mixed, the latter being able to be prepared in a manner known per se.

The new compounds are valuable antibiotics against various gram-positive and gram-negative bacteria and are accordingly used in human and veterinary medicine. The compounds according to the invention Can be used as antibacterial drugs to treat infections caused by gram positive and gram-negative bacteria are generated, for example against Staphylococcus aureus, Escherichia coli, Klebsieila pneumoniae, Bacillus substilis, Salmonella typhosa, Pseudomonas and Bacterium proteus. The antibacterial agents according to the invention can further be used as additives for Animal feed, food preservation and disinfectant used. For example They can be used in aqueous compositions in concentrations ranging from 0.1 to 100 parts per antibiotic Million parts of solution to destroy and inhibit the growth of harmful bacteria on medicinal and dental devices or devices and as bactericides in industrial applications, for example in

- 31 -

809851/0039

15821IA to 27 2 A 56 O

Water-based inoculations, and in the white water of paper mills to inhibit the growth of harmful ones Bacteria are used.

The products of the invention can be used alone or together as an active ingredient in any of the many pharmaceutical Preparations are used. These antibiotics and their corresponding salts can come in capsule form or be used as tablets, powders or as liquid solutions or as suspensions or elixirs. You can orally, administered intravenously or intramuscularly.

The compositions or preparations are preferably in a form that is suitable for absorption by the gastrointestinal Tract is suitable. Tablets and capsules for oral administration can be in unit dosage form and they can be known per se drug excipients such as binders, for example syrup, acacia gum, gelatin, Sorbitol, tragacanth or polyvinylpyrrolidone, fillers, for example lactose, sugar, corn starch, calcium phosphate, Sorbitol or glycine, lubricants, for example magnesium stearate, talc, polyethylene glycol, silicon dioxide, Integrating agents such as potato starch or acceptable wetting agents such as sodium lauryl sulphate contain. The tablets can be coated or coated by processes known per se. Oral liquid preparations can be in the form of aqueous oily suspensions, solutions, emulsions, syrups, elixirs, etc., or they can be used as a dry product for reconstitution with water or another suitable vehicle before Use. Such liquid preparations can contain additives known per se, such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, Aluminum stearate gel or hydrogenated edible oils such as almond oil, fractionated co-

- 32 -

809851/0039

coconut oil, oily esters, propylene glycol or ethyl alcohol, preservatives such as methyl- or propyl-p-hydroxybenzoate or contain sorbic acid. Suppositories contain suppository bases known per se, for example Cocoa, butter or other glycerides.

Preparations for injection can be in unit dosage form in ampoules or in containers holding multiple doses contain, with an added preservative. The preparations can be in the form of suspensions, Solutions or emulsions are present in oily or aqueous vehicles and they can be preparation auxiliaries such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient in powder form for reconstitution with a suitable vehicle, for example sterile pyrogen-free water, available before use.

The preparations can also be used in for absorption through the mucous membranes of the nose and throat or bronchial tissue, and they may conveniently be in powder or liquid form Sprays or inhalants, lozenges or gargles. For medication of the eyes or Ears, the preparations can be present as individual capsules in liquid or semi-solid form, or they can can be used as drops. For topical application, the preparations can be hydrophobic or hydrophilic Base materials are in the form of ointments, creams, solutions or lotions, as a means for brushing or as a powder,

In addition to a carrier, the preparations according to the invention can also contain other ingredients such as stabilizers, Binders, antioxidants, preservatives, lubricants, suspending agents, agents that support the Affect viscosity, or flavor or taste

- 33 -

809851/0039

contain substances and similar agents. The preparations may also contain other active ingredients, thereby giving a wider spectrum for the antibiotic.

For veterinary drugs, for example, the preparations can be used as intramammary preparations in either long-acting or fast-acting ingredients.

The dose to be administered depends to a large extent on the condition of the subject being treated and the weight of the host, the route and frequency of administration. The parenteral route is generally used for generalized infections preferred and the oral route is preferred for intestinal infections. Generally the daily oral dose is from about 15 to about 600 mg of active ingredient per kg of body weight of the subject for one or more applicants. fertilize per day. A preferred daily dose for adult humans is in the range of about 80 to 120 mg of active Component per kg of body weight.

The preparations according to the invention can be in various unit dosage forms, for example in solid or liquid, orally digestible dosage form. The preparations can contain in the unit dose as liquid or solid preparations from 0.1 % to 99 % of active material. The preferred range is from about 10 to 60.55. The formulations will generally contain from about 15 mg to about 1500 mg of the active ingredient. However, it is generally preferred to use a dose amount ranging from about 250 mg to 1000 mg. For parenteral administration, the unit dose generally consists of the pure compound in a slightly acidified sterile water solution or it is in the form of a soluble powder intended for dissolution.

_ 34 _
809851/0039

The following examples illustrate the examples according to the invention Products, processes, preparations and treatment processes. The compounds of the invention are with the previously introduced symbol:

-OR

Th..NH ₂ -COOH

where the three functional groups are listed.

example 1

ΓΟΗ

^Η //

Th H-NH-C-CH- ('

lh

CHO

Preparation of N- (O-Formyl) -D-mandeloylthienamycin sodium salt

To 40 mg of thienamycin in 10 ml of water was subsequently added at 0 ⁰ C 124 mg NaHCO ,, 8 ml of dioxane and then with stirring 1.2 equiv. N- (0-formyl) -l-mandeloyl chloride in the course of 1 min. After a total reaction time of 6 min. The mixture is extracted 3 times with cold ethyl ether. Electrophoresis of an aqueous portion (0.05M, pH 7, aqueous phosphate buffer, 50 V / cm, 20 min.) Shows a 67 / Sige conversion to the desired product. The pH is adjusted to 2.2 with IM Η, ΡΟ ^ solution. The solution is extracted 3 times with ethyl acetate. The ethyl acetate (EtOAc) solution is _{dried over MgSO 1} , and washed twice with 2 equiv

- 35 809851/0039

■ 15821IA _n 272-560

NaHCO ^ solution extracted. The aqueous extract is lyophilized and contains 164 optical density units (ODU) at 302 nm in the UV analysis at pH 7.9, 95 % of which is removed after treatment with hydroxylamine for 1 hour. The yield is 53 %. Electrophoresis, performed as previously described, shows a stain on bioautography 4 cm towards the anode. NMR (Ji ₁ D ₂ O) 1.30 (d, J = 6 Hz, CH ₃ CH)! 2.8-3.7 (m, CH _3), 4.0-4.5 (m, CH ß-lactam), 4.737 HDO; 5.97 (s, C ₆ H ₅ CHOCHO), 7.53 (s, C ₆ H _5), 8.30 (s, C ₆ H ₅ CHOCHO).

Example 2

Th--

"-COONa

Manufacture of N-D-mandeloylthienamycin sodium salt

The title compound is prepared according to the method of Example 1. Before the EtOAc extraction, the aqueous extract can stand at 25 ° C. for 1 hour. Electrophoresis (50 v / cm, 20 min., PH 7, aqueous phosphate, 0.05M) shows a spot on bioautography 4 cm in the direction of the anode. NMR ((f, D ₂ O) 1.50 (d, J = 6 Hz, CH ₃ CH): 2.8-3.8 (m, CH ₂ ), 4.2-4.6 (m, CH β-lactam); 4.96 (s7 ~ HD0); 5.40 (s, C ₆ H ₅ CH).

Example 3 N-propionylthienamycin sodium salt

To 25 mg of thienamycin in 6 ml water at 0 ⁰ C was subsequently added 38.6 mg NaHCO _3, 5 ml of dioxane and then

-36-

809851/0039

15821IA Iß 77? A SRO

Stir 1 equiv. Propionic anhydride in the course of 3 minutes. After 10 minutes, the mixture is extracted 3 times with cold ethyl ether. Electrophoresis of the aqueous (aq.) Extract (0.05M, pH 7, phosphate buffer, 50 v / cm, 20 min.) Shows that no free thienamycin is present. The pH of the aqueous extract is adjusted to 6.8. It contains 600 ODU at 302 nm in the UV analysis, 95 % of which are removed after treatment with hydroxylamine for 1 hour. NMR (ί, D ₂ O) 1.42 (m, CH ₂ CH CH ₃ CH); 2.48 (q, CH ₂ CH ₃ ); 2.86-2.90 (m, CH ₂ ), 4.30-4.70 (m, CH β-lactam), 486 (HDO).

Example 4 N- (methoxyacetyl) thienamycin sodium salt

To 55 mg of thienamycin in 6 ml of water at 0 ° C., 68 mg of NaHCO _7. , 6 ml of dioxane and, with stirring, 1.1 equiv. Methoxyacetyl chloride over a period of 1.5 minutes. The mixture is stirred for an additional 10 minutes. The mixture is extracted 3 times with cold ethyl ether. Electrophoresis of the aqueous extract (0.05M, pH 7, phosphate buffer, 50 V / cm, 20 min.) Shows that no free thienamycin is present. The pH of the aqueous extract is adjusted to 6.8. It contains 105 ODU at 302 nm by UV analysis, 95 % of which is eliminated after treatment with hydroxylamine for 1 hour. NMR (<f, D ₃ O) 1.56 (m, CHCH ₃ ), 2.84-3.60 (CH ₂ ), 3.72 (s, OCH ₃ ), 4.29 (s, OCH ₂ ) , 4.98 (s, HDO).

Example 5 N- (p-nitrobenzyloxycarbonylthienamycin lithium salt

To 220 mg of thienamycin in 60 ml of water at 0 C are added successively 679 mg of NaHCO ,, 60 ml of dioxane and then added Stir 1.1 fiquiv. p-nitrobenzyl chloroformate in the course of 1.5 min. The mixture can then react for 10 min. and

- 37 -

809851/0039

15821IA Vi 27 2 A 56

it is then extracted 3 times with cold ethyl ether. Electrophoresis (0.05M, pH 7, phosphate buffer, 50 V / cm, 20 min.) Shows that no free thienamycin is present. The pH of the aqueous extract is _{adjusted to 2.2 with 1M H, PO 4} ~ solution and the solution is extracted 3 times with EtOAc. The EtOAc extract is _{dried over MgSO 4} , filtered and extracted again with 0.1N LiOH at pH 8.2. The final pH is _{adjusted to 7.0 with IM H 3} PO ₄ and the sample is lyophilized. The yield is 205 mg (54 %).

Example 6

Preparation of M-Cp-nitrobenzyloxycarbonyD-thienamycin p-t-butylbenzyl ester

205 mg of N- (p-nitrobenzyloxycarbonyl) -thienamycin-Li salt, the product of Example 5 in 2 ml of hexamethylphosphoramide (HMPA) are mixed with 0.1625 ml of p-t-butylbenzyl bromide for 2.5 hours treated. The evidence is in HMPA insoluble, but dissolves after 30 min.

The reaction mixture is diluted with ethyl acetate (EtOAc), washed successively with water, aqueous KpHPOu, water, saturated aqueous NaCl, over MgSO ₁ . dried, filtered and evaporated and subjected to preparative thin layer chromatography on silica gel, eluting with 1: 2 CHCl,: EtOAc. The yield is 160 mg (59 %), Rf 0.38, IR (u-film) 2.98 NH and OH 5.63, β-lactam; 5.86 t> reit, ester and urethane: NMR (i, CDCl ₃ ), 1.24 (s, CHCH ₃ , t-butyl), 2.59 ~ 3.27 (m, _# CH ₂ ) 3.83 -4.47 (m, CH β-lactam), 5.15 (s, CH ₂ C ₆ H ₄ NO ₂ ), 5.22 (s, OCH ₂ C ₆ H ₄ , t -butyl), 7.45 and 8.12 (AB quartet, J = 8 Hz C ₆ H ₄ NO ₂ ).

- 38 -

809851/0039

15821IA

Example 7

SO

Production of N-Cp-NitrobenzyloxycarbonyD-thienamycin m-phenoxybenzyl ester _ ^ _________

The procedure described in Example 6 is followed and the title compound is prepared, 1 equiv. Amount of m-phenoxybenzyl bromide being used for the pt-butylbenzyl bromide of Example 6. The yield is 11 % _t IR (μ-film) 3.0 NH ₂ and OH 5.63 β-lactam; 5.86 broad peak _} esters and urethane; NMR (/, CHCl ₃ ) 1.33 (d, CHCH ₃ , J = 6); 2.60-3.62 (m, CH ₂ ), 7.45 and 8.13 (AB quartet, J = 8, C ₆ H ₁₊ NO ₂ ); 7.26 _(s; C ₆ H ₄ OC ₆ H ₅₎ MS m / e 589, 559, 5 ^ 7, I83.

Example 8

Production of

1. N- (0-Formyl) -D-mandeioyl ~ thienamycin-p-t-butylbenzyl ester

2. N- (0-Formyl) -D-mandeloyl-thionaraycin-m-phenoxybenzyl ester 3-N-D-Mandeloyl-thienamycin-p-t-butylbenzyl ester

4. N-D-Mandeloyl-thienamycin-m-phenoxybenzyl ester

5. N-propionyl-thienamycin-p-t-butylbenzyl ester

6. N-propionyl-thienamycin-m-phenoxybenzyl ester

7. N-Methoxyacetyl-thienarnycin-p-t-butylbenzyl ester

8. N-Methoxyacetyl-thienamycin-m-phenoxybenzyl ester

The procedure is as described in Examples 6 and 7 and the title compound is prepared, using the suitable N-acylthienamycin starting materials of Examples 1 to h in an equivalent amount instead of the N- (p-nitrobenzyloxycarbonyl) thienamycin starting material of Examples 6 and 7 be used.

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15821IA 272-560

Example 9

Production of N- (p-methoxybenzyloxycarbonyl) -thienair.ycin pt-butylbenzyl ester

Level A : N- (p-Methoxybenzyloxycarbonyl) -

To 20 mg of thienamycin in 5 ml of water at 0 C are added 105 mg of NaHCO 3 (20 equiv.), 5 ml of dioxane and then 10 equiv. Dropwise with stirring over the course of 1 min. p-methoxybenzyl chloroformate. After 15 minutes the pH is _{adjusted to 7.5 with 1M H, PO 14} and the solution is extracted 3 times with ether. The pH of the aqueous portion is then adjusted at 0 ⁰ C to 2.2 and then 3 times extracted with ethyl acetate (EtOAc). The EtOAc is quickly _{dried over MgSO 1} , filtered and extracted with a few ml of water containing 6.3 mg of NaHCO 3. The aqueous extract is lyophilized. It contains 172 ODU at 303 nm on UV analysis in H ₃ O at pH 7.0, of which 95 % is removed after treatment with hydroxylamine for 1 hour. The yield is 16 mg. Electrophoresis (50 V / cm, 20 min., PH 7 aqueous phosphate, 0.05M) shows a spot on bioautography 4 cm in the direction of the anode. NMR (o ^f , D ₂ 0); 1.49 (d, J = 6 Hz CH ₃ CH); 4.92 (s, HDO); 5.20 (s, OCH ₂ ); 7.13 (d, J = 8 Hz CgH ₁₁ ).

The lithium salt II is prepared in the same way, but the EtOAc solution with 0.1η LiOH to pH 7.8 (instead of aqueous NaHCO3) is extracted. The spectral and electrophoretic properties of II are same as with I.

Stage B : N- (p-Methoxybenzyloxycarbonyl) -thienamycin-pt benzyl ester

Stage A lithium salt (37 mg) in 0.4 ml hexamethylphosphoramide (HMPA) is added for 2.5 hours with 0.033 ml Treated p-t-butylbenzyl bromide. The lithium salt is in

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15821IA SZ 27 2 A 56 O

HMPA insoluble, but dissolves after 15 minutes, reaction time.

The reaction mixture is diluted with EtOAc, then _{washed twice with water, aqueous K 2} HPO _1. , Water and brine, with MgSO ₁ , dried, filtered, evaporated and subjected to thin layer chromatography on silica gel, using 1: 2 CHCl ₃ - EtOAc is eluted. 47 mg of pure II are obtained, Rf = 0.3. IR (µ film): 3.0, NH; 5.63, β-lactam; 5.87 wide, ester and urethane. NMR (<f, CDCl3,): 1.21 (s, Me and t-butyl); 2.6-3.6 (m, CH _3); 3.72 (s, OMe); 3.8-4.4 (m,

β-lactam CH); 4.97 (-OCH ₂ - ^% -OCH,), 5.18 (t-bu); 6.84 and 7.20 (AB Quartet, CgH ^ OMe); 7.32 (s CgHjj-t-Bu). MS: 582, 538, 496.

Example 10

Preparation of N- (p-Methoxybenzyloxycarbonyl) -thienamycin benzhydryl ester

23.5 mg to thienamycin in 5 ml of water is added successively 4 ml dioxane, 62 mg NaHCO, and then added portionwise at 0 ⁰ C under stirring for 4 equiv. p-Methoxybenzyl chloroformate in the course of 4 minutes. After a total reaction time of 10, the pH is adjusted to 7 with IM Η, ΡΟ |, and the mixture is extracted 3 times with ether. Electrophoresis of the aqueous portion (0.05 M, pH 7, aqueous phosphate buffer, 50 V / cm, 20 min.) Shows a 50% conversion to N- (p-methoxybenzyloxycarbonyl) thienamycin. ■

The pH of the aqueous solution is determined with IM H ^ PO ₁ . set at 0 ⁰ C to 2.2 and then 3 times extracted with EtOAc. The EtOAc solution is treated with 50 mg diphenyldiazomethane, evaporated and dissolved in CH, CN. Further di-

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15821IA 53 27 2 A 56

phenyldiazoinethane is added until it remains purple. After 0.5 hours the solution is evaporated and chromatographed on silica gel, eluting with 1: 2 CHCl ^ -EtOAc. 10 mg of pure title compound Rf 0.25 are obtained. IR (µ film): 3.0, NH; 5.63, β-lactam; 5.85, 589, esters and urethane. NMR (, CDCl ₃ ); 1.23 (s, OH); 1.30 (d, J = 6 Hz, CH ₃ CH); 2.6-3.6 (m, CH _2); 3.78 (s, OMe); 5.02 (s, OCH ₂ ); 3.8-4.4 (m, β-lactam CH); 6.9 and 7.35 (AB quartet, J = 9 Hz, C ₆ H ₄ ), 7.3 s CHPh ₃ .

Example 11

Preparation of N- (o-nitrobenzyloxycarbonyl) -thienamycin benzyl ester

Stage A : N- (o-nitrobenzyloxycarbonyl) -thienamycin sodium salt

10 ml of 1: 1 tetrahydrofuran (THP: water) are added to 43 mg of thienamycin at 0 ° C. The mixture is stirred rapidly while 102 mg of NaHCO 3 (10 equiv.) Are added and then 4 equiv. o-Nitrobenzyl chloroformate added. After 30 minutes, the pH is adjusted to 7 with aqueous 25% H, POj, and the solution is extracted 3 times with ether. The aqueous layer is evaporated at 25 ° C. in vacuo and then the pH value is adjusted to 2.2 ^{at 0 ° C.} Solid NaCl is added and the cold acidic solution is extracted 3 times with cold, EtOAc. The EtOAc extracts are combined and quickly backwashed with cold saline, with MgSO ₁ . dried, filtered and with 10 ml of water, the 1.75 kquiv. solid NaHCO ^. contains, back-extracted. The extract is lyophilized in vacuo at 25 ° C. The title compound is obtained.

809851/0039

Stage B : N- (o-nitrobenzyloxycarbonyl) thienamycin benzyl ester

The product of stage A in 7.5 ml EtOAc (from the pH 2.2 extraction) is treated with an excess of phenyldiazomethane (4 ml solution containing 20 mg / ml ether) at 4 ° C. for 2.3 hours . The mixture is concentrated to a wet residue at 2O ⁰ C under reduced pressure. The desired compound is isolated by thin layer chromatography, EtOAc, ether (9: 1). 17.5 mg of N- (o-nitrobenzyloxycarbonyl) thienamycin benzyl ester are obtained.

Example 12

Preparation of N- (o-nitrobenzyloxycarbonyl) -thienamycin p-methoxybenzyl ester

To 70 mg of N- (o-nitrobenzyloxycarbonyl) -thienamycin in 8 ml of EtOAc, 4 ml of p-methoxyphenydiazomethane (9 mg / ml acetonitrile) added at 4 ° C. The weird is stirred for 1.5 hours at 4 C and then to a na ;;: ien paste at reduced Pressure at 20 C concentrated. 42 mg of the title compound are isolated by thin-layer chromatography on silica gel, when eluting with EtOAc: ether (9: 1).

Example 13

Production of N- (o-nitrobenzyloxycarbonyl) -thienamycin-pbromophenacyl ester '

r-0 (TMS)

Th-I-NH (TiMS) ν Th 4- NHC-O-CH., - ^

LCOO (TMS)

OH

NO ₂

- CO-CH ₀ -C-

[where TMS = trimethylsllyl]

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15821IA SS 27 2 A

23 mg of o-nitrocarbobenzyloxychloride and then 0.015 ml of triethylamine are added to 2k mg of Th (TMS) ,, I, prepared according to Example 17, in 0.8 ml of dry THF. After vibromixing for 30 minutes at 25 ° C., the mixture is concentrated to a paste-like residue in a stream of dry N- and washed 3 times with petroleum ether. The residue is suspended in 1 ml of dry THF and Ik mg of p-bromophenacyl bromide are added. Then 0.03 ml of triethylamine are added. After Vibromischen for 30 min. At 25 ° C, the mixture is evaporated to dryness in vacuo at 20 ⁰ C. The residue is dissolved in EtOAc (2 ml) and shaken with 0.3 ml in a pH k buffer for 5 minutes. The organic layer is _{dried over MgSO 1} , filtered and evaporated to a pasty residue. The desired product (kk mg) is isolated by preparative thin layer chromatography on silica gel and eluting with EtOAc: CHCl, (7: 3).

Example 14

Production of N-t-trichloroethoxycarbonyli-thienamycin benzyl ester

Stage A: N- (trichloroethoxycarbonyl) lithium salt

To k0 mg thienamycin in 18 ml 1: 1 THF-HPO are added at 0 ⁰ C under stirring, 225 mg (15.2 Kquiv ..) NaHCO, and then added dropwise with stirring over 2 min, 1.8 eq * Trichloräthylchlorformiat , dissolved in 0.6 ml of THF. After a few minutes, the pH is adjusted to 7.2 with aqueous 25 # iger Η, ΡΟ ^ and the solution is extracted with ether. The pH of the aqueous portion is adjusted to 2.5 at 0 ° C in vacuo after any entrained ether has been removed. Then it is extracted with cold EtOAc. The ethyl acetate extracts are combined, quickly backwashed with cold salt solution ^ with anhydrous MgSO ₁ .

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dried, filtered and back-extracted with 0.01 M LiOH at a pH of 6.8. The aqueous extract is freed from any EtOAc in vacuo and lyophilized. The remaining product contains 936 ODU (39.7 %) according to UV analysis at 302 nm, 90 % of which is removed after treatment with hydroxylamine for 1 hour in 0.05M phosphate buffer (pH 7). The yield is 32 mg. The electrophoresis (50 V / cm, 20 min., PH 7 »aqueous phosphate 0.05M) shows a zone (MB 108, staph. Aureus) 2.4 cm in the direction of the anode in the bioautography. Liquid chromatography C ₁ Q Bondapak (Waters Assoc.) In aqueous THF shows a major peak free of any unreacted thienamycin.

Level B ; N- (trichloroethoxycarbonyl) thienamycin benzyl ester

32 mg of the compound from stage A v / ground in 2 ml of dry, distilled DMF containing 7 % HMPA (dry, pH 6.3), treated with 0.015 ml of benzyl bromide for 2 hours at 15 ° C (the reaction mixture in the The course of the reaction can warm to 25 C). The reaction mixture is diluted with EtOAc, successively with cold H _? 0.1 l aqueous NaHCO, water and cold, saturated, aqueous NaCl solution, washed over MgSO ₁ . dried, filtered, evaporated, and subjected to preparative thin layer chromatography on silica gel, eluting with 1 % CH 4 CN in EtOAc. 10 mg of the title compound are obtained, Rf = 0.63; IR (µ CHCl ₃ ) 5.63, β-lactam; 5.78 and 5.88 wide, ester and urethane. NMR (S , CDCl ₃ ) 1.35 (d, Me); 2.8-3.7 (m, CH _3); 3.51 and 4.27 (dd, J = 6 Hz, β-lactam CH); 4.79 (s, OCH ₂ CCl ₃ ) 5.42 (s, OCH ₂ C ₆ H ₅ ); and 7.41 (m, C ₆ H ₅ ).

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Example 15 ⁵

Production of N-bromoacetylthienamycin methyl and benzyl esters

Level A: N-bromoacety! Thienamycin

To a cooled solution of 28.8 mg of thienamycin and 0.3 g Sodium bicarbonate in 10 ml of water and 8 ml of dioxane are added to a solution of 0.25 g of bromoacetic anhydride with stirring in 2 ml of dioxane in the course of 20 minutes. The pH is kept at 8.0. The mixture is stirred for a further 5 minutes, then covered with a layer of 10 ml of ether, and then the pH value is adjusted to 7 by adding another 8% solution Phosphoric acid. The ether layer is separated off and the aqueous layer is extracted again twice with ether. The aqueous layer is evaporated to 0.5 ml under reduced pressure, diluted to 2 ml with water and to 50 ml XAD-2 resin given.

The column is eluted with water. The first 80 ml are discarded, the next 100 ml are collected. The solvent is changed to 10% THF and an additional 100 ml is collected. The pH of the combined eluates is adjusted to 7, evaporated to 5 ml under reduced pressure and then freeze-dried. The sodium salt of N-bromoacetylthienamycin is obtained in a yield of 60%. UVJ 302 mn.

Stage B : N-bromoacetylthienamycin methyl and benzyl ester

An aqueous solution of the sodium salt is provided with a layer of ethyl acetate at 0 ⁰ C and then the pH value is adjusted to 2. The ethyl acetate phase is separated off and the aqueous phase is extracted with ethyl acetate. The combined ethyl acetate solutions are over

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15821IA Si 27 2 A 56 O

MgSO ₁ , dried and then treated with a solution of diazomethane. The solvents are evaporated and the residue is chromatographed on a silica gel plate. Rf 0.11 in 2: 1 ethyl acetate chloroform. M.p. 118-120 ° C. The mass spectrum shows M at m / e 406 and essential fragments at m / e 362, 320, I83 and

The corresponding benzyl ester is prepared from phenyldiazonemethane in a similar manner. M.p. 142-3 ° C IR 5.65 U and 6.1 µ. Mass spectrum M m / e 482, also m / e 438, 316, 259 and 164.

Example 16

Preparation of N-iBromo-t-butoxycarbonyD-thienamycin-p- bromophenacyl ester

Level A ; Production of N-bromo-t-butoxycarbonyl-O -TMS-thienamycin-TMS ester

16 mg Th (TMS) are dissolved in 0.4 mJ dry tetrahydrofuran, to which 20 μΐ (28 mg, 0.13 mmol) of bromo-t-butyl chloroformate (boiling point 35 ° / 0.9 mm) and 8 μΐ ( 5.67 mg, 0.057 mmol) of triethylamine (redistilled from BaO) are added. The mixture is shaken at 25 ° C. for 20 minutes. Evaporation of the solvent and excess reagent gives the desired crude product. Uv) CH ₃ CO ₂ CH ₂ CH ₃

(E 9000). ^{/ v}

Stage B : Preparation of N- (bromo-t-butoxycarbonyl) -thien amycin

3 mg of the product of stage A are dissolved in 0.5 ml of pH 7 phosphate buffer and 0.1 ml of tetrahydrofuran dissolved. The solution is left to stand at 25 ° C. for 20 minutes. The solution will then passed through a column (5 ml) of Dowex 50 χ 8 (Na form). The eluate fractions are checked with UV. The right ones Fractions are pooled and freeze-dried.

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15821IA 53 "272-560

The desired product is obtained. Uvl ™ * ^ier ^ _{04 nm} (£ = 9300); Electrophoresis at 50 V / cm, 20 min. At pH 7 buffer shows a single bioactive zone migrating 31.5 mm towards the anode.

Step C : Preparation of N- (bromo-t-butoxycarbonyl) -thienamy cin-p-bromophenacyl ester

13 mg of the product of step B (0.022 mmol) are in 0.4 ml Tetrahydrofuran dissolved. 9.6 mg (0.035 mmol) of p-bromophenacyl bromide and 20 μΐ (14.4 mg, 0.14 mmol) triethylamine. The mixture is shaken at 25 ° C. for 30 minutes and then evaporated to dryness. 10 ml of ether are added to the residue and the mixture is washed with 0.2 ml of 0.1M pH 7 phosphate buffer treated.

The organic layer is separated, dried over sodium sulfate, concentrated to 0.5 ml and placed on two 20 × 20 cm, 250 μ silica gel GF tlc plates which are developed with 20 % ethyl acetate in chloroform. Rf = 0.65. The desired product (6.7 mg) is isolated in 42% yield.

Example 17

Preparation of N-Benzyloxycarbonylthienamycin and N-Benzyl oxycarbonylbenzyl carbonic anhydride

[R

A solution of 16.6 mg thienamycin in 4 ml 0.05M pH 7 Phosphate buffer and 2 ml of dioxane in a three-necked flask, the

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equipped with a stirrer, thermometer, pH electrode and a dispensing tip of an automatic titration device, it is cooled to -8 ° C in a methanol-ice bath. The pH is adjusted to 8.2 by adding 0.2N sodium hydroxide in 50% aqueous dioxane. A solution of 0.015 ml of carbobenzyloxy chloride in 2 ml of chloroform is added. The mixture is stirred at -6 ° C., pH 8.2, for 10 minutes, then covered with a layer of ether and then the pH is adjusted to 7 by adding η-hydrochloric acid. The layers are separated by centrifugation and the aqueous phase is re-extracted twice with ether. The aqueous phase is covered with a layer of ethyl acetate and acidified to pH 2. The ethyl acetate is separated off and the aqueous layer is extracted again with ethyl acetate. The combined ethyl acetate layers are washed with saturated sodium chloride solution, dried over magnesium sulfate and filtered. The filtrate is stirred with water and the pH is adjusted to 7 by adding dilute sodium bicarbonate solution. The aqueous phase is separated off and freeze-dried. The sodium salt of N-benzyloxycarbonyl Th. Weight 10 mg (46 50, The UV spectrum ") _m 303 μ, E% 147 (E 6290) shows

/ ^ max

a purity of 80 % . Electrophoresis at 50 V / cm for 20 min. At pH 7, followed by bioautography on S, aureus results in an inhibition zone at +2.5 cm.

The ether extracts of the reaction mixture contain the desired N-benzyloxycarbonylthienamycinbenzy! Carbonic anhydride. UVJ _max 335 mu ..

Example 18 Preparation of N-Benzyloxycarbonylthienamycin Benzyl Ester

N-Benzyloxycarbonylthienamycin (Example 17) in EtOAc prepared according to the method of Example 17, except

809851/0039

Men that an equivalent amount of phenyldiazomethane is added to the dried EtOAc solution from the pH 2 extraction and that the solution is allowed to stand at 4 ° for 2 hours. Evaporation to dryness gives crude N-benzyloxycarbonylthienamycin benzyl ester, which is isolated by thin-layer chromatography Rf 0.24 3: 1 ethyl acetate chloroform. It crystallizes from ether. IR 5.63 U (lacthamcarbonyl); Shoulder 5.8 µ (ester); 5.88 µ (urethane carbonyl). UV, dioxane, '] 318 mμ, E% (2 = 10900) m / e M ⁺ 496.

y \ ΓΠ3-Χ

Example 19

Preparation of N-carbomethoxythienamycin-p-pivaloyloxy benzyl ester

Grade A : N-carbomethoxythienamycin

49 mg thienamycin (148 μΜοΙ) are dissolved in 0.05M pH 7 phosphate buffer (14 ml) and cooled in an ice bath. With stirring, the pH is adjusted to 8.2 using an automatic burette. A solution of methyl chloroformate (46 μΐ, 600 μΜοΙ) in p-dioxane (580 μΐ) is added all at once. A homogeneous solution is obtained. The pH is then maintained at 8.2 using an automatic burette. After 10 minutes, the pH of the solution is adjusted to 7 using dilute phosphoric acid and it is then washed 3 times with an equal volume of diethyl ether. The aqueous solution is then concentrated to 4.5 ml and chromatographed on an XAD-2 resin column. The product is eluted (after elution with water) with an aqueous 5 # strength tetrahydrofuran solution and freeze-dried. 28.9 mg of product are obtained. UV (pH 7 phosphate buffer 0.1n).] _M , _v 303 nm (Z 6450). Electrophoresis (if 5 / biliä 35

(20 min., 0.1 η pH 7 phosphate buffer, 50 V / cm, mobility 3.5 cm to the cathode).

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Stage B : N-carbomethoxythienamycin-p-pivaloyloxybenzyl ester

Following the procedure of Example 16, Step C, and using equivalent amounts of N-carbomethoxythienamycin (as the sodium salt) and p-pivaloyloxybenzyl bromide one N-carbomethoxythienamycin-p-pivaloyloxybenzyl ester,

Example 20

Preparation of N-benzenesulfonylthienamycin ^ -methyl ^ - propen-1-yl ester

, -OH ₀
Th. "

O J-COOR

R = -0-CH ₀ C = CH-2,2

CH ₃

Grade A : N- Benzolsulfonylthienarn yein

52 mg of thienamycin (148 µmol) are dissolved in pH 7 0.1η phosphate buffer (25 ml) and stirred magnetically on an ice bath. The pH is adjusted to 8.2 with 2.5N NaOH using an automatic distribution burette. 227 Ul benzenesulfonyl chloride (226 μπιοί) in 500 ml of p-dioxane are added all at once. The pH is maintained at 8.2 (using the automatic burette) for 30 minutes. The pH is then adjusted to 7.0 with dilute aqueous phosphoric acid. The reaction solution is concentrated to 15 ml and chromatographed on XAD-2 resin (50 ecm). The column is eluted with water and then with 10% aqueous tetrahydrofuran, which elutes the product. The 10% aqueous tetrahydrofuran eluate is concentrated to a third volume and freeze-dried. 28 mg are obtained. The electrophoretic mobility of the product (50 V / cm, 20 min., PH 7 0.05N phosphate buffer) is 3.5 cm in the direction of the cathode. ^ _Ov 303 (E 3650) at pH 7

/ \ IUa, X

01 Phhtff

0.1η phosphate buffer.

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Stage B : N-Benzolsulfonylthienamycin - ^ - methyl ^ -propen- 1-yl-ester ________

One works as described in Example 24 and uses equivalent amounts of N-benzenesulfonylthienamycin (as sodium salt) and 2-methyl-2-propen-1-yl chloride in place of the N-azidoacetylthienamycin sodium salt and p-t-butylbenzyl bromide. This gives the title compound.

Example 21

-OTMS

Th - NHTMS or Th (TMS) ₃
-COOTMS

TMS = Trimethylsilyl Production of the silylated thienamycin

80.0 mg of thienamycin are in ^ G; r.I tetrahydrofuran (THF) suspended and concentrated to 10 ml under an Np atmosphere. 1.0 ml hexamethyldisilazane and 300 μΐ trimethylchlorosilane are admitted. The mixture is reacted for 20 minutes at 25 ° C. with vigorous stirring. The suspension is then centrifuged to remove ammonium chloride. The supernatant solution becomes an oil under nitrogen stream evaporated for further implementation.

Example 22

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

-OH

Th-I-NHR ²
LCOOR ³

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A mixture of 295 mg of p-nitrobenzyloxycarbonyl-thienamycin lithium salt (from Example 5) and 0.4 g of p-nitrobenzyl bromide in 3 ml of hexamethylphosphoramide is stirred at 25 ° C. for 3 hours. The solution is diluted with 50 ml of ethyl acetate and extracted successively with water (3X), pH 7 phosphate buffer and saturated sodium chloride solution. The organic phase is dried over magnesium sulfate and evaporated to 5 nil, the product crystallizing. The crystals are collected and washed with ethyl acetate. 160 mg of Np-nitrobenzyloxycarbonylthienamycin-p-nitrobenzyl ester, melting point 168 ° -17O ° C. are obtained. NMR (CHCl ₃ ) 1.35 / Jd), J = 6 Hz7, 4.0-4.3 and 2.8-3.5 (m), 5.16 / Ts), (carbamate OCH ₂ ] /; 5.24 (AB quartet, J = 14), 7.42 (d), 7.56 (d), 8.16 (d) (J = 9, aromatic) IR 5.65 U (lactam C = O) .

Example 22a

Preparation of N-bromo-t-butyloxycarbonylthienamycin sodium salt

Method A : 190 g of thienamycin are dissolved in 15 ml of 0.1M pH 7.0 phosphate buffer and 15 ml of dioxane and kept at 0.degree. The solution is adjusted to pH 8.5-9.0 with In NaOH and maintained at that while mg of bromo-t-butyl chloroformate is added to the solution over a period of 5 minutes. The mixture is stirred for 30 min., Then neutralized to pH 7.0 with In HCl and extracted with ether. The aqueous layer is separated, concentrated to 10 ml and turned on

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a Dowex 50x8 (Na Form) column (1.5 "x 10") 3.8 χ 25.1 cm, the column with H _? 0 is eluted. 113 mg of the desired product are obtained. Lyophilization of the solution gives a solid product.

Method B: 95 ml thienamycin in 100 ml of 0.1M phosphate buffer and 10 ml dioxane are kept at 0 ⁰ C. The pH of the solution is adjusted to and maintained at 8.5 to 9.0 while 240 mg of bromo-t-butyl chloroformate are added. The mixture is stirred for 30 minutes, then with H, POj. acidified to a pH of 2.0. The acidified solution is extracted twice with 2 × 25 ml of ethyl acetate. The organic layer is separated and back-extracted with 10 ml of NaHCO, solution containing 30 mg of NaHCO-. The aqueous layer contains 30 mg of the desired product. It is lyophilized. The solid product is obtained. NMR (60 MHz, D ₂ O): S 1.26 (d), 1.60 (s), 2.65-3.50 (m), 3.70 (s) and 3.90 - ^, 20 (m). Uv) ° 2 ° 303 nm.

Preparation of N-bromo-t-butyloxycarbonylthienamycin-p nitrobenzyl ester

100 mg of the lyophilized N-bromo-t-butyloxycarbonylthienamycin sodium salt are stirred at 25 ° C. with 300 mg of p-nitrobenzyl bromide in 2 ml of hexamethylphosphoramide for 1 hour. The mixture is diluted with 10 ml of ethyl acetate, then washed well with water. The organic layer is separated, dried over Na SOj and eluted on two 250 μl silica gel GF tlc plates using ethyl acetate as solvent (Rf 0.45). 50 mg of the desired product are obtained,

IR (CDClJ: 1777 (β-lactam) and 1711 cm " ¹ (ester (; UVJ ^

j / VjIicLX

279 nm and 322 nm; NMR (CDCl ₅ , 60 MHz): J 1.38 (d), 1.58 (s), 2.60-3.80 (m), 3.78 (s), 3.90-4.20 ( m), 5.30 (s), 7.55 (d) and 8.30 ppm (d).

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Example 23 Manufacture of O-methylthienamycin

T-OCH-

Th-

-NH-

-COOH

Step A : O-methyl-N- (p-nitrobenzyloxycarbonyl) -thienamycin- (p-nitrobenzyl) ester

To a solution of 135 mg of Np-nitrobenzyloxycarbonyl-thienamycin-p-nitrobenzyl ester in 50 ml of methylene chloride at 0 C, 0.5 ml of Ο, ΟΟβΜ fluoboric acid in ether methylene chloride (3: 1) and then immediately 10 ml of a cooled solution of 0.6M diazomethane in methylene chloride. The diazomethane becomes discolored in 1 minute. The solution is extracted with 10 ml 0.1N pH 7 phosphate buffer, dried and evaporated to a small volume. The solution is poured onto two 20.3 × 20.3 silica gel plates which are developed with 3: 1 sthylacetate chloroform. The band at 3-4.5 cm gives 12 mg of the recovered starting material. The band at 6-8 cm gives 20 mg of crystalline 0-methyl-N- (p-nitrobenzyloxycarbonyl) thienamycin (p-nitrobenzyl) ester. Mp. 172-174 ° C, MS m / e 600 (M ⁺ ), 568, 542, 500, 447, 389, 304 and 59-

Stage B : O-methylthienamycin

A solution of 20 mg of O-methyl-N-p-nitrobenzyloxycarbonylthienamycin-p-nitrobenzyl ester in 2 ml of tetrahydrofuran and 1 ml of ethanol is at 3.5 atm (50 psig) 23 ° C in the presence hydrogenated by 20 mg of platinum oxide for 2.5 hours. The catalyst is filtered off and 1 ml 0.1η pH 7 phosphate buffer is added added to the filtrate. The solution is evaporated to 2 ml under reduced pressure. The mixture is in 5 nil water

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15821IA ft. 272 A 560

water and 5 ml of ethyl acetate taken up and centrifuged. The ethyl acetate layer is removed and the aqueous layer is re-extracted with ethyl acetate and ether and then filtered through Celite. The aqueous solution is applied to a column (20 ml) of XAD-2 resin. The pillar is eluted first with water and then with 10 # tetrahydrofuran. The tetrahydrofuran eluate is concentrated and lyophilized. Essentially pure O-methylthienamycin is obtained.

Example 24 Manufacture of thienamycin-O-phosphate ester

Level A :

To a solution of N- (p-nitrocarbobenzyloxy) thienamycin (p-nitrobenzyl) ester (50 mg) in 5 ml of THF are added at 3 ° C. 30 mg of dibenzyl phosphorochloridate and then 14 μl Triethylamine. The mixture is stirred at 25 ° C. for 2 hours. Then the THF is removed in vacuo. The residue will taken up in methylene chloride and washed with water. The methylene chloride solution is dried over magnesium sulfate and evaporated. The residue is chromatographed on silica gel. O-dibenzylphosphoryl-N-benzyloxycarbonylthienamycin benzyl ester is obtained.

Level B :

A solution of 20 mg of the above product in 10 ml of 80% aqueous dioxane containing 8 mg NaHCO, is in Presence of 20 mg 55% Pd on activated carbon catalyst

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Hydrogenated for 6 hours. The catalyst is filtered off and the filtrate is evaporated to 2 ml. The solution is 2 times extracted with methylene chloride, then concentrated and freeze-dried. The product thienamycin-O-phosphate disodium salt remains behind.

Example 25 Production of Q- (methylcarbamoyl) thienamycin

-OCNHCH ₃

Th NH ₂

-COOH

A solution of 20 mg of N- (p-nitrobenzyloxycarbonyl) thienamycin (p-nitrobenzyl) ester and 20 mg of methyl isocyanate in 5 ml of methylene chloride is stirred at 23 C for 18 hours The solvent is evaporated and the residue is extracted with hexane. The residue, which is insoluble in hexane, becomes chromatographed on silica gel. Essentially, one obtains pure 0- (methylcarbamoyl) -N-p -nitrobenzyloxycarbonylthienamycin p -nitrobenzyl ester. Following the hydrogenation procedure of Example * J, Step B, the product is obtained if the specified Substitution is carried out.

Example 26 Production of O- (methoxymethyl) thienamycin

OCH ₂ OCH ₃

COOH

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809851/0039

Level A ;

A solution of 58 mg of p-nitrobenzyloxycarbonylthienamycin p-nitrobenzyl ester in 5 ml of 1.0 ml of THF and HMPA is cooled to -78 ° C. A 2N solution of phenyllithium (0.1 ml) is added to this solution with stirring, followed immediately by 0.2 ml of methylchloromethyl ether. The mixture is allowed to warm to 25 ° C over a period of 1 hour. 25 ml of methylene chloride are added and the solution is extracted with 0.1 η pH 7 phosphate buffer (25 ml) and water 4 × 25 ml. The methylene chloride solution is evaporated and the residue is triturated with hexane. The residue, which is insoluble in hexane, is chromatographed on silica gel. O-methoxymethyl-N- (p-nitrobenzyloxycarbonyl-thienamycin (p-nitrobenzyl) ester) is obtained.

Level B :

According to the hydrogenation procedure of Example ¹ J, Step B, the title compound is obtained when the indicated substitution takes place.

Example 27

Manufacture of O-methylthienamyci n

-OCH ₃

Th NH ₂

-COOH

Stage A : O-methyl-N-benzyloxycarbonylthienamycin benzyl ester

A solution of 5 mg N-Benzyloxycarbonylthienamycinbenzylester (Example 18) in O, J ml of methylene chloride is cooled to 0 ⁰ C and 0 ^ 1 ml of a solution of 0,006M-fluoboric acid is in 5: 1 ether methylene chloride. After that will be

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Immediately 0.5 ml of Ο, ΙΜ diazomethane in methylene chloride was added. The solution becomes discolored in 1 min. The mixture is stirred with ether and pH 7 phosphate buffer and the ether phase is evaporated. The residue is chromatographed on 2 × 5.08 × 20.3 cm 250 μ silica plates in 35% ethyl acetate chloroform. The band at Rf 0.5 is eluted with ethyl acetate. The measurement of the UV absorption at 318 πιμ shows a 13 # recovery of the initial optical density. Mass spectrum analysis shows m / e M ⁺ 510 and fragments at m / e 410, 59, 333 and 478 which are characteristic of the O-methyl-N-benzyloxycarbonylthienamycin benzyl ester.

Other stage A : O-methyl-M-benzyloxycarbonylthienamycin-

benzyl ester

A solution of 5-N Benzyloxycarbonylthienamycinbenzylester mg in 0.2 ml of dry THF is cooled to -78 ⁰ C. To this solution is added 5 ul Phen.yllithiumlösung (2n) and thereafter immediately 1 μΐ Methylenf luor-oulfonat .The mixture is stirred at -78 ⁰ C for 20 min. And then diluted with 2 ml of ether and 2 ml of 0.01N pH 7 phosphate buffer extracted. The ether layer is evaporated and the residue is chromatographed on 5.08 χ 20.32 cm thin-layer silica gel plates with 1: 1 ethyl acetate chloroform. The band at Rf = 0.63 is isolated. The desired product, O-methyl-N-benzyloxycarbonylthienamycinbenzyl ester, is obtained.

Level B : Q-methylthienamycin

A solution of 0.2 mg of the above product in 0.5 ml of dioxane containing 0.3 % acetic acid is hydrogenated for 3 hours in the presence of 2 mg of palladium oxide catalyst at 3.1 atm. (45 psig) and 25 ° C. The mixture is then shaken with 4 ml of ether and then with 4 ml of water. The UV spectrum of the ether layer (X _O 318) shows a 50% recovery and

! Πα. Χ

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15821IA> /} 272 A 56 O

that of the water layer (λ 285) shows 10 % of the initial absorption. The electrophoresis of a 5 U sample of the aqueous layer at pH 7, 50 V / cm for 20 min. And then the bioautography on plates inoculated with S. aureus shows two zones of inhibition: +0.5 cm (19 mm diameter ) and +4 cm (25 mmol). The slow moving stain is O-methylthienamycin. The faster moving spot is O-methyl-N-carbobenzyloxythienamycin. Essentially pure O-methylthienamycin is obtained from the aqueous phase by chromatography on XAD-2 resin using 10% aqueous THF as the eluting solvent. 0

It

4 0-C-CH ₃ NH ₂ COOH Manufacture of O-acetylthienamycin

Step A : O-acetyl-N- (p-nitrobenzyloxycarbonyl) -thienamycin- (ρ-nitrobenzyl) -ester

0.16 ml of acetic anhydride are added to a solution of 50 mg of N - Cρ - Iί Ltrobenzyloxycarbonyl) thienamycin (p-nitrobenzyl) ester in 0.5 ml of pyridine. The mixture can ^{react at 250 °} C. for 3 hours. Then it is evaporated to dryness under vacuum. The solid residue is dissolved in chloroform and chromatographed on 20.3 x 20.3 cm silica gel plates in 3: 1 ethyl acetate chloroform. The band with an Rf value of 0.55 is isolated. 36 mg of 0-acetyl-N- (p-nitrobenzyloxycarbonyl) thienamycin (p-nitrobenzyl) ester are obtained; Mp 172-175 ° C. NMR (CDCl ₃ ) T, 1, Ul, (d J = 6 Hz CH ₃ ); 2.01 (s, COCH ₃ ), 2.7-3.6 (m) i 4.14 (t of d) (J = 3 and 9 Hz); 5.17 (s, carbamyl _CH2); 5.35 (AB quartet, J = 14 Hz), 7, ^ 5 (d), 7.60 (d), 8.18 (d), (J = 9 Hz, aromatic H) Ms. M ⁺ m / e 628.

Grade B : O-acetylthienamycin

A solution of 20 mg of O-acetyl-N- (p-nitrobenzyloxycarbonyl) -

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thienamycin (p-nitrobenzyl) ester in tetrahydrofuranethanol (2.5: 1) is hydrogenated at 2.81 atm (40 psig) at 23 ° C. in the presence of 20 mg of platinum oxide for 1 hour.
The catalyst is filtered off and the piltrate is diluted with 1 ml of 0, In pH 7 phosphate buffer and evaporated to cloudiness. The mixture is taken up in 5 ml of ethyl acetate and 5 ml of water and then the layers are separated. The ethyl acetate layer is extracted with pH 3 phosphate buffer. The pH of the combined aqueous extracts is adjusted to pH 7 and then extracted with ethyl acetate and with ether. The aqueous phase is concentrated and chromatographed on 4 ml of XAD-2 resin. The column is first eluted with 40 ml of water and then the product
eluted with 10/2 tetrahydrofuran. High pressure liquid chromatography on Co Porosil with 105? THF makes one
Peak with a retention time of 8 min. Versus 5 min. For thienamycin. The THF eluate is evaporated to a small volume (UV 297 μm) and freeze-dried. Man max

receives O-acetylthienamycin as a powder.

Example 29 Preparation of thienamycin-O-methanesulfonyl

Th-

-NH ₂ -COOH

Stage A : N- (p-nitrobenzyloxycarbonyl) -thienamycin-0- methanesulfonyl-p-nitrobenz yl ester

Th-

• -OSO ₂ CH ₃ NHCOCH

-CO ₂ CH ₂

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15821IA yi 272 A

34 ml of methanesulfonyl chloride in CHpClp (0.8 ml) are added dropwise over 5 min. To an ice-cold stirred solution of II7 mg of N- (p-nitrobenzyloxycarbonyl) thienamycin-p-nitrobenzyl ester and 40 mg of triethylamine in 6 ml of anhydrous CHpClp. After stirring for a further 1.5 hours, the reaction mixture is _{diluted with 20 ml of cold CH 2} Cl ₂ , washed with water (3 ° C, 10 ml), 0.1M pH 3 phosphate buffer (3 ° C, 5 ml) and 2 Washed with aqueous NaHCO 3 (3 ° C), over MgSO ₁ . dried and filtered. After evaporation of the solvent in vacuo, p-nitrobenzyl-6- (l-methanesulfonyloxyethyl) -3- / 2- (p-nitrobenzyloxycarbonylamino) -äthyIthio7-7-oxo-l-azabicyclo- / 3.2, £ 7-hept remains -2-en-2-carboxylate (which is otherwise referred to as N- (p-nitrobenzyloxycarbonyl) -thienamycin-O-methanesulfonyl-p-nitrobenzyl ester for reasons of convenience) as an oil.

Stage B : Thienamycin-O-methanesulfonyl

Using the hydrogenation procedure of Example 4, stage B, the title compound is obtained if the indicated substitution takes place in the equivalent amounts.

Example 30 O-sulfothienamycin

rO-SO ₃ ~ Na Th4-NH ₂ -COOH

To a solution of 50 g of N- (p-nitrobenzyloxycarbonyl) -thienamycin- (p-nitrobenzyl) ester in 0.5 ml of pyridine there was 20 mg of pyridine-sulfuric anhydride. The mixture can

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15821IA ^ 272A560

React at 25 for 4 hours. Excess pyridine is then removed under reduced pressure. The residue is with 20 ml of methylene chloride and 0.1% sodium bicarbonate solution Stirred for 30 min. Then the aqueous layer is separated and freeze-dried. The firm one The residue, the O-sulfo-N- (p-nitrobenzyloxycarbonyl) -thienamycin- (p-nitrobenzyl) ester Contains, is dissolved in 20 ml of 1: 1 aqueous dioxane and 4 hours in the presence of 5.0 mg of platinum oxide catalyst at 2.8l atm (40 psig.) Hydrogenated. The catalyst is filtered off and the piltrate is extracted twice with ethyl acetate. The aqueous layer is concentrated to 5 ml and chromatographed on 40 ml of XAD-2 resin. The pillar is with Water eluted and the fraction containing the O-sulfothienamycin sodium salt contains, is extracted and freeze-dried.

Example 31 Manufacture of pharmaceutical preparations

A unit dosage form can be prepared by adding 120 mg of thienamycin-O-phosphate ester disodium salt with 20 mg lactose and 5 mg magnesium stearate mixed and the mixture of 145 mg in a No. 3 gelatin capsule fills. Similarly, if one uses more active ingredient and less lactose, other dosage forms can be used in No. Fill in 3 gelatin capsules, and should it be required, more than 145 mg of total ingredients one can make larger capsules, compressed tablets, and pills. The following Examples explain the production of pharmaceutical preparations:

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15821IA
Tablets

Thienamycin-0-phosphate ester disodium salt Corn starch, U.S.P. Dicalcium Phosphate Lactose, U.S.P.

mg 2724560 mg per tablet mg 125 mg 6th 192 190

The active ingredient is mixed with the dicalcium phosphate, lactose and about half of the corn starch. The mixture is then granulated with a 15 # strength corn starch paste (6 mg) and coarsely sieved. It will dried at M5 C and again through a sieve with a clear mesh size of 1.19 mm sieved. The remainder of corn starch and magnesium stearate is added and the mixture is compressed into tablets which are approximately 1.27 cm in diameter and each 8OO mg to weigh.

Parenteral solution ampoule

Thienamycin O-phosphate ester disodium salt

Diluent: Sterile water for injection

500 mg per tablet

2 ecm

Ophthalmic solution

Thienamycin O-phosphate ester disodium salt

Hydroxypropyl methyl cellulose

Sterile water

100 mg

5 mg

up to 1 ml

Otitic solution

Thienamycin O-phosphate ester disodium salt benzalkonium chloride Sterile water

100 mg

0.1 mg up to 1 ml

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Topical ointment τ (§

Thienamycin-O-phosphate- 100 mg

ester disodium salt

Polyethylene Glycol 4000 USP 400 mg
Polyethylene Glycol 400 USP 1.0 g

In the above preparations, the active ingredient can be used alone or together with other biologically active ingredients, for example with other antibacterial agents such as lincomycin, a penicillin, streptomycin, Novobiocin, gentamicin, neomycin, colistin and kanamycin, or with other therapeutic agents such as probenecid administered.

Manufacture of other raw materials

In addition to thienamycin, its various
Isomers alone or as mixtures serve as starting materials in the preparation of the compounds according to the invention. Some of these isomers are obtained in the fermentation of natural products (see the following remarks). However, through an overall synthesis, all isomers are available (see below) as a mixture of four diastereomers which shows antibacterial activity and which can be broken down by processes known per se. The four diastereoisomers (2 cis, 2 trans) can be separated by chromatography. The splitting of any given d / l pair with optically active acids or bases proceeds after an
known procedures. The absolute configuration of the first identified starting material (I) is 5R 6S 8R.

809851/0039

Production of thienamycin by total synthesis

SCH ₂ CH ₂ NH ₂ COOH

Step A : Preparation of ⁱ l- (2-Acetoxyvinyl) -azetidin-2-one

H ₅ C = CH-CH = CHOC-CH, + O = C = N-SO ₀ Cl 0

CH = CHOCCH.

SO ₂ Cl

Il

CH = CHOCCH ₃

-NH

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 under N 2} in a -20 C bath.

A solution of 2.5 g 1-acetoxybutadiene (22 mmoles) in 2.5 ml anhydrous ether is cooled in a similar manner under N ₂ in a -20 ⁰ C bath.

The chlorosulfonyl isocyanate solution is added dropwise to the acetoxybutadiene solution using a Teflon tube that is immersed in the CSI solution and pressurized _{with N 2.} The addition requires 10 minutes. Little or no discoloration is observed. The reaction mixture is stirred at -20 ⁰ C for 0.5 hours, the solution is clear and has a light yellow color.

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809851/0039

A solution of 2 g of sodium sulfite and 5 g of K ₂ HPO in 20 ml of HpO is used during the above 0.5 hour reaction time
made and chilled in an ice bath. 20 ml of ether are added and the mixture is stirred vigorously in an ice bath. Towards the end of the 30 min. Reaction time, the reaction mixture from the reaction flask, which is kept in the -20 C bath, is transferred again into the vigorously stirred hydrolysis mixture using Np pressure and a Teflon tube. The quick dropwise addition is completed in 5 minutes. The hydrolysis can continue for a further 5 minutes. The hydrolysis mixture has a pH of 6 to 8, preferably a pH of 8.

The phases are separated, leaving a yellowish, orange colored gum with the aqueous phase. The ether phase is used directly with MgSOj. dried. The aqueous / rubber phase is extracted 3 times with 50 ml parts of ether, each part being added to the first ether over MgSO ^.

The dried extracts are filtered and concentrated to 5 ml under a stream of Np.
crystalline at this point.

Np stream concentrated to 5 ml. Part of the product is

A column of 10 g of Baker's silica gel packed in ether is made and the ether concentrate is added
given the top of the column and passed through the column. The flask / solids are added 3 times with 2 ml
Rinsed with ether, whereby the ether is removed with a pipette and placed on the column. Elution begins with ether. The first 25 ml are mostly empty volumes. The next five 10 ml fractions are collected, followed by three 50 ml fractions and all fractions are reduced in volume under a stream of Np. The product crystallizes from fractions 4 to 6, with traces in fractions 3 and 7. The fraction

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15821IA

nen 1 to 3 contain a yellowish, pungent smelling material that gums when standing. Yield: 100 mg as Mixture of the cis and trans isomers.

Stage B: Production of 4- (2-acetoxyethyl) -2-azetidinqn

Il

.OCCH-

_NH

A solution of 10.0 g of 4- (2-acetoxyvinyl) -2-azetidinone (0.065 mol) in 200 ml of ethyl acetate, the 100 mg of 10? Pd / C is hydrogenated on a Parr shaker at 25 C under 2.8 l kg / cm ² (40 psi) hydrogen for 15 min. The mixture is filtered through a pad of Supercel and washed with more ethyl acetate. The combined filtrate is evaporated in vacuo. 10 g of 4- (2-acetoxyethyl) -2-azetidinone are obtained as a crystalline solid. Recrystallization from ether gives white crystals: mp 44-7 °; IR (CHCl ₃ ) 5.66, 5.7 ^; NMR (CDCl ₃ ) T'3.44 (broad s, 1, NH), 5.82 (m, 2, CH ₂ OCOCH ₃ ), 6.29 (m, 1, C-4H), 6.87 ( 1/2 AB pattern that further splits 4-fold by C-4H and NH, 1, J = 12.8 Hz, J = 2.3 Hz, J _NH = 1.0 Hz), 7.93 and 8 , 02 (s on m, total 5, OCOCH ₃ and CH ₂ CH ₂ OCOCH ₃ ).

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

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A solution of 2.24 g (0.01 * 1 mol) of 4- (2-acetoxyethyl) -2-azetidinone in 25 ml of anhydrous methanol with a solution of sodium methoxide (77 mg, 1.4 mmol ) treated in 5 ml of anhydrous methanol. After stirring for 1 hour, the solution is neutralized with glacial acetic acid. After removal of the methanol in vacuo, crude 4- (2-hydroxyethyl) -2-azetidinone remains as an oil. The product is purified by chromatography on silica gel, eluting with 10% MeOH / CHCl 2. 1.55 g of the alcohol are obtained. Melting point 50 ^° C .; IR (CHCl ₃ ) 5.67; NMR (CDCl ₃ )? ' 3.20 (wide, s, 1 NH), 6.24 and 6.28 (m on t, total 3, C-4H and CH ₂ OH), 6.90 (wide, s on 1/2 AB pattern, which further splits 4-fold by C-4H and NH, a total of 2, OH and C-3H, J _em = 13.0 Hz, J. = 4.2 Hz, J _NH = 1.6 Hz), 7, 42 (1/2 AB pattern, which is further split 4-fold by C-4H and NH, 1, C-3H, ^J gem ^{= 13} '° ^Hz > ^J vic ^{= 2} > ^{2 Hz} > ^J NH ^{= ljl Hz)} > ⁸ > ^{l6 (m} > ² > ^C M ₂ CH ₂

Stage D : Production of 8-oxo-2,2-dimethyl-3-oxa-l-azabi cyclo - / ?, 2,07-octane

-NH '

A solution of 1.87 g of 4- (2-hydroxyethyl) -2-azetidinone i (0.016 mol) and 1.69 g of 2,2-dimethoxypropane (0.016 mol) in 25 ml of anhydrous methylene chloride is treated with boron trifluoride etherate (0.201 ml, 0.002 mol) at 25.degree. The resulting solution is stirred for 10 minutes. After removal of the solvent under reduced pressure remains an oil (2.5 g) back. Chromatography of the crude product on silica gel using 2: 1 ethyl acetate / benzene as an eluent gives 1.59 g of 8-oxo-2,2-dimethyl-3-oxa-

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15821IA ti 272A560

l-azabicyclo- / ¥, 2,07-octane as a crystalline pesticide. Recrystallization from ether / hexane gives a product of melting point 60-1 °. IR (CHCl ₃ ) U: 5.73 (β-lactam).

NMR (CDCl ₃ ) Y: 6.02-6.28, m, 2H, C-4 methylene 6.22-6.62, m, IH, C-6 methine 6.90, dd, IH, J _{7 7} = 14 Hz, Jg ₇ = 4.5 Hz C-7 proton cis to C-6H 7, i »7, dd, IH, J _{7 7} = 14 Hz, Zr ₇ = 2 Hz

'j' ⁰ Jl

C-7 proton trans to C-6H 7.82-8.68, m, 2H, C-5 methylene 8.23, s, 3H)
8.57, s, 3Hj ^C " ^{2 methyl}

Stage E : Production of 8-oxo-2,2-dimethyl-7oC- and β- (1-hydroxyethyl) -3-oxa-1-azabicyclo ^ i, 2,0_7-octane

To a solution of 1.1 equiv. freshly prepared lithium diisopropylamide in anhydrous tetrahydrofuran under nitrogen atmosphere at -78 ⁰ C a solution of 8-oxo-2,2-dimethyl-3-oxa-l-azabicyclo-A ^z T, 2 () / - octane in anhydrous tetrahydrofuran which was cooled to -78 ⁰ C. After 2 minutes, the lithium enolate formed is treated with excess acetaldehyde. The solution is stirred for 30 min. At -78 ⁰ C 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 and the crude product is obtained. Purification by chromatography on silica gel using

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^15821IA β 27245 & 0

use of ethyl acetate / benzene gives 8-oxo-2, Z = -aimethyi-7iA- and ß7 (l-hydroxyä.thyl) -3-oxa-l-azabicyclo / ^I f, 2, o7-octane. Values for 8-OXO-2,2-dimethyl-7ß- (1-hydroxyethyl) -3-oxa-1-azabicyclo / 4 ~, 2, () 7-octane: IR (CH ₂ Cl ₂ ) U: 5, 72 1 (β-lactam) NMR (CDCl ₃ ) -y: 5.53-6.43, m, UH, CU methylene +

C-6 methine + C-9 methine

6.90, dd on broad s, 2H, J _{7 g} = 9 Hz

J _{6 7} = 5.5 Hz, C-7 methine + OH *

7.70-8.83, m, 2H, C-5 methylene

8.27, s, 3H)

8.60,

3H

C-2 methyl

8.78, d, 3H, Jg _j10 = 6.5 Hz, C-IO methyl

Values for 8-oxo-2,2-dimethyl-7x - (l-hydroxyäthyD-3-oxa-l-azabicycloyi, 2,0 ^ 7-octane: IR (CHCl ₃ ) M: 2.9 broadly 0-H 5 , 73 β-lactam NMR (acetone-Dg) /: U, 23-3.33, m, C-9 methine + CU

Methylene + C-6 methine

3.33, broad s, OH

2.83, dd, J = 2 Hz, 6 HzJ

2.67, dd, J = 2 Hz, 8 HzJ ^C "'

1.93-1.63, m, C-5 methylene

1.63, s)

_Λ , ._ \ C-2 methyl

1,4U, SJ

1.23, d, J06.5 Hz, C-IO methyl

Stage F : Production of 8-oxo-2,2-dimethyl-7c * - (1-p-nitrobenzyl_2carbonyldioxyethyl) -3-oxa-1-azabicyclo - / ?, 2, £ / -octane

OR

-7 methine

5-17Λ0-Θ3

Under anhydrous conditions at O C one treats a Solution of 8-oxo-2,2-dimethyl-7a .- (1-hydroxyethyl) -3-oxa-lazabicyclo-A4 ~, 2,07-octane (60 mg, 0.302 mol) in 0.6 ml of ether with 19 mg of powdered potassium hydroxide (0.332 mmol). After a P-nitrobenzyl chloroformate (65 mg, 0.302 mmol) was added to the reaction mixture. The mixture is stirred at 25 ° C. for a further 15 hours. The mixture is between IM pH Phosphate buffer and further ether shared. The ether phase is washed with water and brine, over magnesium sulfate dried and filtered. Evaporation of the filtrate under reduced pressure gives 67 mg of a colorless oil. the Purification by preparative thin layer chromatography on silica gel and development with 1: 9 ethyl acetate / benzene gives 8-oxo-2,2-dimethyl-7 * - (1-p-nitrobenzylcarbonyldioxyethyl) -3-oxa-1-azabicyclo- / 4 ~, 2,0-7-octane (40 mg) as a mixture of the diastereomers.

IR (0Η ₂ 01 ₂₎ μ: 5.68 (.beta.-lactam and carbonate), 6.19 and 6.54

(Nitro)
NMR (CDCl ₃ ): 1.67, d, 2H, ArH

2.37, d, 2H, ArH

4.67, s, 2H, ArCH ₂

4.67-5.22, m, CH ₃ CH

5.98-6.25, m, 2H, C-4 methylene

6.25-6.62, m, IH, C-6 methine

6.75-7.12, m, IH, C-7 methine

7.75-8.83, m, 2H, C-5 methylene ^:

8.22, s, 3H, C-2 methyl

8.50-8.58, m, 5H, C-2 methyl + CH ₃ CH

The 7β-diastereoisomers or the 7a- and β-mixture is obtained in an analogous manner.

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Stage G : Production of cis and trans-3- (1-p-nitrobenzylcarbonyl-dioxyethyl) -4- (2-hydroxyethyl) -2-azetidi-

non

OR

NH

R = -C-O-CH.

1.0 g of 8-oxo-3-oxa-2,2-diraethyl-7 "- (l-p-nitrobenzylcarbonyldioxyethyl) -l-azabicyclo- / ¥, 2.07-octane are dissolved in 8 ml of acetic acid and 2 ml of water and heated at 65 ° C for 1.25 hours. Acetic acid and water are reduced under reduced pressure removed and the residue is taken up in benzene and evaporated. Trans-3- (1-p-Nitrobenzylcarbonyldioxyäthyl) -4- (2-hydroxyethyl) -2-azetidinone is obtained as a mixture of the diastereoisomers.

IR (CH “C1 ₀ ) m: 5.67 (β-lactam), 5.72 shoulder, 6.20 and 6.57

NMR (CDCl ₃ )

(Nitro)

1.73, d, 2H, J = 8.5 Hz, ArH 2.43, d, 2H, J = 8.5 Hz, ArH 3.63, broad s, IH, NH
4.37-5.13, m, IH, CH ₃ CH 4.72, s, 2H, ArCH ₂
6.97-6.53, m, IH, CH methine 6.23, t, 2H, J = 5.5 Hz, CH ₂ OH 6.73-6.93, m, IH, C-3 methine 7, 63-8.97, m, 3H, CH ₂ CH ₂ OH 8.53, d, J = 6.5 Hz, CH ₃ CH

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

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15821IA f $ 272 A560

Steps D ¹ , E ', P ¹ and G ¹ as an alternative to steps D, E, F and G for the preparation of 3- (lp-Nitrobenzylcar bonyldioxyäthyl) -4- (2-hydroxyethyl) azetidinone

OR

Levels D ', E ¹

Preparation of l- (2-tetrahydropyranyl) -k - / _ 2- (2 -tetrahydropyranyl) ■ oxyäthyl7- ^: 2-azetidinon

A solution is treated under nitrogen and at 25.degree of 62 mg of 4- (2-hydroxyethyl) -2-azetidinone (0.539 mmol) in 0.5 ml of anhydrous p-dioxane with 0.98 ml of 2,3-dihydropyran (1.08 mmol) and 19 mg of p-toluenesulfonic acid monohydrate (0.10 mmol). The resulting solution is during a

Stirred for 60 minutes and then partitioned between 10 ml of 0.5M pH 7 phosphate buffer and 10 ml of ethyl acetate. The watery Phase is extracted a second time with ethyl acetate. The combined ethyl acetate solutions are washed with brine washed, dried over magnesium sulfate and filtered. The piltrate is evaporated under reduced pressure. Man receives 216 mg of crude product. After purification by preparative thin layer chromatography with development with Ethyl acetate gives 80 mg! - ^ - Tetrahydropyranyl) -4-

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809851/0039

_ / 2- (2-tetrahydropyranyl) -oxyethyl / -2-azetidinone as a

NMR (CDCl ₃ > y: 5.13-5.60, m, OCH

5.83-6.85, m, C-iJH + OCH ₂

6.95, dd, J = 5 Hz and 15 Hz

", _ ,, _T , _TT •," γ- τ, C-3 methylene
7.35, dd, J = 3 Hz and 15 Hz ^J

From 7.62 to 8.95, in, CHCH ₂ CH ₂ CH ₂ CH ₂ + CHCH ₂ CH ₂ O

Production of cis-_ and trans-l- (2-tetrahydropyranyl) -3- (1-hydroxyethyl) -4 - / _ 2- (2-tetrahydropyranyl) -oxyethy 17-2-azetidinone

The procedure is as for the preparation of 8-oxo-2,2-dimethyl-7 · 3 (.- and β- (l-hydroxyethyl) -3-oxa-l-azabicyclo- ^, 2, £ 7-octane from δ -Οχο-2,2-dimethyl-3-oxa-l-azabicyclo- / ¥, 2, £ / -octane described and uses 1- (2-tetrahydropyranyl) -4- / 2- (2-tetrahydropyranyl) -oxyethy ] _: 7-2-azetidinone. A mixture of diastereomers is obtained from both the cis and transit tetrahydropyranyl) -3- (1-hydroxyethyl) -4 - / 2 "- (2-tetrahydropyranyl) oxyethyl 7-2 -azetidinone.

Preparation of cis- and trans-1- (2-tetrahydropyranyl) -3- (1-p-nitrobenzylcarbonyldioxyethyl) -k- / 2- ( 2-tetrahydropyranyl ) -oxyethyl7-2-azetidinone

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15821IA

R = COCHo - <^ \ N— NO

The procedure is as for the preparation of 8-oxo-2,2-dimethyl-7Ä- (1-nitrobenzylcarbonyldioxyethyl) -3-oxa-1-azabicyclo- / ¥, 2, 7-octane from 8-oxo-2,2 -dimethyl-7oC- (1-hydroxyethyl) -3-oxa-1-azabicyclo- / Jf, 2, £ 7-octane and uses trans-1- (2-tetrahydropyranyl) -3- (1-hydroxyethyl) -M - / ß (2-tetrahydropyranyli-oxyathy ^ Z - ^ - azetidinone obtained transl- (2-tetrahydropyranyl) -3- (lp nitrobenzylcarbonyldioxyäthyl) -i | -. ^ 2 ~ - (2-tetrahydropyranyl) -oxyäthy3 _L 7-2-azetidinone The cis diastereoisomers are obtained in an analogous manner.

Production of cis- and trans-3 ~ (1-p-nitrobenzylcarbonyldioxyethyl) -fr- (2-hydroxyethyl) -2-azetidinone

- 76 -

809851/0039

A solution of trans-1- (2-tetrahydropyranyl) -3- (1-pnitrobenzylcarbonyldioxyethyl) -4 - ^? - (2-tetrahydropyranyl) -oxyethyl7-2-azetidinone in methanol is at 25 ° C with 0.1 mol equiv. Treated p-toluenesulfonic acid monohydrate. The solution is stirred for 2 hours and then neutralized with IM pH 7 phosphate buffer. The product is extracted into ethyl acetate. The ethyl acetate solution is washed with brine, over Dried magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure. You get trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone. The cis diastereoisomers are obtained in an analogous manner.

Stage H : Production of ice and trans ^ -3- (1-p-nitro-

benzylcarbonyldioxyethyl) -JJ - / ^, 2-bis- (2- hydroxyethyl) -thioethyl7-2-azetidinone

OR

OR

/ ^ CHO

.NH

OH

A mixture of anhydrous pyridine (0.146 ml, 1.81 mmol) and anhydrous, powdered pyridine is added at 25 ° C. under nitrogen Chromium trioxide (92 mg, 0.916 mmol) in 8 ml of anhydrous aceto

- 77 -

809851/0039

nitrile for a period of 30 min. Stirred. To the emerging 250 mg of dry Supercel and then a solution of 186 mg of trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -U- (2-hydroxyethyl) are added to the dark brown solution. -2-azetidinone (0.550 mmol) in 1 ml of anhydrous acetonitrile. After stirring for 1 hour, the reaction mixture is poured out through a mixed, packed bed 2 g each of silica gel and magnesium sulfate passed. The bed is replenished several times with a total of 30 ml of additional acetonitrile washed. The piltrate is concentrated to a volume of 3 ml at 25 ° C. under reduced pressure. By Thin layer chromatography (silica gel; ethyl acetate / benzene 2: 1) this solution contains a product (Rf = 0.38) that is less is polar than the starting material (Rf = 0.21).

The acetonitrile solution of trans-3- (lp-Nitrobenzylcarbonyldioxyäthyl) -4- (2-oxoethyl) -2-azetidinone, prepared as described above, is under nitrogen and at 0 ⁰ C with 0.386 ml of 2-mercaptoethanol (5.5 mmol) and then immediately treated with boron trifluoride etherate (0.176 ml, 1.43 mmol). After stirring for 15 minutes, this solution is partitioned between aqueous potassium 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 evaporated under reduced pressure. 229 mg of oil are obtained. The product is purified by preparative thin layer chromatography on silica gel with evolution with ethyl acetate. 118 mg of trans-3- (lp-nitrobenzylcarbonyldioxyethyl) - ¹ l - ^ ?, 2-bis- (2-hydroxyethyl) -thioethyl7-2-azetidinone are obtained as a colorless oil. IR (0Η ₂ 01 ₂ ) μ: 5.75 (5.79 shoulder) ß-lactam and carbonate 6.20, 6.55 (nitro)

- 78 609851/0039

NMR (acetone d ^: 1.70, d, J = 8.5 Hz, 2H, ArH

2.28, d, J = 8.5 Hz, 2H, ArH 2.48-2.88, m, IH, NH 4.63, s, ArCH ₂ ] 4.63-5.12, m, CH ₃ CHj ^3H

5.80, t, J = 7 Hz, CH ₂ CH <2 1

5.80-7.45, m, C-4H + C-3H + SCH ₂ CH ₂ OHJ ^13H 7.63-8.33, m, 2H, CH ₂ CH. 8.53, d, J = 6.5 Hz, 3H, CH ₃ CH

The cis diastereoisomers are obtained in an analogous manner.

Alternatively, the mixed diastereoisomers are obtained,

when the starting materials consist of a mixture of the diastereoisomers.

Stage I : Production of_trans-3- (lp-nitrobenzylcarbonyl_- dioxyethyl) -4-72,2-bis- (2-azidoethyl) -thioethy: i / -2-azetidinone

0C00PN3 _0H

^ 'OH

+ 2CH ^ SO ₉ Cl

-NH ^J * TH]

OCOOPNB

.NH

To a solution of 211 mg (MW = 474; 0.445 mmol) of trans-3- (lp-nitrobenzylcarbonyldioxyethyl) -4- / 2,2-bis- (2-hydroxyethyl) thioethyl7-2-azetidinone in 5 ml of tetrahydrofuran (THF) (distilled from lithium aluminum hydride) are given at 0 ⁰ C 103 mg of mesyl chloride (MW = 114; 0.904 mmol) in 1 ml of THF and

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809851/0039

then immediately 134 μΐ triethylamine (MW = IOl; J »= 0.729; 0.967 mmol). The reaction mixture is stirred _{under N 2 for 1 hour.} The triethylamine hydrochloride is filtered off under Np. It is also washed with a few ml of THP. The clear, colorless filtrate is concentrated under a stream of Np and then applied to a high vacuum for 10 min. The dimesylate is immediately stored in 5 ml of DMSO (distilled from CaH _? At 8 mm and over 4A Linde molecular sieves) in the presence of 347 mg of NaN, (MW = 65; 5.34 mmol). After stirring overnight under N _? 10 ml of HpO and 20 ml of ethyl acetate (EA) are added. The layers are separated, the aqueous layer is washed 3 times with 10 ml EA. Each organic layer is backwashed with 10 ml HpO and 10 ml brine. The combined EA layers are dried over anhydrous MgSO ^, filtered and concentrated under a stream of Np. The crude diazide is obtained. Preparative thin-layer chromatography on silica gel gives trans-3- (lp-nitrobenzylcarbonyldioxyethyl) -4- / 2,2-bis (2-azidoethyl) thioethyl.7-2-azetidinone. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

Level J: J_ _H "SCH ₂ CH ₂ N ₃ > _CHN ₂ ToI. > EA / Et ₂ O CO ₂ PNB I ^N SCH-CK ₉ N-
N OH ^{Δ J}
\ Χ + H ₂ O
(C0 ₂ PN3) ₂ 4th
Δ C (OK) ₉
N y ^ζ CO ₂ H + CO ₂ PNB C (OH) ₂ + 2NO ₂ - ^ ^ I> ^/
CO ₂ H 0C00PN3
{ , SCH-CH ₉ N, OCOOPNB
ί SCH CH N
/ N / A ^{2 2 3} O ^ O ^

8098 51/0039

- 80 -

A freshly prepared (H. Davies and M. Schwarz, JO C, 30 , 1242 (1965)) solution of p-nitrophenyldiazomethane (29 mmol) in 150 ml of ether is stirred to a solution of 1.0 g of oxomalonic acid, monohydrate ( MW = 136; 7.35 mmol) in 50 ml ethyl acetate (EA) at 0 ⁰ C. After 2.5 hours, concentrate the yellow solution to approximately half volume on a rotary evaporator with mild heating. It is dried over anhydrous sodium sulfate, filtered and concentrated to an oil as described above. 3.54 g of trans-3- (1-p-Nitrobenzylcarbonyldiosyäthyl) -4- / 2,2-bis- (2-azidoethyl) -thioethyl7-2 are added to the crude p-nitrobenzyl ester in 50 ml of toluene (ToI.) -azetidinone (MW = 524; 6.75 mmol). The reaction mixture is heated in an oil bath while stirring. About one third of the toluene is then allowed to distill off. Toluene (dried over 3A I / I6 "Linde molecular sieves) is added again to adjust the volume to 50 ml. The drying process by azeotropic distillation is repeated three more times. The solution is then _{refluxed under N 2 for} 1 hour. The The drying process using azeotropic distillation is repeated one final time and then refluxed for an additional hour Concentration of the resulting solution under a stream of N ₂ gives crude 1. The crude material is chromatographed on silica gel. Diastereoisomers or

A /

the cis-trans mixture is obtained in an analogous manner.

Level K :

OCOOPNB

SCH ₂ CH ₂ N ₃

+ SOCl ₉ Py. ν

^Z THF * ':

₂ r aB) 2

(CO,

809 85 W8QW9

15821IA

OCOOPNB

/ ι ^ XsCH ₂ CH ₂ N ₃

., N Cl

(CO ₂ PNB) ₂

+ 0 ~ P ao. DMF

XJOOPNB

SCH ₂ CH ₂ N ₃

-γ

(CO,

₂ PNB) ₂

To a solution of 2.80 g of I ^ (MW = 912; 3.07 mmol) in 35 ml THF (distilled from lithium aluminum hydride) is added at -20 ⁰ C 0.3 ml pyridine (MW = 79; J> = 0 , 982; 3.73 mmol) (distilled from NaH and stored over 4A Linde molecular sieves). While stirring and under nitrogen, 0.438 g of thionyl chloride (MW = 119; 3.68 mmol) in 1 ml of THF are added dropwise. The reaction mixture is stirred at ⁰ C and finally 1 hour at 25 ° C under N for 5 min. At -20 ⁰ C, then 1/2 hour at O. The pyridine hydrochloride is under N _? filtered off and washed twice with benzene (dried over 3A I / I6 "Linde Mo-

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809851/0039

lecular sieves) washed. The combined filtrates and _{washings are concentrated under a stream of N 2} with a small volume of benzene with anhydrous MgSO ₁ . Slurried, filtered under N ₂ and then _{concentrated under a stream of N 2.} After applying a high vacuum for 1/2 hour, an oil is obtained. 0.885 g of triphenylphosphine (MW = 262; 3.38 mmol) in 66 ml of 9: 1 dimethylformamide (DMF) / H ₂ O and then 550 mg of K ₂ HPOk (MW = 174; 3j16 mmol) are added to this freshly prepared chlorine compound while stirring ). The reaction mixture is stirred at 25 ° C. for 35 minutes. After dilution with EA and brine, the layers are separated and the aqueous layer is extracted 3 times with EA. The combined EA layers are washed with brine, _{dried over anhydrous MgSO 2+} , filtered and concentrated under a stream of _{N 2.} Raw ^ 2, is obtained. The material is chromatographed on silica gel. 2y is obtained. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

Level L:

OCOOPNB

S-CH ₂ CH ₂ N ₃

V ^H

(CO ₂ PNB)

-CH ₂ CH ₂ N ₃ + Br ₂

GCOOPNB

.N

Br
+ I

SCH ₂ CH ₂ N ₃ SCH ₂ CH ₂ N ₃

(CO ₂ PNB),

8098 51/00

- Ö3 -

(D

(2) NaH, DMF

15821IA

OCOOPNB f

-N

(CO ₂ PNB).

_{0.2 ml Br 2} (MW = 160; f = 3.12; 3.9 mmol) are added to 7.8 ml of pentane (dried over ΊΑ Linde molecular sieves). To the solution of 950 mg of 2 (MW = 896; 1.06 mmol) in 15 ml etop (dried over 3A 0.16 cm Linde molecular sieves) is added at 0 ⁰ C under N ₂ and added dropwise to the above 2,3 ral 0. * <9Μ Br ₂ ~ solution (1.13 mmol). After stirring for 10 minutes at 0 ° C., 114 μl of cyclohexene (MW = 82 ,? = 0.81; 1.13 mmol) are added. After 5 min. At 0 ⁰ C 53 mg 57 / ° NaH are (mg 57% of 53 mg = 30.2, MW = 2 * 4, 1.26 mmol) in mineral oil to the stirred reaction mixture. Then immediately add 14 ml of ice-cold DMF (distilled from anhydrous CaSOj. At 40 mm and stored over 4a Linde molecular sieves). The mixture is stirred at 0 ^° C. under N _{2 for a} further 3 hours. The reaction mixture is poured into a stirred, ice-cold mixture of 2.5 ml IM ΚΗ ₂ Ρ0 |, - 40 ml H ₂ O - 75 ml EA. After separating the layers, the aqueous layer is saturated with NaCl and extracted again with EA. The combined organic layers are extracted once with brine, dried over anhydrous MgSO 4, filtered and concentrated under a stream of _{N 2.} Then they are applied to a high vacuum pump. Crude 3 is obtained. Thin-layer chromatography on silica gel gives 3. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

- QH - 809851/0039

15821IA Level M:

OCOOPNB

SCH ₀ CH ₀ N,

^{2 2}

O ₂ PNB).

CCOOPNB

Br

, chch;

.Br

-SCH ₂ CH ₂ N ₃

(CO ₂ PNB) ₂

+ NaH DMF \

OCOOPNB Br

SCH ₂ CH ₂ N ₃

(CO ₂ PNB) ₂

- 85 -

809851/0039

_{0.2 ml Br 0} (MW = 160, 3.9 mmol) are added to 91.6 ml of pentane (dried over 4A Linde molecular sieves). To 474 mg of 3 (MW = 793; 0.598 mmol) in 13 ml Et ₃ O (dried over 3A 0.16 cm Linde molecular sieves) at 0 ⁰ C is added under N ₂ and added dropwise 1.52 ml of the above 0.42M Brp solution (0.63 mmol). . After 15 min at 0 ⁰ C 33 mg 57% NaH are (£ 57 mg of 33 mg = 18.8; MW = 24; 0.78 mmol) was added to *. Then 6.35 ml of ice-cold DMF (distilled from anhydrous CaSOj, at 40 mm and stored over 4a Linde molecular sieves) are added immediately. The reaction mixture is stirred for 1.5 hours at 0 C, then poured into an ice-cold mixture of 1.6 ml IM KHpPO ^ -20 ml HpO-20 ml EA. The layers are separated and the aqueous layer is saturated with NaCl and extracted again with more EA. The combined organic layers are washed once with brine, dried over anhydrous MgSO ^ and filtered. The filtrate is concentrated under a stream of Np and then applied to a high vacuum pump. Crude 4 ^ is obtained. Preparative thin-layer chromatography on silica gel gives crude 4. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

Stages:

OCOOPNB Br

CH ₂ CH ₂ N ₃ + N

809851/0039

- 86 -

15821IA

DMSO

OCOOPNB

.N

272A560

—Sch ₂ ch ₂ n ₃

(C0 ₂ PN3),

To 210 mg 4, (MW = 871; 0.241 mmol), dissolved in 0.5 ml DMSO (distilled from CaHp at 8 mm and stored over 4A Linde molecular sieves), 40 mg are added at 25 ° C. with stirring 1,5-diazobicyclo- / ^), 4, £ 7-undec-5-ene (distilled at 80 C / 2 mm) (MW = 152; 0.263 mmol) in 0.7 ml of dimethyl sulfoxide (DMSO). The solution is stirred under Np for 4 hours, then to a stirred ice-cold mixture of 0.48 ml IM KHpPOj., 7 ml of HpO and 10 ml of EA are given. After separating the layers, the aqueous layer is extracted again with EA. The combined organic layers are washed once with brine, over anhydrous MgSOj. dried, filtered and concentrated under a stream of Np. They are then applied to a high vacuum pump. Crude 5 is obtained. Preparative thin-layer chromatography on silica gel gives 5 »the cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Level 0:

OCOOPNB

SCH ₂ CH ₂ N ₃ + LiI s-collidine

(CO ₂ PNB),

- 87 809851/0059

15821IA

CH ₂ CH ₂ N ₃

CO ₂ PNB

A solution of 187 mg 5 (MW = 791; 0.236 mmol) in 2.5 ml s-

A-I

Collidine (distilled from powdered KOH at 30 mm pressure) is added to 45 mg anhydrous LiI (dried for few hours at 100 ⁰ C over P ₂ O ₁ - under vacuum) (MW = 134; 0.036 mmol). With stirring under N _2, the reaction mixture is heated in an oil bath at 120 ⁰ C. After a total of 25 minutes, the reaction mixture is cooled to 25 ° C., diluted with CHpCl and placed in a round bottom flask and concentrated under a stream of Np and then in a high vacuum. The residue is between 10 ml EA and 1.8 ml IM KHpPO ₂ . divided into 10 ml of water and then the aqueous layer is extracted twice with EA. The combined organic layers are extracted with brine, dried over MgSO 4, filtered and concentrated _{under a stream of N 2.} Of crude 6. The preparative thin layer chromatography on silica gel gave mixed 6. The cis diastereoisomers or the cis-trans-Ge is obtained in an analogous manner.

Level P:

SCH ₂ CH ₂ N ₃ CO ₂ PNB

809851/0039

15821IA

DMSO

100

/ (Mg 34; MW = 6L2; 0.055 mmol) to a solution of the mixed diastereoisomers 6 in 0.2 mo DMSO (distilled from CaH ₃ mm at 8 and stored over 4A Linde molecular sieves) is added with stirring 9.5 Ul 1, 5-DIaZObICyCIo-ZUr, ¹ *, 07-undec-5-en (distilled at e 80 ° C / 2 mm) (MW = 152; = 1; 0.0625 mmol). The solution is stirred under Np for 15 min., Diluted to a total of 1 ml total volume with CHCl and immediately poured onto 2-1000μ silica gel plates. The product band gives 7 as a mixture of the cis and trans diastereoisomers.

Level Q:

SCH ₂ CE ₂ N ₃

H ₂ MO psi

SCH ₂ CH ₂ NH ₂

In the presence of 61 mg PtO-, 61 mg 7 (MW = 612; 0.1 mmol) in 6 ml dioxane, 6 ml THF, 3 ml H ₂ O are hydrogenated at 2.81 kg / cm (40 psi) for 4 hours . After concentrating in vacuo to the cloud point, the aqueous mixture is extracted with ethyl acetate. The water layer is concentrated in a small volume and on a column

- 89 -

809851/0039

15821IA _ηΰ 27 2 A 56 O

applied from 100 g of XAD-2 resin. After eluting with HpO and after removing the first fraction, be the fractions containing the product are lyophilized. 8 is obtained as a mixture of the cis and trans diastereoisomers.

The following procedure for enzymatic N-deacylation of thienamycin applies to all isomers of thienamycin, especially to the specifically defined N-acetyl isomers 89OA and 89OA ,, which are described below.

Deacetylation of N-acetylthienamycin

A liquid (w / v) suspension of fertile turf soil is made prepared by suspending 1 g of the turf soil in 100 ml of sterile phosphate buffer-saline solution, the phosphate buffer-saline solution has the following composition:

Phosphate buffer saline solution

NaCl 8.8 g

IM phosphate buffer, pH 7.5 * 10 ml

Distilled H _? 0 1000 ml

* 1M phosphate buffer, pH 7.5

16 ml of IM KH ₂ PO ₂₁ are mixed with 84 ml of IM K ₂ HPO _{i (} . The pH of the phosphate buffer is adjusted to 7.5 by adding small amounts of either IM KHpPOj or IM.

Aliquots of this liquid base soil suspension are used Used to produce 1Ox, 1OOx and 1OOOx dilutions.

1 ml portions of the base suspension, and 1 ml portions of the 1ox, LooX and lOOOx dilutions are added to 2 ml portions of sterile 1, above Agarlösungen ^ i8 at ⁰ C. The mixtures are quickly drawn over the surface of sterile Petri dishes

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809851/0039

with a diameter of 85 mm and containing 20 ml of medium A, given. Medium A has the following composition composition:

Medium A

KH PO. ^ 0 s.

K ₂ HPO ₁₁ 7.0 g

MgSO ₁₁ 0.1 g

Distilled H ₂ O 1000 ml

N-acetylethanol- 8 5 ml

amine solution * '

♦ N-acetylethanolamine solution N-acetylethanolamine is diluted with 10 times the amount of water and sterilized on a membrane. This solution is added after the treatment in the autoclave.

For solid media : 20 g agar are added.

The Petri dishes are incubated at 28 ° C. for 18 days. A well isolated colony is picked and placed on a petri dish which contains the medium B. The medium B has the following composition:

Medium B

Tomato paste 40 g

Ground wheat flour 15 g

Distilled H ₂ O 1000 ml

pH: adjusted to 6 using NaOH

For solid media : 20 g agar are added.

An individual clone is selected and grown on a medium A slant at 28 ° C for 2 days. Part of the Growth of this slant is recorded on the surface of slants made from medium B.

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809851/0039

stroked. These slants are incubated at 28 ° C. for 2 days. This culture is called Protaminobacter ruber identified and was in the depository of Merck & CO., Inc., Rahway, N.J./USA designated as MB-3528. A sample was obtained from Agriculture Research Service, Deposed U.S. Department of Agriculture. Deposit * number NRRL B-81 ^ 3.

A portion of the growth on the slant of Protaminobacter ruber MB-3528 is used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of medium C was used. The medium C has the following composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Maxsoak- ^

on a moist basis ° ⁶

Distilled H ₂ O 1000 ml

pH: adjusted to 7 with NaOH or HCl

N-acetylethanolamine- 8.5 ml

solution *

♦ N-acetylethanolamine solution

N-acetylethanolamine is diluted with 10 times the amount of water and membrane sterilized. This solution will give after the autoclaving züge->.

The flask is shaken at 28 ° C on a 220 rpm (5.1 cm vibration amplitude) shaker for 4 days. A 25 ml portion of the flask is centrifuged at 8000 rpm for 15 minutes. The supernatant solution is removed and the cells on the surface of the media solids are scraped off in 0.5 ml of 0.05M potassium phosphate buffer pH 1, k. The resulting suspension is using ultrasonic separation

- 91a 809851/0039

15821IA

a Branson instrument model LS-75 sonifier with a 1.27 cm probe at setting 4 for 4.15 sec intervals, the suspension being cooled in ice water during and after the separation. A 10 μl portion of the sonicate is mixed with 25 μl of solution containing 840 μg / ml N-acetylthienamycin in 10 mM potassium phosphate buffer pH 7 and incubated at 28 ° C. overnight. Comparative samples containing only antibiotic and buffer alone and cells treated with ultrasound and buffer without antibiotic are also used. After incubation overnight at 28 ⁰ C 2 μΐ quantities are on cellulose TLC plates coated applied in the EtOHrH O _3, be developed 70:30. After air drying, the TLC plate is placed on a Staphylococcus aureus ATCC 6538P test plate for 5 min.

The test plates are produced as follows: an overnight growth of the test organism Staphylococcus aureus ATCC 6538P in nutrient broth + 0.2 % yeast extract is diluted with nutrient broth + 0.2 % yeast extract to a suspension which shows a transmittance of 60 % at a wavelength of 660 nm. This suspension is added to Difco nutrient agar at 37 ° C. to 48 ° C. supplemented with 2.0 g / l Disco yeast extract. A composition is thus obtained which contains 33.2 ml of suspension per liter of agar. 40 ml of this suspension are poured into 22.5 cm × 22.5 cm Petri dishes and these plates are cooled and stored at 4 ° C. until they are used (maximum 5 days).

The TLC plate is removed and the experimental plate is incubated at 37 ° C. overnight. In addition to the unreacted bioactive N-acetylthienamycin spots at Rf = 0.7-9.89 a bioactive spot is observed at Rf = 0.44-0.47, which is due to the thienamycin Control incubation mixes of antibiotic plus buffer,

- 92 -

809851/0039

1582IiA ^ s 272 A

Cell sonicate plus buffer and antibiotic plus buffer, to which cell sonicates were added before application to the TLC plate, give none at RF = 0.44-0.47 bioactive material.

Preparation of 89OAI, a defined isomer

QH

SCH ₂ CH ₂ NHCCH ₃ COOH

A tube from 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 sterile Davis salts of the following composition:

Davis salts

Sodium citrate 0.5 g

7.0 g

3.0 g

SO ₁₁ 1.0 g

7H ₂ O 0.1 g

Distilled H ₃ O 1000 ml

This suspension is used to inoculate four medium A slants of the following composition:

Medium A

Glycerin 20.0 g

Primary yeast 5.0 g

Fish meal 15.0 g

Distilled H ₂ O 1000 ml

Agar 20.0 g

pH: adjusted to 7 using NaOH

- 93 809851/0039

15821IA * 700

The inoculated slants are incubated for one week at 27-28 ° C and then at 4-6 ° C until used stored.

One third of the growth of these three slants is used for inoculation of 9 250 ml Erlenmeyer flasks, the Deflectors containing 50 ml of medium B of the following composition are used:

Medium B

Yeast autolysate (Ardamin) 10.0 g

Glucose 10.0 g

Phosphate buffer * 2.0 ml

TH ₂ O 50 mg

Distilled H ₃ O 1000 ml

pH: adjusted to 6.5 using HCl or NaOH

Ardamin: Yeast Products Corporation

♦ Phosphate buffer solution

KH ₂ PO ₁₁ 91.0 g

Na ₂ HPO ₁₁ 95.0 g

Distilled H ₃ O 1000 ml

The seed flask is shaken for 1 days at 27-28 ⁰ C on a 220 rpm shaker (5.1 cm amplitude of oscillation). The flask and contents are stored stationary for 1 day at 4 ° C.

33 21-Erlenmeyer production flasks, each with 250 ml of medium

C contain 8 ml per flask of growth

inoculated into the inoculation flask. The medium C has the following composition:

- 94 -

809851/0039

15821IA

Medium C SfQy.

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl ₂ * 6H ₂ O 5.0 mg

Distilled H ₃ O 1000 ml

pH: adjusted to "J, 2-7, H using NaOH

After inoculation, the product flask at 24 ⁰ C un ter movement on a 212 rpm shaker (5.1 cm amplitude of vibration) are incubated for 4 days. The yield is withdrawn from the flasks and their activity is determined using standard Salmonella gallinarum MB-1287 and Vibrio percolans ATCC 8461 experimental plates using 1.27 cm experimental disks immersed in centrifuged broth samples. The samples are diluted with 0.02M phosphate buffer, pH 7.0, if necessary. The results are summarized below:

Withdrawal of the yield after hours 96

pH 7.2

Salmonella gallinarum

(mm zone) 29.5

Vibrio percolans 1/10 dilution

(mm zone) 31

890 experimental units 40

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

7 ml of 0.1M are added to the combined supernatant solutions neutral EDTA and then the entire sample is absorbed on a Dowex-1 χ 2 (Cl ~) column 0.297-0.149 mm (50-100 mesh), The layer dimensions are 5.1 x 25 cm. A flow rate of 40 ml / min is used. The pillar will with 500 ml of deionized water containing 5 ml of IM Tris-HCl buffer

- 95 -

809851/0039

15821IA ίοι 27 2 A 56 O

fer pH 7.0 and 25 μΜ neutral EDTA contains 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. Fractions of 100 ml each are collected, starting with the first occurrence of salt at the column exit. Bioactivity occurs in fractions 1-10 with a peak at fraction 2. Fractions 2-5, which contain 17% of the applied activity, are pooled.

The combined fractions are concentrated to 110 ml by rotary evaporation under reduced pressure. The pH is adjusted to 5.8 by adding 4.2 ml of IM HCl. The adjusted concentrate is placed on a column
made of XAD-2 resin, layer dimensions 3.8 χ 50 cm, applied beforehand with 3 1 6O55 aqueous acetone and then with 3 1 deionized water, 3 1 5% (w / v) sodium chloride and 1 1 25% ( w / v) sodium chloride was washed in deionized water. The concentrate applied can run off down to the level of the layer. The antibiotic is eluted with deionized water at a flow rate of 15 ml / min. 22 fractions of 100 ml each are collected, starting with counting from the first application of the sample. Bioactivity occurs in fractions 6-22 with a peak at fractions 9 and 10. Fractions 9-20 are pooled for further processing. Fractions prepared similarly are combined and all material is concentrated to 70 ml on a rotary evaporator under reduced pressure.

The concentrate is absorbed on a column which is absorbed with a Dowex 1x4 (Cl ") resin that passes through a sieve with a mesh size of 0.037 mm, the layer dimensions being 2.2 × 27 cm ^ The column is
washed with 50 ml deionized water and the antibiotic

- 96 -

809851/0039

15821IA , φ 27 2 A 56 O

tikum is eluted with 2 1 0.07OM NaCl + 0.005M NH ₁₁ Cl + Ο, ΟΟΟΙΜ NH, in deionized water at a flow rate of 2 ml / min. Fractions of 9.5 ml are collected.

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

The combined fractions 146-157 are on a rotary evaporator concentrated to 3 ml under reduced pressure. By adding 5 Hl IM NH, the pH of the concentrate is adjusted set to 6.5. The concentrate is applied to a (2.2 × 70 cm) column of Bio-Gel P-2 (0.074-0.037 mm) which washed with 20 ml of 5M NaCl and 500 ml of deionized water became. After the concentrate has run off to the level of the layer, rinsing liquid of 1 ml each is deionized twice Water is applied and can run off to the level of the layer. The column is then filled with deionized water in a Flow rate of 0.6 ml / min, eluted. Fractions of 2.9 ml are collected.

The main peak of the antibiotic is eluted at fractions 63-70. The factions 64 and 65 are for the further Work-up united.

The combined fractions 64 and 65 are on a rotary evaporator evaporated under reduced pressure to 2 ml and then frozen in a bowl and in a 14 ml ampoule lyophilized with screw cap for 8 hours. 2.25 mg of essentially pure antibiotic 89OAI, the N-acetyl group, are obtained is split off as described above. The free base is obtained:

Item ⁹ O 0 ~ 39

Preparation of 890A3, a defined isomer

* 3

W '» e. nnv

A tube from 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 sterile Davis salts of the following composition:

Davis salts

Sodium citrate 0.5 g

K ₂ HPO ^ 7.0 g

KH ₂ PO ₁₁ 3.0 g

(NH ₄ ) ₂ SO ₄ 1.0 g

MgSO ₄ -7H ₂ O 0.1 g Distilled H ₂ O 1000 ml

This suspension is used to inoculate four slants of Medium A of the following composition

Medium A

Glycerin 20.0 g

Primary yeast 5.0 g

Fish meal 15.0 g

Distilled H ₂ O 1000 ml

Agar 20.0 g

pH: adjusted to 7.2 using NaOH

incubated and then stored at 4-6 C until used

The inoculated slant cultures are incubated for 1 week at 27-28 ° C and then gelagi at 4-6 ° C (no longer than 21 days).

- 98 809851/0039

IN THE

A third of the growth from four slant cultures is used to inoculate 12 250 ml Erlenmeyer flasks with Deflectors, each 50 ml of medium B of the following composition contain, uses:

Medium B

Yeast autolysate (Ardamin) 10.0 g

Glucose 10.0 g phosphate buffer * 2.0 ml

TH ₂ O 50 mg

Distilled H ₂ O 1000 ml

pH: adjusted to 6.5 using Hcl or NaOH

Ardamin: Yeast Products Corporation

♦ Phosphate buffer solution

KH ₂ PO ₁₁ 91.0 g

Na ₂ HPO ₁ , 95.0 g

Distilled H ₃ O 1000 ml

The seed flasks are shaken for 1 day at 27-28 ⁰ C. on a 220 rpm shaker (5.1 cm amplitude of oscillation). The flask and contents are stored stationary for 1 day at 4 ° C.

44 2 1 Erlenmeyer production flasks, each with 200 ml of medium C are inoculated at 8 ml per flask of growth from the inoculation flask. Medium C has the following Composition:

Medium C

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl ₂ -6H ₂ O 5.0 mg

Distilled H ₃ O 1000 ml

809851/003

15821IA

pH: adjusted to 7.2-7.4 using NaOH

After inoculation, the production flasks are placed at 24 ° C with stirring on a 212 rpm shaker (5.1 cm Vibration amplitude) incubated for 4 days and 5 hours. The yield is taken from the flask and the activity is using standard Salmonella gallinarum MB-1287 and Vibro percolans ATCC 8461 test plates. Man uses 1.27 cm experimental disks immersed in centrifuged broth samples. The samples are given where appropriate diluted with 0.02M phosphate buffer, pH 7.0. The results are listed below:

Removal of the yield after 101 hours, pH 7.2

Salmonella gallinarum

(mm zone) 35

Vibrio percolans 1/10 dilution

(mm zone) 34

890 experimental units 121

A total of 7.0 1 of the total broth obtained from this fermentation obtained, are cooled to 3 ° C and centrifuged in 200 ml portions at 900 rpm for 15 minutes each. To the united 1.7 ml of 0.1 H neutral EDTA are added to the supernatant solutions, and the batch is then kept at 3 ° C.

The above fermentation is repeated under identical conditions with the exception that the 44 21 Erlenmeyer production flasks can be inoculated with 7 ml per flask of growth from the inoculation flask. The pH value and the test results are summarized below:

- 100 -

809851/0039

15821IA ^ ₇₃ 27 2 A 56 O

Removal of the yield after 101 hours, pH 7.3

Salmonella gallinarum

(mm zone) 38

Vibrio percolans 1/10 dilution

(mm zone) 39

890 experimental units 92.8

A total of 7.4 liters of the total broth obtained in this fermentation is cooled to 3 ° C. and divided into 200 ml portions centrifuged at 9000 rpm for 15 min. 1.8 ml of 0.1M neutral EDTA are added to the combined supernatant solutions.

The supernatant solutions of the centrifuged broths obtained from the two fermentations above in this example received, are combined to a total of 13 1.

The combined supernatant solutions are passed through a column of Dowex-1 χ 2 (Cl "), 0.297-0.149 mm resin with layer dimensions 4.7 x 50 cm at a flow rate of 60 ml / min. The column is v / ird with 1 1 deionized Water washed and the antibiotic is given 5 1 a 5 $ - igen (w / v) NaCl solution, the 0.01M tris-HCl buffer, pH 7.0, and contains 25 μΜ neutral EDTA, eluted. Fractions of 220 ml are collected at a flow rate of 50 ml / min, and the fractions are transferred to Salmonella gallinarum MB-I287 plates checked.

The antibiotic activities appear in fractions 3-26 with a peak in fractions 5 and 6. Fractions 5-9 are pooled for further treatment. The pH of these combined fractions is 7.8 and the combined fractions contain 24 % of the initial bioactivity, determined on Salmonella gallinarum MB-I287 plates.

- 101 -

809851/0039

15821IA ^^ 27 2 h 56 Π

The combined fractions 5-9 are to 150 mL on a Rotary evaporator concentrated at reduced pressure. They are placed on a column (4.9 x 47 cm Layer dimensions) from XAD-2 applied, previously with 5 1 60 / Sigem (v / v) aqueous acetone and then with 5 1 deionized water 50 g / l NaCl in deionized Water was washed. The sample is applied in 20 ml portions, each time running the column until can run down to the layer level. After completion of the application, 3 20 ml parts of deionized water applied and allowed to run down to the level of the layer. The sample is mixed with deionized water in a Flow rate of 20 ml / hin, eluted. All processes on the XAD column are carried out at room temperature (24 ° C). The eluted fractions are immediately after they were removed, quickly cooled in an ice bath. The fractions 95-230 ml are collected.

The antibiotic activity occurs in fractions 2-21, determined by tests on Salmonella gaLlinarum MB-1287 Plates, with a peak at fractions 5-7 (this corresponds to 510-895 ml of the eluted volume if one counts from the first time you use deionized water). Fractions 6-21 are pooled.

The combined fractions 6-21 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 applied to a column (2.2 cm χ 21 cm layer dimensions) made of Dowex-1 χ 4 (Cl) resin, which is passed through a sieve with a mesh size of 0.037 mm passes through (-400 mesh). The column is washed with 20 ml of deionized water and then the Antibiotics with 2 1 0.07M NaCl + 0.005M NH ^ Cl + 0.000IM NtU in deionized water at a flow rate of 1.6 ml /

- 102 -

809851/0039

272 * 560

Min. Eluted. Fractions of 8 ml each were collected. The fractions 157-179 have the greatest activity and are united.

These pooled fractions are on a rotary evaporator concentrated to 7 ml under reduced pressure. The pH value is increased to 7.5 by adding 20 μΐ IM NaOH set. The solution is further concentrated to 5 ml and applied to a column (2.2 x 75 cm) Bio-Ge] P-2, 0.07 ^ -0.037 mm applied. The sample is washed into the column layer with two rinses of 1 ml each of deionized water and eluted with deionized water at a flow rate of 0.6 ml / min. There are 10 fractions of 3.3 ml, then 60 fractions of 2.65 ml and then 10 fractions of 2.0 ml collected.

The pH is adjusted by adding 1.5-2.5 µl of 0.1M NaOH of the fractions were adjusted to a value within a range of .7j5 ~ 8.0. The parliamentary groups 62, 63, 6 * J and 65 are frozen individually in l'l rnl glass ampoules and lyophilized and stored at -20 C in a vacuum.

For the isolation of the antibiotic 89OA3, that of 89OAI is free, the relatively higher resistance of the antibiotic 89OA3 to decomposition is used Penicillinase like this:

The Bio-Gel fraction 63 is combined with the fractions 61, 66 and 67 from the Bio-Gel column. 0.2 ml of IM tris-HCl buffer, pH 7.5, and 0.2 ml of penicillinase (Difco "Bacto-Penase") are added to these four combined fractions. After 113 minutes at 23 ^° C., a further 0.2 ml of penicillinase is added. After a further 7 hours at room temperature, a further 0.2 ml of penicillinase are added. After a further 2 hours at room temperature

- 103 -

809851/0039

the reaction mixture is cooled in an ice bath. That The quenched reaction mixture is diluted to 15 ml by adding 5 nil of deionized water.

- 1 - The reaction mixture is absorbed on a Dowex χ 4 (Cl) resin column, whereby the resin passes through a sieve with a mesh size of 0.037 mm, layer dimensions 2.15 x 40 cm. The antibiotic 89OA3 is treated with 0.07M NaCl + 0.005M NH ₂₊ Cl + Ο, ΟΟΟΙΜ NH, in deionized water at a flow rate of 3 ml / min. eluted. Fractions of 13.8 ml each are collected. Fractions 187-200 are merged.

The combined fractions are concentrated to 4 ml on a rotary evaporator under reduced pressure The concentrate is absorbed onto a 2.2 × 70 cm column of Bio-Gel P-2, 0.07 ^ -0.037 now (200-400 mesh). The antibiotic 89OA3 is eluted with deionized water at a flow rate of 0.6 ml / min. There will be 30 parliamentary groups of 3.3 ml and then 50 fractions of 2.65 ml collected.

Fractions 66-70 are pooled and the pooled samples are made up to 1.5 ml on a rotary evaporator concentrated under reduced pressure. The concentrate is frozen and lyophilized. 5.4 mg of solids are obtained, which contain the antibiotic 89OA plus residual salts. The N-acetyl group is split off as described above, where the free base is obtained:

CH ₂ CH ₂ NH ₂ COOH

- End of description -

- 104 -

809851/0039

Claims (3)

Merck & Co ,, Inc, l5ök! LIA 27 2 A 560 Claims Λ. Connection of the structural formula: SCH ₂ CH ₂ NH ₂ ¹ COOH and their non-toxic, pharmaceutically acceptable salts; Ii wherein R is independently acyl, alkyl, aryl, aralkyl, alkenyl and Means alkynyl. 2. A compound according to claim 1, wherein the group R denotes wherein X denotes 0 or S ; η is an integer selected from 0 or 1 ; RH, amino, alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl or heteroaralkyl means. 4 3. A compound according to claim 1, wherein the group R "^ ^C 4- ^(CH 2 ⁾ n ^ZR ! means, in which X means 0 or S, R H, amino, mercapto, Hydroxy, alkylamino, dialkylamino, alkyl, alkylthio, aryl - 1 - 809851/0039 OTRIGlNAL INSPECTED thio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, aralkyl, Is acylcycloalkyl, heteroaryl or heteroaralkyl; η is an integer selected from 0, 1, 2, 3 or 4, i m means an integer of 0 or 1 j means Z O, Is S, N or carbonyl; except when Z is oxygen, R is not mercapto or hydroxy, when Z is sulfur, R is not amino or hydroxy, and when Z is nitrogen, R is not mercapto means. k. A compound of Claim 1 wherein the group R denotes wherein X denotes 0 or S; η is an integer selected from 0 or I ₁ ; R is H, amino, mercapto, hydroxy, alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl or heteroaralkyl; R 'is amino, hydroxy, azido, carbamoyl, guanidine, acyloxy, halo, sulfamino, tetrazolyl, sulfo, carboxy, carbalkoxy, phosphono, alkoxy or allylthio; with the exception that both of the substituents R and R 'cannot be hydroxy, amino or mercapto. 4 5 · The compound of claim 1, wherein the group R T «'Η (O) η or Il denotes wherein X denotes 0 or S; R H, amino, mercapto, 809851/0039 Hydroxy, alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aralkoxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl or heteroaralkyl denotes jm and η denote integers and are independently selected from 0 or 1; Y (ΓΉ, -NRp or R denotes where M is selected from hydrogen, alkali and alkaline earth metal cations or an organic base; Y ¹ and Y "are independently selected from the group (ΓΈ, -NRp or Rj Y ^t and Y" together with the phosphorus atoms to which they are attached, can form cyclic esters and amides. 6. A compound according to claim 1, wherein the group R -M- denotes in which m and η denote integers selected from 0 to 5, ^ ρ denotes 0 or 1; A 0, NR ¹ (R 'denotes hydrogen or lower alkyl having 1 to 6 carbon atoms), S or A denotes a single bond ; and Y is selected from the group: 1. Amino or substituted amino
-N (R) ₂ and -N (R) ₃ wherein R is independently selected from hydrogen; N (R ') ~ (R ¹ is hydrogen or lower alkyl having 1 to 6 carbon atoms); Lower alkyl and lower alkoxyl of 1 to carbon atoms; Lower alkoxy-lower alkyl, in which the alkoxy moiety contains 1 to 6 carbon atoms and the alkyl moiety contains 2 to 6 carbon atoms; Cycloalkyl and cycloalkylalkyl, in which the cycloalkyl moiety has 3 to carbon atoms and the alkyl moiety has 1 to 3 carbon atoms 809851/0039 contains atoms; and wherein two groups R together with the nitrogen atom to which they are attached form a ring Can form 3 to 6 atoms. 2. Amidino and substituted amidino -N = CN (R)
j
R. wherein the groups R are independently selected from: hydrogen; N (R ¹ ) ₂ (R ¹ is hydrogen or lower alkyl of 1 to 6 carbon atoms); Lower alkyl and lower alkoxyl of 1 to 6 carbon atoms; Lower alkoxy lower alkyl wherein the alkoxy moiety contains 1 to 6 carbon atoms and the alkyl moiety contains 2 to 6 carbon atoms (where, when the lower alkoxy lower alkyl group is attached to the carbon the alkyl moiety contains 1 to 6 carbon atoms); Cycloalkyl and cycloalkylalkyl, in which the alkyl moiety Contains 1 to 3 carbon atoms; and where two groups R together with the atoms to which they are attached can form a ring with 3 to 6 atoms. 3. Guanidino and substituted guanidino -NH-CN (R) ₂
NO where R has the definition given in 2. above. i |. Guanyl and substituted guanyl -C = NR
NO), where R has the definition given under 2. above 809851/0039 5. Nitrogen-containing, mono- and bicyclic heterocycles (aromatic and non-aromatic) with 4 to 10
Ring atoms in which the hetero atom or atoms in addition to nitrogen are selected from oxygen and sulfur. 7. A compound according to claim 2, wherein the group R means where R is benzyl, p-hydroxybenzyl, 4-amino-4-carboxybutyl, methyl, cyanomethyl, 2-pentenyl, n-amyl, n-heptyl, ethyl, 3- and 4-nitrobenzyl, phenethyl, ß, ß-diphenylethyl ,
Methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, 3,5-dimethyl-4-isoxazolyl, 3-butyl-5-methyl-4-isoxazolyl, 5-methyl-3 ~ phenyl-4-isoxazolyl, 3- (2-chlorophenyl) -5-methyl-4-isoxazolyl, 3- (2,6 dichlorophenyl) -5-methyl-4-isoxazolyl, D-4-amino-4- carboxybutyl, D - ^ - N-Benzoylamino-il-carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, (3-pyridyl) -methyl, 2-ethoxy-l-naphthyl, 3-carboxy-2-quinoxalinyl, 3- (2,6-dichlorophenyl) -5- (2-furyl) -4-isoxazolyl,
3-phenyl-4-isoxazolyl, 5-methyl-3- (4-guanidinophenyl) -4-isoxazolyl, 4-guanidinomethylphenyl, 4-guanidinomethylbenzyl, 4-guanidinobenzyl, 4-guanidinophenyl, 2,6-dimethoxy-4-guanidinophenyl, o-sulfobenzyl, p-Carboxymethylbenzyl _s p-Carbamoylmethylbenzyl, m-Pluorobenzyl, m-bromobenzyl, p-chlorobenzyl, p-methoxybenzyl, 1-naphthylmethyl, 3-Isothiazolylmethyl, 4-Isothiazolylmethyl, 5-Isothiazolylmethyl, Guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4-methoxy-5-isoxazolylmethyl, 4-methyl-5-isoxazolylmethyl, 1-imidazolylmethyl, 2-benzofuranylmethyl, 2-indolylmethyl, 2-phenylvinyl, 2-phenylethinyl, 1-aminocyclohexyl, 2-aminomethyl and 3-thienyl , 2- (5 ~ nitrofuranyl) -vinyl, phenyl- o-methoxyphenyl, o-chlorophenyl, o-phenylphenyl, p-aminomethylbenzyl, 1- 809851/0039 (5-cyanotriazolyl) methyl, difluoromethyl, dichloromethyl, Dibromomethyl, 1- (3-methylimidazolyl) 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- (1,2,5-thiadiazolyJ-methyl, 3- (4-methoxy-1,2,5-thiadiazolyl) methyl, 2-purylmethyl, 2- (5-nitrofuryl) methyl, 3-Fury! Methyl, 2-thienylmethyl, 3-thienylmethyl, tetrazolylmethyl, benzamidinomethyl and means cyclohexylamidinomethyl. 8. A compound according to claim 3> wherein the group R means, in which the molecular part - (CHp) ZR of the acyl group is selected from the group which contains: allylthiomethyl, phenylthiomethyl, butyl mercaptomethyl, -chlorocrotyl mercaptomethyl, phenoxymethyl, phenoxyethyl, phenoxybutyl, phenoxybenzyl, diphenoxymethyl, dimethylmethoxymethyl, dimopethyl methoxymethyl, diphenoxymethyl, dimopethyl methoxymethyl, diphenoxymethyl, dimopethyl methoxymethyl, diphenoxymethyl, dimopethyl methoxymethyl, diphenoxymethyl, dimopethyl methoxymethyl, 4-phenoxymethyl, phenylthiomethyl, phenylthiomethyl , 4-pyridylthiomethyl, p- (carboxymethyl) -phenoxymethyl, p- (carboxymethyl) -phenylthiomethyl, 2-thiazolylthiomethyl, p- (sulfo) -phenoxymethyl, p- (carboxymethyl) -phenylthiomethyl, 2-pyrimidinylthiomethyl, phenethylthiomethyl 5,6,7,8-tetrahydronaphthyl) oxymethyl, N-methyl-4-pyridiniumthio, benzyloxy,
Methoxy, fithoxy, phenoxy, phenylthio, amino, methylamino, dimethylaminOj pyridiniummethyl, trimethylammoniummethyl, cyanomethylthiomethyl, trifluoromethylthiomethyl, 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 1-pyridylbutyl, 1-imidazolyl-3-imidazolyl, 1-pyridazolyl, 3-imidazolyl-3-pyridazolazol, 1-pyridinium-3-imidazolazol, 1-pyridinium-3-imidazolyl-3-imidazolyl-3-pyridiniummethyl, trimethylammoniummethyl -Pyrrolopropyl and 1-pyrrolobutyl. 809851/0039 ^15821IA > 27 ^ 4 560 9. A compound according to claim H, wherein the group R CHRR ¹ means in which the moiety -CHRR 'of the acyl group is selected from the group containing:! X-aminobenzyl,-amino- (2-thienyl) -methyl, χ- (methylamino) -benzyl, ίχ-aminomethylmercaptopropyl, & - Amino-3- or 4-chlorobenzyl, A-amino-3- or Jj-hydroxybenzyl, • 5 <-amino-2, ¹ <-dichlorobenzyl, Ot-amino-3, ^ - dichlorobenzyl-DC -) - ivhydroxybenzyl, cx -Carboxybenzyl, o (-amino- (3-thienyl) -methyl, D - (-) - fc-amino-3-chloro- ^ l-hydroxybenzyl, <X -amino-cyclohexylJ-methyl, i \ - (5- Tetrazolyl) benzyl, 2-thienylcarboxymethyl, 3-thienylcarboxymethyl, 2-furylcarboxymethyl, 3-furylcarboxymethyl,. ^ - sulfaminobenzyl, 3-thienylsulfaminomethyl, iX- (N-methylsulfamino) benzyl, D - (-) - 2-thienyl, D - (-) - 0 (-Guanidinobenzyl, 0 <-Guanylureidobenzyl, <* ■ -Hydroxybenzyl, o <-Azidobenzyl, o.-Fluorobenzyl, 4- (5-Methoxy-1,3-oxadiazolyl) -aminomethyl, ^ - (5-methoxy-1,3-oxadiazolyl) -hydroxymethyl, 4- (5-methoxy-1,3-sulfadiazolyl) -hydroxymethyl, 4- (5-chlorothienyl) aminomethyl, 2- (5-chlorothienyl) -hydroxymethyl, 2- (5 ~ ChlorthienyD-carboxymethyl, 3- (1,2-thiazolyl) -aminomethyl, 3- (1,2-thiazolyl) -hydroxymethyl, 3- (1,2-thiazolyl) -carboxymethyl, 2-thiazolylaminomethyl, 2-thiazolylhydroxymethyl, 2 -Thiazolylcarboxymethyl, 2-benzothienylcarboxymethyl, 2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl, o \ - sulfobenzyl, cx-phosphonobenzyl, ov-diethylphosphono and (X, -monoethylphosphono. 10. A compound according to claim 6, wherein the group R is selected from the group comprising: -T- 809851/0039 15821IA 272A560 O NH O NH O N I » ₂ CH ₂ NHC-CH ₃ - -CCH ₂ CH ₂ NHC-CH ₃ , O NH ff © -L ₂ CH ₂ NHlI-H, -CCH ₂ CH ₂ CH ₂ N (CH ₃ ) O NH I '* -CCH ^ CH-CH-N (CK,) -, -CCH ₂ CH ₂ NHC-NH ₂ , 2 2 2 3 2 Q NH 9 Π S / I ₂ CH ₂ N (CH ₃ ) ₂ , ^ ₂ JC _n .η ₂ . NH N O CH ₂ CH ₂ C-NH ₂ , η 4 - · CCK ₉ -O-CH-C ^{2 2} 11. A compound according to claim 1, wherein the group R is selected from the group containing: pormyl, propionyl, Butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, Methoxycarbonylj ethoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, Phenylthiocarbonyl, 3-aminopropionyl, 4-aminobutyryl, N-methylaminoacetyl, Ν, Ν-dimethylaminoacetyl, Ν, Ν, Ν-trimethylaminoacetylj 3- (N, N-dimethyl) -aminopropionyl, 3 ~ (N, N, N-trimethyl) -aminopropionyl, N, N, N-triethylaminoacetyl, Pyridinium acetyl, guanylthioacetyl, guanidinoacetyl, 3-guanidinopropionyl, N -methylguanidinopropionyl, Hydroxyacetyl, 3 "hydroxypropionyl, acryloyl, propinoyl, malonyl, phenoxycarbonyl, amidinoacetyl, Acetamidinoacetyl, 809851/0039 Amidinopropionyl, Acetamidinopropionyl, Guanylureidoacetyl, Guanylcarbamoyl, Carboxymethylaminoacetyl, SuIfoacetylaminoacetyl, Phosphonoacetylaminoacetyl, N ^ -Dimethy1 · aminoacetamidinopropionyl, ureidocarbonyl, Dimethylaminoguanylthioacetyl, 3- ^(l-methyl-i J-pyridinium) propionyl, 3- (5-aminoimidazol-l-yl ) -propionyl, 3-methyl-l-imidazolium acetyl, 3 ~ sydnonylacetyl, o-aminomethylbenzoyl, o-aminobenzoyl, | 1 II OCH ₃ \\ -P (OCH ₃ ) ₂ , -P (OCH ₃ ) ₂ ,? ^ P η S S -PN (CH ₃ J ₂ , -P IN (CH ₃ J ₂ I ₂ , -PN (CH ₃ J ₂ * \) Na ONa 2 | 12. A compound according to claim 1, wherein R is selected from the group which contains: SuIfο, phosphono, carbamoyl, Methylsulfonyl, sulfamoyl, dimethylsulfamoyl, N-methylcarbamoyl, Bromoacetyl, hydroxyacetyl, aminoacetyl, dimethylaminoacetyl, trimethylammoniumacetyl, amidinoacetyl, guanidinoacetyl, Methoxyacetyl, guanylacetyl, guanylthioacetyl, Phosphamoyl, phosphonothioyl, thiocarbamoyl, methoxymethyl, Hydroxyethyl, methoxyethyl, dimethylaminomethyl, dimethylaminoethyl, Methylthiomethyl, amidinomethyl and guanidinoethyl. 13. A compound according to claim 1 of the structural formula: 809851/0039 15821IA where vT is hydrogen or an alkali metal cation and its non-toxic, pharmaceutically acceptable salts, 14. A method for producing a compound according to claim 1, characterized in that a compound the structure: COOR * wherein R 'and R are easily removable protecting groups, with an alkylating or acylating reagent comprising the group H
R results, treated. 15. Pharmaceutical preparation, characterized in that that it contains a compound according to claim 1 and a pharmaceutical carrier therefor. 16. Pharmaceutical preparation, characterized in that that they are in unit dosage form a therapeutically effective amount of a compound of claim 1 and a pharmaceutically contains acceptable carrier therefor. -10- 809851/0039