CH628051A5 - Process for the preparation of N-alkylated derivatives of thienamycin - Google Patents

Description

The invention relates to a process for producing new N-alkylated thienamycin derivatives, namely N-alkyl, N, N-dialkyl and N, N, N-trialkyl derivatives of the new antibiotic thienamycin. Such compounds and their pharmaceutically acceptable salt, ester and amide derivatives are effective as antibiotics.

Thienamycin (I) is described and claimed in U.S. Patent 3,950,357. This patent is hereby incorporated into the disclosure because thienamycin can be used as a starting compound in the preparation of the new compounds:

Nos. 634 006, 634 298 and 634 294, all of which were filed on November 21, 1975 and which are described and claimed in the corresponding continuation-in-part applications. These applications are hereby incorporated into the disclosure because they describe useful starting compounds and also methods for converting the N-alkylated thienamycins which can be prepared according to the invention into the carboxyl-, O-, and carboxyl- and O-derived forms, which are also known as antibiotics are usable.

The N-alkylated thienamycin derivatives which can be prepared according to the invention have the following formula: sch2ch2nh2

cooh

OR

A

N.

sch2ch2nr5r6r7

coxr

(I)

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

Other preferred starting materials for the preparation of the new compounds have the following formulas or are more simply designated by the symbol - OR4

Th

CH2CH2NH2

cooh

Ia ch2ch2nh2

coxr

Ib sch2ch2nh2 coxr

Ic

-NR5R6R7

ii l-COOM

30 where «th» symbolizes the bicyclic core of thienamycin.

The invention therefore relates to a process for the preparation of new N-alkylated thienamycin derivatives of the formula

OR

A

40

e

N.

sch2ch2nr5r6r7 coxr

(I)

wherein R3, X and R have the meaning given below. Starting compounds Ia, Ib and Ic, which are also suitable as antibiotics, are described in the US patent applications Serial and their pharmacologically acceptable salts, where R5, R6 and R7 are each independently a hydrogen atom, 45 not all of the radicals R5, R6 and R7 at the same time hydrogen atoms, a respectively substituted or unsubstituted lower alkyl radical with 1 to 6 carbon atoms, alkenyl radical with 2 to 10 carbon atoms or alkynyl radical with 2 to 10 carbon atoms, a ring-substituted or unsubstituted-50 ied cycloalkyl, cycloalkenyl, cycloalkenylalkyl or Cycloalkylakyl radical with 3 to 6 ring carbon atoms and 1 to 6 carbon atoms in the alkyl chain, an aryl radical with 6 to 10 carbon atoms, an aralkyl radical with 6 to 10 ring carbon atoms and 1 to 6 carbon atoms in the alkyl chain 55 or a mono- or bicyclic heteroaryl or heteroaralkyl radical 4 to 10 ring atoms, of which one or more oxygen, S represent tickstoff and / or sulfur, and 1 to 6 carbon atoms in the alkyl chain, the ring or chain substituents each having at least one chlorine, bromine, 60 iodine or fluorine atom, an azido, cyano or amino group, a mono-, di- or trialkyl-substituted amino group, where the alkyl group has 1 to 6 carbon atoms, a hydroxyl group, an alkoxy group with 1 to 6 carbon atoms, a thioalkyl group with 1 to 6 carbon atoms, a carboxyl-65 or oxo group, an alkoxycarbonyl group with 1 to 6 carbon atoms in the alkoxy part, one Acyloxy radical having 2 to 10 carbon atoms, a carbamoyl group, a mono- or dialkylcarbamoyl group, the alkyl radicals in each case 1 to 4

628 051

4th

Have carbon atoms which represent thiocyanine group —SCN or the nitro group,

R4 is a hydrogen atom or an acyl, alkyl, aryl, aralkyl, alkenyl or alkynyl radical,

X represents an oxygen or sulfur atom or a radical of the formula NR ', in which R' = H or R,

R is a hydrogen atom, an alkyl radical with 1 to 10 carbon atoms, a phenacyl group or a nucleus-substituted phenacyl group, the substituent being a chlorine, bromine or fluorine atom or an alkyl radical with 1 to 6 carbon atoms,

an alkoxyalkyl radical, the alkoxy part being open-chain or cyclic and having 1 to 6 carbon atoms and the alkyl part likewise having 1 to 6 carbon atoms, an alkanoyloxyalkyl radical having 2 to 12 carbon atoms, a halogen or perhalogenalkyl radical, where halogen is chlorine,

Is bromine or fluorine and the alkyl chain has 1 to 6 carbon atoms, an alkenyl radical with 2 to 10 carbon atoms, an alkoxycarbonyloxyalkyl radical with 3 to 14 carbon atoms, a dialkylaminoacetoxyalkyl radical with 4 to 21 carbon atoms, an alkanoyloamide alkyl radical with 2 to 13 carbon atoms, an aralkyl radical, wherein the alkyl portion has 1 to 3 carbon atoms and the aryl portion has 6 to 10 carbon atoms, a mono- or bicyclic heteroaralkyl radical having 4 to 10 ring atoms and 1 to 6 carbon atoms in the alkyl portion, the hetero atom being an oxygen, sulfur or nitrogen atom, a nucleus-substituted aralkyl or heteroaralkyl radical, where the substituent is chlorine, fluorine, bromine or iodine or a lower alkyl radical with 1 to 6 carbon atoms, a lower alkanoyloxy radical with 1 to 6 carbon atoms, a lower alkoxy radical with 1 to 6 carbon atoms, a mono- or bicyclic heterocyclylal-kylrest, the heterocycle 4 bi s has 10 atoms and the heteroatom is an oxygen, sulfur or nitrogen atom and the alkyl portion contains 1 to 6 carbon atoms, an aryl or nucleus-substituted aryl radical with 6 to 10 ring carbon atoms, the core substituent being a hydroxyl group, a lower alkyl radical with 1 to 6 carbon atoms, a chlorine, fluorine or bromine atom or an alkylthioalkyl radical having 2 to 12 carbon atoms, a cycloalkylthioalkyl radical having 4 to 12 carbon atoms or an acylthioalkyl radical, the acyl content being 2 to 10 carbon atoms and the alkyl content Have 1 to 6 carbon atoms.

The process according to the invention for the preparation of the new compounds is characterized in that a compound of the formula sch2ch2nh2

coxr

<f

or an N-alkyl derivative of such a compound is reacted with an N-alkylating agent suitable for introducing the substituents R5, R6 and R7 and, if appropriate, the compounds obtained are converted into the corresponding pharmacologically acceptable salts.

Preferred connections that can be produced according to the invention also include embodiments of the following structure:

I — OR3

Th

- NR5Rf'R7 .COXR

ii wherein the basic symbols have the meaning given above and X are oxygen, sulfur or NR '(R1 = H or R) and R can be selected, for example, from the group consisting of hydrogen, 5 customary blocking groups, such as trialkylsilyl, acyl and the pharmaceutically acceptable salt, ester and amide groups, as are known for bicyclic β-lactams in antibiotics. The definition of R is given in great detail below.

10 R3 means: 1.) acyl (in general the group OR3 can be referred to as an ester group); or 2.) R3 is selected from alkyl, aryl, alkenyl, aralkyl and the like (so that the OR3 group can generally be referred to as ether). R3 can also mean hydrogen. The term 15 "acyl" is intended to include, by definition, the alkanoyles including derivatives and analogues thereof, such as the thio analogs in which the carbonyl oxygen is replaced by sulfur, and also sulfur and phosphoracyl analogs, such as substituted sulfonyl, sulfinyl and sulfenyl radicals, and substituted P (III and 20 V) residues, such as substituted phosphorous, phosphorus, hypophosphorous and phosphinous residues. Such acyl residues are further defined, as are the residues (2nd, top), which represent the preferred ether embodiment.

There is a constant need for new antibiotics. Unfortunately, there is no static effectiveness for a given antibiotic because continued use on a broad scale of such an antibiotic selectively increases the formation of resistant strains and pathogens.

In addition, the known antibiotics have the disadvantage that they are only effective against certain types of microorganisms. Therefore, research continues on new antibiotics.

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

It is therefore an object of the present invention to provide a method for producing a new class of antibiotics which have the basic core structure of thienamycin but which are characterized in that they are N-alkylated derivatives thereof. These antibiotics are active against a wide range of pathogens, for which both gram-positive bacteria are representative, such as S. aureus, Strep. pyogenes and B. subtilis, and gram-negative bacteria, such as E. coli, Proteus morganii, Klebsiella, are representative.

With regard to the description of compounds of structure II, these preferred embodiments are those in which R5, R6 and R7 are selected from hydrogen, low-50-trigalkyl and alkenyl having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, allyl and the like; Benzyl and nucleus-substituted benzyl such as p-tert-butylbenzyl and the like; He-teroaralkylene, such as 4-pyridylmethyl, 2-furylmethyl, 2-thienyl-methyl and the like, and R3, X and R have the meanings given above - particularly preferred embodiments are those in which R3 is hydrogen, X is oxygen and R is hydrogen .

There are no particularly critical conditions when carrying out the process according to the invention and a large number of the known N-alkylation processes can be used. The identity of the N-alkylating agent depends on the choice within the limits given by the definition of R5, R6 and R7. The N-alkylation can be carried out in a large number of solvent systems which are inert or essentially inert to the desired course of the reaction. Suitable solvents include a polar solvent such as water, lower alkano-yl such as ethanol, dioxane, tetrahydrofuran (THF), acetonitrile,

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Hexamethylene phosphoramide (HMPA), dimethylformamide (DMF) and the like and mixtures (especially aqueous mixtures) of the abovementioned solvents; as well as non-polar solvents such as benzene and halogenated hydrocarbons such as methylene chloride, chloroform and the like.

The reaction is typically carried out at temperatures in the range from -40 to +50 ° C. for 15 minutes to 5 hours. In general, the reaction is carried out in the presence of an acid acceptor such as propylene oxide, magnesium oxide, potassium carbonate and the like. The preferred N-alkylating agents include active halides, sulfate esters and Michael addition reagents. The following reagents are representative of such alkylating agents: methyl iodide, allyl bromide, bromoacetone, phenacyl bromide, benzyl bromide, ethyl chloroacetate, propargyl bromide, 2-bromoethyl ethyl ether, dimethyl sulfate and methyl fluorosulfonate, chloromethyl thiocyanomethyl, cycloblidomethylsulfonylmethyl, chloro-ethyl-sulfonate, chlorofluoromethyl-dibonomethyl-sulfonyl-methyl-sulfonate, chlorofluoromethyl-sulfonyl-methyl-sulfonyl-methyl-sulfonyl-methyl-sulfonyl-methyl-sulfonate, cyclofluoromethane-2-methylphenylmethyl-sulfonyl-methyl-sulfonyl-methylphenol-2-chloromethane-2-methylboronyl-methyl-sulfonyl-methyl-sulfonyl-methylphenyl-methyl-dib-2-chloro-methyl-2-chloromethane-2-methylbenzyl-2-chloro-methyl-2-methyl-3-chloro-2-methyl-3-methyl-2-chloromethyl -Chlormethylpyridine, acylonitrile, methyl methyl acrylate, nitroethylene and the like.

The monoalkyl embodiments can be made in a variety of different ways. A convenient starting material is tris-trimethylsilylthien-amycin (Th (TMS) 3). If it is desired that R3, R or R3 and R have a meaning other than hydrogen, suitable derivatives of the starting materials, such as Ia, Ib or Ic (above) can be used. The reaction can be carried out in any of the non-protic solvents listed above in the presence of the N-alkylating agent of choice (R'X ', where R' =

R5, R6 or R7 and X 'is halogen or sulfate). The desired product is usually obtained by aqueous hydrolysis following the N-alkylation step. The following reaction diagram describes this procedure:

r — OTMS

Th

• OTMS

- NHTMS

R'X '

Th

10 COOTMS

Th (TMS) 3

• NR'TMS

15

hydrolysis

Th

- COOTMS OH

NHR '

COOH

20th

wherein TMS, R 'and X' have the meaning given above.

A second scheme for preferred production of the monoalkyl embodiments includes the N-alkylation of N-substituted thienamycin, in which the substituent is an easily removable, large group (R °), such as an aralkyl group, e.g. a substituted or unsubstituted group such as: benzyl, benzylhydryl (-CH (C6H5) 2) and trityl (-C (C6H5) 3), wherein the ring substituent on the aralkyl is halogen, nitro, lower alkyl, lower alkoxyl and the like. The following reaction diagram describes this scheme:

Th

- OR3 - NHR °

R'X '

Th.

COXR 1

[—OR3

"Hi / cat. ^ -NR R -> Th

• COXR 2

OR3 ■ NR '

- COXR

3rd

40

where all symbols have the meaning given above.

With reference to the above diagram, the starting material X 5 can be prepared, for example, from a reaction of thienamycin or a derivative thereof with an aralkyl halide, with the N-alkylating agent of choice R X ', as previously described, with 45 the N, N- Dialkyl intermediate 2 obtained. The aralkyl-N-substituent R ° is easily removable, with 3 being obtained by hydrogenolysis. Suitable conditions for this final cleavage step include hydrogenation of 2 ^ in a solvent such as ethanol under hydrogen (1 to 4 atmospheres) in the presence of a catalyst such as platinum, palladium or oxides thereof. The end product of this reaction is finally 3 ,, the N-mono-lower alkyl. However, the N, N-di-lower alkyl-thienamycin is present together. Such contaminating by-products can be separated by chromatographic methods and the level of contamination can be reduced by using 1 equivalent or less of the alkylating agent R'X '.

A third preferred method for producing the N-monoalkyl species, particularly the N-lower alkyl species, is similar to the procedure previously described except that the starting material is JjaN, N-Diaralky! -Thie-namycin. The production of this starting material is described below. The following reaction diagram describes this procedure:

Th

-OR3 - NR ° R ° • COXR

R'X '

Th la

OR3

Hi / cat -r,

■ NR R R - »Th

COXR

OR3 • NR 'COXR

where all symbols have the meaning given above.

It should be noted that this scheme for the preparation of N-lower alkyl thienamycins is not complicated by the coproduction of N, N-din Lower alkyl thienamycins. A fourth preferred method which is particularly

c, 5 is suitable for the preparation of N-lower alkyl-thienamycin species includes the N-alkylation of a Schiff base of thienamycin. The following diagram shows this reaction:

628 051

Th

- OR3

-N = C-0

i

H

-COXR

R'X '

Th

6

■ OR3

-N = C-0 I

R

-COXR

Hydrolysis or hydrogenation

0 = phenyl

Th

- OR3 -N-R '

- COXR 6

Herein all symbols have the meaning given above and in addition 0 means phenyl, R and R3 can be tri-methylsilyl and X can be oxygen. The preferred Schff'sehe base is that which can be obtained by reacting thienamycin with benzaldehyde or a nucleus-substituted benzaldehyde. In this process, it is not critical how the Schiff base was made and its manufacture is described in U.S. Patent Application Serial No.

634 297 and their continuation-in-part application, which has the internal file number 15833IA. This US patent application is incorporated here for disclosure purposes because it describes the preparation of the starting compound 4. The reaction of 4 with an alkylating agent R'X 'gives the intermediate 5, which can give the desired N-lower alkyl-thienamycin species 6 on aqueous hydrolysis or catalytic hydrogenolysis.

The new N, N-dialkyl-thienamycin derivatives can be made in a variety of ways. The N-substituents can be the same («symmetrical») or different («asymmetrical»). The symmetrical type can be prepared from the starting compound 1, with an excess of the alkylating agent used. In this process, only the N, N-disubstituted product is obtained.

Preferred hydrogenation of the resulting intermediate to cleave the N-aralkyl substituent usually gives the desired N-dialkyl-thienamycin species in the manner previously described.

In general, the N, N-dialkyl-thienamycin species can be prepared by direct alkylation when the alkylation reaction is carried out in water, a protic solvent, or a mixture thereof without protecting the carboxyl group. However, if this direct alkylation is carried out in an aprotic solvent, such as HMPA, the free carboxyl group is generally esterified. In cases where ester formation is not desired, the carboxyl group is preferably blocked by a common, easily removable carboxyl blocking group. (Preparation of such carboxyl blocked thienamycin species will be described later.) It should be noted that the direct N-alkylation of thienamycin generally results in a mixture of products consisting of the mono-, di- and trialkyl species . The relative proportions result from steric reasons which are determined by the size of R 'of the alkylating agent and the amount of reagent used. If R 'is small (less large), such as methyl or ethyl, the N, N, N trialkyl species predominate. If the size of R 'increases, the mono- and dialkyl species predominate.

The symmetric type can be prepared either by reductive alkylation with an aldehyde (R "CHO, where —CH2R" = R5, R6 or R7) or by dialkylation of an ester of thienamycin ir ': 1s of an alkyl halide or sulfate and desired subsequent cleavage the ester group by standard methods such as hydrogenation.

The asymmetric types can be made by reductive alkylation of a monoalkylthienamycin species with a suitable aldehyde or by alkylation of

Tris-trimethylsilyl-N-monoalkyl-thienamycin with an alkylating agent of choice in the presence of an acid acceptor such as propylene oxide, K2C03 or MgO. Again, any of the starting materials Ia, Ib or Ic can be used in order to obtain the corresponding O, carboxyl or O and carboxyl derivatives. The following diagram describes the above reactions:

20th

Th

OTMS ■ NR'TMS '—COOTMS

R "X '

Acid acceptor

-OTMS Th --NR'R "-COOTMS rOH

hydrolysis

Th-

NR'R "'COOH

30 wherein all symbols have the meaning given above and R 'can be the same or not the same R' 'and where R' and

35

R "are selected from Rs, R6 and R7; rOR3

Th-

• NH2 L-COOR

R'X '

40

MgO

Hydrogenation H2 / Pd

Th-

Th.

r

• N (R ') 2 -COOR OR3 ■ N (R') 2 1-COOH

Here R has the meaning given above and, in the event of a final cleavage, represents an easily removable blocking group, such as benzyl or p-nitro-50 benzyl; R3 has the meaning given above, R '= R5, R6 or R7;

55

Th-

-OR3 • NH2 KXDOR

1.) R "CHO

Th-

OR3

2.) H2 / Pt

Herein -CH2R "has the meaning R5, R6 or R7 and R.

• N (CH2R ") 2 LCOOR

60 and R3 have the meaning given above.

rOR3 rOR3

Th-

65

nh2

L-COOR

R'X 'HMPA

or

Th -N (R ') 2 -COOR

7

628 051

Th-

rOR3

• NH2 LCOOH

R'X 'HMPA

Th-

rOR3 N (R ') 2 l-COOR'

Here R3 has the meaning given above; R '= R5, R6 or R7; X '= halide or sulfate; R has the meaning given - the particular case that R = H has been described here. Again, it should be noted that ester formation can be avoided when working with the free acid if the alkylation is carried out in water or a protic solvent.

The new N, N, N-trialkyl-thienamycin derivatives can preferably be prepared from thienamycin, an O, carboxyl or O and carboxyl derivative thereof, or from the N, N-dialkyl species by alkylation with an alkyl halide or sulfate. The carboxyl group can be protected by a conventional blocking group such as benzyl or p-nitrobenzyl. However, it should be noted here that R3- and R-substituted N, N-dialkylthienamycins are valuable substrates when it is desired to prepare compounds of the following structure:

Th-

rOR3

©

-NR5R6R7 -COXR

A ©

in which the non-critical counter anion A® has been previously identified. The following diagram shows this last procedure:

Th—

• OTMS -NR5R6 -COOTMS

raw

1.) R7X '

2.) hydrolysis

Th--

© c ^,

-NR5R6R7

LCOO ©

wherein all substituents are given above.

Identification of the rest -COX R

In the general description of the compounds (II) obtained according to the invention, the radical designated by the group -COX'R means, inter alia, -COOH (X is oxygen and R is hydrogen) and all radicals which are known to be effective pharmaceutically acceptable Esters, anhydrides (R is acyl) and amide residues in bicyclic ß-lactam antibiotics, such as in the cephalosporins and penicillins and their analogues in the core.

Suitable radicals (R) include conventional protective or carboxyl blocking groups. The term "blocking group", as used herein, is used in the same manner as used in U.S. Patent 3,697,515, which is hereby incorporated by reference into the disclosure. New pharmaceutically acceptable thienamycin derivatives which can be prepared according to the invention and which come under this class are given below. Suitable blocking esters therefore include those as given in the following list, which is representative, but in no way represents an exhaustive list of the possible ester groups in which X = O and R are given:

(i) R = CRaRbRc, wherein at least one of Ra, Rb and Rc is an electron donor, for example p-methoxyphenyl,

2,4,6-trimethylphenyl, 9-anthryl, methoxy, CH2SCH3, tetrahydrofur-2-yl, tetrahydropyran-2-yl or fur-2-yl. The remaining Ra, Rb and Rc groups can be hydrogen or organic substitution groups. Suitable ester groups of this 5 type include p-methoxybenzyloxycarbonyl and 2,4,6-tri-methylbenzyloxycarbonyl.

(ii) R = CRaRbRc, wherein at least one of the Ra, Rb and Rc groups is an electron attractive group, for example benzoyl, p-nitrophenyl, 4-pyridyl, trichloromethyl, tribromo-

ic thyl, iodomethyl, cyanomethyl, ethoxycarbonylmethyl, arylsulfonylmethyl, 2-dimethylsulphoniummethyl, o-nitrophenyl or cyano. Suitable esters of this type include benzoyl-methoxycarbonyl, p-nitrobenzyloxycarbonyl, 4-pyridylmeth-oxycarbonyl, 2,2,2-trichloroethoxycarbonyl and 2,2,2-tribromo-15 ethoxycarbonyl.

(iii) R = CRaRbR °, wherein at least two of the Ra, Rb and Rc radicals are hydrocarbons, such as alkyl, for example methyl or ethyl or aryl, for example phenyl and the rest of the Ra, Rb and Rc group, if present, are hydrogen

20 means. Suitable esters of this type include tert-butyloxycarbonyl, tert-amyloxycarbonyl, diphenylmethoxycarbonyl and triphenylmethoxycarbonyl.

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

25 silyl esters of this category of blocking groups can be easily prepared from halosilanes or silazanes of the formula:

R43SiX '; R42SiX'2; R43SiNR42; R43SiNHCOR4;

R43Si - NH • CO • NH • SiR43; R4NH • CO • NH4 • SiR43; or 30 R4C (OSiR43); HN (SiR43) 2, wherein X 'is halogen, chlorine or bromine and the different groups R4, which may be the same or different, represent hydrogen atoms or alkyl, for example methyl, ethyl, n-propyl, isopropyl; Aryl, for example phenyl; or aralkyl, for example a benzyl-35 group.

More generally, new, pharmaceutically acceptable carboxyl derivatives are those derived from the reaction of N-acylated thienamycin (1) with alcohols, phenols, mercaptaenes, thiophenols, acylating agents and the like, whereby compounds (2, above), which are then converted to derivatives to give the R3 group of the new compounds (II, above). For example, interesting esters and amides are the compounds of the formula II (above) with the following groups in the 2-position: —COXR, where -45 in X is oxygen, sulfur or NR '(R' = H or R), and R is alkyl means with 1 to 10 carbon atoms, straight or branched, such as methyl, ethyl, tert-butyl, pentyl, decyl and the like; Carbonylmethyl, including phenacyl, p-bromophenacyl, tert-butylphenacyl, acetoxyace-50 tylmethyl, pivaloxyacetylmethyl, carboxymethyl and its alkyl and aryl esters, a-carboxy-a-isopropyl; Aminoalkyl including 2-methylaminoethyl, 2-diethylaminoethyl, 2-acetamidoethyl, phthalimidomethyl, succinimido-methyl; Alkoxyalkyl, in which the alkoxy part has 1 to 10 and preferably 1 to 6 carbon atoms and can be branched, straight-chain or cyclic, and in which the alkyl part has 1 to 6 carbon atoms, such as methoxymethyl, ethoxymethyl, iso-propoxymethyl, decyloxymethyl, Ethoxypropyl, decyloxyptenyl, cyclohexyloxymethyl and the like; Alkanoyloxyalkyl, 60 wherein the alkanoyloxy part is straight or branched and has 1 to 6 carbon atoms and the alkyl part has 1 to 6 carbon atoms, such as acetoxymethyl, pivaloyloxymethyl, acetoxyethyl, propionyloxyethyl, acetoxypropyl and the like. Haloalkyl, in which halogen is chlorine, bromine, fluorine or iodine 65 and the alkyl part is straight-chain or branched and has 1 to 6 carbon atoms, for example 2,2,2-trichloroethyl, trifluoroethyl, 2-bromopropyl, diiodomethyl, 2-chloroethyl, 2-bromoethyl and the like; Alkenyl with 1 to 10 carbon atoms, that

628 051

is either straight-chain or branched, for example allyl,

2-propenyl, 3-butenyi, 4-butenyl, 4-pentenyl, 2-butenyl,

3-pentenyl, 3-methyl-3-butenyl, methallyl, 1,4-cyclohexadien-1 -yl-methyl and the like; Alkynyl having 1 to 10 carbon atoms, which can be either straight-chain or branched, for example 3-pentenyl, propargyl, ethynyl, 3-butyl-l-yl and the like; Alkanoyl, which can be either straight chain or branched, having 1 to 10 carbon atoms, such as pivaloyl, acetyl, propionyl and the like; Aralkyl or Iieteroalkyl, in which the alkyl has 1 to 3 carbon atoms, and hetero means that

1 to 4 heteroatoms are present, selected from the group consisting of O, S or N, such as benzyl, benzhydryl and substituted benzyl or benzhydryl, for example benzyl or benzhydryl, substituted by 1 to 3 substituents, such as benzyl, phenoxy, halogen, lower alkyl, Lower alkanoyloxy with 1 to 5 carbon atoms, lower alkoxy, hydroxy, nitro, blocked carboxy or combinations thereof, for example p-chlorobenzyl, o-nitrobenzyl, 3,5-dinitrobenzyl, p-methoxy-benzyl, m-benzoylbenzyl, p-tert-butylbenzyl , in-phenoxybenzy !, p-benzoylbenzyl, p-nitrobenzyl, 3,5-dichloro-4-hydroxy-benzyl, p-methoxycarbonylbenzyl, p-methoxybenzhydryl, p-carboxybenzyl, the latter either as a free acid, as an ester or is present as the sodium salt, 2,4,6-trimethylbenzyl, p-pivaloyloxybenzyl, p-tert.-butoxycarbonylbenzyl, p-methyl-benzyl, p-benzoyloxybenzyl, p-acetoxybenzyl, p-2-ethylhexanoylbenzyl, p-ethoxycarbonylbenzyl, p-benzoylthiobenzyl, p-benzamidobenzyl, o-pivaloyloxybenzyl, m-piva loyloxybenzyl, p-isopropoxybenzyl, p-tert-butoxybenzyl and also the cyclic analogues thereof, 2,2-dimethyl-5-coumaranmethyl, 5-indanyimethyl, p-trimethylsilylbenzyl, 3,5-bis-t-butoxy-4 -hydroxybenzyl; 2-thienylmethyl, 2-furylmethyl, 3-tert-butyl-5-isothiazole methyl, 6-pivaloyloxy-3-pyridazinylethyl, 5-phenylthio-1-tetra / olylmethyl or the like (the term lower alkyl or lower alkoxy in this context is a chain of 1 to 4 carbon atoms); or phthalodyl; or phenylethyl, 2- (p-methylphenyl) ethyl and the aryl-thioalkyl analogs, aryloxyalkyl, where aryl is preferably a phenyl ring with 0 to 3 substituents, preferably 0 or 1 substituents in the ortho or para position and the alkyl 1 to Has 6 carbon atoms, for example (4-methoxy) phenoxymethyl, phenoxymethyl, (4-chloro) phenoxymethyl, (4-nitro) phenoxymethyl, (4-benzyloxy) phenoxymethyl, (4-methyl) -phenoxymethyl, (2-methoxy) -phenoxymethyl, (1-phen-oxy) -ethyl, i4-amino) -phenoxymethyl, (4-methoxy) -phenyl-thioinethyl, (4-chloro) -phenylthiomethyl, phenylthioethyl; Aryl, where Ary! Phenyl, 5-indanyl or substituted phenyl with 0 to 3 substituents, preferably 0 to 1 substituents in the ortho or para position, for example (4-methyl) phenyl, (4-hydroxy) phenyl, (4-tert .-Butyl) phenyl, p-nitro-phenyi, 3,5-dinitrophenyl or p-carboxyphenyl, the latter being present either as the free acid or as the sodium salt; Aralkenyl, wherein aryl is phenyl or alkenyl having 1 to 6 carbon atoms, such as 3-phenyl-2-propenyl; Aralkoxyalkyl, wherein aralkoxy is benzyloxy and the alkyl has 1 to 3 carbon atoms, such as benzyloxymethyl, (4-nitro) -benzyloxyme-thyi, (4-chloro) -ber.zyloxymethyl; Alkylthioalkyl, in which the alkyl part has 1 to 10 and preferably 1 to 6 carbon atoms, but which can be branched, straight-chain or cyclic and the alkyl part has 1 to 6 carbon atoms, such as methylthioethyl, ethylthioethyl, cyclohexylthiomethyl, decylthiobutyl, methylthiopropyl, isopropylthioethyl , Methylthiobutyl and the like.

In addition to the esters (and thioesters) listed above, amides may also be present, the R '

means where X is an -N group. Typical examples of such amides are those in which R 'is selected from the group consisting of hydrogen, methyl, ethyl, phenyl, p-meth-

oxyphenyl, benzyl, carboxymethyl, methylthioethyl and heteroaryl; The grouping -COXR also includes anhydrides in which R is acyl, for example benzyloxycarbonyl, ethoxycarbonyl, benzoyl and pivaloyl. 5 The most preferred -COXR radicals are those in which, relative to structure II, X is oxygen, sulfur or NR '(R' is selected from the group consisting of hydrogen and lower alkyl); and R is selected from the group consisting of: lower alkyl, lower alkenyl such as methallyl, 10 3-methylbutenyl, 3-butenyl, and the like; Methylthioethyl; Benzyl and substituted benzyl such as p-tert-butylbenzyl, m-phenoxybenzyl, p-pivaloyloxybenzyl, p-nitrobenzyl and the like; Pi valoyloxy methyl, 3-phthalidyl and acetoxymethyl, propionyloxymethyl, acetylthiomethyl, pivaloylthiomethyl, Alis lyl, 4-butenyl, 2-butenyl, 3-methyl-2-butenyl, phenacyl, acet-oxyacetylmethyl, methoxymethyl, p-acetoxyzylbenzyl , p-isopropoxybenzyl, 5-indanyImethyI, 5-indanyl, benzyloxymethyl, ethylthioethyl, methylthiopropyl, meth-oxycarbonyloxymethyl, ethoxycarbonyloxymethyl, dimethyl-20 aminoacetoxymethyl, crotonolacton-3-yI and acetamidomethyl.

Identification of R3

In a general presentation of the new compounds 25 in structure II, the radical R3, in addition to hydrogen, means 1.) acyl (in general the group OR3 is referred to as an ester); or 2.) R3 is selected from alkyl, aryl, aralkyl and the like, so that the group -OR3 can be referred to as an ether. For the ester version (1), R3 is selected 30 from the following definitions for acyl residues (p = 1). In the so-called ether version (2.) of the present invention, R3 is selected from the same acyl radicals in which O X

II II

35 the carbonyl group -C- or generally -C— is omitted (p = 0); so that R3 is selected from the following radicals, in which all symbols are designated below:

'-OL II)

Xx / X>

I "

p- (CH2) nZR "-Vc / p-CHR" R '"

45

X

C / p-CHR4R5 -MH / p- (CH2) m-A- (CH2) n-Y

Therefore, with respect to the definition of R3, the acyl radical can be, inter alia, a substituted or unsubstituted aliphatic, aromatic or heterocyclic, araliphatic or heterocyclylaliphatic carboxylic acid radical, a substituted or unsubstituted carbamyl radical or a carbothio-55 acid radical. A group of acyl residues can be represented by the general formula:

X

60 -C —R ",

where X = O or S and R "is hydrogen; amino; substituted amino, such as alkyl- and dialkylamino, wherein the alkyl radical comprises 1 to 6 carbon atoms; substituted-65 ized or unsubstituted radicals, such as: straight-chain or branched-chain alkyl radicals, wherein the alkyl radical comprises 1 to 6 carbon atoms; mercapto, aryloxy, which typically comprises 6 to 10 carbon atoms; alkenyl or alkynyl

628 051

groups typically comprising 2 to 6 carbon atoms; Aryl such as phenyl; Aralkyl such as benzyl; Cycloalkyl, which typically comprises 3 to 6 carbon atoms; or a heteroaryl or heteroaralkyl group (mono- or bicyclic), in which the alkyl group typically comprises 1 to 3 carbon atoms and the heterocyclic ring typically contains 4 to 10 atoms and the hetero atom or heteroatoms are selected from O, N and S; the aforementioned groups can be unsubstituted or they can be substituted by radicals such as OH, SH, SR (R is lower alkyl or aryl such as phenyl), alkyl or alkoxy groups having 1 to about 6 carbon atoms, halogen such as Cl, Br, F and J, cyano, carboxy, sulfamino, carbamoyi, sulfonyl, azido, amino, substituted amino, such as alkylamino including quaternary ammonium, in which the alkyl groups have 1 to 6 carbon atoms, haloalkyl, such as trifluoromethyl, carboxyalkyl, carbamoylalkyl, N-substituted carbamoylalkyl, wherein the alkyl group of the above four radicals comprises 1 to about 6 carbon atoms, amidino, guanidino, N-substituted guanidino, guanidino lower alkyl and the like. Typical examples of such acyl groups, which can be mentioned here, are those in which R "is benzyl, p-hydroxybenzyl, 4-amino-4-carboxybutyl, methyl, cyanomethyl, 2-pentenyl, n-amyl, n- Heptyl, ethyl, 3- or 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-isoxazolvl, 5-methyl-3-pheny 1-4-isoxazoly 1, 3- (2-chlorophenyl) -5-methyl-4-isoxazolyl, 3- ( 2,6-dichlorophenyl) -5-methyl-4-isoxazolyl, D-4-amino-4-carboxybutyl, D-4N-benzoylamino-4-carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl, in-aminobenzyl , p-Dimethylaminobenzyl, (3-pyridyl) methyl, 2-ethoxy-1-naphthyl, 3-carboxy-2-quinoxaiinyl, 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-guani-dinophenyl, 2,6-dimethoxy-4-guanidino, o-sulfobenzyl, p-carboxymethylbenzyl, p-carbamoylmethylbenzyl, m-fluoro-benzyl, m-bromobenzyl, p-chlorobenzyl, p-methoxybenzyl,

1-naphthylmethyl, 3-isothiazolylmethyl, 4-isothiazolylmethyl,

5-isothiazolylmethyl, guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4-methoxy-S-isoxazolylmethyl, 4-methyl-5-isoxazolylmethyl, 1-imidazolylmethyl, 2-benzofuranyimethyl, 2-phenolylmethyl, 2-indolylmethyl, 2 Phenylethynyl, 1-aminocyclohexyi, 2- and 3-thienylarninomethyl, 2- (5-nitrofuranyl) vinyl, phenyl, o-methoxyphenyl, o-chlorophenyl, o-phenylphenyl, p-aminomethylbenzyl, l- ( 5-cyanotriazolyl) ~ methyl, difluoromethyl, dichloromethyl, dibromomethyl, 1- (3-methylimidazolyl) methyl, 2- or 3- (5-carboxymethylthienyl) methyl, 2- or 3- (4-C ' arbamoylthienyl) methyl, 2- or 3- (5-methylthienyl) methyl, 2- or 3- (methoxythienyl) methyl, 2- or 3- (4-chlorothienyl) methyl, 2- or 3- ( 5-sulfothienyl) methyl, 2- or 3- (5-carboxythienyl) methyl, 3- (1,2,5-thiadiazolyl) methyl, 3- (4-methoxy-1,2,5 -thiadiazoIyl) -mcthyl, 2-furyl-methyl, 2- (5-nitrofuryl) -methyl, 3-furylmethyl, 2-thienylmethyl, 3-thienylmethyl, tetrazolylmethyl, benzamidinomethy! and cyclohexylam idinomethyl.

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

X

-C (CH2) "ZR",

wherein X is O or S and n is 0 to 4 and Z is oxygen, sulfur, carbonyl or nitrogen and R "has the meaning given above. Typical members of the substituent

- (CH2) nZR ",

which should be mentioned here are allylthiomethyl, phenylthio-methyl, butyl mercaptomethyl, a-chlorocrotyl mercaptome-5 thyl, phenoxymethyl, phenoxyethyl, phenoxybutyl, phenoxy-benzyl, diphenoxymethyl, dimethylmethoxyethyl, dimethyl-butoxymethyl, dimethylphenoxymophen, 4-guanophenyl Pyridylthiomethyl, p- (carboxymethyl) phenoxyrne-methyl, p- (carboxymethyl) phenylthiomethyl, 2-thiazolylthiomethyl, p- (sulfo) phenoxymethyl, p- (carboxymethyl) phenylthiomethyl, 2-pyrimidinylthiomethyl, phenethyl , l- (5,6,7,8-tetrahydronaphthyl) oxomethyl, N-methyl-4-pyridylthio, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio. Amino, methylamino, dimethylamino, pyridiniummethyl, tri-15methylammoniummethyl, cyanomethylthiomethyl, trifluoromethylthiomethyl, 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 3-imidazolylethyl, 3-imidazolylopropyl, 1-pyridylolipidol, 1-pyridyloloyl and 1-pyrrolobutyl.

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

X

25th

-C-CHR "

R "'

be in which R "has the meaning given above and R '" is a radical, such as amino, hydroxy, azido, carbamoyi, guanidino, amidino, acyloxy, halogen, such as chlorine, fluorine, bromine, iodine, Sul-30 famino, tetrazolyl , Sulfo, carboxy, carbalkoxy, phosphono and the like. Typical members of the substituent

-CHR "

I.

35 R '"

which should be mentioned here are a-aminobenzyl, a-amino (2-thienyl) methyl, a- (methylamino) benzyl, a-aminomethyl mercaptopropyl, a-amino-3- or -4-chlorobenzyl, a-amino-3- or -4-hydroxybenzyl, a-amino-2,4-dichlorobenzyl, a-amino-3,4-dichlorobenzyl, D (-) - a-hydroxybenzyl, a-carboxybenzyl, o.- Amino- (3-thienyl) methyl, D (-) - a-amino-3-chloro-4-hydroxybenzyl, c.-amino- (cyclohexyl) -methyl, a- (5-tetrazolyI) benzyl, 2-thienylcarboxylmethyl, 3-thienylcarboxymethyl, 2-furylcarboxymethyl, 3-furylcarboxymethyl, a-sulfaminobenzyl, 3-thienylsulfaminomethyl, a- (N-methylsulfamino) -benzyI, D (-) - 2-thienylguanidinomethyl, D (- ) -a-guanidinobenzyl, a-guanylureidobenzyl, a-hydroxybenzyl, a-azidobenzyl, a-fluorobenzyl, 4- (5-methoxy-1,3-oxadiazolyl) aminomethyl, 4- (5-methoxy-1,3-oxadiazolyl ) -hydroxymethyl, 4- (5-methoxy-l, 3-sulfadiazolyl) hydroxymethyl, 4- (5-chlorothienyl) aminomethyl, 2- (5-chlorothienyl) hydroxymethyl, 2- (5-chlorothienyl) - carboxymethyl, 3- (1,2-thiazolyl) aminomethyl, 3- (1,2-thiazolyl) - hydroxymethyl, 3- (l, 2-thiazolyl) carboxymethyl, 2- (l, 4-thiazolyl) ami-55 nomethyl, 2- (l, 4-thiazolyl) hydroxymethyl, 2- (l, 4- Thiazolyl) carboxymethyl, 2-benzothienylaminomethyl, 2-benzothienyl-hydroxymethyl, 2-benzothienylcarboxymethyl, a-sulfobenzyl, a-phosphonobenzyl, a-diethylphosphono and a-monoethyl-phosphono.

Other interesting acyl residues in this class where X = oxygen are:

O

45

65

-CCHR3R4,

wherein R3 and R4 are defined below. R3 means hydrogen, halogen, such as chlorine, fluorine, bromine, iodine, amino,

628 051

10th

Guanidino, phosphono, hydroxy, tetrazolyl, carboxy, sulfo or sulfamino and R4 means phenyl, substituted phenyl, a mono- or bicyclic heterocyclyl containing one or more oxygen, sulfur or nitrogen atoms in the ring, such as furyl, quinoxalyl, thienyl, quinolyl , Quinazolyl, thiazolyl, thiothiazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl and the like, substituted heterocycles, phenylthio, phenyloxy, lower alkyl having 1 to 6 carbon atoms, heterocyclic or substituted heterocyclic thio groups or cyano. The substituents on the groups R3 and R4 can be halogen, carboxymethyl, guanidino, guanidinomethyl, carboxyamidomethyl, aminomethyl, nitro, methoxy or methyl. R3 is selected from the group consisting of hydrogen, hydroxy, amino or carboxy and R4 selected from the group consisting of phenyl or a 5- or 6-membered heterocyclic ring with one or two sulfur, oxygen or nitrogen heteroatoms, such as Tetrazolyl, thienyl, furyl and phenyl, the following acyl radicals are typical examples: phenylacetyl, 3-bromophenylacetyl, p-aminomethylphe-nyiaceiyl, 4-carboxymethyIphenylacetyl, 4-carboxyamidome-thylphenylacetyl, 2-furylacetyl, 5-nitrolacetyl, 5-nitrolacetyl 3-furyl-acetyl, 2-thienylacetyl, 5-chloro-2-thienylacetyl, 5-methoxy-2-thienylacetyl, a-guanidino-2-thienylacetyl, 3-thienylacetyI,

2- (4-methylthienyl) acetyl, 3-isothiazolylacetyl, 4-methoxy-3-isothiazolylacetyl, 4-isothiazolylacetyI, 3-methyl-4-isothiazoIyl-acetyl, 5-isothiazolylacetyl, 3-chloro-5-isothiazolylacetyl, 3- Methyl-l, 2,5-oxadiazolyIacetyl, l, 2,5-thiadiazolyl-4-acetyl,

3-methyl-l, 2,5-thiadiazolylacetyl, 3-chloro-l, 2,5-thiadiazolyl-acetyl, 3-methoxy-l, 2,5-thiadiazolylacetyl, phenylthioacetyl,

4-pyridylthioacetyl, cyanoacetyl, l-tetrazolylacetyl, a-fluorophenylacetyl, D-phenylglycyl, 4-hydroxy-D-phenylglycyl, 2-thienylglycyl, 3-thienylglycyl, phenylmalonyl, 3-chlorophenyl-3-malienylyl, 2-thyl-malonyl, 2-thyl ThienyImaIonyl, a-Phospho-nophenylacetyl, a-Aminocyclohexadienylacetyl, a-Sulfamino-phenylacetyl, a-Hydroxyphenylacetyl, a-Tetrazolylphenyl-acetyl and a-SuIfophenylacetyl.

The acyl radical can also be selected from sulfur (1,) -

and phosphorus (2) residues:

40

(O) m (X) n

II I

-S-Y -P-Y '

II I

(O) n Y "45

phino) thienamycin, Q- (dihydroxyphosphino) thienamycin di-sodium salt, 0- (dimethoxyphosphinyl) thienamycin, 0- (di-methoxyphosphinothioyl) thienamycin, 0- (dibenzyloxyphos-phinyl) thienamycin and 0- (dihydroxyphosphinyl) thienamycin -5 cindisodium salt.

An acyl class of particular interest is those acyl radicals which are selected from the group consisting of customary known N-acyl blocking or protecting groups, such as carbobenzyloxy, ring-substituted carbobenzyloxy, such as o- and io p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy, chloroacetyl tyl, bromoacetyl, phenylacetyl, tert-butoxycarbonyl, trifluoro-acetyl, bromoethoxycarbonyl, 9-fluorenylmethoxycarbonyl, dichloroacetyl, o-nitrophenylsulfenyl, 2,2,2-trichloroethoxycarbonyl, bromotert-butoxycarbonyl, phenoxyacetyl; Non-acy-15-lated protecting groups, such as tri-lower alkylsilyl, for example trimethylsilyl and tert-butyldimethylsilyl, are also of interest.

The following radicals are preferred in accordance with the aforementioned definition of acyl: formyl, propionyl, 20 butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, phenylthiocarbonyl, 3-aminopropionyl, 4-aminobaminoacyl, N , N, N-dimethylaminoacetyl, N, N, N-tri-methylaminoacetyl, 3- (N, N-dimethyl) aminopropionyl,

3- (N, N, N-TrimethyI) aminopropionyl, N, N, N-triethylaminoacetyl, pyridiniumacetyl, guanidinoacetyl, 3-guanidinopropionyl, N3-methylguanidinopropionyI, hydroxyacetyl, 3-hydroxypropionyl, acryloyl, propynoyl, Malonyl, Phenoxycarbonyl, Amidinoacetyl, Acetamidinoacetyl, Amidinopropionyl, Acetaminopropionyl, Guanylureidoacetyl, Guanylcarbamoyl, Carboxymethylaminoacetyl, Phospho-noacetylaminoacetyl, N3-Dimethylaminoacyl

4-pyridinium) propionyl, 3- (5-aminoimidazol-l-yl) propionyl, 3-methyl-l-imidazoliumacetyl, 3-sydnonylacetyl, o-aminomethylbenzoyl, o-aminobenzoyl, sulfo, phosphono,

25th

30th

35

O S S II 11 II

-P (OCH3) 2, -P (OCH3) 2, -P

s o

OCH,

\

O

ONa

-P [N (CH3) 2] 2, -P-N (CH3) 2, -P [N (CH3) 2] 2, ^ ONa

S

55

where with respect to 1,> m and n are integers which are selected between 0 and 1 and Y ° = O © M®, -N (R ") 2 and R"; where M © is selected from hydrogen, alkali cations and organic bases and R ″ has the abovementioned meaning, for example alkyl, alkenyl, aryl and heteroaryl. With respect to 2, X = 0 or; n = 0 or 1; and Y 'and Y "are selected from the groups consisting of O © M ©, -N (R' ') 2, R" and ZR ", in which all symbols have the meaning given above, and for example R" and ZR " typically have the following meanings: alkyl, alkenyl, aryl, heteroaryloxy, Y'and Y ", including the R" groups, which can be connected to one another to form cyclic esters, ester amide and amide functions. Typical examples for X are 0- (methylsulfonyl) thienamycin, 0- (p-nitrophenylsulfonyl) thienamycin, 0- (p-chlorophenylsulfinyl) thienamycin, 0- (o-nitrophenylsulfenyl) thienamycin, O-sulfamoylthiena- 65 mycin, O-dimethylsulfamoylthienamycin and the sodium salt of thienamycin-O-sulfonic acid Typical examples of 2 are 0- (dimethoxyphosphino) thienamycin, O-dibenzyloxyphos-

-P-N (CH3) 2 ONa

A particularly preferred class of acyl radicals are terminally substituted acyls in which the substituent is a basic group which can be substituted or unsubstituted: amino, amidino, guanidino, guanyl and nitrogen-containing mono- and bicyclic heterocycles (aromatic and non-aromatic), in which the heteroatom or the heteroatoms are selected from oxygen and sulfur in addition to nitrogen. Such substituted acyls can be represented by the following formula:

O

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

are characterized wherein m and n are integers from 0 to 5;

11

628 051

A is O, NR '(R' is hydrogen or lower alkyl of 1 to 6 carbon atoms), S or A is a single bond; and Y is selected from the following groups:

1.) Amino or substituted amino:

-N (R) 2 and -N (R) 3,

wherein the values for R are independently selected from: hydrogen; N (R ') 2 (R' 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 alkyl group comprises 1 to 6 carbon atoms and the alkyl group has 2 to 6 carbon atoms; Cycloalkyl and cycloalkylalkyl, in which the cycloalkyl group comprises 3 to 6 carbon atoms and the alkyl group has 1 to 3 carbon atoms, and where two R groups can be connected to one another via the N atom to which they are bonded to form a ring with 3 up to 6 atoms.

2.) Amidino and substituted amidino:

-N = C-N (R) 2,

I.

R

wherein the values for R are independently selected from the groups: hydrogen; N (R ') 2 (R' is hydrogen or lower alkyl of 1 to 6 carbon atoms); Lower alkyl and lower alkoxyl having 1 to 6 carbon atoms, lower alkoxy lower alkyl, wherein the alkoxyl group contains 1 to 6 carbon atoms and the alkyl group contains 2 to 6 carbon atoms (if the lower alkoxy lower alkyl group is bonded to a carbon atom, the alkyl group comprises 1 to 6 carbon atoms); Cycloalkyl and cycloalkylalkyl, wherein the alkyl group comprises 1 to 3 carbon atoms; two R groups can be linked to each other with the atom to which they are attached to form a ring with 3 to 6 atoms;

3.) Guanidino and substituted guanidino:

_NH-C-N (R) 2,

I.

NO

where R has the definition given under 2.

4.) Guanyi and substituted guanyl:

-C = NR,

!

NO. 2

where R has the definition as previously given in 2.

5.) Nitrogen-containing mono- and bicyclic heterocycles (aromatic and non-aromatic) with 4 to 10 core atoms, in which the hetero atom or the hetero atoms are selected from nitrogen and oxygen apart from nitrogen. Such heterocycles are typically illustrated by the following list of residues (R 'is H or lower alkyl with 1 to 6 carbon atoms):

55

60

A

7

- <0

V

J

The following preferred specific acyl residues that fall into this class are additionally representative and are preferred:

30th

40

©,

No

O

nh

-CCH, CH, NHC-CH,

O

NH

-CCH2CH2NHC-H

O

NH

-CCH2CH2NHC-NH2

O

-CCH2CH2N (CH3) 2

O

©

-CCH2CH2N (CH3) 3

O NH

II I

-CCH2CH2C-NH2

O NH

II I

-CCH2CH2CH2NHC-CH3

O

II ©

-CCH2CH2CH2N (CH3) 3

O

II

-CCH2CH2CH2N (CH3) 2

O

NH

-cch2s-c

\

NH,

628 051

12th

O

S

NH

-CCH.S-CH, -C

\

NH,

O

-C CH -.- O-CH? C

NH

\

NH2

However, it is to be understood that any acyl group can be used in the practice of the invention and is included in the invention.

IS

25th

Preferred preparation of the starting materials Ia, Ib and Ic:

The starting materials described above are simply made from an N-protected thienamycin (X), such as an N-acylated thienamycin (X), 20

- OH

Th -! - NR'R2 -COOH

I.

wherein R 'and R2 are selected from hydrogen and the aforementioned acyl radicals. Preferably R1 is hydrogen and R2 is an easily removable blocking group such as: carbobenzyloxy, ring-substituted carbobenzyloxy such as o- and p-nitrocarhobenzyloxy, p-methoxycarbobenzyloxy, chloro-acetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl, trifluoro-acetyl, broir .ethoxycarbonyl, 9-fluorenylmethoxycarbonyl, dichloroacetyl, o-nitrophenylsulfenyl, 2,2,2-trichloroethoxycaronyl, bromo-t-butoxycarbonyl, phenoxyacetyl; Non-acyl-protected groups, such as tri-lower alkylsilyl, for example tri-methylsilyl and tert-butyldimethylsilyl, are also of interest. The most preferred N-blocking groups are substituted and unsubstituted carbobenzyloxy:

35

40

R ~

O

- - c - OCH

cr ")

n where n is 0 to 2 (n = 0, R '- hydrogen) and R' is lower alkoxy or nitro and bromine-tert-butoxycarbonyl.

The final N deblocking in the manufacture of Ia, Ib or Ic is carried out by a variety of known methods, including hydrolysis or hydrogenation; if hydrogenation is used, suitable conditions include a solvent such as a lower alkano-yl in the presence of a hydrogenation catalyst such as palladium, platinum or oxides thereof.

The N-acylated intermediate [X, above] is prepared by treating thienamycin (I) with an acylating agent, for example an acyl halide or an acyl anhydride such as an aliphatic, aromatic, heterocyclic, araliphatic or heterocyclic aliphatic carboxylic acid halide or anhydride. Other acylating agents that can also be used are, for example, mixed carboxylic anhydrides and in particular lower alkyl esters of mixed carboxylic carboxylic anhydrides; carboxylic acids in the presence of a carbodiimide, such as 1,3-di-cyclohexylcarbodiimide, and an activated ester of carboxylic acids, such as p-nitrophenyl ester, are also suitable.

These N-acylated thienamycin starting materials are fully described in U.S. Patent Application Serial No. 634,291 dated November 21, 1975 and the Continuation-in-part U.S. Application which coexists with the present application for the same Applicant has been filed with the internal designation 15 775 YA. These applications are hereby incorporated into the description.

The acylation reaction can be carried out at temperatures in the range from about -20 to about 100 ° C, but preferably temperatures in the range from -9 ° C to 25 ° C are used. Any solvent in which the reactants are soluble and which is substantially inert can be used, for example polar solvents such as water, alcohols and polar organic solvents, generally those such as dimethylformamide (DMF), hexamethylphosphoramide (HMPA), acetone, Dioxane, tetrahydrofuran (THF), acetonitrile, heterocyclic amines, such as pyridine, ethyl acetate, aqueous mixtures of the aforementioned, and also halogenated solvents, such as methylene chloride and chloroform. The reaction is preferably carried out for a time ranging from about 5 minutes to a maximum of 3 hours, but a reaction time of about 0.5 to about 1 hour is generally sufficient. The following equation describes this process using a carboxylic acid halide; however, it goes without saying that the same products can be obtained by using a carboxylic acid anhydride or another functional equivalent acylating agent:

SCH2CH2NH2 COOH

Acylhalosenjd

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

When performing the implementation described here, it is

CH2CH2NR1R2 COOH

60 generally not necessary to protect the 2-carboxy group or the 1 'hydroxy group; in cases where the acylating agent is extremely water sensitive, it is sometimes advantageous to carry out the acylation in a non-aqueous solvent system. Triorganosilyl (or tin) -65 derivatives of thienamycin quickly give the tris-triorganosilyl derivatives, for example tris-trimethylsilylthienamycin Th (TMS) 3:

13

628 051

- OTMS

Th

NHTMS

- COOTMS

These derivatives, which are readily soluble in organic solvents, can be easily prepared by allowing thienamycin with an excess of hexamethyldiio.

silazan and a stoichiometric amount of trimethylchlorosilane at 25 ° C with vigorous stirring under an N2 atmosphere. The resulting NH4C1 is usually removed by centrifugation and the solvent is removed by evaporation to give the desired silyl derivative.

The intermediate starting product Ic can be produced according to the following scheme; It should be noted, however, that direct esterification without protecting the amino group also

Th

OH

NR'R2

Th

COOH

OH

,, Unblocking NR'R2 5 »Th

COX R

OH NH2

COXR

i Ib in which all symbols have the meaning given above.

In general, the transition (1_ Ib) is effected by customary techniques of the prior art. These procedures include:

1.) Reacting X (or I) with a diazoalkane such as diazomethane, phenyldiazomethane, diphenyldiazomethane, and the like in a solvent such as dioxane, ethyl acetate, acetonitrile and the like, at temperatures between 0 ° C and reflux for a period of a few minutes up to 2 hours.

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

3.) Reaction of X with an alcohol such as methanol, ethanol, benzyl alcohol and the like. This reaction can be carried out in the presence of a carbodiimide condensing agent such as dicyclohexylcarbodiimide and the like.

25th

Ib same. Suitable solvents at temperatures between 20 ° C. and the reflux temperature and reaction times of 15 minutes to 18 hours include CHC13, CH3C1, CH2C12 and the like.

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

5.) Reaction of labile esters of X> such as trimethylsilyl ester, dimethyl tert-butylsilyl ester or the like with RX 'in which X' is halogen, such as bromine or chlorine, and R has the meaning given above, in a solvent such as THF, CH2C12 and the like at temperatures between 0 ° C and reflux for 15 minutes to 16 hours. This implementation takes place, for example, according to the following scheme:

35

Th

OTMS

N-acylation NHTMS -> Th

COOTMS 3

mild hydrolysis

OTMS

NR'TMS VereStemng, Th

COOTMS

OTMS NR1TMS

COOR

Th

OH NHR1

COOR

wherein TMS means triorganosllyl, such as trimethylsilyl, and all 55 other symbols have the meaning given above.

The new amides can be prepared in a simple manner by reacting the acid anhydride of Ib (X = O, R = acyl) with ammonia or with the selected amine, for example the aforementioned alkyl, dialkyl, aralkyl 60 or heterocyclic amines.

The previously outlined schemes for the esterification are known for the related bicyclic β-lactamantibiotics and generally for organic syntheses, and it should be stated here that there are no particular critical conditions with regard to the reaction parameters in the preparation of the N-acylated and carboxyl derivatives Ib suitable as starting compounds for the present invention are required.

The starting compounds Ia and Ic can be prepared in a simple manner by any of the known esterification or etherification reactions of the secondary alcohol group of Ib. Such methods preferably include:

OH F OR3

Th

NR'R2

COXR Ib

Th

NR'R

COXR

628 051

14

1.) For the preparation of the ether embodiments, the acid catalyzed reaction of Ib is carried out with a diazoalkane such as diazomethane, phenyldiazomethane, diphenyldiazomethane and the like in an inert solvent such as dioxane, tetrahydrofuran (THF), halogenated hydrocarbons such as CH2C12, ethyl acetate and the like in the presence of a catalytic amount of a strong acid or Lewis acid such as toluenesulfonic acid, trifluoroacetic acid, fluoroboric acid, boron trifluoride and the like at temperatures between

- 78 ° C and 25 ° C for a few minutes to 2 hours. 10th

2.) For the preparation of the ether embodiments, the reaction of Ib is carried out with an alkylating agent, such as an active halide, for example methyl iodide, benzyl bromide, m-phenoxybenzyl bromide and the like; Al-alkyl sulfonate such as dimethyl sulfate, diethyl sulfate, methyl fluorosulfonate and the like in the presence of a strong base capable of forming an alcoholate anion of Ib. Suitable bases include alkali and alkaline earth oxides and hydroxides, alkali alkoxides such as potassium tert-butoxide, tert. Amines such as triethylamine, alkali alkyls and aryls such as phenyllithium and 20 alkali amides such as sodium amide. Suitable solvents include an inert anhydrous solvent such as tert-buta-nol, dimethylformamide (DMF), THF, hexamethylphosphoramide (HMPA), dioxane and the like at temperatures of

- 78 ° C to 25 ° C for a period of a few minutes to 4 hours.

3.) For the preparation of the ester embodiments, the reaction of Ib with any of the acyl radicals listed above takes place in their acid form. This reaction can be carried out in the presence of a carbodiimide condensing agent such as a dicyclohexylcarbodiimide and the like. Suitable solvents include an inert solvent such as CHCl3, CH2C12, DMF, HMPA, acetone, dioxane and the like at temperatures from 0 ° C to 60 ° C for a period of 15 minutes to 12 hours. 35

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

Suitable acyl halides and anhydrides include: acetic anhydride, bromoacetic anhydride, propionic anhydride, benzoyl chloride, phenylacetyl chloride, azidoacetyl chloride, 2-thienylacetyl chloride, 2-, 3- and 4-nicotinyl chloride, p-nitro-benzoyl chloride, 2,6-dimethoxybenzoyl Guanidino-phenylacetyl chloride, hydrochloride, methanesulfonyl chloride, dibenzyl phosphorochloridate, dimethylthiophosphorochloridate, 2-furoylethyIcarboxylic acid anhydride, methyl chloroformate, bis (p-nitrobenzyl) phosphorochloridate and the like.

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

Intermediate 2 can then be N-deblocked as previously described to give starting compounds Ic.

Ic is preferably produced from Ic by deblocking the carboxyl group:

Th

OR3

NR'R2

COXR

Th

OR3

nh2

COXR

Th

OR3 NH2

COOH

Ic

Yes

45

The starting material Ia can be obtained simply and preferably when X is oxygen and R is an easily removable carboxyl protecting or blocking group (see above). The starting material Ia is e.g. made by deblocking by a variety of known procedures 50 including hydrolysis and hydrogenation. If the preferred carboxyl blocking groups are used (below), the preferred deblocking process is hydrogenation, in which the intermediate (Ic or 2) is in a solvent such as a lower alkanoyl in the presence of a hydrogenation catalyst such as palladium, platinum or oxides thereof. is hydrogenated.

In this connection it should be noted that the suitable c-blocking groups »R include the subgroups which are defined as previously as aralkyl, haloalkyl, alkanoyloxyalkyl, alkoxylalkyl, alkenyl, substituted alkyl or 60 aralkoxyalkyl and also alkylsilyl, in which the alkyl group 1 to 10 Has carbon atoms. Suitable “blocking groups” R include, for example, benzyl, phenacyl, p-nitrobenzyl, methoxymethyl, trichloroethyl, trimethylsilyl, tributyltin, p-methoxybenzyl and benzhydryl. These blocking groups are preferred because they are generally recognized as easily removable blocking groups in the prior art relating to the cephalosporins and penicillins.

The preferred carboxyl blocking groups are benzyl and substituted benzyl:

R ') n where n = 0 to 2 (n = 0, R' = H) and R 'mean lower alkoxyl or nitro.

The new products of formula (II) form a large number of pharmacologically acceptable salts with inorganic and organic bases, which include, for example, metal salts which are derived from alkali and alkaline earth metal oxides, carbonates or bicarbonates and salts which are derived of primary, secondary or tertiary amines, such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylenediamines, N, N-diaralkyl-lower alkylenediamines, aralkylamines, amino-substituted-lower alkanols, lower, amino-polyamino, lower, alkanol-amino, lower, alkanolamino and guanidino-substituted lower alkanoic acids and nitrogen

15 628 051

holding heterocyclic amines. Typical examples include coating agents, in the white water of paper mills, to inhibit the salts derived from sodium hydroxide, sodium growth of harmful bacteria.

carbonate, sodium bicarbonate, potassium carbonate, potassium hydro- The products produced according to the invention can be used alone xid, calcium carbonate, trimethylamine, triethylamine, piperidine, or in combination with active constituent morpholine, quinine, lysine, protamine, arginine, procain, ethanol-5 oils in a variety of pharmaceutical preparations.

amine, morphine, benzylamine, ethylenediamine, N, N'-dibenzyl- These antibiotics and their corresponding salts can be used in ethylenediamine, diethanolamine, piperazine, dimethylaminoätha- in the form of capsules or as tablets, as a powder or as a liquid, 2-amino- 2-methyl-l-propano !, thiophylline, N-methylge solutions or used as suspensions and elixirs glucamine and the like. Acid addition salts, for example. They can be administered orally, intravenously or intramuscularly - hydrofluoric acid, hydrobromic acid, sulfuric acid, io.

Nitric acid, p-toluenesulfonic acid and methanesulfonic acid. The compositions are preferably in a sol-

can be used in cases where the acyl residue form is prepared to contain a basic group absorbed in the gastrointestinal tract. beers. Tablets and capsules for oral administration

The salts can be mono-salts, such as the monosodium salt, can be in single dosage form and can be obtained by containing an equivalent of sodium hydrois usual excipients, such as binders, for example reacting xid with an equivalent of product II, or syrup , Gum arabic, gelatin, sorbose, tragacanth or Po-mixed Disalze. Such salts are obtained by using an equilvinylpyrrolidone; Fillers, for example lactose, sugar, valent a base with a divalent cation, such as calcium hy maize starch, calcium phosphate, sorbose or glycerin; Lubricant hydroxide, reacted with an equivalent of product II. The medium, for example magnesium stearate, talc, polyethylene obtained according to the invention are pharmacologically acceptable, glycol, silicon dioxide; Disintegrants, for example potato-acceptable non-toxic derivatives, which act as active ingredients in fine starch or suitable humectants, such as single-dose formulations suitable for sodium sulfate for pharmaceutical sulfate. The tablets can be coated in a known manner, see products can be used. You can also join in. Oral liquid preparations can be combined in the form of aqueous drugs to achieve a wider range of oily suspensions, solutions, emulsions, Siru activity spectrum. 25 pens, elixirs and the like are present or can be

The new thienamycin derivatives obtained according to the invention are produced so that they are then water vate valuable antibiotics with an activity against different or another suitable carrier material before their various gram-positive and gram-negative pathogens, such as Ba turn, are turned on can. Such liquid preparation-cillus subtilis, Salmonella schottmuelleri and Proteus vulgaris. The usual additives, such as suspending agents, the free acids and in particular their salts, such as the amine, for example sorbose, syrup, methyl cellulose, glucose / sugar salts and metal salts, in particular the alkali and alkaline earth metal salts, rup, gelatin, hydroxyethyl cellulose, Carboxymethyl cellulose, are useful bactericides and can be used to contain aluminum stearate gel or hydrogenated edible oils, harmful bacteria on medical and dental technology, for example to remove almond oil, fractionated coconut oil, diesters, pro equipment to separate microorganisms containing pylene glycol or ethyl alcohol; Preservative and for therapeutic use in humans and animals. For 35 tel, for example methyl or propyl p-hydroxybenzoate or the latter use, especially pharmaceutical sorbic acid. Suppositories contain the usual salts, suitable for this purpose, with inorganic or organic substances, for example cocoa butter or other gly bases, using those that are already cerine.

knows from use in the administration of penicilli compositions for injections can be administered in single

nen and cephalosporins. For example, salts, such as Al 4 solutions, can be prepared in the form of ampoules or used in potassium and alkaline earth metal salts and primary, secondary and tertiary containers with added preservatives for multiple amine salts for this purpose. Dosage of these salts. The compositions can be in the form of Su-can with pharmaceutically acceptable liquid or solid liquids, solutions or emulsions in oil or aqueous vehicles to form suitable single-dose formulations carrier materials and they can be formulating agents such as Su-processed such as pills, tablets, capsules, suppositories Retention agents, stabilizing agents and / or dispersing agents, syrups, elixirs and the like, these containing administrations. Alternatively, the active ingredients can be in the form of dosage forms according to known methods of the prior art, so that they can be prepared using a suitable carrier technique. material, for example sterile, germ-free water, can be turned on before use.

The new compounds are new, valuable antibiotics, 50 The compositions can also be used in suitable forms which are active against various gram-positive and gram-negative {for the abation by the mucous bacteria and therefore the human and veterinary skins of the nose and neck and bronchial tissues and they are used in medicine. The new compounds can also be prepared in the form of powdery or therefore antibacterial drugs for the treatment of liquids for inhalations or in infections caused by gram-positive and 55 form of pastules or preparations for brushing the neck. For Gram-negative bacteria, for example, medical use on the eyes or ears, gen Staphylococcus aureus, Escherichia coli, Klebsiella pneu - the preparations as special capsules in liquid or semi-momae, Bacillus subtle, s, Salmonella typhosa Pseudomonas and liquid form or as a door and the. Bactenum proteus. The bactericals obtained according to the invention can be used for tical applications can also be used as additives for feed, hydrophobic or hydrophilic bases as ointments, creams, and for food preservatives and as formulations, formulations or powders. infectious agent. For example, they can be used in aqueous compositions in concentrations in the range from 0.1 to 100. In addition to a carrier material, the parts of the antibiotic obtained according to the invention can be used per million parts of solution , Binders, antioxidants, preservative-dental and dental equipment to destroy agents, lubricants, suspending agents, viscosity agents or or to inhibit, and also as bactericides in industrial flavors and the like. Furthermore, applications, for example in the case of water-based materials, can also have other active substances in the compositions

628 051 16

Components are present to use a broader spectrum of the anti-benzyl bromide, one obtains N, N-bis-p-methoxybenzyl-biotic activity. thienamycin-p-methoxybenzyl ester.

For veterinary medicine, the compositions can be used, for example, as preparations which can be inserted inside the breast, Example 2

5 N-p-nitrobenzyl-thienamycin-p-nitrobenzyl ester and N, N-bis formulated with either a fast or slow release effect. p-nitrobenzyl-thieno.mycin-p-nitrobenzyl ester

The doses to be administered depend on a solution of 71 mg of thienamycin in 2 ml of dimethyl sulfate.

Significant part of the condition of the treated patient from oxide is with a solution of 27 mg triethylamine in 0.27 ml and of the weight of the patient, the route of administration methylene dichloride and then with 56 mg of p-nitrobenzyl and the frequency of administration, the parenteral 10 bromide added. The mixture is stirred for 30 min at 25 ° C. and route is preferred for general infections and the oral solution is then mixed with 7 ml of methylene dichloride and 7 ml of 0.1 pH route for infections of the intestine. Generally contains 9 buffers. The organic phase is separated off, washed with water and a daily oral dosage of about 15 to about 600 mg of active sodium chloride solution and evaporated. The back component per kg of body weight of the patient at one or more times is administered on a 20.32 x 20.32 cm (8 x 8 in.) Large 1000 administrations per day. A preferred daily 15 jx-Si02 plate is chromatographed, the development with dosage for adults is in the range of about 80 to 120 ethyl acetate. The band or strip at 0.5 to 2.5 mg of active ingredient per kg of body weight. cm is extracted with ethyl acetate. Here you get the N-p-

The present compositions can be in various nitrobenzyl-thienamycin-p-nitrobenzyl esters; UV> 267 ny to which single dosage forms are administered, for example Sh (shoulder) 320 m ^; relative extinction * 2 The band, when in solid or liquid orally ingestible dosage forms 20 4; 5 b | f 7 cm, provides the "N, N-bis-p-mtrobenzyl-thienamycin-p-forms. The compositions per single dose, Whether it is nitrobenzyl ester, UV A.max 267 nifi Sh 320 m [x, relative liquid or solid, should be about 0.1% to 99% of active material (absorption) 2.1.

included, with the preferred range between about 10 and

60% lies. The compositions generally contain Example 3

about 15 mg to about 1500 mg of active ingredient; However, in the general manufacture of N, N-dimethylthienamycin it is preferred to use a dosage amount in the range of about 250 to 1000 mg. In the case of parenteral administration, the single dose is generally the pure compound in a slightly acidified aqueous sterile solution or in the form of a soluble powder which is intended to be dissolved.

The following examples are intended to illustrate the compounds, the method and the pharmaceutical compositions of the invention in more detail;

however, they should not be interpreted in a restrictive sense. ^ 12.7 mg of thienamycin, which was dissolved in 2 ml of 0.07% aqueous formaldehyde solution, are hydrogenated at a pressure of 1.81 kp / cm 2 (40 psi) in the presence of 14 mg of platinum oxide

Iletr'.e.lung of N, N-di-p-teit.-butylbenzyl-thienamycin-p-tert.- catalyst. After 4 hours, the solution is brought to a pH

Value of 7.0 and chromatographed on a column containing 15 ml of 40 XAD-2 resin. The column is eluted with water. A fraction is collected which contains a mixture of thienamycin and N, N-dimethylthienamycin. Analytical high pressure liquid chromatography on C18-Porasil with 10% tetrahydrofuran in water as solvent gives 45 two peaks with retention times of 1.75 and 2.5 minutes.

Th oh ch3 ch3

cq2h butylbenzyl ester

Th

OH N (R) 2

CO, R

R = -GH,

C (CH3) 3

40 mg of thienamycin are stirred for 3 hours in 0.6 ml of hexamethylphosphoramide (HMPA) with 0.0275 ml of p-tert-butylbenzylbro-mid. The thienamycin dissolves within 30 minutes. The reaction mixture is then diluted with ethyl acetate and washed successively with aqueous K2HP04, water (twice) and sodium chloride solution. The ethyl acetate layer is dried over magnesium sulfate, filtered and evaporated. The residue is chromatographed by thin layer chromatography on silica gel eluting with 5 <: r methanol in CHC13. This gives 4 mg of the pure desired compound (Rf about 0.6); IR (yy ,, film): 3.0, oh; 5.62, β-lactam; 5.90, ester; Nuclear magnetic resonance (NMR)

ib CKCI3): 1.29 (s, t-Bu), 2.6 to 3.3 (m, CH, groups), 3.57 (s, NCTL), 3.8 to 4.4 (m, β-lactam CH groups), 5.23 (s, OCH2), 7.29 (s, aryl); MS: m / e 710,666,624, 367,335,322 (t-Bu = tert-butyl).

According to the same method, but using instead of p-tert-butylbenzyl bromide the equivalent amount of p-methoxy-

Example 4

50 Preparation of thienamycin benzyl ester stage A:

N- (p-nitrobenzyloxycarbonyl) thienamycin sodium salt

43 mg of thienamycin are mixed with 0 ml of tetrahydrofuran / water (1: 1) at 0 ° C. 102 mg (10 equivalents) of sodium bicarbonate are added to the mixture with rapid stirring, and 4 equivalents of chloroformic acid o-nitrobenzyl ester are then added dropwise with stirring over the course of 2 minutes. After 30 minutes, the pH is adjusted to 7 with 25% aqueous phosphoric acid and the solution is extracted three times with ether. The aqueous portion is adjusted to a pH of 2.2 at 0 ° C and with 500 mg of solid NaC! transferred. The cold, acidic solution is extracted three times with cold ethyl acetate. The combined extracts are quickly washed back with cold sodium chloride solution, dried over magnesium sulfate and filtered. The filtrate is back extracted with 10 ml of water, to which 1.75 equivalents of solid NaHCO have been incorporated. The extract is freeze-dried at 20 ° C in a vacuum; you get the desired connection.

17th

628 051

Level B:

N- (p-nitrobenzyloxycarbonyl) thienamycin benzyl ester

The product from stage A) in 7.5 ml of ethyl acetate is reacted for 2 hours at 4 ° C. with an excess of phenyldiazomethane (4 ml of a solution containing 20 mg / ml of ether) 5. The mixture is then evaporated to a wet residue at 20 ° C. under reduced pressure. The desired compound is isolated by thin layer chromatography eluting with ethyl acetate / ether (9: 1); 17.5 mg of N- (p-nitrobenzyloxycarbonyl) thienamycin io benzyl ester are obtained.

Level C:

Thienamycin benzyl ester

The compound from stage B) is dissolved in ethanol, 35 mg of PtO 2 is added and the mixture is hydrogenated for 45 minutes at a pressure of 3.51 kp / cm 2 (50 lbs.). Then the reaction mixture is centrifuged, the liquid is decanted and, after evaporation of the preparative thin layer chromatography on silica gel, it is subjected to elution with methanol / chloroform 20 (1: 4).

Example 5

Production of N, N-dimethyl-thienamycin

A mixture of 18 mg of thienamycin benzyl ester, 14 mg of 25 methyl iodide and 4 mg of MgO in 2 ml of hexamethylphosphoramide is stirred for 1 hour at 25 ° C. and then poured into hexane. The resulting precipitate of crude N, N-dimethyl-thien-amacin benzyl ester is taken up in ethyl acetate and purified by thin layer chromatography on silica gel. 30th

A solution of 10 mg of N, N-dimethyl-thienamycin-benzyl ester in 1 ml of dioxane / water (4: 1) is stirred for 2 hours at a pressure of 2.81 kp / cm2 (40 psi) in the presence of mg hydrogenated palladium oxide. The catalyst is then filtered off and the filtrate is shaken with a mixture of 5 ml of 35 ethyl acetate and 5 ml of 0.0 in pH 7 phosphate buffer. The aqueous phase is separated off, strongly concentrated and freeze-dried. This gives N, N-dimethylthienamycin.

40

Example 6

N-benzyl and N, N-dibenzyl-thienamycin

A mixture of 150 mg of thienamycin, 300 ml of benzylbro-mid and 200 mg of sodium bicarbonate in 10 ml of 80% aqueous ethanol is stirred at 23 ° C. for 5 hours. The solution obtained is evaporated to 2 ml under reduced pressure, diluted with 5 ml of water and extracted with ether. The aqueous layer is chromatographed on a column containing 100 ml of XAD-2 resin. The unreacted thienamycin is removed by elution with water. Elution with 50 increasing concentrations of tetrahydrofuran yields a fraction containing N-benzylthienamycin and then a fraction containing N, N-dibenzylthienamycin. The products are isolated by freeze drying.

The same method, but using the equivalent amount of allyl bromide instead of 55 benzyl bromide, gives N-AlIIyl- and N, N-DialIyI-thienamycin.

Example 7

N-ethyl-N, N-bis-p-methoxybenzyl-thienamycin-p-methoxy-60 benzyl ester iodide

A solution of 100 mg of N, N-bis-p-methoxybenzylthien-amycin-p-methoxybenzyl ester and 0.5 ml of ethyl iodide in 1 ml of dimethylformamide is stirred at 25 ° C. for 2 hours. The unreacted reaction components and the excess solvent are then evaporated off under reduced pressure and the residue is rubbed with ether. You get the solid product.

Example 8

N, N-dimethyl-N-benzylthienamycin

A solution of 200 mg of N-benzylthienamycin in 5 ml of 50% aqueous dioxane is adjusted to a pH of 8.4 (titrated). Then 0.5 ml of dimethyl sulfate in 2 ml of dioxane is added with stirring for 10 minutes. The pH is kept at 8.4 by adding in sodium hydroxide solution using an automatic titration device. The mixture is then stirred for a further hour, then diluted with 10 ml of water, adjusted to a pH of 7 and extracted with ether. The aqueous phase is concentrated to 5 ml and chromatographed on 200 ml of XAD-2 resin. The column is eluted with water and then with 20% aqueous tetrahydrofuran. The N, N-dimethyl-N-benzyl-thienamycin is isolated from the tetrahydrofuran eluate by freeze-drying.

Example 9 N-methyl-thienamy a-benzyl ester

A solution of 50 mg of N-methyl-thienamycin in 1 ml of water and 1 ml of dioxane is cooled to 0 ° C. and adjusted to a pH of 5 with in sulfuric acid. 37 mg of phenyldiazomethane in 0.5 ml of dioxane are then added over the course of 5 minutes, the pH being kept at 5 to 5.5 using an automatic titration device. The mixture is then diluted with 5 ml of water and extracted with ether. The aqueous phase is overlaid with ethyl acetate, cooled and adjusted to a pH of 2.5. The ethyl acetate phase is centrifuged off, the aqueous phase is adjusted to pH 8.0 with sodium bicarbonate and extracted twice with ethyl acetate. The extracts are combined and evaporated. The product is isolated by preparative thin layer chromatography on silica gel using chloroform / methanol (5: 1) as solvent.

The same method, but starting from N, N-dimethyl-thienamycin, gives N, N-dimethyl-thien-amycin-benzyl ester.

Example 10 N, N, N-Trimethylthienamycin

A solution of 150 mg of thienamycin in 7.5 ml of 0.1 in pH 7 phosphate buffer and 7.5 ml of dioxane is adjusted to a pH of 8.4, 1 ml of dimethyl sulfate is added and the mixture is stirred rapidly for 40 minutes, the pH is kept at 8.4 by adding sodium hydroxide solution. Then the solution is extracted twice with ether. The aqueous phase is evaporated to 4 ml and applied to a column containing 200 ml of XAD-2 resin. The column is eluted with water and the course of the separation is monitored by high-pressure liquid chromatography on C18-Bondapak resin using 10% aqueous acetonitrile. The product has 1.2 times the retention time of thienamycin. The fractions containing the product are combined, evaporated and freeze-dried. 41 mg of N, N, N-trimethylthienamycin are obtained. Electrophoresis (50 V / cm, 2 hours, pH 7 buffer) results in a bioactive zone which moves 6 cm in the direction of the cathode. The NMR spectrum (60 MHz, D20) shows a strong methyl singlet at 6.78 t with an integral 3X side chain methyl doublet at 8.7 t; UVXmax298 mjx, E% 185.

According to the above method, but using 0.25 ml of dimethyl sulfate and allowing the reaction to proceed for 15 minutes, a mixture is obtained which contains N-methyl and N, N-dimethylthienamycin and is separated by column chromatography.

Example 11

Production of N, N, N-trimethylthienamycin

A suspension of 20 mg N, N-dimethyl-thienamycin in

628 051

18th

10 ml of tetrahydrofuran is mixed with 0.2 ml of hexamethyldisilizane and 0.1 ml of trimethylchlorosilane in a nitrogen atmosphere with stirring. The mixture is stirred vigorously at 23 ° C. for 20 minutes and then centrifuged. The supernatant solution is evaporated under reduced pressure. The remaining oil is dissolved in 1 ml of tetrahydrofuran, 0.05 ml of methyl iodide is added with vigorous stirring and the mixture is stirred for 30 minutes. Then 5 ml of ethyl acetate and 5 ml of 0.1 in pH 4 phosphate buffer are added, the mixture is stirred for a further 15 minutes at 25 ° C. and then adjusted to a pH of 7. The aqueous layer is then separated, concentrated to 1 ml and applied to a column containing 20 ml of XAD-2 resin. It is eluted with water and then with 10% tetrahydrofuran. A fraction containing N, N, N-trimethylthienamycin is obtained, which gives a solid product by freeze-drying.

Example 12

N, N-dimethylthienamycin pivaloyloxymethyl ester

A solution of 30 mg of N, N-dimethylthienamycin and 20 25 mg of pivaloylmethyl bromide in 0.2 ml of hexamethylphosphoramide was stirred at 23 ° C. for 1 hour. Then 5 ml of ethyl acetate are added and the mixture is extracted successively with aqueous sodium bicarbonate solution, water and saturated sodium chloride solution. The organic phase is dried, strongly concentrated and chromatographed on a 20.32 X 45.72 cm (8 X18 in.) Large 1000 j.i-SiO 2 plate using chloroform / methanol (5: 1) as solvent. The strip containing the N, N-dimethyI-thienamycin-pivaloyImethyl ester is scraped off and eluted with ethyl acetate. 30th

Example 13

N, N-Dimethylthienamycin-3-methyl-2-butenyl ester hydrochloride

A solution of 30 mg of N, N-dimethylthienamycin in 35 0.5 ml of 3-methyl-2-butenyl alcohol, which contains 3.6 mg of HCl, is mixed with 21 mg of dicyclohexylcarbodiimide and stirred at 23 ° C. for 1 hour. The dicyclohexylurea is then filtered off, the filtrate is evaporated and the residue is rubbed with ether. A solid material remains, which contains N, N-dimethyl 40-thienamycin-3-methyl-2-butenyl ester hydrochloride.

According to the above method, but using methylthioethanol instead of 2-methyl-2-butenol, N, N-dimethylthienamycin methylthioethyl ester is obtained. 45

Example 14

O-A cetyl-N, N-dimethylthienamycin

100 mg of N, N-dimethyl-thienamycin are introduced into a mixture of 0.3 ml of acetic anhydride and 1 ml of pyridine. 50 The mixture is left to react at 23 ° C. for 3 hours and then evaporated to dryness in vacuo. The solid residue is dissolved in water and the solution is chromatographed on 100 ml of XAD-2 resin. After elution with water, the product is eluted with 10% tetrahydrofuran. The fractions containing the 0-acetyl-N, N-di-55 methylthienamycin are combined, evaporated and freeze-dried.

Example 15

Thienamycin benzyl ester 60

A solution of 47 mg of thienamycin in 1 ml of water and 1 ml of dioxane is cooled to 0 ° C. and adjusted to a pH of 5 with in sulfuric acid. Then 37.2 mg of phenyldiazomethane in 0.5 ml of dioxane are added in the course of 2 minutes, the pH being kept at 5 with the aid of an automatic titration device. After a further 5 minutes, 5 ml of water are added and the mixture is extracted with ether. The aqueous phase is overlaid with ethyl acetate, cooled and adjusted to a pH of 2.5. The ethyl acetate phase is separated off, the aqueous phase is adjusted to a pH of 8 with sodium bicarbonate and extracted twice with ethyl acetate. The two extracts are combined and dried over anhydrous magnesium sulfate. Thin layer chromatography on silica gel using methanol / chloroform (1: 5) gives a single ninhydrin positive tail at Rf 0.24. The UV spectrum of the ethyl acetate solution shows a maximum (Xmax) at 318 m [x with an optical density of 250.

Example 16

N, N-Dimethyl-0-sulfothienamycin benzyl ester

A solution of 39 mg of N, N-dimethyl-thien-amycin-benzyl ester in 0.3 ml of pyridine is mixed with 17 mg of sulfur trio-oxide / pyridine. The mixture is stirred at 25 ° C for 3 hours. The excess pyridine is then evaporated off under reduced pressure, the residue is taken up in 5 ml of water to which 10 mg of sodium bicarbonate have been incorporated and extracted once with ethyl acetate. The aqueous solution is concentrated to 2 ml and chromatographed on 50 g of XAD-2 resin. The fractions containing the N, N-dimethyl-0-sulfothienamycin-benzyl ester are combined, evaporated and freeze-dried.

Example 17

N, N-dimethyl-osulfothienamycin sodium salt

24 mgN, N-dimethyl-0-sulfothienamycin-benzyl ester in the form of a solution in 1 ml of water containing 5 mg of sodium bicarbonate are for 2 hours at 23 ° C and a pressure of 1 atm in the presence of 20 mg of palladium oxide hydrated. The catalyst is then filtered off and the filtrate is chromatographed on 20 g of XAD-2 resin. The fractions containing the N, N-dimethyl-O-sulfothienamycin sodium salt are combined, evaporated and freeze-dried.

Example 18

O-formyl-N, N-dimethylthienamycin benzyl ester

A solution of 100 mg of N, N-dimethyl-thienamycin-benzyl ester in 1 ml of pyridine is mixed with a mixture of 100 mg of formic acid and 200 mg of acetic anhydride. The mixture obtained is stirred for 2 hours at 25 ° C. and then the excess reagents are removed under reduced pressure. The residue is taken up in ethyl acetate and the product is isolated by thin layer chromatography on silica gel using ethyl acetate / chloroform (1: 1) as solvent.

Example 19

O-formyl-N, N-dimethylthienamycin

50 mg of O-formyl-N, N-dimethyl-thienamycin-benzyl ester in the form of a solution in 2 ml of 90% ethanol are for 4 hours at 23 ° C and a pressure of 1 atm in the presence of 50 mg of 10% palladium -Active carbon hydrated. The catalyst is then filtered off, the filtrate is evaporated and the residue containing the O-formyl-N, N-dimethyl-thienamycin is purified by chromatography on XAD-2 resin.

Example 20

The following new compounds are prepared by the methods explained in the examples above and in the description:

Oh"

A

é

SCH2CH2NR5R6R7

.COXR

a0

19th

628 051

connection

X

r3

R

r5

r6

r7

A

î)

0

H

-ch2-o-c-ch3

H

ch3

ch3

ch3

Cl

il o

2)

0

ch3

-

ch3

ch3

ch3

-

3)

0

so3h

Well ch3

ch3

ch3

-

4)

0

H

-

ch3

ch3

c2h5

-

5)

0

h h

c2h5

ch3

-

-

6)

0

h h

c2h5

c2h5

-

-

7)

0

H

-ch2-0-ch2c (ch3) 3

-ch2ch = ch2

h h

Cl

B)

0

h h

-ch2ch2ch3

H

-

-

9)

0

h h

-ch02

H

-

10)

0

h h

-ch02

ch3

-

H)

0

h h

-c-03

ch3

-

12)

0

H

-

ch02

ch3

Cil;

-

13)

0

H

-ch2-ch = ch2

-ch2-ch = ch2

h h

Cl

14)

0

H

-ch2-c = ch2

1

-ch2-c = ch2

1

h h

CI

1

ch3

J

ch3

15)

0

H

-ch2ch2n (c2h5) 2

ch3

h h

hso4

16)

0

-ch2och3

p-nitrobenzyl

-ch2och3

-ch2och3

-

-

17)

s h

-ch2ch2ch3

-ch2ch = chch3

h h

Cl

18)

0

h h

-ch2ch2ch = ch2

H

-

-

19)

0

h h

-ch2 "0

H

-

-

ft

20)

0

-coch3

m h

-cw h

-

-

il o

21)

0

h h

-ch2ch2ch2ch2-

-

-

22)

0

H

-ch2ch2n- (ch3) 2

-ch2ch2-n- (ch3) 3

H

h hpo4

23)

0

h h

-ch2-c-ch3

II

H

-

-

II

O

24)

0

h h

-ch2ch2 (och3) 2

H

-

-

25)

0

h h

-ch2-c = ch h

-

-

26)

0

h h

-ch2ch2o-ch2ch2-

H

-

-

27)

0

H

-ch2c-0

II

-ch2c-0

II

ch3

-

-

11

O

II

O

28)

0

h ch2s-ch3

-ch2sch3

-ch2sch3

-

-

29)

0

H

-ch2-0-c (ch3) 3 II

-ch2-o-c (ch3) 3

II

H

-

-

II

O

II

O

30)

0

Si (CH3) 3

Si (CH3) 3

-ch2ch2c = n h

-

-

31)

0

H

H

-ch2ch2-c-och3

II

H

-

u

. O

32)

0

H

H

-o-n02

H

-

33)

34)

35)

36)

37)

0

-c-ch2nh2 h

O

o h o h o h o h h

h h h

"no2

-c2h5

ycu2 -CHr <I ch2

-ch (ch3) 2

-ch2ch2ch2ch3 -ch2ch-ch3 I

ch,

-c2h5

> ch2

-ch2- / i ch2

h h h h

ch, coo

628 051

20th

connection

X

R3

R

38)

O

H

H

39)

O

H

H

40)

O

H

H

41)

O

H

H

42)

O

H

H

43)

O

H

H

44)

O

H

H

45)

O

H

H

46)

O

H

H

47)

O

H

H

48)

O

H

H

49)

O

H

H

50)

O

H

H

51)

O

H

H

52)

O

H

H

53)

O

H

H

54)

O

H

H

55)

O

H

H

56)

O

H

H

57)

O

H

H

58)

O

H

H

59)

O

H

H

60)

O

H

H

61)

O

H

H

62)

O

H

H

63)

O

H

H

64)

O

H

H

r5

-chch2ch3

I.

ch3

-ch2ch2ch2ch2ch3 -ch-ch2ch2ch3 I

ch3

-ch2ch-ch2ch3 I

ch3

-ch2ch2ch-ch3 I

ch3

-ch-ch-ch3 I I ch3 ch3

-ch2-c (ch3) 3

-ch2ch2ch2ch2ch2ch3

-chch2ch (ch3) 2 I

ch3

-ch2ch = ch2 -ch2ch-ch3 I

ch3

-ch-ch = ch2 I

ch2

-ch2ch = ch-ch3 -ch2-ch2-ch = ch2 I

ch3

-ch-ch = ch2 I

Q> h5

-ch2-ch = ch-ch2ch3 -ch2ch = ch-ch2ch2ch3

_ch, -c = ch-ch2ch3 I

ch3

-ch2-ch2-ch = ch-ch3 ch2ch = ch-ch-ch3

ch,

- <3

-ch2ch2-

o o

-ch2-ch =

n

-ch

-ch r6 h h h h

h h

h h h h

h h

h h

h h h h h h h

h h h

h h

H

21st

Connection X R3

65)

66)

67)

68)

69)

o h o h o h o h o h r h h h h h

R5

-ch2- CJ

0 '

-ch2-ch = ch-0 —ch ~ —ô och-

-ch,

-ch.

r6 h h h h

-ch2ch = ch2 I

ch3

628 051

R7 A

70)

71)

72)

73)

74)

75)

76)

77)

78)

79)

0 = phenyl o h o h o h o h o h o h o h o h o h o h h h h h h

H

h h

h h

-ch3

-ch3

-ch3 -ch3

-ch3 -ch3 -ch3 -ch3 -ch3 -ch3

-ch2ch = ch-ch3 -ch-ch = ch2 I

ch3

-ch = ch-ch2ch3 -ch2-c = ch2-ch3 I

ch3

-ch2-ch = ch-ch-ch3 -I

ch,

-ch ch o

-O-0

-ch, -ch = ch-0

45

Production of thienamycin by total synthesis

Manufacture of raw materials

In addition to thienamycin, it is obvious to the person skilled in the art

that its various isomers alone or as mixtures as

Starting materials in the production of the inventive

Connections can be used. Some of these isomes- 50

Ren are available from natural fermentation products

(see below). However, all isomers can be obtained by total synthesis (see below) in the form of a mixture of 4

reoisomers which have bactericidal activity and which can be separated by customary technical processes

nen. The 4 diastereoisomers (2 eis, 2 trans) can be separated chromatographically. The separation of a given d / 1 pair with optically active acids or bases is carried out according to customary methods. It should be noted

that the absolute configuration of the first identified level 60:

transition material (I) 5R 6S 8R. Preparation of 4- (2-acetoxyvinyl) azetidin-2-one

, sch2ch2nh2 cooh

628 051

22

U

H, C = CH-CH = CHOC-CH3 + O = C = N-S02CI-

II

O

0

ch = ch0cch,

so2ci

0

/

/

0

ch = ch0cch.

1

• 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 N2 in a bath at -20 ° C.

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

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

A solution of 2 g of sodium sulfite and 5 g of K2HP04 in 20 ml of H20 is prepared during the aforementioned 0.5 hour reaction time and cooled on an ice bath; 20 ml of ether are added to the mixture with vigorous stirring on an ice bath. After the end of the 30-minute reaction time, the reaction mixture, again using N2 pressure and a polytetrafluoroethylene tube, is transferred from the reaction flask, which is kept in a bath of −20 ° C., to the vigorously stirred hydrolysis mixture. The quick, dropwise addition is complete after 5 minutes. The hydrolysis is allowed to take place for a further 5 minutes. The hydrolysis mixture has a pH of 6-8, preferably pH 8.

15

25th

35

40

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

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

A column of 10 g of Baker's silica gel, packed in ether, is made and the ether concentrate is added to the top and allowed to run through. The residue in the flask is rinsed three times with 2 ml of ether, pipetted off each time and added to the column. Then it is eluted with ether.

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

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

0

, occtu

_nh

A solution of 4- (2-acetoxyvinyl) -2-azetidinone (10.0 g, 0.065 mol) in 200 ml of ethyl acetate containing 100 mg of a 10% palladium / C catalyst is hydrogenated in a Parr shaker at 25 ° C under a pressure of 2.8 kg / cm2 over 15 minutes. The mixture is filtered through a bed of Supercel and washed with additional ethyl acetate. The combined filtrate is washed in vacuo to give 4- (2-acetoxyethyl) -2-azetidinone (10.0 g) as a crystalline solid. When recrystallizing from ether, white crystals are obtained: mp 44 to 47 ° C; IR (CHCl3) (t 5.66, 5.74; KMR (CDC13) t3.44

0

(broad s, 1, NH), 5.82 (m, 2, CH2OCOCH3), 6.29 (m, 1, C-4H, 6.87 (1/2 AB pattern is further divided into four by C-4H and NH, 1, Jgem = 12.8 Hz, J = 4.5 HHnh = 1.9 Hz, 7.38 (1/2 AB pattern is still divided fourfold by C-4H and NH, 1, Jgem = 12.8 Hz, J = 2.3 Hz, JNH = 1.0 Hz). 7.93 and 8.02 (s on m, total 5, OCOCH3 and CH2CH2OCOCH3, respectively).

Level C:

60 Preparation of 4- (2-hydroxyethyl) -2-azetidinone

55

23

628 051

A solution of 4- (2-acetoxyethyl) -2-azetidinone (2.24 g, 0.014 mol) in 25 ml of anhydrous methanol with a solution of sodium methylate (77 mg, 1.4 mmol ) treated in 5 ml of anhydrous methanol. After stirring for one hour, the solution is neutralized with glacial acetic acid. Removal of the methanol in vacuo gives the crude 4- (2-hydroxyethyl) -2-azetidinone as an oil. The product is chromatographed on silica gel by eluting with 10% methanol / CHClj to give 1.55 g of the alcohol: mp 50 ° C; IR (CHC13) h 5.67; KMR (CDCl3) t3.20 (broad s, 1, NH), 6.24 and 6.28 (m at t, total 3, C-4H and CH? QH

accordingly), 6.90 (broad s at 1/2 AB pattern further splits into four by C-4H and NH, total 2, OH and C-3H accordingly, Jgem = 13.0 Hz, Jvic = 4.2 Hz , Hnh = 1.6 Hz), 7.42 (1/2 AB pattern further splits into four by C-4H and NH, 1, s C-3H, Jgem = 13.0 Hz, Jvic = 2.2 Hz , JNH = 1.1 Hz), 816 (m, 2, CH2CH2OH).

Level D:

io Preparation of 8-oxo-2,2-dimethyl-3-oxa-l-azabicyclo [4,2,0] octane y

Oh

-nh

A solution of 4- (2-hydroxyethyl) -2-azetidinone (1.87 g, 20 KMR (CDC13) t: 0.016 mol) and 2,2-dimethoxypropane (1.69 g, 0.016 mol) in 25 ml of anhydrous methylene chloride is treated with boron trifluoride etherate (0.201 ml, 0.002 mol) at 25 ° C. The solution obtained is stirred for 10 minutes. Removal of the solvent under reduced pressure gives an oil 25 (2.5 g). Chromatography of the crude product on a silica gel column using 2: 1 ethyl acetate / benzene as the eluent gives 8-oxo-2,2-dimethyl-3-oxa-l-azabi-cyclo- [4,2,0] -octane (1 , 59 g) as a crystalline solid. Recrystallization from ether / hexane gives a product with 30 F. 60 to 61 ° C.

6.02-6.28, m, 2H, C-4 methylene 6.22-6.62, m, IH, C-6 methine 6.90, dd, 1H, J7i7 = 14 Hz, J6.7 = 4 , 5 Hz C-7 proton ice to C-6H 7.47, dd, 1H, J7.7 = 14 Hz, J6> 7 = 2 Hz C-7 proton trans to D-6H 7.82-8.68, m, 2H, C-5 methylene 8.23, s, 3H! c_2 Methyle

8.57, s, 3H |

ir (CHC13) | ì: 5.73 (ß-lactam)

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

ho

A solution of 8-0x0-2,2-dimethyl-3-oxa-l-azabicyclo- [4 , 2,0] octane in anhydrous tetrahydrofuran, which has been cooled to -78 ° C, added. After 2 minutes, the lithium eno 50 lat obtained is treated with excess acetaldehyde. The solution is stirred at -78 ° C for 30 minutes and then poured into water. The aqueous phase is saturated with sodium chloride and extracted with ethyl acetate. The combined ethyl acetate solutions are dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give the crude product. By purification by means of chromatography on silica gel using ethyl acetate /

8-Oxo-2,2-dimethyl-7a and ß- (l-hydr-oxyethyl) -3-oxa-l-azabicyclo [4,2,0] octane are obtained in benzene. eo

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

IR (CH2C12) h: 5.72h (β-lactam)

KMR (CDC13) t: 5.53-6.43, m, 4H, C-4 methylene + C-6 methine + C-9 methine 6.90, dd broad s, 2H, J7.9 = 9 Hz J6 ) 7 = 5.5 Hz, C-7 methine + ÒH

65

7.70-8.83, m, 2H, C-5 methylene 8.27, s, 3HI c_2 methyl 8.60, s, 3H)

8.78, d, 3H, J910 = 6.5 Hz, C-10 methyl

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

IR (CHC13) | ì: 2.9 broad O-H

5.73 ß-lactam KMR (acetone - d6) ô: 4.23-3.33, m, C-6 methine 3.33, broad s, OH

2.83, dd, J = 2 Hz, 6 Hz c_7 methine

2.67, dd, J = 2 Hz, 8 Hz 1.93-1.63, m, C-5 methylene 1.63, sì c_2 meth le

1.40, s I

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

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

628 051

24th

->

r fi0ch2-0

<=>

• no,

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

IR (CH2C12) and: 5.68 (β-lactam and carbonate), 6.19

and 6.54 (nitro)

; KMR (CDCI3):

1.67, d, 2H, ArH 2.37, d, 2H, ArH 4.67, s, 2H, ArCH2 4.67-5.22, m, CH3CH 5.98-6.25, m, 2H, C-4 methylene 6.25-6.62, m, 1H, C-6 methine 7.75-8.83, m, 2H, C-5 methylene 8.22, s, 3H, C-2 methyl 8, 50-8.59, m, 5H, C-2 methyl + CH3CH

25th

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

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

• R =

O

-c-0-ch.

/ "^ V-KO

8-Oxo-3-oxa-2,2-dimethyl-7a- (lp-nitrobenzylcarbonyl-dioxyethyl) -l-azabicyclo- [4,2,0] -octane (1.0 g) is dissolved in 8 ml acetic acid and 2 ml of water dissolved and heated to 65 ° C 50 for 1.25 hours. The acetic acid and water are removed under reduced pressure and the residue is taken up in benzene and evaporated to give trans-3- (lp-nitro-benzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone as a mixture of the diastereoisomers . 55

IR (CH2Cl2) u: 5.67 (β-lactam), 5.72 shoulder, 6.20

and 6.57 (nitro)

KMR (CDCI3): 1.73, d, 2H, H = 8.5 Hz, ArH

2.43, d, 2H, J = 8.5 Hz, ArH 3.63, broad s, 1H, NH 60

4.37-5.13, m, 1H, CH3CH 4.72, s, 2H, ArCH2

6.07-6.53, m, 1H, C-4 methine 6.23, t, 2H, J = 5.5 Hz, CH2OH 6.73-6.93, m, IH, C-3 methine 7, 63-8.97, m, 3H, CH2CH2OH 8.53, d, J = 6.5 Hz, CH3CH

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

Stage D ', E, F' and G 'as an alternative to stages D, E, Fund G for the production of 3- (l-p-nitrobenzylcarbonyldioxy-ethyl) -4- (2-hydroxyethyl) -azetidinone

25th

628 051

or

O

r = -coch

Levels D ', E', F and G '

Preparation of l- (2-tetrahydropyranyl) -4- [2-tetrahydropyranyl) oxyethyl] -2-azetidinone

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

35 KMR (CDC13) t: 5.13-5.60, m, OCH

5.83-6.85, m, C-4H + OCH2 6.95, dd, J = 5 Hz and 15 Hz C-3 methylene 7.35, dd, J = 3 Hz and 15 Hz 40 7.62- 8.95, m, CHCH2CH2CH2CH2

+ chch2ch2o

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

If you work in the for the production of 8-oxo-2,2-cis and trans-l- (2-tetrahydropyranyl) -3- (l-hydroxyethyl) -4-

dimethyl-7a and β- (1-hydroxyethyl) -3-oxa-1-azabicyclo- [2- (2-tetrahydropyranyl) -oxyethyl] -2-azetidinone.

[4,2,0] octane from 8-oxo-2,2-dimethyl-3-oxa-l-azabicyclo-65

[4,2,0] -octane described way and uses l- (2-tetra preparation of cis and trans-l- (2-tetrahydropyranyl) -3- (l-hydropyranyl) -4- [2- (2nd -tetrahydropyranyl) -oxyethyl] -2-acetidi- p-nitrobenzylcarbonyldioxyäthyl) -4- [2- (2-tetrahydropyranyl) -non, so you get a diastereomeric mixture of both oxyäthyl] -2-azetidinone

628 051

26

3rd

/ ^ y r

ÌOCH,

^ wNO,

The procedure is the same as for the production of 8-oxo-2,2-dimethyl-7a- (lp-nitrobenzylcarbonyldioxyethyl) -8-oxa-l-azabicyclo [4,2,0] octane from 8-oxo -2,2-dimethyl-7- (l-hydroxyethyl) -3-oxa-l-azabicyclo [4,2,0] octane and uses Trans-l- (2-tetrahydropyranyl) -3- (l-hydroxyethyl ) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone, this gives trans-l- (2-tetra-

20th

25th

hydropyranyl) -3- (l-p-nitrobenzylcarbonyldioxyethyl) -4- [2- (2-tetrahydropyranyI) -oxyethyl] -2-azetidinone. The cis-diastereoisomer is obtained in an analogous manner.

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

A solution of trans-l- (2-tetrahydropyranyl) -3- (lp-nitrobenzylcarbonyldioxyäthyl) -4- [2- (2-tetrahydropyranyl) -oxyethyl] -2-azetidinone in methanol at 25 ° C is with a 0 , 1 molar equivalent of p-toluenesulfonic acid monohydrate treated. The solution is stirred for 2 hours and then neutralized with pH 7 phosphate buffer. The product is extracted into ethyl acetate. The ethyl acetate solution is washed with salt water, dried over magnesium sulfate and filtered. The

45

or

Oh

0

//

jnh

The filtrate is evaporated under reduced pressure to give trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone. The cis diastereoisomer is obtained in an analogous manner.

Stage H. Preparation of cis- and trans-3- (l-p-nitrobenzylcarbonyldi-oxyethyl) -4- [2,2-bis (2-hydroxyethyl) thioethyl] -2-azetidinone r

î ^

zZX-_zxch0 _nh

r '

v

0

coch,

■ - <

■ NO,

27th

628 051

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

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

The acetonitrile solution of trans-3- (lp-nitrobenzylcarbo-nyldioxyäthyI) -4- (2-oxoäthyl) -2-azetidinone, which has been prepared as above, is with nitrogen at 0 ° C with 2-mercaptoethanol (0.386 ml , 5.5 mmol) and immediately thereafter treated with boron trifluoride etherate (0.176 ml, 1.43 mmol). After stirring for 15 minutes, this solution is divided between aqueous dipotassium hydrogen phosphate (1.5 g in 4 ml of water) and 12 ml of ethyl acetate. The aqueous phase is used a second time

Extracted ethyl acetate. The combined ethyl acetate solutions are washed with brine, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give 229 mg of an oil. The product is purified by preparative thick-layer chromatography on silica gel and developed with ethyl acetate, 118 mg of trans-3- (lp-nitrobenzylcarbonyldioxyäthyl) -4- [2,2-bis- (2-hydroxyethyl) -thioethyl] -2-azetidinone as receives a colorless oil.

IR (CH2C12) h: KMR (acetone-d6) r:

5.75 (5.79 shoulder) β-lactam and carbonate 6.20.6.55 nitro 1.70, d, J = 8.5 Hz, 2H, ArH 2.28, d, J = 8.5 Hz , 2H, ArH 2.48-2.88, m, 1H, NH 4.63, s, ArCH2 ì

4.63-5.12, m, CH3CH / 3H

13H

20 5.80, t, J = 7 Hz, CH2CH <"

5.80-7.45, m, C-4H + C- 3H + SCH2CH2OH 7.63-8.33, m, 2H, CH2CH 25 8.53, d, J = 6.5 Hz 3H, CH3CH

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

Stage I. Preparation of Trans-3- (l-p-nitrobenzylcarbonyldioxyäthyl) - 4- [2,2 ~ bis- (2-azidoäthyl) -thioäthyl] -2-azetidinon ocoopn3

À

. Oh

X v ßE

jnh

+ 2CHoS0 '/ iC 1 NaN-} \

3 2 ~ THF ^ "T5HSCF

ocoopnb

/

c ^ -

nh

To a solution of 211 mg (molecular weight = 474; 0.445 55 mmol) of trans-3- (lp-nitrobenzylcarbonyldioxyäthyl) -4- [2,2-bis- (2-hydroxyethyl) -thioethyl] -2-azetidinone in 5 ml of tetrahydrofuran (THF) (distilled from lithium aluminum hydride) at 0 ° C., 103 mg of mesyl chloride (molecular weight = 114; 0.904 mmol) are added to 1 ml of tetrahydrofuran and immediately 60 (134 ml xl triethylamine (molecular weight 101; e = 0.729; 0.967 mmol) The reaction mixture is stirred for 1 hour under N2. The triethylamine hydrochloride is filtered under N2 and washed with a few ml of additional THF. The clear colorless filtrate is concentrated under a stream of N2 and then pumped under high vacuum for 65 minutes. The dimesylate is immediately dissolved in 5 ml of DMSO (distilled from CaH2 at 8 mm and stored over a 4A Linde molecular sieve) in the presence of 347 mg of NaN3 (molecular weight = 65; 5.34 mmol). After stirring overnight and under N2, 10 ml H20 and 20 ml of ethyl acetate (EA) were added n separated and the aqueous layer is washed three times with 10 ml of EA, each organic layer being washed again with 10 ml of H2O and 10 ml of saline. The combined ethyl acetate layers are dried over anhydrous magnesium sulfate and filtered and concentrated under a stream of N2 to give the crude diazide. Preparative thin layer chromatography on silica gel gives trans-3- (l-p-nitrobenzylcarbonyIdioxyäthyl) -4- [2,2-bis- (2-azidoäthyl) -thioäthyl] -2-azetidinone. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

628 051

Level J:

28

co ~ h i 2

c (0h)

co2h

-f 2M2-y> ^ VCKN2 EA ^ Et2 °

c0opnb

! 2nd

c (0h)?

I.

c02pnb

-f

CH2CH2N3 sch2ch2k3

Toi.

■>

0c00pnb h

n oh

, <* Y

(c02pnb) 2

sck2ch2n3 sch2CH2ìnt3

+ n20

pnb

2 X.

V

no.,

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

35

40

on an oil bath, allowing about 1/3 of the toluene to be distilled off. Toluene (dried over a 3 A1 / 16 "Linde molecular sieve) is again added to make up the volume to 50 ml and the azeo drying process is repeated three times. The solution is then refluxed under N2 for one hour and the azeo drying process becomes a final one Repeated times and the reflux treatment is continued for another hour. When the solution obtained is concentrated under a stream of N2, crude X is obtained. The crude material is chromatographed on silica gel, giving X. The cis-diastereoisomers or the cis-trans-mi schung is obtained in an analogous manner.

45

Level K:

0C00PN3

| , sch..ch, n- XsCH2CH2N3

/ V0H + socl * -gf->

(c02fnb) 2

29

628 051

sch0ch0no / Â 2. 5

sch2ch2n3

+ 03P

aq. dmf

ocoopnb

/ S

S

O'

SCH-CH-N-y Z Z 3

sch2ch2n3

"\/H

(io2? NB),

To a solution of 2.80 g of X (molecular weight = 912; 3.07 mmol) in 35 ml of THF (distilled from lithium aluminum hydride) at -20 ° C, 0.3 ml of pyridine (molecular weight = 79; e = 0.982; 3, 73 mmol) (distilled from NaH and stored over a 4A Linde molecular sieve). While stirring under N2, 0.438 g of thionyl chloride (molecular weight = 119; 3.68 mmol) in 1 ml of THF are added dropwise. The reaction mixture is stirred under N2 at -20 ° C for 5 minutes and then at 0 ° C for 1/2 hour and finally at 25 ° C for 1 hour. The pyridine hydrochloride is filtered off under N2 and washed twice with benzene (dried over a 3A1 / 16 "Linde molecular sieve). The combined filtrates and washing solutions are concentrated under a stream of N2, slurried in a small volume of benzene with anhydrous MgSO4, under N2 filtered and then concentrated under a stream of N2

40

Oil under high vacuum for 1/2 hour. To this freshly prepared chlorine compound, 0.885 g of triphenylphosphine (molecular weight = 262; 3.38 mmol) in 66 ml of 9: 1 dimethylformamide (DMF) / H2Ö and then 550 mg of K2HP04 (molecular weight = 174; 3.16 mmol) are added. The reaction mixture is stirred at 25 ° C for 35 minutes. After dilution with EA and saline, the layers are separated and the aqueous layer is extracted three times with EA. The combined EA layers are washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of N2 to give crude 2. The material is chromatographed on silica gel, giving 2. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Level L:

ocoopnb s-ch2ch2n3

S-CH2CH2N3 + Br2

Et20 / C5H12

4th

628 051

30th

OCOOPNB

Br sch2ch2n3

SCÌ ^ Cj ^ Ì ^

(1)

(2) NaH, BKI

OCOOPNB

f

SCH2CH2N3

(C02PNB) 2

"+

S CH.

Sr.

0.2 ml of Br2 (molecular weight = 160; e = 3.12; 3.9 mmol) are added to 7.8 ml of pentane (dried over a 4A Linde molecular sieve). To a solution of 950 mg 2 (molecular weight = 896; 1.06 mmol) in 15 ml of diethyl ether (dried over a 3A1 / 16 "Linde molecular sieve) is added dropwise at 0 ° C and under N2 with stirring with 2.3 ml of The above 0.49m Br2 solution (1.13 mmol) After stirring for 10 minutes at 0 ° C., 114 μl of cyclohexene (molecular weight = 82, e = 0.81; 1.13 mmol) are added, and after 5 minutes at 0 ° C. 53 mg of 57% NaH (57% of 53 mg = 30.2 mg, molecular weight 24.1.26 mmol) in mineral oil are added to the stirred reaction mixture, and 14 ml of ice-cold DMF (distilled from anhydrous CaS04 at 40 mm and stored over ei30

35

40 ..

4A Linde molecular sieve). Stirring is continued under N2 at 0 ° C for 3 hours. The reaction mixture is poured onto a stirred ice-cold mixture of 2.5 ml of KH2P04-40 ml HzO-75 ml EA. After separation of 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 MgSO4, filtered and concentrated under a stream of N2 and then pumped under high vacuum to give the crude 3. Preparative thin-layer chromatography on silica gel gives 3. The cis-diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

Level K:

OCOOPNB

sch2ch2n3

4- Br2 ether-VC5H12 ^

31

628 051

OCOOPNB

B:

I / Br

XHCH,

\

SCÎI2CH2N3

1

(C02PN3) 2

■ + ■ NaH

BlfF

\

OCOOPNB

SCH ^ CH ^ u ^

(C02PNB) 2

0.2 ml of Br2 (molecular weight = 160; 3.9 mmol) are added to 9.16 ml of pentane (dried over a 4A Linde molecular sieve). To 474 mg 3 (molecular weight = 793; 0.598 mmol) in 13 ml of diethyl ether (dried over a 3A 1/16 "Linde molecular sieve) at 0 ° C. under N2, 1.52 ml of the abovementioned 0 are added dropwise with stirring. 42m Br2 solution (0.63mmol).

After 15 minutes at 0 ° C., 33 mg of 57% NaH (57% of 33 mg = 18.8 mg; molecular weight = 24; 0.78 mmol) are added 40 and immediately thereafter 6.35 ml of ice-cold DMF (distilled from anhydrous CaS04 at 40 mm and stored over a 4A Linde molecular sieve). The reaction mixture is stirred for IV2 hours at 0 ° C., then poured into a stirred ice-cold mixture of 1.6 ml in KH2P04-20 ml H20 and 20 ml EA. The layers are separated and the aqueous layer is saturated with NaCl and extracted again with further EA. The combined organic layers are washed once with salt water, dried over anhydrous magnesium sulfate and filtered. The filtrate is concentrated under a stream of N2 and then placed under a high vacuum to give the crude. Preparative thin layer chromatography on silica gel gives 4. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Stages:

628 051

32

0c00pn3

dmso

-sch2CH2N3

(co2pnb).

To 210 mg of 4 (molecular weight = 871; 0.241 mmol), dissolved in 0.5 ml of DMSO (distilled from CaH2 at 8 mm and stored over a 4A Linde molecular sieve), 40 mg of l, 5-diazobicyclo- [5,4,0] -undec-5-ene (distilled at 80 ° C / 2 mm) (molecular weight = 152; 0.263 mmol) in 0.7 ml of dimethyl sulfoxide (DMSO). The solution is stirred for 4 hours under N2 and then added to a stirred ice-cold mixture of 0.48 ml of KH2P04.7 ml of HzO and 10 ml of EA. After separating the layers, the

Level O:

15 aqueous layer again extracted with EA. The combined organic layers are washed once with salt water and then dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of N2 and then placed under a high vacuum to give the crude 5. Preparative thin layer chromatography on silica gel gives 5_. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

20th

SCH0CH9No + LxI

2 2 3

s-collidine

(c02pnb) 2

CH2CH2N3

H .C02PNB

To a solution of 187 mg 5, (molecular weight = 791; 0.236 mmol) in 2.5 ml of s-collidine (distilled from powdered KOH at 30 mm pressure), 45 g of anhydrous LiJ (dried for a few hours at 100 ° C. over P205 under vacuum) (molecular weight = 134; 0.336 mmol). The reaction mixture is heated to 120 ° C. on an oil bath while stirring under N2. After a total of 25 minutes, the reaction mixture is cooled to 25 ° C., diluted with CH2C12 and transferred to a round bottom flask in order to concentrate under a stream of N2 and under high vacuum. The backlog is between

10 ml EA and 1.8 ml In KH2P04 divided into 10 ml H20 and then the aqueous layer is extracted two more times with EA. The combined organic layers are extracted with salt water, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of nitrogen to give crude 6. Preparative thin layer chromatography on silica gel gives 6. The cis-diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Level P:

33

628 051

O

OCOPNB

SCH2CH2ÎÎ3

cq2fnb

+

DMSO

sch2ch2n3 c02? nb

7

To a solution of the mixed diastereoisomers 6, (34 mg; molecular weight = 612; 0.055 mmol) in 0.2 ml DMSO (distilled from CaH2 at 8 mm and stored over a 4A Linde molecular sieve) 9.5 (xi l , 5-Diazobicyc! O- [5,4,0] -undec-5-ene (distilled at - 80 ° C / 2 mm) (molecular weight = 152, e = 1; 0.0625

mmol). The solution is stirred under nitrogen for 15 minutes and diluted to a total volume of 1 ml with CHC13 and immediately added to 2 to 1000 ^ silica gel plates. The 35 band of the product shows 7 as a mixture of cis and trans diastereoisomers.

OCOQPNB

/ Z \ ___ ZZ \ - S CH 7 CH2N3

c02pn3

H ^ / 2.8 kg / cn '

SCH9CHvNK?

In the presence of 61 mg Pt02, 61 mg 7 (molecular weight = 612; 0.1 mmol) in 6 ml dioxane, 6 ml THF, 3 ml H20 are hydrogenated with hydrogen for 4 hours at a pressure of 2.8 kg / cm2 . The reaction mixture is filtered through a Celite filter and washed with 2 ml of 0.1N pH 7 phosphate buffer. After concentrating in vacuo to the cloud point, the aqueous mixture is extracted with ethyl acetate. The aqueous layer is concentrated to a small volume and placed on a column of 100 g XAD-2 resin. When eluting with HzO and discarding the initial fractions, the fractions containing the product are freeze-dried, giving 8 ^ as a mixture of cis and trans diastereoisomers.

N-acetyl isomers 890A and 890A3, described below.

55 Deacetylation of N-acetylthienamycin

A 1% (weight / volume) suspension of fertile turf soil is made by adding 1 g of the turf soil to 100 ml of sterile phosphate buffer saline, the phosphate buffer saline having the following composition:

60

Phosphate buffer saline NaCl

Distilled water in phosphate buffer, pH 7.5 "*

8.8 g 10 ml 1000 ml

The procedure described below for the enzymatic N-deacylation of thienamycin can be used for all isomers of thienamycin, especially for the pronounced ones

+ In the phosphate buffer, pH 7.5

16 ml in KH2P04 are mixed with 84 ml in K2HP04. The pH of the phosphate buffer is increased by adding a small

628 051

34

Quantity of either Im KH2P04 or Im K2HP04 set to 7.5.

Aliquots of this 1% stock earth suspension are prepared for the preparation of 10 X, 100 X and 1000 X dilutions.

1 ml portions of the stock suspension or 1 ml portions of the 10 X, 100X and 1000 X dilutions are added to 2 ml portions of sterile, 1.0% agar solutions at 48 ° C. The mixture is quickly poured over the surface of sterile petri dishes 85 mm in diameter containing 20 ml of medium A. Medium A has the following composition:

Medium A

kh, po4 k2hpo4

MgSO,

distilled h20 N-acetylethanolamine solution4

3.0 g 7.0 g 0.1 g 1000 ml 8.5 ml

+ N-acetylethanolamine solution

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

For solid media: add 20 g agar.

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

Medium B

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

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

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

phosphate buffer, pH 7.4. The suspension obtained is subjected to ultrasound treatment using a Branson Instruments Model LS-75 Sonifier with a 1/2 inch probe at setting 4 with four 15 second 5 den intervals while the suspension is cooled in ice water during and between interruptions becomes. A 10 | xl portion of the sound-treated product is mixed with 25 2 ml of solution containing 840 (xg / ml of N-acetylthienamycin in 10 mmol of potassium phosphate buffer, pH 7) and incubated overnight at io 28 ° C. Controls, which the antibiotic and Buffer alone and sonicated cells and antibiotic-free buffers were also made After incubation overnight at 28 ° C., 2-xl amounts are placed on cellulose-coated thin-layer chromatography plates which were developed in ethanol: H20.70: 30 After air drying, the thin layer chromatography plate is placed on a plate infected with Staphylococcus aureus ATCC 6538P for 5 minutes.

The infected plates were prepared as follows: The overnight growth of the organism Staphylococcus aureus ATCC 6538P used for the infection in meat broth plus 0.2% yeast extract is diluted to a suspension with meat broth plus 0.2% Hefe extract, which has a 60% transmission at a wavelength of 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g / 1 Difco yeast extract at 37 to 48 ° C to obtain a composition containing 33.2 ml of the suspension per liter of agar . 40 ml of this suspension are poured onto Petri dishes, 22.5 cm X 22.5 cm, and these plates are cooled 30 and kept at 4 ° C until used (5 days maximum).

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

40

45

50

Production of 890A h a special isomer

Medium C

Dextrose

Pharmamedia

Liquid from soaked corn (wet basis) distilled water pH: adjusted to 7 with NaOH or HCl N-acetylethanolamine solution +

20 g 8 g 5 g

1000 ml 8.5 ml

60

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

The flask is shaken at 28 ° C and 220 rpm on a vibrator for 4 days. A 25 ml portion from the flask is centrifuged at 8000 rpm for 65 minutes. The supernatant is removed and the cells on the surfaces of the solid medium are scraped off and made into a 0.5 ml 0.05m potassium

O «

sch2ck2nhcch3 cooh

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

Davis salt sodium citrate k2hpo4 kh2po4 (nh4) 2so4

MgS04 • 7H20 distilled water

0.5 g 7.0 g 3.0 g 1.0 g 0.1 g 1000 ml

35

628 051

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

Medium A Glycerin primary yeast fish meal distilled water agar

20.0 g 5.0 g 15.0 g 1000 ml 20.0 g pH: adjusted to 7.2 using NaOH. The inoculated cultures are incubated for one week at 27-28 ° C. and then until used at 4 Store at -6 ° C.

A 1/3 portion of the growth of three cultures is used to inoculate 9 stoppered Erlenmeyer flasks containing 50 ml of Medium B of the following composition:

10th

Medium B

Yeast autolysate (+ ardamine) glucose

Phosphate buffer ++ MgS04 • 7H20 distilled water

10.0 g 10.0 g 2.0 ml 50 mg 1000 ml

20th

pH: adjusted to 6.5 using HCl or NaOH

+ Ardamine: Yeast Products Corporation

+ + Phosphate buffer solution kh2po4

Na2HP04 distilled water

91.0 g 95.0 g 1000 ml

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

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

Medium C

Tomato paste 20.0 g primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl2 • 6H20 5.0 mg distilled water 1000 ml pH: adjusted to 7.2-7.4 by NaOH After the inoculation, the production flasks are incubated at 24 ° C. with shaking on a vibrator which is operated at 212 rpm and for 4 days. The flasks are then emptied and the contents checked for activity using standard Salmonella gallinarum MB 1287 and Vibrio percolans ATCC 8461 plates using 1.25 cm discs immersed in the centrifuged broth samples. If necessary, the samples are diluted with 0.02m phosphate buffer, pH 7.0. The results are given below:

35

45

50

55

60

Harvest Age hours h 96 pH 7.2

Salmonella gallinarum (mm zone) 29.5 Vibrio percolans 1/10 dilution

(mm zone) 31

890 test units 40 (

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

7 ml of 0.1 m neutral EDTA are added to the combined supernatant liquids and the entire sample is placed on a Dowex-1 X 2 (Cl ~~), 50-100 mesh column with bed dimensions of 5.1 X 25 cm and one Flow rate of 40 ml / min. The column is washed with 500 ml of deionized water, containing 5 ml in Tris-HCl buffer, pH 7.0, and 25 | xMol neutral EDTA. The antibiotic is eluted with 1 liter of deionized water containing 50 g of sodium chloride, and the column is then washed with 300 ml of deionized water. 100 ml fractions are collected starting from the first appearance of salt at the column exit. The bioactivity is present in fractions 1 to 10 with a peak in fraction 2. Fractions 2 to 5, which contain 17% of the activity applied, are pooled.

The pooled fractions are concentrated to 110 ml by means of a rotary evaporator under reduced pressure and the pH is adjusted to 5.8 by adding 4.2 ml in HCl. The concentrate set in this way is placed on a column of XAD-2 with a bed dimension of 3.8 × 50 cm, which had previously been washed with 3 160% aqueous acetone (volume / volume) and then with 3 1 of deionized water, 3 1 of 5% (weight / volume) sodium chloride and 1125% (weight / volume) sodium chloride in deionized water. The concentrate used is drained to the level of the bed. The antibiotic is eluted with deionized water at a flow rate of 15 ml / min. 22 fractions of 100 ml each are collected, calculated from the first application of the sample. The bioactivity appears in fractions 6 to 22 with a peak in fractions 9 and 10. Fractions 9 to 20 are combined for further processing. Similar fractions are pooled and the pooled fractions are concentrated to 70 ml using a rotary evaporator under reduced pressure.

The concentrate is placed on a Dowex-1 X 4 (Cl ~) 400 mesh column with a bed dimension of 2.2 X 27 cm. The column is washed with 50 ml deionized water and the antibiotic eluted with 210.070m NaCl + 0.005m NH4CI + 0.0001m NH3 in deionized water at a flow rate of 2 ml / min. 9.5 ml fractions are collected.

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

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

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

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

628 051

36

sch2ch2nh2 cooh

Production of 890A3, a special isomer io oh

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

Medium C

Tomato paste 20.0 g primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl2 • 6H20 5.0 mg distilled water 1000 ml pH: adjusted to 7.2-7.4 with NaOH

0

_n_

\ _sch2ch2hhcch3 LLjjoûh

15

0 ^

A tube of a lyophilized culture of Streptomyces 20 flavorgriseus MA-4434 (NRRL 8139) is opened aseptically and the contents are suspended in a tube containing 0.8 ml of a sterile Davis saline solution of the following composition:

Davis salt sodium citrate k2hpo4 kh2po4 (nh4) 2so4

MgS04 • 7H20 distilled water

0.5 g 7.0 g 3.0 g 1.0 g 0.1 g 1000 ml

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

Medium A glycerin primary yeast fish meal distilled water agar pH: adjusted to 7.2 under NaOH

20.0 g 5.0 g 15.0 g 1000 ml 20.0 g

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

A 1/3 portion of the growth from 4 cultures is used to inoculate 12 plugged 250 ml Erlenmeyer flasks containing 50 ml Medium B of the following composition:

Medium B

Yeast autolysate (+ ardamine)

glucose

Phosphate buffer + +

MgS04 • 7H20 distilled H20

pH: adjusted to 6.5 under HCl or NaOH + Ardamine: Yeast Products Corporation

+ "Phosphate buffer solution kh, po4

Na2HP04 distilled water

10.0 g 10.0 g 2.0 ml 50 mg 1000 ml

91.0 g 95.0 g 1000 ml

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

25th

30th

35

40

45

55

60

65

After the inoculation, the production flasks are shaken at 24 ° C. for 4 days and 5 hours on a shaking machine which is operated at 212 rpm. The flasks are then emptied and assayed for activity using standard Salmonella gallinarum MB 1287 and Vibro percolans ATCC 8461 sample plates using 1.25 cm sample disks immersed in the centrifuged samples of the broth. The samples are diluted with 0.2m phosphate buffer, pH 7.0, if necessary. The results are given below:

Harvest Age hours h 101

pH 7.2

Salmonella gallinarum (mm zone) 35

Vibrio percolans 1/10 dilution (mm zone) 34

890 sample units 121

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

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

The above fermentation is repeated under identical conditions except that the 44 2-liter ErIen-meyer flasks are inoculated with 7 ml per bottle of growth from the bottles containing the cultures; the pH and the test results obtained are given below:

Harvest Age hours h 101 pH 7.3

Salmonella gallinarum (mm zone) 38 Vibro percolans 1/10 dilution

(mm zone) 39

890 sample units 92.8

A total of 7.4 l of the entire broth obtained from this fermentation are cooled to 3 ° C. and centrifuged for 15 minutes in 200 ml portions at 9000 rpm. One gives to the combined protruding solutions

1.8 ml 0.1m neutral EDTA.

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

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

37

628 OSI

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

The combined fractions 5 to 9 are concentrated to 150 ml by means of a rotary evaporator under reduced pressure and placed on a column (4.9 cm X 47 cm) of XAD-2, which had been washed beforehand with 5 160% (volume / volume - 10 \ umen) aqueous acetone and then with 5 1 deionized water and 5 150 g / 1 NaCl in deionized water. The sample is applied in 20 ml increments, soaking the column to bed level each time. When application is complete, three 20 ml portions of deionized water are added and each time allowed to flow to bed level. The sample is eluted with deionized water at a flow rate of 20 ml / min. All measures using the XAD column are carried out at room temperature (24 ° C) and the eluted fractions are quickly cooled in an ice bath immediately after collection. Fractions from 95 to 230 ml are collected.

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

The pooled fractions 6 to 21 are concentrated to 68 ml 30 under reduced pressure on a rotary evaporator and then diluted to 112 ml by adding deionized water. The concentrate is placed on a column (2.2 cmX21 cm) of Dowey-1 X4 (Cl-) 400 mesh. The column is washed with 20 ml deionized water and the antibiotic is eluted with 210.07m NaCl + 0.005m 35 NH4CI + 0.0001m NH3 in deionized water at a flow rate of 1.6 ml / min. Fractions of 8 ml each are collected. Fractions 157 to 179 are the most active and are pooled.

These combined fractions are concentrated 4o on a rotary evaporator under reduced pressure to 7 ml, the pH is adjusted to 7.5 by adding 20 | xl in NaOH. The solution is further concentrated to 5 ml and placed on a column (2.2 X 75 cm) of Bio-Gel P-2,200 to 400 mesh. The sample is washed in the column bed with two washes of 1 ml of deionized water each and eluted with deionized water at a flow rate of 0.6 ml / min. Ten fractions of 3.3 ml and then 60 fractions of 2.65 ml and ten fractions of 2.0 ml are collected. 50

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

For the isolation of the antibiotic 890A3 from 890A! Take advantage of the relatively higher resistance of the 890A3 antibiotic to degradation by penicillinase in the following ways:

Bio-Gel fraction 63 is combined with fractions 61, 66 and 67 from the Bio-Gel column. 0.2 ml in Tris-HCl buffer, pH 7.5 and 0.2 ml penicillinase (Difco "Bacto-Penase") are added to these four combined fractions. After 113 minutes at 23 ° C a further 0.2 ml penicillinase are added. After a further 7 hours at room temperature, a further 0.2 ml of penicillinase are added, and after a further 2 hours at room temperature the reaction mixture is cooled on an ice bath. The finished raction mixture is diluted to 15 ml by adding 5 ml of deionized water.

The reaction mixture is adsorbed on a Do-wex ~ 1X 4 (Cl ~) 400 mesh column, bed dimension 2.15 X 40 cm. The antibiotic 890A3 is eluted with 0.07m NaCl + 0.005m NH4Cl + 0.0001m NH3 in deionized water at a flow rate of 3 ml / min. Fractions of 13.8 ml are collected in each case. Fractions 187 to 200 are pooled.

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

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

/

OH

0

tf-

I.

.n_

rSCH2CH2NH2 cooh c

Claims (4)

628 051 2nd PATENT CLAIMS 1. Process for the preparation of new N-alkylated derivatives of thienamycin of the formula or 5 s 7 SCH2CH2NR R R (I) coxr 10th and their pharmacologically acceptable salts, where R5, R6 and R7 are each independently a hydrogen atom, 15 not all of the radicals R5, R6 and R7 are simultaneously hydrogen atoms, a substituted or unsubstituted lower alkyl radical having 1 to 6 carbon atoms, alkenyl radical having 2 to 10 carbon atoms or alkynyl radical with 2 to 10 carbon atoms, each ring-substituted or unsubstituted cycloalkyl, cycloalkenyl, cycloalkenylalkyl or cycloalkoalkylalkyl radical with 3 to 6 ring carbon atoms and 1 to 6 carbon atoms in the alkyl chain, an aryl radical with 6 to 10 Carbon atoms, an aralkyl radical with 6 to 10 ring carbon atoms and 1 to 6 carbon atoms in the alkyl chain 25 or a mono- or bicyclic heteroaryl or hetero-aralkyl radical with 4 to 10 ring atoms, one or more of which represent oxygen, nitrogen and / or sulfur, and 1 to 6 carbon atoms in the alkyl chain, the ring or chain substituents each have at least one chlorine, bromine, iodine or fluorine atom, an azido, cyano or amino group, a mono-, di- or trialkyl-substituted amino group, the alkyl radical having 1 to 6 carbon atoms, a hydroxyl group, an alkoxy radical with 1 to 6 carbon atoms, a thioalkyl radical with 1 to 6 carbon atoms, a carboxyl or oxo group, an alkoxycarbonyl radical with 1 to 6 carbon atoms in the alkoxy part, an acyloxy radical having 2 to 10 carbon atoms, a carbamoyl group, a mono- or dialkylcarbamoyl group, the alkyl radicals each having 1 to 4 carbon atoms, the thiocyanine group —SCN or 40 representing the nitro group, R4 is a hydrogen atom or an acyl, alkyl, aryl, aralkyl, alkenyl or alkynyl radical, X represents an oxygen or sulfur atom or a radical of the formula NR ', in which R' = H or R, 45 R represents a hydrogen atom, an alkyl radical having 1 to 10 carbon atoms, a phenacyl group or a nucleus-substituted phenacyl group, the substituent being a chlorine, bromine or fluorine atom or an alkyl radical having 1 to 6 carbon atoms, an alkoxyalkyl radical, the alkoxy part being open-chain or 50 is cyclic and has 1 to 6 carbon atoms and the alkyl portion also has 1 to 6 carbon atoms, an alkanoyloxyalkyl radical with 2 to 12 carbon atoms, a halogen or perhaloalkyl radical, where halogen is chlorine, Is bromine or fluorine and the alkyl chain has 1 to 6 carbon atoms, an alkenyl radical with 2 to 10 carbon atoms, an alkoxycarbonyloxyalkyl radical with 3 to 14 carbon atoms, a dialkylaminoacetoxyalkyl radical with 4 to 21 carbon atoms, an alkanoylamidoalkyl radical with 2 to 13 carbon atoms , an aralkyl radical, where the alkyl moiety has 1 to 3 60 carbon atoms and the aryl moiety has 6 to 10 carbon atoms, a mono- or bicyclic heteroaralkyl radical with 4 to 10 ring atoms and 1 to 6 carbon atoms in the alkyl moiety, the hetero atom being an oxygen, sulfur or Represents nitrogen atom, a nucleus-substituted aralkyl or 65 heteroaralkyl radical, the substituent being chlorine, fluorine, bromine or iodine or a lower alkyl radical having 1 to 6 carbon atoms, a lower alkanoyloxy radical having 1 to 6 Carbon atoms, a lower alkoxy radical with 1 to 6 carbon atoms, a mono- or bicyclic heterocyclylalkyl radical, where the heterocycle has 4 to 10 atoms and the hetero atom is an oxygen, sulfur or nitrogen atom and the alkyl portion contains 1 to 6 carbon atoms , an aryl or core-substituted aryl radical having 6 to 10 ring carbon atoms, the core substituent being a hydroxyl group, a lower alkyl radical having 1 to 6 carbon atoms, a chlorine, fluorine or bromine atom or an alkylthioalkyl radical having 2 to 12 carbon atoms, a cycloalkylthioalkyl radical with 4 to 12 carbon atoms or an acylthioalkyl radical, the acyl moiety having 2 to 10 carbon atoms and the alkyl moiety having 1 to 6 carbon atoms, characterized in that a compound of the formula sch2CH2NH2 coxr or an N-alkyl derivative of such a compound is reacted with an N-alkylating agent suitable for introducing the substituents R5, R6 and R7 and, if appropriate, the compounds obtained are converted into the corresponding pharmacologically acceptable salts. 2. The method according to claim 1, characterized in that a compound of the formula ch2ch2hhch3 cooh manufactures. 3. The method according to claim 1, characterized in that a compound of the formula oh sch2ch2n (ch3) 2 cooh manufactures. 4. The method according to claim 1, characterized in that one has a compound of the formula © sch-ch9n (ch-j, a Cu »• COOH in which A ° means a pharmacologically acceptable anion. 3rd 628 051