CS250658B2 - Method of carbapeneme derivatives' production - Google Patents

Description

The present invention relates to a novel process for the preparation of carbapenem derivatives bearing at position 2 a substituent corresponding to the general formula -S-A-R ⁴⁴ wherein:

A represents a cyclopentylene group, a cyclohexylene group or a (C 2 -C 6) alkylene group optionally substituted by one or two (C 1 -C 4) alkyl radicals, and

R ¹⁴ is defined below, an aromatic or nonaromatic heterocyclic radical containing a quaternized nitrogen atom, bonded to the radical A by quaternized nitrogen atom.

Carbapenem derivatives produced by the process of the present invention are disclosed and protected in U.S. Patent Application Serial Nos. 366,910, 471,379 and 389,652, filed April 9, 1982, March 8, 1983, and U.S. Pat. On June 18, 1932 by Choung U. Kim and Peter F. Misco jr.

U.S. patent application Ser. No. 366,910 and its continuation-in-part application filed March 8, 1983, which partially corresponds to German patent application No. 3,312,533, disclose the preparation of carbapenem antibiotics of formula IA

in which

R ⁸ represents a hydrogen atom and R ¹ represents a radical selected from the group consisting of hydrogen and optionally substituted C 1 -C 10 alkyl, alkenyl and alkynyl groups, C 3 -C 6 cycloalkyl and cycloalkylalkyl groups in the C 1 -C 6 cycloalkyl moiety carbon in the alkyl, phenyl, aralkyl, aralkenyl and aralkynyl moiety wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains up to 6 carbon atoms, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl groups wherein the heteroatom or heteroatoms are selected from the group consisting of one up to four oxygen, nitrogen and sulfur atoms, and wherein the alkyl moiety attached to the heterocyclic moiety contains 1 to 6 carbon atoms, wherein said substituent or substituents of the above-mentioned moieties are selected from the group consisting of alkyl groups of 1 to 6 carbon atoms optionally substituted amino, halogen, hydroxyl or carboxyl, halogen atoms, -OR ³ radicals,

O —OCNR ³ R ⁴

O

II — CN ³ R ⁴ —nr ³ r ⁴

NR $ //

---- _x \ SS, L * —so ₂ nr ³ r ⁴ o —NHCNR ³ R ⁴

O

RŠCNR ⁴ - —co ₂ r ³ = 0 o

II —OCR ³ —SR ³

O

II —SR ⁹ o

—SR ⁹

II o

-CN, -N ₃ -0S0 _{3 R} ³ -OSO 2 R ^3, -NR ³ SO 2 R ^4, -NR ³ C == NR ⁴

R ^{3 -} NR ³ CO ² R ⁴ and -NO 2 in which

R ³ and R ⁴ are each independently hydrogen, alkyl, alkenyl or alkynyl of up to 10 carbon atoms each, cycloalkyl, cycloalkylalkyl or alkylcycloalkyl of 3 to 6 carbon atoms in the cycloalkyl ring and 1 to 6 carbon atoms in the alkyl moiety , a phenyl, aralkyl, aralkenyl or aralkynyl group wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains up to 6 carbon atoms, a heteroaryl, heteroarylalkyl, heterocyclyl or heterocyclylalkyl group wherein the heteroatom or heteroatoms are selected from one to four oxygen atoms, nitrogen and sulfur and the alkyl moiety attached to the heterocyclic moiety contains 1 to 6 carbon atoms; or

R ³ and R ⁴ together with the nitrogen atom to which at least one of these residues is attached form a five- or six-membered nitrogen-containing heterocyclic ring, and

R ⁹ is as defined above for R ³ except hydrogen, or

R ¹ and R ⁸ together form an alkylidene group having 2 to 10 carbon atoms, optionally substituted by a hydroxyl group,

A represents a cyclopentylene group, a cyclohexylene group or an alkylene group having 2 to 6 carbon atoms, optionally substituted by one or more alkyl groups having 1 to 4 carbon atoms,

R ² represents a hydrogen atom, an anionic negative charge or a conventional readily cleavable carboxyl function protecting group, provided that when R ³ represents a hydrogen atom or a protecting group, a charge-balancing anion is also present, and

-Nj represents an optionally substituted mono-, bi- or polycyclic aromatic heterocyclic radical containing at least one nitrogen atom in the ring and attached to the radical A by a nitrogen atom in the ring, and their pharmaceutically acceptable salts as described in the following reaction scheme:

R ⁶ hl diphenyl chlorophosphate

0 ’'N -

COOR (IH)

R

HS-A-OH wherein A is C 5 -C 6 alkylene or cycloalkylene of ^:

(IN)

S-A-OH

COOR ^Z> methanesulfonyl chloride

sa-axis _from ch ₃

COOR ', Θs-Á-i

- COOR (II)

1 ' _A & e (X g X

O - buffer anion)

O ¹

R * H

S ~ A ~ N), X®

¹ R (IA)

H

^S * ' ^{, A} " ^N O (Γ N \ _c00f 2 2 (IA)

Further, a process for the preparation of carbapenem antibiotics of the general formula IB has already been described

COOR (IB)

in which

R ⁸ represents a hydrogen atom and R ¹ represents a radical selected from the group consisting of hydrogen and optionally substituted alkyl, alkenyl and alkynyl groups of up to 10 carbon atoms, cycloalkyl and cycloalkylalkyl groups of 3 to 6 carbon atoms in the cycloalkyl moiety and 1 to 6 carbon atoms an alkyl, phenyl, aralkyl, aralkenyl and aralkynyl moiety wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains up to 6 carbon atoms, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl groups wherein the heteroatom or heteroatoms are selected from one to one four oxygen, nitrogen and sulfur atoms, and wherein the alkyl moiety attached to the heterocyclic moiety contains 1 to 6 carbon atoms, wherein said substituent or substituents of the above-mentioned moieties are selected from the group consisting of alkyl groups of 1 to 6 carbon atoms optionally substituted amino, halogen, hydroxyl or carboxyl, halogen atoms, radicals of the formula -OR ³

O

II -NHCNR3R ⁴

O

R ³ CNR ⁴ - —CO ₂ R ³ = O

O —OCR ³ —SR ³

O —SR ⁹

O

II —SR ⁹

O —CN

_-N3 -OSO3R ³ -OSO ₂ R ^3, -NR ³ -SO ₂ -NR ³ R ⁴ C = NR ⁴

R ^{3 —} NR ³ CO ₂ R ⁴ a

-NO ₂ in which —OCNR ³ R ⁴

O

II — CN ³ R ^{4 —} NR ³ R ⁴

NR ⁵

—SO ₂ NR ³ R ⁴

R ³ and R ⁴ are each independently hydrogen, alkyl, alkenyl or alkynyl of 1 to 10 carbon atoms, cycloalkyl, cycloalkylalkyl of 3 to 6 carbon atoms in the cycloalkyl ring and 1 to 6 carbon atoms of alkyl a phenyl, aralkyl, aralkenyl or aralkynyl group wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains 1 to 6 carbon atoms, a heteroaryl, heteroarylalkyl, heterocyclyl or heterocyclylalkyl group wherein the heteroatom or heteroatoms are selected from one to four atoms oxygen, nitrogen and sulfur and the alkyl moiety attached to the heterocyclic radical contains 1 to 6 carbon atoms, or

250

R ³ and R ⁴ together with the nitrogen atom to which at least one of these residues is attached form a five- or six-membered nitrogen-containing heterocyclic ring, and

R ⁹ is as defined above for R ³ except hydrogen, or

R ¹ and R ⁸ together form an alkylidene group having 2 to 10 carbon atoms, optionally substituted by a hydroxyl group,

R @ ¹ represents an optionally substituted alkyl, alkenyl or alkynyl group containing from 1 to 10 carbon atoms, a cycloalkyl or cycloalkylalkyl group having from 3 to 6 carbon atoms in the cycloalkyl moiety and from 1 to 6 carbon atoms in the alkyl, phenyl, aralkyl, aralkenyl or an aralkynyl group wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains 1 to 6 carbon atoms, a heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylalkyl group wherein the heteroatom or heteroatoms in the heterocyclic moieties are selected from the group consisting of 1 to 4 oxygen, nitrogen and sulfur atoms and the alkyl moiety attached to the heterocyclic radical contains 1 to 6 oxygen atoms, wherein the above-mentioned optionally substituted radicals R ⁵ carry one to three substituents independently selected from the group consisting of alkyl groups of 1 to 6 carbon atoms, optionally subs. substituted with amino, fluorine or chlorine, carboxyl, hydroxy or carbamoyl, fluorine, chlorine and bromine, radicals —OR ³ —OCO ₂ R ³ —OCOR ³ —OCONR ³ R ⁴ —OSO ₂ R ³ —NR ³ R ⁴

R ³ CONR ⁴ - —NR ³ CO2R ⁴ —NR ³ CONR ³ R ⁴ —nr ³ so2r ⁴ —SR ^{3 8}

O t

—S — R ⁹ —SO ₃ R ³ —COaR ³ —CONR ³ R ⁴ —CN phenyl optionally substituted by one to three fluorine, chlorine and bromine atoms, alkyl of 1 to 6 carbon atoms, —OR ³ , - NR ³ R ⁴ , -SO a R ³ , -CO a R ³ or -CONR ³ R ⁴ , wherein R ³ , R ⁴ and R ⁹ in the radicals R ⁵ are as defined above, or R ⁵ represents a divalent phenylene group or (C až-C) alkylene bonded to the residue

to form a bridged polycyclic group

A represents a cyclopentylene group, a cyclohexylene group or an alkylene group having 2 to 6 carbon atoms, optionally substituted with one to four alkyl groups,

R ² represents hydrogen, an anionic charge or a negative conventional readily removable carboxyl protecting group provided that when R ² is hydrogen or a protecting group, there is also present a charge balancing anion, and

means an optionally substituted mono-, bi- or polycyclic non-aromatic heterocyclic radical containing at least one nitrogen atom in the ring and bonded to radical A with a nitrogen atom in the ring to form a quaternary ammonium group, and pharmaceutically acceptable salts thereof according to the following reaction scheme:

diphenyl chlorophosphate

HS-A-OH wherein A represents an alkylene group or a cycloalkylene group having 5 to 6 carbon atoms

Methanesulfonyl chloride

R ^R R ⁸ H

Λ — Ν (VI)

S-A-OSO 2 CH 2

COOR ^Z '

Ziix

S-A-I

0 '

N · (II) 'COOR ^J

optionally removing the protecting group

In accordance with prior art reaction schemes, the starting material of formula III is reacted with an diphenyl chlorophosphate in the presence of a base in an inert organic solvent in the presence of a base to form an intermediate of formula IV. The intermediate of formula IV is then reacted with mercaptan of formula HS-A-OH in an inert organic solvent and in the presence of a base to form an intermediate of formula V. This intermediate of formula V is acylated with methanesulfonyl chloride in an inert organic solvent in the presence of a base to which is reacted with an iodide ion source in an inert organic solvent to provide an intermediate of formula (II). The intermediate of formula (II) is reacted with the desired amine in an inert organic solvent in the presence of silver ions to form a quaternized product of formula (IΓΒA) or (ΓΒ) from which the protecting group can then be removed to give the corresponding unprotected carbapenem (IA or IB).

The above described process has several disadvantages. For example, the process described above is carried out in several reaction stages, the number of which would be advantageous to reduce. Also, the overall yield of the reaction is rather low and the quaternization step is carried out with the already prepared carbapenem derivative. It would therefore be desirable to have a new process for the preparation of compounds of the formula (IA) or (IB)

1) involve fewer reaction steps,

(2) provide higher yields;

3) allowed the preparation of a quaternized amine first and then its coupling to the carbapenem nucleus at a later stage of the synthesis; and

4) could be used to facilitate the preparation of quaternary amino derivatives using a wide variety of amines such as spherically hindered amines and amines with low pK _b values.

The present invention relates to a novel process for the preparation of carbapenem derivatives of the general formula I in which:

R ⁸ represents a hydrogen atom and

R ¹ represents a radical selected from the group consisting of hydrogen and optionally substituted alkyl, alkenyl and alkynyl groups of 1 to 10 carbon atoms, cycloalkyl and cycloalkylalkyl groups of 3 to 6 carbon atoms in the cycloalkyl moiety and 1 to 6 carbon atoms in the alkyl, phenyl moiety aralkyl, aralkenyl and aralkynyl groups wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains 1 to 6 carbon atoms, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl groups containing up to 18 ring members wherein the heteroatom or heteroatoms are selected from the group consisting of one up to four oxygen, nitrogen and sulfur atoms and wherein the alkyl moiety attached to the heterocyclic moiety contains 1 to 6 carbon atoms, wherein said substituent or substituents of the above-mentioned moieties are selected from the group consisting of alkyl groups of 1 to Θ carbon atoms, optionally substituted amino, halogen, hydroxyl or carboxyl group, halogen atoms, radicals of the formula -OR ³

O — QCNR ³ R ⁴

O — CN ³ R ^{4 —} NR ³ R ⁴

NR ⁵

SO ₂ NR ³ R ⁴ o

-A-R ^ τΎ

-M- \ o

COOR ^{4 —} NHCNR ³ R ⁴

O

II

R ³ CNR ⁴ -

- about ₂ r ³ (I) = 0

O —OCR ³ —SR ³

O

II —SR ⁹ o

—SR ⁹

II o

—CN —n ₃ —oso ₃ r ³ —oso2r ^{3 —} NR ³ SO2R ' ^{1 —} NR ³ C == NR ⁴

R ³ —NR ³ CO2R ⁴ and —NO2 in which

R ³ and R ⁴ are each independently hydrogen, alkyl, alkenyl or alkynyl of 1 to 10 carbon atoms, cycloalkyl, cycloalkylalkyl or alkylcycloalkyl of 3 to 6 carbon atoms in the cycloalkyl ring and 1 to 6 carbon atoms in the alkyl a phenyl, aralkyl, aralkenyl or aralkynyl group wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains from 1 to 6 carbon atoms, a heteroaryl, heteroarylalkyl, heterocyclyl or heterocyclylalkyl group containing up to 18 ring members wherein the heteroatom or heteroatoms are selected from comprising one to four oxygen, nitrogen and sulfur atoms and the alkyl moiety attached to the heterocyclic moiety contains 1 to 6 carbon atoms, or

R ³ and R ⁴ together with the nitrogen atom to which at least one of these residues is attached form a five- or six-membered nitrogen-containing heterocyclic ring, and

R ⁹ is as defined above for R ³ except hydrogen, or

R ¹ and R ⁸ together form an alkylidene group having 2 to 10 carbon atoms, optionally substituted by a hydroxyl group,

A represents a cyclopentylene group, a cyclohexylene group or an alkylene group having 2 to 6 carbon atoms, optionally substituted by one or two alkyl groups having 1 to 4 carbon atoms,

R ³ represents a hydrogen atom, an anionic negative charge or a conventional readily cleavable carboxyl function protecting group, provided that when R ³ represents a hydrogen atom or a protecting group, a charge-balancing anion is also present, and

R ¹⁴ represents a quaternizable nitrogen containing an aromatic or non-aromatic heterocyclic radical attached to radical A via a ring nitrogen atom to form a quaternary ammonium group selected from the group consisting of pyridine, optionally substituted with one or two C 1 -C 4 alkyl, C 1 alkoxy groups C 1 -C 4 alkylthio, C 1 -C 4 alkylthio, C 1 -C 4 hydroxyalkyl or amino groups, imidazole radical substituted with C 1 -C 4 alkyl and / or C 1 -C 4 alkylthio, piperazine radical substituted once , two or three C 1 -C 4 alkyl groups, a C 1 -C 4 alkyl morpholine moiety, a C 1 -C 4 alkyl thiomorpholine moiety, and a C 1 -C 4 alkyl thiomorpholine-S-oxide moiety at and pharmaceutically acceptable salts thereof, characterized in that the intermediate of formula IV

(IV) wherein

R ¹ and R ⁸ have the abovementioned meaning,

R ² 'represents a conventional, readily removable carboxyl protecting group and

L is a conventional leaving group, reacted with a thiol of formula VII

HS — A — R ¹⁴ Χ “(VII) in which

A and R ¹⁴ have ^the abovementioned meaning and

X represents a balancing anion, in an inert solvent in the presence of a base, to form a carbapenem derivative of formula f

R ^e Η

SAR í ¹ *

Ni.

CQOR (Γ) in which

R @ ¹ , R @ ⁸ , R @ 1, A, R @ ¹⁴ and X @ ^- are as defined above and optionally cleave the carboxyl protecting group R @ ² 'to give the corresponding unprotected compound of formula I or a pharmaceutically acceptable salt thereof.

Carbapenem derivatives of formula (I) are potent antibacterial agents or intermediates useful in the preparation of such agents.

The compounds of formula I above contain a carbapenem core of the formula

and thus may be named as 1-carb-2-penem-3-carboxylic acid derivatives.

Alternatively, the above compounds may be considered to contain the basic structural core of the formula

Ι7Ί —N

and name them as derivatives of 7-oxo-1-azabicyclo [3.2.0] hept-2-enecarboxylic acid. Although the invention encompasses compounds with relative stereochemistry of protons at both the 5 and 6 positions of both cis and trans, preferred compounds nevertheless have the stereochemistry of 5R, 6S (trans), as is the case with thienamycin.

The compounds of formula (I) may be unsubstituted at the 6-position or substituted at that position by the substituents described above for other carbapenem derivatives. More specifically, R ⁸ may be hydrogen and R ¹ may be hydrogen or one of the substituents listed, for example, in European Published Application No. 38,869 (see definition of R ₆ ). Alternatively, R ⁸ and R ^{1 may be taken} together to form a C 2 -C 10 alkylidene or C 2 -C 10 alkylidene group substituted with, for example, a hydroxy group.

Individual general terms in the meaning of symbols R ^! and R ⁸ have the following meanings:

a) The aliphatic alkyl, alkenyl and alkynyl groups may have a straight or branched chain of 1 to 10 carbon atoms, preferably 1 to 6 and most preferably 1 to 4 carbon atoms. When these groups form part of more complex substituents, for example cycloalkylalkyl, heteroaralkyl or aralkenyl substituents, in such cases they contain alkyl, alkenyl and alkynyl groups preferably of 1 to 6, most preferably 1 to 4 carbon atoms.

b) The term "heteroaryl" refers to mono-, bi- and polycyclic aromatic heterocyclic groups containing up to 18 ring members, including 1 to 4 oxygen, nitrogen or sulfur atoms, preferably five- or six-membered heterocyclic rings, such as thienyl, furyl, thiadiazolyl, oxadiazolyl, triazolyl, isothiazolyl, thiazolyl, imidazolyl, isoxazolyl, tetrazolyl, oxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, pyrazolyl and the like.

c) The term "heterocyclyl" includes mono-, bi- and polycyclic saturated or unsaturated non-aromatic heterocyclic radicals containing up to 18 ring members, including 1 to 4 oxygen, nitrogen or sulfur atoms, preferably five- or six-membered heterocyclic radicals, such as morpholinyl, piperazinyl, piperidyl, pyrazolinyl, pyrazolidinyl, imidazolidinyl, imidazolinyl, pyrrolinyl, pyrrolidinyl and the like.

d) Halogens are chlorine, bromine, fluorine and iodine, preferably chlorine or bromine.

The term "conventional, easily cleavable protecting group for a carboxyl function" refers to a known ester group which can be used to protect the carboxyl function during the chemical reactions described below, and which can be cleaved, if desired, by methods causing no significant destruction of the remainder of the molecule. , for example by chemical or enzymatic hydrolysis, treatment with chemical reducing agents under mild conditions, irradiation with ultraviolet radiation or catalytic hydrogenation. examples of such ester protecting groups include benzhydryl, p-nitrobenzyl, 2-naphthylmethyl, allyl, benzyl, trlchloroethyl, silyl, such as trimethylsilyl, phenacyl, p-methoxybenzyl, acetonyl, o-nitrobenzyl, 4-pyridylmethyl and 1-alkyl groups; up to 6 carbon atoms such as methyl, ethyl or tert-butyl. These protecting groups include those that are hydrolyzed under physiological conditions, such as the pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl groups.

Particularly preferred carboxyl protecting groups are a p-nitrobenzyl group which is readily cleaved by catalytic hydrogenolysis and an allyl group which can be cleaved by a tetra (triphenylphosphine) palladium catalyzed reaction.

The above-mentioned pharmaceutically acceptable salts include non-toxic acid addition salts, for example salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid and the like, and salts with organic acids such as maleic acid, acid acetic acid, citric acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, lactic acid, gluconic acid and malic acid. The compounds of formula I, in the form of acid addition salts, may be represented by the formula pharmaceutically acceptable salts, useful as antibacterial agents. The remaining compounds of formula (I) are valuable intermediates which can be converted to the aforementioned biologically active compounds.

According to a preferred embodiment, compounds of formula (I) are prepared wherein R ⁸ is hydrogen and R ¹ is hydrogen, ethyl,

CH ₃

CH—,

CH ₃

CH ₃ OH ₃ or ₃

OH

CH ₃ CH—.

Preferred compounds of this subgroup are those wherein R ¹ is (R ² = hydrogen or a protecting group) wherein X ¹ is an acid anion. The anion X may be selected so as to obtain a pharmaceutically acceptable salt for therapeutic application, but in the case of the intermediate compounds of formula I X may also be a toxic anion. In such a case, the ion may subsequently be removed or replaced with a pharmaceutically acceptable anion to form an effective end product suitable for therapeutic use.

If acidic or basic groups are present in the R @ ¹ radical or the quaternized R @ ¹⁴ radical, the compounds according to the invention can also form suitable salts with bases or acids on these functional groups. These include, for example, acid addition salts for basic groups and salts with metals (for example, sodium, potassium, calcium and aluminum), ammonium salts and salts with non-toxic amines (for example with trialkylamines, procaine, dibenzylamine, 1-efenamine, N- benzyl- (3-phenethylamine, N, N'-dibenzylethylenediamine and the like) in the case of acidic groups.

Compounds of formula I wherein R ² is hydrogen, an anionic charge or an ester hydrolyzable under physiological conditions, and their

OH

CH ₃ , CH ₃ , with the most preferred compounds having the absolute configuration of 5R, 6S, 8R.

In accordance with another preferred embodiment, the invention includes compounds of formula I wherein R ¹ and R ⁸ together form an alkylidene radical of formula

HOCH ₂

C =

H ₃ C

The alkylene or cycloalkylene radical A in the compounds of formula I may be a cyclopentylene radical

cyclohexylene or C 2 -C 6 alkylene optionally substituted by one or more C 1 -C 4 alkyl substituents. Preferred A substituents are cyclopentylene, cyclohexylene or alkylene of formula

RiO Ri2

I I —c — c—

I I

R 11 _R 13 wherein R ¹⁰ , R ¹¹ , R ¹² and R ¹³ are each independently hydrogen or C 1 -C 4 alkyl. According to a preferred embodiment, they are those compounds of formula I wherein A is -CH ₂ CH _2; - —chch ₂ -,

I ch ₃ or "CH ₂ CH".

CH; j

In the case of certain compounds of formula I bearing a cycloalkylene or branched alkylene group as defined by A, one or more other asymmetric carbon atoms may be present resulting in the formation of diastereoisomers. The invention includes mixtures of these diastereoisomers as well as individual pure diastereoisomers.

One preferred group of R ¹⁴ substituents is represented by the formula represented by an optionally substituted heteroaryl radical as defined above containing at least one ring nitrogen atom and attached to the carbon atom of radical A via a ring nitrogen atom to form a quaternary ammonium group.

Preferred compounds of the above group are those wherein A is a group

-CH ₂ CH ₃ -, —ch ₂ CH- ₃ or —CH ₂ CH — ch ₃ and either

a) R ¹ and R ⁸ together form the radical of the formula

HOCH ₂ \

C = h ₃ c or

b) R ⁸ is hydrogen and R ¹ is hydrogen, CH ₃ CH ₃ -,

CH ₃ \

CH— ch ₃

_CH3 OH \ ic / or CH ₃

1 * 9

OH

CH ₃ CH—

Particularly preferred are those in which R ⁹ represents a hydrogen atom and R ¹ represents a residue

OH

CH ₃ CH—.

preferably compounds having the absolute configuration of 5R, 6S, 8R.

In accordance with a particularly preferred embodiment, the invention provides a process for preparing those compounds in which the residue is present

-o represents a radical of formula

in which

R ⁵ and R ^{6 are each} independently hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, substituted hydroxy, C 1 -C 4 alkylthio, or amino.

Of this subgroup, those compounds in which A is -CH ₂ CH ₂ -CH ₃ -CH ₂ - and R ¹ and R ⁸ together form a grouping are most preferred.

HOCH ₂ \

C = /

h ₃ c or

b) R ⁸ represents a hydrogen atom and R ¹ represents a residue

CH ₃ CH ₃ -,

CH _t \

CH— /

ch ₃

CH ₃ OH

CH ·} or

OH

CH _S CH—.

Particularly preferred are those in which R ⁸ is hydrogen and R ¹ is a radical

OH

Preferably, CHfCH- is a compound having the absolute configuration of 5R, 6S, 8R.

In accordance with another preferred embodiment, the invention provides a process for the preparation of compounds wherein:

-O

means a radical of the formula | ^\! “·> - SCh * or —ch ₂ ch— ch ₃ and where either

Of this subgroup of compounds, those are those wherein A is —ch ₂ ch ₃ —ch ₂ ch ₂ -

-ch ₂ chch ₃ and where will it be

(a) R ¹ and R ² together form a group

HOCH ₂ \

C = /

h ₃ c or

b) R ⁸ represents a hydrogen atom and R ¹ represents a group

CH ₃ CH ₂ CH ₃ \

CH— /

. CH ₃

CH ₃ OH \ lc— /

ch ₃ or

OH

AND

CH ₃ CH—.

Particularly preferred are those wherein R ⁸ is hydrogen and R ¹ is a group

OH

CH ₃ CH - preferably compounds having the absolute configuration of 5R, 6S, 8R.

In accordance with another preferred embodiment, the invention provides a process for the preparation of those compounds in which it is a pyridinium residue.

Of this subgroup of compounds are those preferred compounds wherein the symbol A is -CHCH ₂ - CH ₃

-ch ₂ ch ₂ -

or —CH ₂ CH - CH ₃ and wherein either R ¹ and R ⁸ together form a group

HOCHi \

C = /

H ₃ c or

b) R ⁸ represents a hydrogen atom and R ¹ represents a hydrogen atom, a group

CH ₃ CH ₂ _ ch ₃ \

CH— /

ch ₃

CH ₃ OH \ lc— /

^CH 3 or

OH

CH ₃ CH—.

Particularly preferred are those wherein R ⁸ is hydrogen and R ¹ is a group

OH

CH ₃ CH— (4)

CH OH ' ^CH ? ^CH 2 ^N CO is preferably a compound having the absolute configuration of 5R, 6S, 8R.

In accordance with the most preferred embodiment, the invention describes a method of preparing compounds of formula (5) ^CH I ^CH Z ^N \ ^ /

OH

(6)

SCH, (7) wherein - A ^CH 2 ^CH 2 ^N

(8) represents residue (1) (9) (2)

OCH _and (10)

(Π)

R or S diastereoisomers (12) in which

R ¹⁶ represents a C 1 -C 4 alkyl group and a residue

(13)

represents a piperazine, morpholine, thiomorpholine or thiomorpholine-S-oxide moiety which may be substituted as described above.

In accordance with a preferred embodiment, the invention describes a process for the manufacture of those compounds in which

(14) represents a radical of formula, N

CH,

R, R or S, S diastereoisomers on two asymmetric carbons of the cyclohexyl group

+ HH i V. / ch ₃ cHi;

\ rr \

-N N-CH * ¹ 4 \ _J $ _CH3 \ \ / - \ '

CHa

R ² represents a hydrogen atom, an anionic negative charge, or a conventional readily cleavable carboxyl function protecting group, provided that when R ² is a hydrogen atom or a protecting group, a balancing charge is also present, and pharmaceutically acceptable acid addition salts thereof.

Another group of quaternized R ¹⁴ substituents can be represented by the general formula in which

Y represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.

Of this subgroup those compounds are preferred in which A represents a radical CH ₃ -CHCHa-

-CH ₂ CH-, CH _3, or - _(CH2) "- wherein n is an integer of 2, 3 or 4, then even more preferred are those compounds wherein A is the radical -CH _{2 CH 3} - CH ₂ CH ₂ CH ₂ - —chch ₃ -

CH ·, ch ₃ \

CH— /

CH ₃

_CH3 OH \ LC. / ch ₃ or '

OH

CH ₃ CH—.

Particularly preferred are those in which R ⁸ is hydrogen and R ¹ is a radical

OH

CH ₃ CH - especially compounds with the absolute configuration of 5R, 6S, 8R.

According to an even more preferred embodiment, the invention provides a process for the preparation of those compounds in which the residue or represents a group --CH ₂ CH - CH ₃ and most preferably those in which A represents a residue

CH *>

-CH ₂ CH ₂ CH ₂ cm

CH, and wherein either (a) R ¹ and R ⁸ together form a group

H0CH ₂ '3' CH

CH * \ ^J / - \

0 = h ₃ c or (b) R ⁸ represents a hydrogen atom and R ¹ represents a hydrogen atom, a group

CH ₃ CH ₂ CHCH ₂ - ch ₃

Preferred compounds from this preferred group below are those compounds wherein A constitutes radical or CH ₃

Ch —ch ₃ ch— ch ₃

CH—

Z ch ₃

CH ₃ OH

NI c—

CH ₃ or

- (CH ₃ ) _n where n is a number of 2, 3 or 4, more preferred are those in which A is a group

OH

CH ₃ CH—.

Particularly preferred are those compounds wherein R ⁸ is hydrogen and R ¹ is -CH ₂ CH ₂ - -CH ₂ CH ₂ CH ₂ -

OH

CH ₃ CH— or —chch ₂ ch _3, and in particular substances with an absolute configuration of 5R, 6S, 8R.

According to a most preferred embodiment, the invention comprises a process for the preparation of compounds of the formula

OH

1, (R)

O * ^N 'C-OOR ² ' —ch ₂ ch — ch ₃ and most preferably those compounds in which A represents a group in which the grouping

CH ₂ CH ₂ - and where either (and R ¹ and R ⁸ together form an H ₂ CH ₂ moiety ₎

means the rest

0 = h ₃ c or (b) R ⁸ represents a hydrogen atom and R ¹ represents a hydrogen atom, a group ch ₃ ch ₂ -n: p

CH%

-NS (a) - (both α- and β-diastereoisomers)

ch ₃

CHi, \ 4 ~ λ x n <z.bo -N Ň \ _J x

CH, CH ₂ and R ² represent a hydrogen atom, an anionic negative charge or a conventional readily cleavable carboxyl function protecting group, wherein when R ² represents a hydrogen atom or a protecting group, a balancing ion is also present, and their pharmaceutically acceptable addition salts with acids.

Certain products within the scope of formula (I) may be formed as optical isomers as well as mixtures of epimers. It should be noted that the invention encompasses both these optical isomers and mixtures of epimers. For example, when the substituent at the 6-position is a hydroxyethyl group, the substituent may have both R and S configurations and the invention includes both the resulting isomers and mixtures of epimers.

Intermediates of formula (IV) are used in the process of the invention

W

«(IV).

which are described, for example, in European Published Application No. 38,869 and which can be prepared by the general methods described therein. The symbol L represents a conventional leaving group (designated by X in European Patent Application No. 38,869), such as chlorine, bromine, iodine, benzenesulfonyloxy, p-toluenesulfonyloxy, p-nitrobenzenesulfonyloxy, methanesulfonyloxy, trifluoromethanesulfonyloxy, triethyloxyphenoxy, triethyloxyphenoxy, triethyloxyphenoxy, triethyloxyphenoxy, triethyloxyphenoxy. group is diphenoxyphosphinyloxy. Intermediates of formula IV is generally prepared in situ by reacting an intermediate of formula III, _R j and CR

"CO 07 RO:

in which

R ¹ , R ⁸ and R ² are as defined above, with a suitable acylating agent of the general formula

R ° —L

A preferred intermediate of formula IV wherein L is a dlphenoxyphosphinyloxy group may be prepared by reacting a ketoester of formula III in an inert organic solvent such as methylene chloride, acetonitrile or dimethylformamide with an approximately equimolar amount of diphenyl chlorophosphate in the presence of a base such as diisopropylethylamine, triethylamine. dimethylaminopyridine and the like, at a temperature ranging from about -20 ° C to +40 ° C, most preferably at about 0 ° C. The intermediate of formula (IV) may optionally be isolated, but is conveniently used as a starting material for the process of the invention without isolation and purification.

In the process according to the invention, the carbapenem intermediate of formula IV is reacted with a quaternary amine-thiol of formula VII

HS-A-R ^at X '(VII) wherein

A represents a cyclopentylene group, a cyclohexylene group or a C2-6 alkylene group optionally substituted by one or two C1-4 alkyl groups, most preferably a cyclopentylene group, a cyclohexylene group or a radical of the formula

R10R12 [I —C — C—

Ø ¹¹ R ¹³ where

R ^10, R ^n, R ⁿ and R ¹³ independently of one another are hydrogen or alkyl having 1 to 4 carbon atoms,

X ^- represents a balancing anion of a strong acid, such as the anion Cl -, Br ^- , CH ₃ SO ₃ ^- , CF ₃ SO ₃ ^- or

R ¹⁴ is the above-defined aromatic or nonaromatic heterocycle containing a quaternized nitrogen atom.

This reaction is carried out in an inert solvent such as acetonitrile, acetonitrile / dimethylformamide, tetrahydrofuran, tetrahydrofuran / water, acetonitrile / water or acetone, in the presence of a base. The nature of this base does not play a decisive role, but the best yields are obtained using a non-nucleophilic tertiary amine base such as diisopropylethylamine, 1,8-diazabicyclo [5,4,0] undec-7-ene, 1,5-diazabicyclo [4,3, O] non-5-ene or C 1 -C 4 trialkylamine in each alkyl moiety, such as triethylamine, tributylamine or tripropylamine.

The reaction of the intermediate IV with the thiol VII can be carried out over a wide temperature range, for example at -15 ° C to room temperature, but is preferably carried out at a temperature of about -15 ° C to +15 ° C, most preferably at about 0 ° C.

The carbapenem compound formed by the reaction of quaternary aminothiol of formula VII with intermediate IV will contain a buffering anion [e.g., (C _Fi H O) ₂ PO ₂ ^-, Cl ^-, or the anion contained in the quaternary thiol] which may at this stage to replace it with another balancing anion, for example one that is more suitable from a pharmaceutical standpoint. This substitution is carried out in a conventional manner. Alternatively, the equivalent anion can be removed in a subsequent deprotection step. If the quaternized carbapenem compound and the balancing anion form an insoluble product, it can crystallize from the reaction mixture immediately after its formation and can be isolated in pure form by simple filtration.

After the formation of the desired carbapenem product, the R ² 'protecting group of the carboxyl function in the compound of formula Γ may optionally be cleaved by conventional methods such as solvolysis, chemical reduction or hydrogenation. When a protecting group cleavable by catalytic hydrogenation, such as a p-nitrobenzyl, benzyl, benzhydryl or 2-naphthylmethyl group, is used, such an intermediate of formula (Γ) may be used in a suitable solvent such as dioxane, water and ethanol, tetrahydrofuran, diethyl ether. and a buffer, in a mixture of tetrahydrofuran, aqueous potassium monohydrogen phosphate and isopropanol, and the like, in the presence of a hydrogenation catalyst such as palladium on carbon, palladium hydroxide, platinum oxide, etc., at a temperature of 0 to 50 ° C for about 0.24 to Apply hydrogen for 4 hours at a pressure of 0.1 to 0.4 MPa. If R ² 'is, for example, an o-nitrobenzyl group, it can also be cleaved by photolysis. Such protecting groups, such as 2,2,2-trichloroethyl, can be removed by a slight reduction with zinc. The allyl protecting group may be cleaved using a catalyst consisting of a mixture of a palladium compound and triphenylphosphine in a suitable aprotic solvent such as tetrahydrofuran, methylene chloride or diethyl ether. Similarly, other conventional carboxyl function protecting groups may be cleaved by methods known in the art. Finally, as mentioned above, those compounds of formula Γ wherein R ² 'is a physiologically hydrolysable ester moiety such as acetoxymethyl, phthalidyl, indanyl, pivaloyloxymethyl, methoxymethyl, and the like can be administered directly to the patient, thus, without leaving the protecting group, since such esters are hydrolyzed in vivo under physiological conditions.

If the radical R ¹ and / or R ^8, or quaternized nucleophile ^R14 bonded to the group A contains a functional group that can be 11a malfunction intended course of reaction, it may be a functional group protected by a suitable protecting group, and then if removed to regenerate the desired functional group . Suitable protecting groups and procedures for their introduction and removal are well known to those skilled in the art.

As with other β-lactam antibiotics, the compounds of formula (I) may be converted into pharmaceutically acceptable salts which are equivalent to the free compounds for the purposes of this invention by known methods. For example, the compound of formula I wherein R ² is an anionic charge, dissolved in a suitable inert solvent and the solution is added an equivalent amount of a pharmaceutically acceptable acid. The desired acid addition salt may be isolated in a conventional manner, for example by solvent precipitation, lyophilization, and the like.

If the compound of formula (I) contains further basic or acidic functional groups, further addition salts with pharmaceutically acceptable bases and pharmaceutically acceptable acids can be prepared analogously by known methods.

A compound of formula I wherein R ² is a hydrogen atom or an anionic charge, or a pharmaceutically acceptable salt thereof, can also be converted by conventional methods to the corresponding compound wherein R ² is a physiologically hydrolyzable ester moiety, or a compound of formula I wherein R 2 ² is a conventional carboxyl-protecting group, may be converted to the corresponding compound wherein R ³ represents a hydrogen atom, an anionic charge, or a physiologically hydrolyzable ester moiety, or a pharmaceutically acceptable salt thereof.

The process for the preparation of the starting thiols of the above general formula (II) is the subject of our related Czechoslovak patent application AO No. 256,686.

The carbapenem derivatives of general formula I wherein R ² is hydrogen, an anionic charge or a physiologically hydrolyzable carboxyl protecting group, and pharmaceutically acceptable salts thereof, are potent antibiotics active against various Gram-positive and Gram-negative bacteria and can be used for example as feed additives growth stimulating animals, as food and feed preservatives, as bactericides for industrial applications, for example, in water-based coatings and in white waters from paper machines to inhibit the growth of harmful bacteria, and as disinfectants to kill or inhibit the growth of harmful bacteria at medical and dental facilities. However, the disclosed compounds are particularly useful for the treatment of infectious diseases in humans and other animals caused by Gram-positive and Gram-negative bacteria.

The pharmaceutically active compounds produced by the process of the present invention may be used alone or may be formulated into pharmaceutical compositions containing, in addition to the active carbapenem component, a pharmaceutically acceptable carrier or diluent. The compounds can be administered by a variety of routes, of which oral, topical or parenteral (intravenous or intramuscular injection) is of crucial importance.

The pharmaceutical compositions may be in solid form (such as in the form of capsules, tablets, powders, etc.) or in liquid form (such as in the form of solutions, suspensions or emulsions). Formulations for injection, which are preferred preparations, may be presented in unit dosage form in ampoules or in containers containing multiple unit doses, and may contain excipients such as suspending agents, stabilizers, and dispersing agents. These compositions may be in a ready-to-use form or in powder form, which upon reconstitution is reconstituted with a suitable vehicle, such as sterile water.

The dosage employed depends largely on the active ingredient, the nature of the dosage form, the route of administration, the condition of the patient, the site of infection and the organism to be controlled. The choice of the preferred dosage and route of administration is, of course, a matter for the attending physician, but in general, the compounds of the invention may be administered to a mammal in need of treatment at about 5 to 200 mg / kg / day. The total daily dose is generally administered in several divided doses, for example three to four times daily.

The invention is illustrated by the following non-limiting examples.

Example 1

Preparation of 3- (2- (1-pyridinium) ethylthiilo) -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

A. 1- (2-mercaptoethyl) pyridinium methanesulfonate

+ MsOH ° C ~ ------— hours

(Ms = methanesulfonyl)

To a suspension of pyridimum methanesulfonate in pyridine, prepared with cooling by dropwise addition of 1.95 ml (0.03 mol) of methanesulfonic acid to 8.0 ml (0.099 mol) of pyridine, was added 1.96 ml (0.033 mol) of ethylene sulfide. The resulting mixture was stirred at 55 ° C for 16 hours and then concentrated under reduced pressure to a thick syrup which was mixed with a few milliliters of water. The resulting solution was loaded onto a x -ondapak (40 x 16 cm) column

C-18 and elute the column with water. Lyophilization of the appropriate fractions yielded 6.5 g (91%) of a colorless syrup-like product.

IR (film): in _max

300—2 600 (wide belt, SH),

635 (pyridinium residue),

490, 1200 (sulfonate residue),

1068, 1060, 1045, 791, 780 cm @ -1) @ ¹ H-NMR (CDCl3) .delta.

2.32 (3H, singlet, CH 3 SO 3 -),

2.61, 2.70, 2.73, 2.82 (1H, part B of system A ₂ B, SH),

3.07 (2H, multiplet [with D ₂ O 3.08 (2H, triplet, J = 6.5 Hz)], CH <> Sj,

4.76 (2H, triplet, J = 6.5 Hz, CH ₂ N ^+), 8.19 (2H, m, Hm pyridinium group)

8.6 (1H, multiplet, Ho of the pyridinium residue),

9.08 (2H, double doublet, J = 6.8 Hz, J = 1.4 Hz, Ho of the pyridine residue).

UV (water: A _max 206 (ε 5 230),

258 (ε 3,760) nm.

Method A

Β. 1- (2-mercaptoethyl) pyridinium chloride

permutite S — 1

Cl

-z HS

An aqueous solution of 9.4 g (0.04 mol) of crude 1- (2-mercaptoethyl) pyridinium methanesulfonate was applied to a column (2.5 x 41 cm) of permutite S-1 (Cl) ion exchanger. The column is eluted with water at a rate of 0.5 ml per minute, the appropriate fractions are combined and lyophilized. 7.0 g (100%) of a yellowish syrup product are obtained, which product is used as is in the following stage.

¹ H-NMR (deuterium oxide, values <5):

3.22 (2H, m, multiplet, CH, S),

4.88 (m, CH ₂ N +),

8.18 (2H, multiplet, Hm of pyridinium)],

8.7 (III, multiplet. Hp of pyridinium residue),

9.0 ppm (2H, multiplet, Ho of the pyridinium residue).

Method B

To ice-cooled pyridine (5.6 mL, 70 mmol) was added pyridine hydrochloride (4.05 g, 35 mmol) and ethylene sulfide (2.1 mL, 35 mmol). The mixture was heated to 65 ° C and stirred for 75 minutes while switching to a two-phase system. The lighter phase was removed, the oily residue was washed five times with 10 ml of ether each time and dried under high vacuum. The title compound was obtained (yield 90-100%), which was used in the next step without further purification.

C. p-Nitrobenzyl-3- [2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate chloride

1) NEt (iPr) ₂

OH

COOPNB

O

II

2) ClP (OPh) ₂

4) NEt (iPr) ₂

(PNB - p-nitrobenzyl Et = ethyl iPr = isopropyl)

A solution of 6.09 g (17.5 mmol) of p-nitrobenzyl-6α- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2-carboxylate in 20 ml of acetonitrile was cooled to + 5 ° C under nitrogen and 3.65 ml (21.0 mmol) of diisopropylethylamine and 4.34 ml (21.0 mmol) of diphenyl chlorophosphate were gradually added thereto. The resulting mixture was stirred at 5 ° C for 30 minutes, then cooled to -5 ° C, and a solution of crude 1- (2-mercaptoethyl) pyridinium chloride (4.3 g, 24 mmol) in 1.0 mL was added first. of diisopropylethylamine (3.65 ml, 21.0 mmol) was added dropwise. The reaction mixture was stirred at 0 ° C for 1 hour, then cooled to -30 ° C and stirred for an additional 15 minutes. minutes. The solid material was filtered off and washed with cold (-30 ° C) acetonitrile. 5.77 g (65%) of the desired product is obtained.

IR (nujol) max max

300 (OH)

1775 (G = O / 3-lactam)

690 (C = O of p-nitrobenzyl ester),

630 (pyridinium residue),

605 (phenyl p-nitrobenzyl ester),

1515 (NO _2),

335 ^cm-1 _(and NO).

^f H-NMR (perdeuterodimethylsulfoxide, δ in ppm):

1.17 (3H, doublet, J = 6.1 Hz, CH 3 CHOH),

3.2 to 3.75 (5H, H-4, H-6, _CH2 S),

3.75-4.5 (2H, H-5, CH 3 CHOH),

4.92 (2H, broad triplet, J - 6.5 Hz, CH ₂ N ^+),

5.18 (1H, doublet, J = 4.9 Hz, OH),

5.37 (center of AB-quartet, _{J, b} = 14.2 Hz, CH ₂ P-nitrobenzylic residue)

7.69 (2H, doublet, J = 8.7 Hz, Ho of the p-nitrobenzyl residue),

8.24 (doublet, J = 8.7 Hz, Hm of p-nitrobenzyl residue),

8.0-8.4 (4H, Hm of p-nitrobenzyl residue, Hm of pyridinium residue),

8.66 (1H, multiplet, Hp of the pyridinium residue),

9.17 (2H, broad doublet, J = 5.5 Hz, Ho of pyridinium residue).

The filtrate was combined with the washings and diluted with 150 mL of ether. The supernatant was decanted off and the resinous residue was dissolved in 40 ml of water containing sufficient acetonitrile to dissolve any solid material. Load the solution onto a μ-bondapak C-18 column (3 x 10 cm), eluting first with 150 ml of a mixture of 10% acetonitrile and 90% water and then with 100 ml of a mixture of 50% acetonitrile and 50% water. Appropriate fractions were combined, the acetonitrile was evaporated in vacuo and the residue was lyophilized. A yellowish powder product is obtained. The NMR spectrum of this product indicated the presence of the title compound, in admixture with p-nitrobenzyl-3- [2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-diphenylphosphate. -azabicyclo [3.2.0] hept-2-ene-2-carboxylate (2: 1). The powdered product was dissolved in a minimum amount of water and passed through a column (1.5 x 21 cm) of permeability S-1 (Cl ^- ) using water as the eluent. Lyophilization of the appropriate fractions gave 1.8 g (20%) of the title compound.

D. p-Nitrobenzyl-3- [2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- diphenylphosphate en-2-carboxylate

ČOOPNB

1) Net (iPr) ₃

O

II

2) ClP (OPh) ₂

3)

MóO

A solution of 0.174 g (0.50 mmol) of p-nitrobenzyl-6- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2-carboxylate in 2 mL of acetonitrile was added in It was cooled to 0 ^° C under nitrogen atmosphere and diisopropylethylamine (0.105 mL, 0.60 mmol) and diphenyl chlorophosphate (0.124 mL, 0.60 mmol) were added sequentially. The solution was stirred at 0 ° C for 30 minutes, then a solution of 0.170 g (0.72 mmol) of 1- (2-mercaptoethyl) pyridinium methanesulfonate in 0.6 mL of acetonitrile and 0.105 mL (0.60) was gradually added. mmol) of diisopropylethylamine. The reaction mixture was stirred at 0 ^° C for 15 minutes, then diluted with 7 mL cold water (0 ° C) and poured onto a column (1.5 x 6.4 cm) of μ-bondapak C-18 adsorbent. The column is eluted with a mixture of acetonitrile (25-50%) in water (75-50%). Appropriate fractions were combined, the acetonitrile was evaporated in vacuo and the residue was lyophilized. 0.33 g (92%) of a yellowish powder product is obtained.

IC (KBr technology):

^ niax

600-3000 (OH),

1765 (C = O / Haktam)

690 (C = O of p-nitrobenzyl ester j,

625 (pyridinium residue),

585 (phenyl),

1510 (NO ₂ ),

330 (NO)

885 cm ^-1 (NO).

NMR-NMR (perdeuterodimethylsulfoxide, δ in ppm):

1.16 (3H, doublet, j = 6.2 Hz, CH 3 CHOH), 4.87 (2H, broad triplet, J = 6.6 Hz, CH ₂ Si), 5.37 (center of AB-quartet, J _{and ab} = 14.3 Hz,

CH ₂ P-nitrobenzylic residue],

6.7-7.5 (phenyl),

7.68 (doublet, J = 8.8 Hz, Ho of the p-nitrobenzyl residue),

8.23 (doublet, J = 8.8 Hz, Hm of p-nitrobenzyl residue),

8.0-8.3 (multiplet, Hm of pyridinium residue),

8.4-8.8 (1H, Hp of pyridinium residue), 9.09 (2H, double doublet, j = 6.7 Hz, j = 1.3 Hz, H of pyridinium residue).

E. 3- [2- (1-pyridinium ethylthio) -6- [1- (R-hydroxyethyl) -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

Method A

10% Pd / coal,

H ₂ , tetrahydrofuran, ether

To a solution of p-nitrobenzyl-3- [2- (1-pyridinyl) ethylthio] -6a- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclof 3 diphenylphosphate (0.16 g, 0.22 mmol) 2,0] Hept-2-ene-2-carboxylate in 10 ml of wet tetrahydrofuran is added 10 ml of ether, 16 ml of 0.05 M pH 7.4 buffer based on potassium dihydrogen phosphate and sodium hydroxide and 0.16 g of 10% The resulting mixture was hydrogenated at 25 DEG C. under 50 psi for 1 hour, the phases were separated and the organic phase was extracted twice with 3 ml of water each time, and the aqueous solutions were combined, washed twice with 10 ml of ether each time and applied. on a column (1.5 x 6.2 cm) μ-bondapak C-18 (after removing the last traces of organic solvents by evaporation in vacuo).

The column is eluted with water and the appropriate fractions are lyophilized. 0.062 g (84%) of a yellowish powder product was obtained.

IR (KBr): in _max

700-3000 (OH),

755 (C = O / J-lactam)

630 (pyridinium residue),

590 cm ^-1 (carboxylate).

1 H-NMR (CDCl 3, δ values in ppm): 1.22 (3H, doublet, J = 6.4 Hz, CH ₃ CHOH),

2.92 (doublet, J = 9.1 Hz, H-4),

2.97 (doublet, J = 9.1 Hz, H-4),

3.20 (double doublet, J = 2.5 Hz, J = 6.1 Hz, H-6),

3.44 (triplet, J = 6.0 Hz, _CH2 S),

3.93 (double doublet, J = 9.1 Hz, J = 2.5 Hz, H-5),

4.82 (triplet, J = 6.0 Hz, CH _and N +),

8.04 (multiplet, Hm of pyridinium residue),

8.5 (multiplet, Hp of pyridinium residue),

8.82 (double doublet, J = 3.2 Hz, J = 1.1 Hz, Ho of the pyridinium residue).

UV (water):

^ max

259 (ε 5,800),

296 (ε 7,030) nm.

τι / 2 = 13.5 hours (measured at ΙΟ ^-4 M in phosphate buffer pH 7.4 at 36.8 ° C).

Method B

Cl

10% Pd / carbon, H ₂

N

tetrahydrofuran, ether, pH 7.2 buffer

To a solution of 5.77 g (11.4 mmol) of p-nitrobenzyl-3- [2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo chloride [3,2,0] hept-2-en-2-carboxylate in 170 ml of 0.2 M potassium dihydrogen phosphate and sodium hydroxide buffer (pH 7.22) is added 30 ml of tetrahydrofuran, 30 ml of ether and 7 g of 10% palladium on carbon. the resulting mixture for 1 hour hydro ₃ shall pay at 2'2 ^C C at a pressure of 0.28 MPa, and then filtered through diatomaceous earth. filter aid was washed twice with 15 ml of water, the filtrate is combined with The aqueous phase is separated, washed three times with 100 ml of ether each and loaded onto a column (4.5 x 20 cm) of p-bondapak C-18 (after removal of the organic solvents by evaporation under vacuum). Elution of the column with water first and then 1% aqueous acetonitrile and lyophilization of the appropriate fractions gave 2.48 g (65%) of the title compound as a white solid, m.p. The analytical data of this product were those obtained for the compound prepared according to Method A.

Example 2

Preparation of 3- (2- (1- (3,5-dimethylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate

A. 1- (2-mercaptoethyl) -3,5-dimethylpyridinium methanesulfonate

To a suspension of 3,5-lutidinium methanesulfonate in 3,5-lutidine, prepared by adding 0.65 ml (0.010 mol) of methanesulfonic acid to 2.51 ml (0.022 mol) of cold 3,5-iutidine, is added 0.655 ml (0.011 mol) ) ethylene sulfide. The resulting mixture was stirred at 55 ° C under nitrogen for 24 hours, then cooled to 23 ° C and diluted with 5 mL of water and 5 mL of ether. The organic layer was separated and the aqueous solution was washed six times with 4 ml of ether each time. The residual ether is removed by evaporation in vacuo and the aqueous solution is applied to a column (2.5 xx 6.0 cm @ 3 μ-bondapak C-18 which is eluted with water. Lyophilization of the appropriate fractions gives 2.4 g (91%) of a colorless syrup. product.

IC (film). Vniax

520

628 (pyridinium residue),

600, 1 495, 1 325, 1 305, 1 283, 1200 (sulphonate),

040, 938, 765, 680 ^cm-first

Dd-NMR (perdeuterodimethylsulfoxide, ppm):

2.31 (3H, s, CH ₃ SO ₃ -),

2.47 (6H, singlet, CH ₃ on pyridinium residue),

2.57, 2.66, 2.69, 2.78 (1H, part B of the system)

A, B, SH),

3.06 (2H, multiplet [with deuterium oxide added (2H, triplet, J = 6.5 Hz)], CH> S},

4.65 (2H, triplet, J - = 6.5 Hz, CH ₂ N +), 8.34 (1H, s, Hp of pyridinium group)

8.79 (2H, singlet, H ¹ pyridinium residue).

UV (water): A _msx

271 (ε 4,860) nm.

For C ₁₀ H ₁₇ NO ₃ S ₂ · 0.5 H ₂ 0 calculated:

H, 6.66; N, 5.14; S, 23.54. Found:

H, 6.49; N, 5.17; S, 24.18.

B. p-Nitrobenzyl-3- [2- (1- (3,5-dimethylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.1] diphenylphosphate O] hept-2-ene-2-carboxylate

1) NEt (iPr) ₂

O

2) ClP (OPh) ₂

3)

4) Net (iPr) ₂

To a solution of 0.523 g (1.50 mmol) of p-nitrobenzyl 6α- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2-carboxylate in 6 1.0 ml of acetonitrile, cooled to 0 DEG C., was first treated with 0.314 ml (1.8 mmol) of diisopropylethylamine and then 0.373 ml (1.8 mmol) of diphenylchlorophosphate under nitrogen, and the reaction mixture was stirred for 30 minutes. solution of 0.493 g (1.87 mmol) of 1- (2-mercaptoethyl) -3,5-dimethylpyridinium methanesulfonate in 1.9 ml of acetonitrile and 0.314 ml (1.8 mmol) of diisopropylethylamine was added and the resulting mixture was stirred at 0 for 1 hour. ° C, then diluted with 26 ml of cold water (0 ° C) and applied to the column (7.0 x 3.5 cm) μ-bondapak C-18, eluting with 25 to 50% acetonitrile and 75 to 50% acetonitrile. 50% water, the appropriate fractions were combined and lyophilized to give 1.01 g (90%) of the title compound as a yellowish powder.

IR (KBr): Aniax

700—3100 (OH),

778 (C = O / Mactam)

700 (C = O of p-nitrobenzyl ester),

635 (pyrldinium residue),

595 (phenyl)

1521 (NO _2),

335 (NO ₂ ),

895 cm @ ^-1 _(NO2).

Ή-NMR (perdeuterodimethylsulfoxide, δ in ppm):

_1,16 (3H, doublet, J = 6.1 Hz, CH ₃ CH0H), 2.43 (s, _CH3 on pyridinium moiety)

4.75 (2H, multiplet, CH ₂ N ^+),

5.38 (center of AB-quartet, J _a , _(l = 14.3 Hz,

CH _{2 of} p-nitrobenzyl radical), θ, θ-7,5 (10H, multiplet, phenyl),

7.70 (2H, doublet, J = 8.7 Hz, Ho of the p-nitrobenzyl residue),

8.0-8.5 (3H, multiplet, Hp of pyridinium residue, Hm of p-nitrobenzyl residue),

8.82 (2H, singlet, Ho of the pyridinium residue).

UV (water): A _max 270 (ε 11 570),

306 (ε 7,343) nm.

Analysis: for C ₃₇ H ₃₈ N ₃ O ₁₀ SP. H ₂ O calculated:

H, 5.26; N, 5.48; S, 4.18. Found:

H, 5.05; N, 5.22. S, 4.34.

C. 3- (2- (1- (3,5-dimethylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept- 2-ene-2-carboxylate

tetrahydrofuran, ether, buffer

To a solution of p-nitrobenzyl-3- [2- (1- (3,5-dimethylpyridinium) -1-ethylthio] -6- [1- (R) -hydroxyethyl] -7- (0,600 g, 0.80 mmol) diphenylphosphate oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 36 ml tetrahydrofuran can be added 36 ml ether, 44 ml 0.05 M potassium dihydrogen phosphate buffer and sodium hydroxide (pH 7.4) and 0.60 g of 10% palladium on carbon. The resulting mixture was hydrogenated at 23 ° C under 50 psi for 1.25 hours, the organic layer was separated and extracted twice with 5 ml of buffer each time. Combine the aqueous layers, filter through a pad of diatomaceous earth, wash the filter aid with 40 ml of ether, remove the last traces of organic solvents in vacuo and apply to a column (2,5 x 10,0 cm) μ-bondapak C-18. Elution of the column with water and lyophilization of the appropriate fractions gave 0.186 g (64%) of the title compound as a yellowish powder.

IR (KBr): _{ν max}

700-300 (OH),

760 (C = O / J-lactam)

595 cm @ ^-1 (carboxylate).

^j H-NMR (deuterium oxide, δ in ppm):

1.21 (3H, doublet, J = 6.3 Hz, CH 3 CHOH),

2.45 (6H, singlet, CH _:( on pyridinium residue),

2.81 (doublet, J = 9.2 Hz, H-4),

2.96 (doublet, J = 9.2 Hz, H-4),

3.22 (double doublet, J = 2,6 Hz, J = 6,2 Hz, H-6),

3.40 (triplet, J = 6.2 Hz, _CH2 S),

3.84 (double doublet, J = 9.2 Hz, J = 2.6 Hz, H-5),

4.15 (multiplet, CH ₃ CHOH),

4.71 (triplet, J = 6.2 Hz, CH ₂ N +),

8.21 (1H, singlet, Hp of the pyridinium residue),

8.46 (2H, singlet, Ho of the pyridinium residue).

UV (water): A _max 279 (ε 8,345),

296 (ε 7,714) lim.

[Α] D + 40.7 ° (c = 0.53, water).

τι / ₂ - 16.9 hours (measured at a concentration of 10 'M in phosphate buffer pH 7.4 at 36.8 ° C).

Example 3

Preparation of (5R, 63) -3 - ((2- / 3-hydroxymethylpyridinio / ethyl) thio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept -2-ene-2-carboxylate

A. 3-Hydroxymethyl-1- (2-mercaptoethyl) pyridinium trifluoromethanesulfonate

CŘLOH

CH _Z OW / O> + cf ₃ Sat ₃ h + cf ₃ Sat ₃

Κ 2.91 ml (0.030 mol) of 3-pyridine-methanol was added dropwise first, 1.327 ml (0.015 mol) of trifluoromethanesulfonic acid and then 0.89 ml (0.015 mol) of ethylene sulfide. The resulting homogeneous mixture was heated to 50-70 ° C under nitrogen in an oil bath for 20 hours, then taken up in 15 ml of water and extracted five times with 5 ml of dichloromethane each time. The aqueous phase is concentrated in vacuo and the residue is applied to a reverse phase column (C, ₈ ). Elution of the column with water and evaporation of the appropriate fractions gave a pale yellow oily material which, after new chromatography, gave an almost colorless oil. After drying in vacuo over phosphorus pentoxide, 4.50 g (94%) of the product is obtained as a viscous oil.

IC (film): v _max 3450 (s, OH),

560 (w, SH) cm ^-1 .

¹ H-NMR (perdeuteroacetone, δ in ppm):

9.10—8.05 (multiplet, 4H, aromatic protons),

5.01 (triplet, J = 5.5Hz, 2H, N-CH3),

4.93 (s, 2H, -CH ₂ OH);

4.43 (broad singlet, 1H, —OH),

3.43 to 3.18 (multiplet, 2H, _S-CH2), 2.34 to 2.10 (m, 1H, SH).

B. p-Nitrobenzyl- (5R, 6S) -3- [2- (3-hydroxymethylpyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.1] diphenylphosphate O] hept-2-ene-2-carboxylate

To a solution of 0.174 g (0.50 mmol) of p-nitrobenzyl- (5R, 6S) -6- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2- of carboxylate in 2 ml of dry acetonitrile was added 0.096 ml (0.55 mmol) of diisopropylethylamine under nitrogen at 0 ° C. After the dropwise addition of 0.114 ml (0.55 mmol) of diphenyl chlorophosphate, the reaction mixture was stirred at 0 ° C for 30 min before a solution of 0.223 g (0.70 mmol) of 3-hydroxymethyl-1- (2-m pyridinium trifluoromethanesulfonate in 0.50 ml acetonitrile and then 0.122 ml (0.70 mmol) diisopropylethylamine The reaction mixture was allowed to react at 0 ° C for 30 minutes, then concentrated in vacuo and the residual yellow gum residue was removed. water, whereby to dissolve was added required amount of acetonitrile. the resulting solution was applied to a reversed phase (C _L8) which was eluted with 15% acetonitrile: water. Lyophilization of the appropriate fractions afforded 0.305 g (81%) of product as a beige solids.

IC (KBr-technique): in _mf1X 3,420 (broad signal, OH),

1775 (0 = O-lactam residue),

695 (-CO ₂ PNB) cm ^1st

¹ H-NMR (perdeuteroacetone, δ values in ppm):

9.44—7.72 (multiplet, 8H, aromatic protons),

7.22-6.91 (multiplet, 10H, diphenylphosphate), 5.53, 5.27 (AB-quartet, J = 14 Hz, 2H, benzyl group),

5.04 (triplet, J = 7.4 Hz, 2H, _N-CH2), 4.75 (s, 2H, _CH2 OH);

4.5 to 3.1 (m, 8H), 1.21 (doublet, J = 6.3 Hz, 3H, CH _3).

C. (5R, 6S) -3- [2- (3-Hydroxymethyl-pyridinio) -ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate

To a solution of p-nitrobenzyl- (5R, 6S) -3- [2- (3-hydroxymethylpyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-diphenylphosphate (0.145 g, 0.194 mmol) azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 10 ml tetrahydrofuran containing 5 drops of water was added 6.0 ml 0.05 M phosphate buffer (pH 7.4), 0.145 g 10% palladium on carbon and 10 mL of ether. The mixture was hydrogenated in a Parr apparatus at 50 psi for 1 hour and then filtered through a pad of diatomaceous earth. The filter cake is washed with a small amount of water and ether, the aqueous phase is separated and extracted three times with ether. The aqueous solution is cooled to 0 ° C and adjusted to pH 7.0 with a pH 7.4 buffer. The residual volatiles were removed in vacuo and the aqueous solution was applied to a reverse phase column (C _I8) which was eluted with water. Lyophilization of the appropriate fractions gave 36 mg (51%) of the product as a pale yellow solid.

After further purification by reverse phase high performance liquid chromatography, 31 mg (41%) of the product was obtained as a solid.

IR (KBr): in _max

300 (broad signal, OH),

755 (Τ-lactam carbonyl),

590 ^cm-1 ₍₂ --COO -).

Ή-NMR (deuterium oxide, δ in ppm): 8.78-7.94 (multiplet, 4H, aromatic protons),

4.83 (triplet, J = 6.0 Hz, 2H, _N-CH2)

4.83 (s, 2H, CH ₃ OH)

4.16 (doublet of quartets, J = J ‘= 6,2 Hz),

IH, H-1 ‘),

3.98 (doublet of triplets. J = 9.1 Hz, J = 2.6)

Hz, IH, H-5)

3.75-3.20 (multiplet, 3H),

3.20 to 2.65 (m, 2H), __1,22 (doublet, J = 6.4 Hz, 3H, CH _3).

UV (water): A _raax 294 (ε _7,614 ),

266 (ε 6,936) nm.

ie / 2 = 14.0 hours (pH 7.4, 36.8 ° C).

Example 4

Preparation of (5R, 6S) -3- [2- (4-hydroxymethylpyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate

A. 4-Hydroxymethyl-1- (2-mercaptoethyl) pyridinium trifluoromethanesulfonate

In ^N '^) ^ ^CH 2p ^{H +} + / -> ^hs ch ^ ch ₍

To a solution of 1.635 g (0.015 mol) of 4-pyridinmethanol in 10 ml of dichloromethane under nitrogen at 0 ° C was added dropwise 1.327 ml (0.015 mol) of trifluoromethanesulfonic acid, rapidly precipitating a light brown oily material. An additional 1.635 g (0.015 mol) of 4-pyridine-methanol was added to the mixture, and the solvent was evaporated under reduced pressure. To the oily residue was added 0.891 ml (0.015 mol) of ethylene sulfide and the resulting mixture was heated in an oil bath at about 60 ° C for 3 hours. The reaction mixture is taken up in 15 ml of water and the aqueous solution is washed five times with 5 ml of dichloromethane each time. After removal of the residual organic solvent by evaporation in vacuo, the remaining aqueous solution was applied to a reverse phase ( _C18 ) column which was eluted with water. Evaporation of the appropriate fractions gave an oily material which was further dried in vacuo over phosphorus pentoxide to give 4.64 g (97%) of the product as a colorless oil.

IR (film): in _Iliax 3,455 (s, OH),

565 (w, SH) cm -1.

⁴ H-NMR (perdeuteroacetone, δ in ppm):

9.07, 8.18 (AB-quartet, J = 6.8 Hz, 4H, aromatic protons),

5.03 (s, 2H, _CH2 OH);

4.96 (triplet, J = 6.5 Hz, 2H, _N-CH2), '4.09 (br s, 1H, -OH)

3.5-3.1 (multiplet, 2H, _S-CH2);

2.25 (broad singlet, 1H, -SH).

B. p-Nitrobenzyl- (5R, 6S) -3- (2- (4-hydroxymethylpyridinio) ethylthio) -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3-diphenylphosphate] , 2.0) hept-2-ene-2-carboxylate

To a solution of 0.348 g (1.0 mmol) of p-nitrobenzyl- (5R, 6S) -6- [1- (R'-hydroxyethyl) -3,7-dioxo-1-azabicyclo [3.2.0] heptane- Of 2-carboxylate in 5 ml of dry acetonitrile, under nitrogen at 0 [deg.] C., is dropped first with 0.191 ml (1.1 mmol) of diisopropylethylamine and then with 0.228 ml (1.1 mmol) of diphenyl chlorophosphate. A solution of 0.447 g (1.4 mmol) of 4-hydroxymethyl-1- (2-mercaptoethyl) pyridinium trifluoromethanesulfonate in 1 ml of acetonitrile and then 0.191 ml (1.1 mmol) of diisopropylethylamine was added thereto for 40 minutes. After a reaction time of 20 minutes at 0 ° C, the reaction mixture is filtered and the filtrate is concentrated in vacuo, the residue is taken up in a minimum volume of a mixture of equal parts of acetonitrile and water and applied to a reverse phase column (C, ₈ j. Elution with 25% aqueous acetonitrile and subsequent lyophilization of the appropriate fractions 0.353 g (47%) of a cream colored product is obtained as a solid.

IR (KBr): _mnx 3,240 (broad signal, OHJ,

775 C- = O / 3-lactam,

695 (- CO ₂ PNB) cm ^-1 .

´ Ή-NMR (perdeuteroacetone, values in ppmj:

9.24—7.84 (multiplet, 8H, aromatic protons],

7.4 - 6.9 (multiplet, 10H, diphenylphosphate), 5.52, 5.24 (AB-quartet, J = 14 Hz, 2H, benzyl residue),

5.15-4.80 (multiplet, 4H)

4.45-3.05 (multiplet, 7H),

1.35 (doublet, J = 6.6 Hz, 3H, CHCH3).

C. (5R, 6S) -3- (2- (4-Hydroxymethyl-pyridinioethylthio) -7- (1- (R-hydroxyethyl) -7-oxo-1-azabicyclo [3.2.0] hept) -2-ene-2-carboxylate

«

A mixture of 0.348 g (0.465 mmol) of diphenylphosphate β-nitrobenzyl- (5R, 6S) -3- [2- (4-hydroxymethylpyrldinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1- azabicyclo [3.2.0] hept-2-ene-2-carboxylate and 0.35 g of 10% palladium on carbon in 11 ml of 0.05 M phosphate buffer (pH 7.4), 5 ml of tetrahydrofuran and 10 ml of ether are added. Hydrogenated at 50 psi for 1.25 hours.

The resulting mixture was filtered through a pad of diatomaceous earth and the aqueous phase was washed three times with ether. The pH of the aqueous solution was adjusted to 7.0 using an additional buffer of pH 7.4, the remaining volatiles were evaporated in vacuo and the aqueous residue was applied to a reverse phase column (C, ₈ ). Elution with 2% aqueous acetonitrile followed by lyophilization gave a tan solid which, after repeated chromatography (reverse phase _C18 / water), yielded 0.060 g (36%) of the desired product as a yellowish solid.

IR (KBr): _max

400 (broad signal, OH),

755 (C O 3 ^L lactam),

1590 (-CO ₂ -) cm ^1st

1 H-NMR (deuterium oxide, values <S in ppm):

8.73, 7.96 (AB-quartet, J = 6.8 Hz, 4H, aromatic protons),

4.93 (s, 2H, _CH2 OH);

4.77 (triplet, J = 6.0 Hz, 2H, _N-CH2)

4.15 (doublet of quartets, J = J ‘= 6.3 Hz, 1H, H-1‘),

3.96 (doublet of triplets, J = 9.2 Hz, J = 2.6 Hz, 1H, H-5),

3.65-3.20 (multiplet, 3H),

3.13 - 2.62 (multiplet, 2H),

1.21 (doublet, J = 6.3 Hz, 3H, CHC _i).

UV (water): A _max 295 (ε 6 880),

256 (ε 5,595),

224 (ε 8111) nm.

τ _{1 / 2ι} = 14.5 hours (pH 7.4, 36.8 ° C).

Example 5

Preparation of 3- (2- (1- (2-methylpyridinium)) ethylthio] -6α- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene -carboxylate

A. 1- (2-mercaptoethyl) -2-methylpyridinium methanesulfonate

5 * C

HsOH

HS

MsO ™

250858

To a suspension of 2-methylpyridinium methanesulfonate in 2-methylpyridine prepared by adding 0.65 mL (0.010 mol) of methanesulfonic acid to 2.17 mL (0.022 mol) of cold 2-methylpyridine was added 0.655 mL (0.011 mol) of ethylene sulfide. The reaction mixture was stirred under nitrogen at 55 ° C for 21 hours, then cooled to 23 ° C and diluted with 5 mL of water. The aqueous solution was washed six times with 4 ml of ether each time, evaporated in vacuo to remove the last traces of organic solvents and applied to a column (2.5 x 10.0 cm) of μ-bondapak C-18. The column is eluted with water, the appropriate fractions are combined and lyophilized. This afforded 2.13 g (85%) of the title compound.

IR (film): y _max

520 (SH)

623 (pyridinium residue),

574, 1 512, 1 485, 1 412, 1 195 (sulphonate),

038 cm- ¹ .

@ 1 H-NMR (CDCl3 + .delta., values < S in ppm):

2.37 (3H, singlet, CH ₃ SO ₃ -),

2.83 (3H, s, CH ₃ on pyrldiniovém residue)

3.09 (2H,) = 6.9 Hz, _CH2 S),

4.71 (2H, triplet, J = 6.9Hz, CH, N &lt; + &gt;),

7.93 (2H, multiplet, Hm of pyridinium residue j,

8.44 (1H, multiplet, Hp of the pyridinium residue),

8.89 (1H, multiplet, Ho of the pyridinium residue).

UV (water): ^max 266 (ε 3,550) nm.

B. p-Nitrobenzyl-3- (2- (1- (2-methylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] diphenylphosphate hept-2-ene-2-carboxylate is NEt (iPr) ₂

OH

CGOPHB

2) ClP (OPh)

3)

HS ‘10

4) NEt (iPr)

O

II (PhO) 4 PO

To a solution of 0.523 g (1.50 mmol) of p-nitrobenzyl 6α- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2-carboxylate in 6 mL of acetonitrile, cooled to 0 [deg.] C., is added first under nitrogen atmosphere 0.314 ml (1.80 mmol) of diisopropylethylamine and then 0.373 ml (1.80 mmol) of diphenyl chlorophosphate. A solution of 0.530 g (2.16 mmol) of 1- (2-mercaptoethyl) -2-methylpyridinium methanesulfonate in 18 ml of acetonitrile and then 0.314 ml of (1, 8 mmol) of diisopropylethylamine. The reaction mixture is stirred at 0 ° C for 1 hour, then diluted with 26 ml of cold water (0 ° C) and loaded onto a μ-bondapak C-18 (3.5 x 7.0 cm) column.

Elution of the column with a mixture of 25% acetonitrile and 75% water, and 50% acetonitrile and 50% water followed by lyophilization of the appropriate fractions gave 1.06 g (96%) of the title compound as a yellowish powder.

IR (KBr): _n , _ax 3 650-3 100 (OH),

700 (C = O / 3-lactam),

695 and 1690 (C = O-p-nitrobenzyl ester), 1630 (pyridinium residue),

595 (phenyl)

1518 (NO ·,)

335 (NO),

890 cm ^-1 (NO,).

@ 1 H-NMR (CDCl3, <5 ppm):

1.15 (3H, doublet, J = 6.1 Hz, CH 3 CHOH),

2.87 (singlet, CH ₃ on pyridinium residue),

3.6-4.4 (2H, m, H-5, GH ₃ CHOH), 4.75 (2H, m, CH ₂ N ^+),

5.37 (center of AB-quartet, J = 14 Hz, _CH2 P-nitrobenzylic residue)

6.5-7.4 (10H, multiplet, phenyl),

7.70 (2H, doublet, J = 8.8 Hz, Ho of the p-nitrobenzyl residue),

8.0 (2H, multiplet, Hm of pyridinium residue),

8.24 (2H, doublet, J = 8.8 Hz, Hm of p-nitrobenzyl residue),

8.50 (1H, multiplet, Hp of the pyridinium residue),

8.95 (1H, broad doublet, J = 6.1 Hz, Ho of the pyridinium residue).

UV (water):

265 (ε 11,990),

314 (ε 8,020) nm.

C. 3- (2- (1- (2-Methylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl) -7-oxo-1-azabicyclo [3.2.0] hept-2-ene -carboxylate

10% Pd / C, H ₂ tetrahydrofuran, ether, buffer

To a solution of 0.66 g (0.90 mmol) of diphenylphosphate p-nitrobenzyl-3- [2- (1- (2-methylpyridinium)) ethylthio] -6a- [1- (R) -hydroxyethyl] -7 -oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 34 mL of wet tetrahydrofuran was added 34 mL of ether, 16.5 mL of 0.15 M potassium dihydrogen phosphate buffer and sodium hydroxide (pH) 7.22) and 0.66 grams of 10% palladium on carbon. The resulting mixture was hydrogenated at 23 degrees Celsius at 50 psi for 1.25 hours, then the organic layer was separated and extracted twice with 6 ml of buffer each time. Combine the aqueous layers, filter through a pad of diatomaceous earth, wash the filter aid with 40 ml of ether, evaporate the organic solvents in vacuo and apply the aqueous residue to a μ-bondapak C-18 (2.5 x 10 cm) column. Elution with water and lyophilization of the appropriate fractions gave 0.098 g (31%) of the title compound as a yellowish powder.

IR (KBr):

^ max

650—3100 (OH)

1755 (0 = 0 / β-lactam),

630 (pyridinium residue),

595 cm ^-1 (carboxylate).

* H-NMR (deuterium oxide, δ in ppm):

1.20 (3H, doublet, J = 6.3 Hz, CH 3 CHOH),

2.83 (singlet, CH ₃ on pyridine residue),

2.7-3.1 (5H, H-4, CH ₃ on pyridinium residue),

3.1 to 3.7 (3H, multiplet, CH ₂ S, H-6)

3.90 (double doublet, J = 9.1 Hz, J = 2.6

Hz, H-5)

3.1 (multiplet, CH ₃ CHOH),

4.78 (triplet, J = 6.2 Hz, CH ₂ N ^+),

7.8 (2H, multiplet, Hm of pyridinium residue),

8.3 (1H, multiplet, Hp of the pyridinium residue),

8.65 (1H, multiplet, Ho of the pyridinium residue).

UV (water): Λ _π , " _χ 268 (ε 9 350),

296 (ε 8,840) nm.

[α] _D ²³ - + 41 ° (c - 0.5, water).

t 1/2 = 15.0 hours (measured at ¹⁰ µM in phosphate buffer pH 7.4 at 36.8 ° C).

Example 6

Preparation of 3- [2- (1- (4-methylpyridinium)) ethylthio] -6α- [1 (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-en-2 -carboxylate

A. 1- (2-mercaptoethyl) -4-methylpyridinium methanesulfonate

+ · N ^e C ______ \ hours

HS

To a suspension of 4-picolinium methanesulfonate in 4-picoline, prepared by adding 0.65 mL of methanesulfonic acid (0.010 mol) to 2.14 mL (0.022 mol) of 4-picoline, was added with cooling 0.655 mL (0.011 mol) of ethylene sulfide. The reaction mixture was stirred at 55 ° C for 24 h under nitrogen, then cooled to 23 ° C and diluted with 5 mL water and 10 mL ether. The organic layer was separated, the aqueous layer was washed five times with 5 ml of ether each time, and after removal of the last traces of ether under reduced pressure, loaded onto a column (2.5 x 10 cm), µ-bondapak C-18.

The column was eluted with a mixture of 15% acetonitrile and 85% water and the appropriate fractions were lyophilized. 2.66 g (100%) of a colorless syrup-like product are obtained.

ID (film):

500 (SH)

640 (pyridinium residue),

572, 1 520, 1 478, 1 200 (sulphonate),

040, 833 and 768 cm ¹ .

¹ H-NMR (perdeuterodimethylsulfoxide, S-value in ppm):

2.31 (3H, singlet, CH 3 SO 3 -),

2.62 (singlet, CH _:( on pyridinium residue),

2,2-2,9 (4H, SH, CH · on pyridine residue),

3.04 (2H, multiplet, CH.S),

4.68 (2H, triplet, J = 6.4 Hz, CH ₂ N +),

8.01 (2H, doublet, J 6.6 Hz, pyridine Hm),

8.89 (2H, doublet, J = 6.6 Hz, Ho of pyridinium residue).

UV (water): A _max 256 b 4100),

221 (ε 7,544) nm.

Β. 1- (2-mercaptoethyl) -4-methylpyridinium p-toluenesulfonate

To a suspension of 1.72 g (0.01 mol) of p-toluenesulfonic acid in 6.5 ml of benzene was added 1.17 ml (0.012 mol) of 4-picoline. The resulting mixture was stirred at 23 ° C under nitrogen for 30 minutes, then 0.65 ml (0.011 mol) of ethylene sulfide was added and the mixture was stirred at 75 ° C for 24 hours. After an additional 0.65 ml (0.011 mol) of ethylene sulfide was added, stirring was continued at 75 ° C for 24 hours, then the reaction mixture was cooled to 23 ° C and diluted with 5 ml of water and 8 ml of ether.

The aqueous layer was separated and washed with ether (3 x 8 mL). The last traces of organic solvents were removed by evaporation in vacuo and the residue was chromatographed on a µ-bondapak C-18 column using water as eluent. It is obtained

2.94 g (90%) of the title compound as a colorless syrup.

IR (film): p _max 2 510 (SH),

640 (pyridinium residue),

595, 1582, 1475, 1200 (sulfonate), 1031, 1010, 818 cm ^-1 .

PH-NMR (perdeuterodimethylsulfoxide, δ in ppm):

2.29 (3H, singlet, CH ₂ on pyridinium residue),

2.61 (singlet, CH ₃ Ph),

2.4-2.8 (4H, SH, CH ₃ Ph),

3.03 [2H, multiplet (adding deuteriumoxldu gave triplet, J - 6.4 Hz at 3.04), _CH2 S], OH

4.68 (2H, triplet,] = 6.4 Hz, CH ₂ N +), 7.11, 7.49 (4H, two doublets) = 7.9 Hz, phenyl),

8.00 (2H, doublet, J = 6.5 Hz, pyridinium Hm),

8.89 (2H, doublet, J = 6.5 Hz, Ho of pyridinium residue).

UV (water):

λπίβχ

256 (ε 4,315),

222 (ε 17,045) nm.

C. p-Nitrobenzyl-3- [2- (1- (4-methylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo (3.2.0) diphenylphosphate hept-2-ene-2-carboxylate 1) NEt (iPr) ₂

2) ClP (OPh) ₃ coopnb

To a solution of 0.522 g (1.5 mmol) of p-nitrobenzyl 6α- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2-carboxylate in 6 mL of acetonitrile, After cooling to 0 DEG C., diisopropylethylamine (0.314 mL, 1.8 mmol) was added, followed by diphenyl chlorophosphate (0.373 mL, 1.9 mmol) under a nitrogen atmosphere. A solution of 0.539 g (2.16 mmol) of 1- (2-mercaptoethyl) -4-methylpyridinium methanesulfonate in 1.8 ml of acetonitrile and then 0.314 ml (1.8 mmol) was added dropwise. diisopropylethylamine. The resulting mixture was stirred at 0 ° C for 1 hour, then diluted with 24 mL of cold water (0 ° C) and loaded onto a 8-bondapak C-18 column (2.5 x 8.5 cm).

The column is eluted first with a mixture of 25% acetonitrile and 75% water (100 ml) and then with 100 ml of a mixture of 50% acetonitrile and 50% water. Lyophilization of the appropriate fractions gave 0.91 g (83%) of the title compound as a yellowish powder.

IR (KBr): in _max

700—2 800 (OH),

770 (C = O / 3-lactam)

1700 (C = O of p-nitrobenzyl ester),

640 (pyridinium residue),

595 (phenyl)

520 (NO ₂ )

340 (NCM)

890 cm '~ (NO _2).

⁴ H-NMR (CDCl 3, δ 'in ppm):

1.16 (3H, doublet, _J = 6.2 Hz, CH ₃ CHOH), 2.61 (singlet, CH ₃ pyridine residue), 3.1-3.7 (3H, multiplet, H-6, CH ₂ S) ,

3.7- 4.4 (2H, m, H-5, _CH3 CHOH)

4.79 (2H, broad triplet,) = 6.3 Hz,

CH, N + J

5.17 (doublet, J = 4.9 Hz, OH),

5.37 (center of AB-quartet,) == 14.1 Hz, CH ₂ P-nitrobenzylic residue)

6.7-7.4 (10H, multiplet, phenyl),

7.69 (2H, doublet,) = 8.8 Hz, Ho of the p-nitrobenzyl residue),

8.00 (2H, doublet, J = - 6.5 Hz, Hm of pyridinium residue),

8.23 (2H, doublet,) = 8.8 Hz, Hm of the p-nitrobenzyl residue),

8.92 (2H, doublet, J = 6.5 Hz, Ho of pyridinium residue).

UV (water): A _max found:

262 (ε 10 835), 56.89% C, 5.13 θ / ο H, 5.19 θ / ο N, 4.41 θ / ο S.

311 (ε 9,670) nm.

D. 3- [2- (l- / 4-methylpyridinium /) ethylthio] Analysis for C3 H3 _{G 6} N ₃ O ₁₀ SP. O 1.5 H _{2 liters} -6a- [l- (R) -hydroxyethyl] -7-oxo-azabicyclo [3.2.0] hept-2-ene-2-carboxylate Calculated:

% C, 56.84;% H, 5.17;% N, 5.52;% S, 4.21; OH

10% Pd / C, H ₂ tetrahydrofuran, ether, buffer

To a solution of p-nitrobenzyl-3- [2- (1- (4-methylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1 diphenylphosphate 0.587 g (0.80 mmol) -azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 30 ml wet tetrahydrofuran was added 30 ml ether, 14.7 ml 0.15 M potassium dihydrogen phosphate buffer and sodium hydroxide (pH 7.22). and 0.59 grams of 10% palladium on carbon. The resulting mixture was hydrogenated at 23 degrees Celsius at 50 psi for 1.25 hours, then the organic layer was separated and extracted twice with 6 ml of buffer each time. Combine the aqueous extracts, filter through a pad of diatomaceous earth, wash three times with 20 ml of ether each time, evaporate the organic solvents under reduced pressure and apply the residue to a column (2.5 x 10 cm μ-bondapak C-18). water and lyophilization of the appropriate fractions gave 0.136 g (49%) of the title compound as a yellowish powder.

IR (KBr-tech): v ,,,. ,,

700-3,000 (OH),

770 (C = O / 3-lactam)

642 (pyridinium residue),

592 cm ^-1 (carboxylate).

1 H-NMR of deuterium oxide. values in ppm):

1.19 (311, triplet, J = 6.3 Hz, CH-CHOH),

2.59 (3H, singlet, CH ₃ on pyridinium residue),

2.84 (doublet, J = 9.1 Hz, H-4),

2.90 (doublet, J = 9.1 Hz, H-4),

3.0-3.6 (3H, multiplet, CH ₂ S, H-6)

3.86 (double doublet, J = 9.1 Hz, J = 2.6 Hz, H-5), __ 4.1? (m, CH3.CHOH) '4.5-4.9 (CH ₂ N + masked by HOD)

7.80 (2H, doublet, J = 6.6 Hz, Pyridyl residue play),

8.58 (2H, doublet, J = 6.6 Hz, Ho of pyridinium residue).

UV (water): A _max 256 (ε 5,510),

262 (ε 5,360),

296 (ε 7,050) nm.

[α] _D ³³ = 3 + 20.8 ° (c = 0.48, water).

Ti / τ = 12.8 hours (measured at a L0 ^{- 1} M concentration in pH 7.4 phosphate buffer at 36.8 ° C).

Example 7

Preparation of (5R) -3- [2- (4-methylthiopyridinio) ethylthio] - (6S) - [(1R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene 2-carboxylate

CH

A. 4-Methylthiopyridine

The preparation of this compound has been described by Kin ?; and

Ware in J. Chem. Soc., 873 (1939). The procedure described in the above-cited work is described below.

SH

1) McI / EtOH

2) Na-OH

SM <z, (Me = methyl, Et = ethyl)

5.55 g (50.0 mmol) of 4-mercaptopyridine (Aldrich) are dissolved in 50 ml of boiling absolute ethanol and the insoluble material is removed by filtration through a pad of diatomaceous earth. The filtrate was heated to redissolve the solids, and after cooling to about 50 ° C, 3.17 mL (51.0 mmol) of methyl iodide (Aldrich) was added in one portion. The resulting mixture was allowed to crystallize. Filtration of the solid afforded 6.77 g (26.7 mmol) of the title compound as the hydroiodide. The yield was 53.5%.

NMR-NMR (deuterium oxide, δ in ppm):

2.70 (3H, singlet, --SCHJ and

7.65-7.77-8.35-8.48 (411, type A ₂ B ₂ , aromatic protons).

IR (nujol): in _llaxa

615, 1585 (aromatics) and

780 cm -1.

UV (water): λ _{ω; ιχ}

227 (ε 2.02 χ 10 ⁴ a

298 nm (ε 1.64 x 10 ⁴ ).

6.33 g (25.0 mmol) of the above hydroiodide are dissolved in 40 ml of water, the insoluble material is filtered off and washed with 10 ml of water. 5 g of sodium hydroxide in pellets are added to the filtrate at 0 to 5 ° C, the aqueous layer is saturated with sodium chloride and extracted three times with 25 ml of diethyl ether each time. The combined organic extracts were washed twice with brine, dried (MgSO 4) and evaporated. 2.92 g (23.4 mmol) (total yield 50%) of the title compound are obtained.

Ή-NMR (deuterochloroform, δ values in ppm):

2.48 (3H, s, - _SCH3) and

7.03-7.13-8.38-8.48 ppm (4H, type A ₂ B ₂ , aromatic protons).

IR (film): in _mnx

580 and 800 cm ^-1 .

B. 4-Methylthio-N- (2-mercaptoethyl) pyridimurethane sulfonate

SMc50-60 ° C

-Y

SM &

To 0.65 ml (10.5 mmol) of freshly distilled methanesulfonic acid was slowly added 2.75 g (22.0 mmol) of 4-methylthiopyridine while cooling in ice. To the resulting solid mixture was added 0.66 mL (11.0 mmol) of freshly distilled ethylene sulfide (Aldrich) and the resulting mixture was heated at 50-60 ° C for 21 h. as the reaction proceeds, the solids pass into solution. After cooling, the reaction mixture was dissolved in 5 ml of water and the solution was washed five times with 4 ml of diethyl ether each time. The cloudy aqueous layer was filtered through silica and the filtrate was purified by reversed phase silica gel column chromatography (10 g _C18 micro bondapack) using water as eluent. Fractions of 10 ml are collected. Fractions 2 and 3 were combined and purified again by reverse phase column chromatography. From fraction 2, 1.258 g (4.48 mmol; yield 42.6%) of the title compound was obtained as a viscous oil.

NMR-NMR (perdeuterodimethylsulfoxide,

CFT — 20, values of δ in ppm):

2.32 (3H, singlet, CH ₃ SO ₃ -),

2.72 (3H, singlet, —CH ₃ ),

2.68 (1H, multiplet, SH);

2.9-3.2 (3H, multiplet, -CH ₂ S-)

4.59 (2H, triplet, J = 6.4 Hz, - CH ₂ N +), 7.97 (2H, doublet, J = 7.2 Hz, aromatic protons), and

8.72 (2H, doublet, J = 7.2 Hz, aromatic protons).

250638

IR (neat) in _Inax 1630, 1200 (br, ⁺ _-SO3)

785 and 770 cm ^-1 .

C. (5R) -p-Nitrobenzyl-3- [2- (4-methylthiopyridino) ethylthio] - (GS) - [(1R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.1] chloride O] hept-2-ene-2-carboxylate

OH

1) CIPP (OPh),

2)

HS

/

S Ha

OH

MsO

With a HaK solution of 475 mg (1.36 mmol) of (5R) -p-nitrobenzyl-3,7-dioxo- (6S) - [(1R) -hydroxyethyl] -1-azabicyclo [3.2.0] heptane- ( 2R) -carboxylate and 0.24 ml (1.4 mmol) of diisopropylethylamine in 5 ml of acetonitrile were added under nitrogen at 0-5 ° C 0.29 ml (1.41 mmol) of diphenyl chlorophosphate. The mixture was stirred at 0-5 ° C for 30 minutes, then an oil suspension of 4-methylthio-N- (2-mercaptoethyl) pyridinium methanesulfonate (678 mg, 1.45 mmol, 60% purity) in 1 mL was added. 5 ml of acetonitrile and then 0.24 ml (1.4 mmol) of diisopropylethylamine. The resulting mixture was stirred at 0-5 ° C for 1 hour. Immediately after addition of the base, a yellowish precipitate formed, which was filtered off and washed with 3 ml of cold acetonitrile to give 413 mg of a yellowish solid. The latter, by trituration with 10% aqueous methanol (5 ml), 341 mg (0.618 mmol, yield 45.4%) of the title compound as white crystals, mp 118-120 ° C ⁿ

NMR-NMR (perdeuterodimethylsulfoxide,

CFT — 20, values of δ in ppm):

.1,16 (3H, doublet, J = 6.1 Hz, _1'-CH3);

2.72 (3H, singlet, —SCHJ,

3.1-3.7 (5H, multiplet),

3.7-4.3 (2H, multiplet),

4.71 (2H, triplet, J = 6.3Hz, -CH-, N ⁺ ),

5.15 (1H, doublet, I = 4.9Hz, OH),

5.20-5.35-5.40-5.55 (2H, AB-quartet, CO ₂ CH ₉ —Ar),

7.70 (2H, doublet, J = 8.8 Hz, nitrophenyl proton protons),

7.97 (2H, doublet, J = 7.0 Hz, pyridine residue protons),

8.25 (2IT, doublet, J = 8.8 Hz, nitrophenyl proton protons) and

8.76 (2H, doublet, J = 7.1 Hz, pyridinium protons).

IR (nujol): _{δ, 11.1K,} 3,250 (OH),

775 (t-lactam),

700 (ester) a

625 cm ^-1 (pyridinium residue).

UV (absolute ethanol):

308 nm (ε 4.47 x 10 '·).

[α] D ²⁵ + 24.8 ° (c = 0.5, methanol).

Analysis: for C ₂₄ H ₂₀ N ₃ O ₀ S ₂ Cl. H ₃ O calculated:

% C, 50.50;% H, 4.95;% N, 7.37.

H, 4.72; N, 6.89.

D. (5R) -3- [2- (4-Methylthiopyridinio) ethylthio] - (6S) - [(1R) -Hydroxyethyl] -7-oxo-1-azabicyclo (3.2.0. Hept-2-ene) -2-carboxylate

OH

330 mg (0.688 mmol) of (5R) -p-nitrobenzyl-3- [2- (4-methylthlopyridinio) ethylthio] - (6S) - [(1R) -hydroxyethyl] -7-oxo-1-azabicyclo [3, 2.0] hep-2-ene-2-carboxylate was dissolved in 31.5 ml tetrahydrofuran and 31.5 ml 0.05 M phosphate buffer pH 7.40 (Fisher) and the solution was diluted with 31.5 ml diethyl ether.

380 mg of 10% palladium on carbon (Engelhard) was added to the solution, and the mixture was hydrogenated in a Parr apparatus for 1 hour at room temperature under 50 psi. The aqueous layer was filtered through a pad of diatomaceous earth to remove the catalyst, which was then washed twice with 5 ml of water each time. The filtrate was combined with the washings and washed twice with 30 ml of ether each time. The aqueous layer was vacuum stripped of all organic solvent residues and purified by column chromatography through 13 g of adsorbent reverse phase (C | microhondapak _8), which was eluted with water. Fractions in which the absorption band at 307 nm appears at about 307 nm are combined (ca. 1 liter) and lyophilized to give 127 mg (0.334 mmol, 48.5% yield) of the title compound as a yellowish powder.

hr1-NMR (deuterium oxide, CFT-20, δ in ppmj):

1.20 (3H, doublet, J = 6.4 Hz, 1'-CH ₃ ),

2.64 (3H, singlet, —SCH _{: i} ),

2.81 (2H, multiplet, -SCH ₂ -),

3.19 (1H, doublet, J ₍ ; i = 6.1 Hz,

Yo — s = 2.6 Hz, 6-H),

3.32 (2H, doublet, J = 11 Hz, J = 5.5 Hz, protons at position 4),

3.92 (1H, double triplet, J = 9.2 Hz, J5-0 = 2.6 Hz, 5-H),

4.1 (1H, multiplet, 1H);

4.61 (2H, triplet, J = 5.9 Hz, -CH ₂ N ⁺ ),

7.70 (2H, doublet, J = 7.1 Hz, aromatic protons) a

8.40 (2H, doublet, J = -7,11-aromatic protons).

IR (KBr): at _max 3,400 (OH),

750 (/ Haktam)

630 (pyridinium residue) and 1590 cm ^-1 (carboxylate).

UV (water): _λmax 231 (ε 9,800) and 307 nm (ε 25,000).

[ _.alpha. ] _D @ ²³ = + 3.14 DEG ( _C = 0.5, water).

Example 8

Preparation of 3- [2- (3-methoxy-1-pyridiniumethylthio) -6α- [Γ- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-en-2- carboxylate

OM <xA. 1- (2-mercaptoethyl) -3-metboxypyridinium methanesulfonate

250638

To 698 mg (6.4 mmol) of 3-methoxypyridine, pre-cooled to 5 ° C, was added dropwise 0.216 ml (3.05 mmol) of methanesulfonic acid and 0.19 ml (3.2 mmol) of ethylene sulfide. The mixture was heated at 60 ° C for 18 hours, then cooled to 20 ° C, diluted with 10 mL of water and washed with ether (3 x 10 mL). The aqueous phase is evaporated for 15 minutes under high vacuum and then loaded onto a reverse phase column (C _J8).

The title compound is eluted with water. The appropriate fractions were combined and evaporated under high vacuum. 61.6 mg (yield 76.3%) of the desired thiol is obtained.

IR (dichloromethane): in _11lax

550 (w, SH);

620, 1600, 1585 cm ^-1 (m, aromatics).

@ 1 H-NMR (CDCl3, .delta. in ppm):

8.90-7.90 (4H, multiplet, C-H aromatic residue),

4.72 (2H, triplet, J = 6.6 Hz, CH ₂ N ^+), 4.01 (3H, s, _OCH3);

3.5-3.0 (multiplet, covered with CH ₂ S),

2.66 (1H, doublet, J - 9.5 Hz, J -

7.5 Hz, SH) a

2.31 (3H, s, CH ₃ SO ₃ -).

B. p-Nitrobenzyl-3- (2- (3-methoxy-1-pyridinium chloride) ethylthio) -6- [1'- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept ^: 2-ene-2-carboxylate

To a cold solution (0 ° C), 1.04 g (3 mmol) of p-nitrobenzyl-6 '- [1'- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] of heptane-2-carboxylates in 12 mL of acetonitrile was added dropwise 0.63 mL (3.6 mmol) of diisopropylethylamine and 0.75 mL (3.6 mmol) of diphenylchlorophosphate, and the mixture was stirred at 0 ° C for 30 min. The resulting enol phosphate was treated with 1.14 g (4.30 mmol) of 1- (2-mercaptoethyl) -3-methoxypyridinium methanesulfonate in 7 mL of acetonitrile and 0.63 mL (4.30 mmol) of diisopropylethylamine, and the mixture was stirred for 30 min. The mixture was stirred for 30 minutes and then cooled to -10 ° C for 30 minutes. The precipitated solid product is filtered off, washed with 2 ml of cold acetonitrile and dried. This gave 1.32 g (82% yield) of the title compound.

IR (nujolj: in _max 3320 (m, OH)),

1780, 1765 (s, β-lactam),

700, 1695 (m, C = O ester) and 1520 cm ^-1 (s, NO ₂ ).

¹ H-NMR (perdeuterodimethylsulfoxide, values <S in ppm):

9.01 (1H, broad singlet, aromatic H-3),

8.75 (1H, broad doublet, J = 5.4 Hz, aromatic H-6),

8.35-7.95 (4H, multiplet, aromatic residue protons),

7.70 (2H, doublet, J = 7.7 Hz, aromatic residue protons),

5.37 (2H, center of AB-quartet, J = 13 Hz, _CH2 PNB)

5.17 (1H, doublet, J = 4.9Hz, OH),

4.87 (2H, triplet, J = 6.3 Hz, CH ₃ -N +), 4.35 to 3.75 (2H, m, H-5 and H-1 '), 4.00 (3H, singlet , OCH ₃ ),

3.56 (part of the triplet, J = 6.3 Hz, _CH2 S),

3.5-3.20 (3H, multiplet, H-6, H-3) and 1.16 (3H, doublet, J = 6.1 Hz, CH ₃ CHO).

C. 3- (2- (3-Methoxy-1-pyridinium) ethylthio) -6- [1'- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate

A solution of 600 mg (1.12 mmol) of p-nitrobenzyl-3- [2- (3-methoxy-1-pyridinium chloride) ethylthio] -6- [1 '- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3,2,0] hept-2-ene-2-carboxylate in 25 ml tetrahydrofuran, 25 ml ether and 25 in 10 M phosphate buffer (pH 7.4) in the presence of 1.1 g of 10 ^G / b palladium on carbon was hydrogenated in a Parr apparatus at 0.28 MPa for 1 hour. The mixture was diluted with ether and the aqueous phase was filtered through a cured filter paper. The aqueous layer was washed with ether (2 x 20 ml), the volatiles were removed in vacuo and applied to a reverse phase silica gel column. The title compound is eluted with water containing 2 and 5% acetonitrile. Appropriate fractions were combined and lyophilized. A yellow solid is obtained which, after purification by HPLC, yields 150 mg (38%) of the desired penemarboxylate.

IR (nujol):

^ max

1750 (s, C = O (3-lactam) a

580 cm ^-1 (s, carboxylate).

@ 1 H-NMR (CDCl3) .delta.

8.55-8.30 (2H, multiplet, H-2, H-6 aromatic residue),

8.17-7.75 (2H, multiplet, H-3, H-4 aromatic residue),

4.77 (2H, triplet, J = 5.9 Hz, CH ₂ .N +), 4.10 (1H, part 5 signals, J = 6.3 Hz, H-1 '), 3.97 (3H, singlet, OCH ₃ ),

3.85, 3.82 (2 lines, part of double triplet. J = 2.6 Hz, part H-5),

3.42 (2H, triplet, J = 5.9 Hz, CH ₂ -S)

3.25 (1H, doublet, J = 6.1 Hz,

J = 2.6 Hz, H-6)

2.99-2.60 (2H, 6 lines, part H-4) a

1.20 (3H, doublet, J = 6.4 Hz, CH _3).

UV (water, c = 0.05): A, _nax 290 (ε 10 517),

223 (ε 6,643) nm.

Tip 20 hours (0.1 M phosphate buffer pH 7.4, 37 ° C).

Example 9

Preparation of (5R, 6S) -3- [2- (3-methylthiopyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azablicyclo [3.2.0] hept-2- en-2-carboxylate

A. 3-Methylthio-1- (2-mercaptoethyl) pyridinium chloride

S Ma.

HOí

SM &

To a solution of 3.00 g (0.016 mol) of 3-methylthiopyridine in 10 ml of ether was added 15 ml of 1N hydrochloric acid and the mixture was shaken well. The aqueous phase was separated, washed with 10 ml of ether and evaporated. The remaining hydrochloride was dried under vacuum over phosphorus pentoxide. To the white solid hydrochloride was added 1.88 g (0.015 mol) of 3-methylthiopyridine and 0.80 ml (0.015 mol) of ethylene sulfide, and the mixture was heated in an oil bath at 55-65 ° C under nitrogen for 15 hours.

A slightly cloudy oil is formed which is taken up in 125 ml of water and the solution is washed with dichloromethane. The aqueous solution is concentrated to a volume of about 25 ml and homogeneous by addition of a few drops of acetonitrile. The resulting aqueous solution was applied to a reversed phase (C _J8) which was eluted with water. Evaporation of the appropriate fractions gave 2.66 g (80%) of the desired product as a pale yellow viscous oil.

IR (film) _{:? Llux}

410 cm ^-1 (wide band, Sil).

@ 1 H-NMR (CDCl3-deuterium oxide, <5 ppm):

8.88-7.88 (multiplet, 4H, aromatic protons),

4.70 (triplet, J 6.5 Hz, 2H, kCl ·),

08 (unbalanced triplet, J = 6.5 Hz, 2H,

S — Cil ·,),

2.64 (singlet, 3H, S-CH _; J.

The starting 3-ethylthiopyridine was prepared as described by J. A, Zoltewiez and C. Nisi in J. Org. Chem. 34, 765 (1969).

B. p-Nitrobenzyl- (5R, 6S) -3- [2- (3-methylthiopyridinio) ethylthio) -6- (1- (R) -hydroxyethyl] -7-oxo-1-azabicycloyl chloride 3,2,0 hept-2-ene-2-carboxylate

OH

CtyPNB

O

A solution of 0.522 g (1.50 mmol) of p-nitrobenzyl- (5R, 6S) -6- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptan-2 The carboxylate in 7 mL of dry acetonitrile was cooled to 0 ° C and 0.287 mL (1.65 mmol) of diisopropylethylamine was added dropwise.

0.342 ml (1.65 mmol) of diphenyl chlorophosphate was added dropwise, the mixture was cooled to 0 ° C for 30 minutes, then 0.313 ml (1.80 mmol) of diisopropylethylamine and then a solution of 0.398 g (1 ml) were added dropwise. (80 mmol) of 3-methylthio-1- (2-mercaptoethyl) -pyridinium chloride in 0.70 mL of dry dimethylformamide precipitate precipitated from the reaction mixture about one minute after addition, and after cooling to -10 ° C for 10 min, a solid orange colored solid was obtained. The solid residue was filtered off, washed with acetonitrile, then with acetone and finally dried in vacuo to give 0.455 g (55%) of a cream colored solid.

The combined filtrates were evaporated to a yellow oily residue which was taken up with a minimum amount of acetonitrile and the solution was cooled to 0 ° C for 30 minutes. Filtration of the resulting mixture yielded an additional 0.139 g of product as a pale yellow solid. The total yield was 0.594 g (72%).

IR (KBr): at _max 3,345 (broad band, —OH),

770 (carbonyl / 3-lactam),

680 (-CO _and PNB) cm ^-1 .

@ 1 H-NMR (CDCl3, .delta. In ppm):

8.98-7.96 (multiplet, 411, pyridinium aromatic protons),

8.20-7.65 (AB-quartet, J = 7.0 Hz, 4H, aromatic protons of the p-nitrobenzyl residue),

5.53 - 4.80 (multiplet, 4H),

4.3-3.7 (multiplet, 2H);

3.6-3.25 (multiplet, 6H),

2.66 (s, 3H, _S-CH3);

1.16 (doublet, J = 6.0 Hz, 3H, CH _3).

C. (5R, 6S) -3- [2- (3-Methylthiopyridinio) ethylthlo] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylate

To a mixture of 0.551 g (1.0 mmol) of p-nitrobenzyl- (5R, 6S) -3- [2- (3-methylthiopyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo- 1-azabicyclo [3.2.0] hept-2-ene-2'-carboxylate and 0.55 g of 10% palladium on carbon in 25 ml of 0.05 M phosphate buffer (pH 7.4) are added with 5 ml of tetrahydrofuran and 25 ml of ether. The mixture was then hydrogenated in a Parr apparatus at 50 psi for 1 hour. The reaction mixture was filtered through a pad of diatomaceous earth and the filter cake was washed with water and ether. The aqueous phase was separated and washed three more times with ether. Residues of organic solvents were evaporated in vacuo, the aqueous solution is saturated aqueous sodium hydrogenuhličitanein adjusted to pH 7.0 and immediately loaded onto a column of adsorbent with a reverse phase (C | _8). Elution with water followed by lyophilization of the appropriate fractions gave 0.25 grams of a bright yellow solid, which was purified by reverse phase high pressure liquid chromatography to give 0.210 grams (55%) of the product as a yellowish solid.

IR (KBr): v _max 3400 (broad band, —OH),

755 ((3-lactam carbonyl),

590 (-CO ₂ -) cm ^'1.

¹ H-NMR (deuterium oxide, δ values in ppm): 8.60-7.76 (multiplet, 4H, aromatic protons),

4.76 (triplet, J = 5.8 Hz, 2H, _N-CH2)

4.13 (doublet of quartets, J = J ‘= 6.3 Hz,

1H, Η-Γ)

3.95 (doublet of triplets, J = 9.0 Hz, J * = 2.8

Hz, 1H, H-5),

3.45 - 2.75 (multiplet, 5Hj,

2.59 (singlet, 3H, S-CH3),

1.20 (doublet, J = 6.4 Hz, 3H, CH _3).

UV (water): A _max 296 (ε 8,509),

273 (ε 13,005),

231 (ε 11,576) nm.

η _{/ 2} = 20 hours (pH 7.4, 36.8 ° C).

Example 10

Preparation of 3- (2- (1- (2,6-dimethylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo (3.2.0) hept-2- en-2-carboxylate

A. 1- (2-mercaptoethyl) -2,6-dimethylpyridinium methanesulfonate

-to

100 ^P C 42 hours © / - ⁷ MsO '

A mixture of 19.2 ml (0.165 mol) of 2,6-dimethyipyridine and 3.27 ml (0.050 mol) of methanesulfonic acid was stirred for 15 minutes, then 4.17 ml (0.070 mol) of ethylene sulfide was added thereto, and the resulting mixture was stirred under nitrogen. stirred at 100 ° C for 1 hour. After cooling to 25 ° C, the reaction mixture is diluted with 45 ml of ether and 30 ml of water, the two layers are separated and the organic layer is extracted twice with 5 ml of water each. Combine the aqueous phases, filter through a pad of diatomaceous earth. Wash twice with 15 ml of ether each time, remove the last traces of organic solvents in vacuo and apply to a column (3.0 x 12 cm @ 3) - bondapak G-18.

The column is eluted with a mixture of 3% acetonilril and 97% water. Lyophilization of the appropriate fractions afforded 2.5 g of a syrupy impure title compound which, after purification by high-pressure liquid chromatography on a .alpha.-bondapak C-18 column, afforded 0.90 g (7%) of the title compound.

IR (film): in _max

520 (SH)

640 and 1625 (pyridinium residue),

585, 1490, 1200 cm ^-1 (sulfonate residue).

* H-NMR (perdeuterodimethylsulfoxide + + deuterium oxide, δ in ppm):

2.36 (3H, s, CH ₃ SO ~)

4.62 (2H, m, CH ₂ N ^+),

7.74 (2H, multiplet, Hm of the pyridinium residue),

8.24 (1H, multiplet, Hp of the pyridinium residue).

LJV (water:

272 (ε 4080 µm).

B. p-Nitrobenzyl-3- [2- (1- (2,6-dimethylpyridinium)) ethylthio] -6- (1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2] diphenylphosphate O] hept-2-enecarboxylate

250858

To a solution of 0.058 g (t, 89 mmol) of p-nitrobenzyl-Ga- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo | 3,2,0] Heptane-2-carboxylate in 0 ml of acetonitrile cooled to 0 degrees Celsius was added 0.394 ml (2.26 mmol) of diisopropylethylainine and 0.468 ml (2.26 mmol) of diphenyl chlorophosphate under nitrogen. After stirring for 30 minutes, a solution of 0.720 g (2.73 mmol) of 1- (2-mercaptoethyl) -2,6-dimethylpyridinium methanesulfonate in 3 mL of acetonitrile was added followed by 0.394 mL (2.26 mmol). diisopropylethylamine. The resulting mixture was stirred at 0 ° C for 2 hours, then diluted with 7Π ml of water at 0 ^° C and loaded onto a column (2.5 x 9.0 cm). Elution of the column with acetonitrile / water mixtures and lyophilization of the appropriate fractions gave 0.92 g (65%) of the title compound.

IR (KBr): _niax

700-3000 (OH),

765 (C ---- O / L-lactam),

690 (C O p-nitrobenzyl ester),

620 (pyridinium residue),

590 (phenyl),

1517 (NO.)

1. 330 (NO.),

880 cm ^-1 (NO ;,).

⁴ µl - NMR (perdimethylsulfoxide, δ in ppm):

_J. 15 (3H, doublet, J = 6.2 Hz, CH ₃ CHOH)

2.7- 3.7 (11H, CH _{2 l} S-CH2 _3i pyridinium residue, H-4, H-6),

3.7-4.4 (2H, CH 3 CHOH, H-5),

4.7 (2H, multiplet, ClHN + j,

5.14 (1H, doublet, J = 4.5 Hz, OH),

5.37 (center of AB-quartet, J = 13.2 Hz, CH ₂ P-nitrobenzylic residue)

6.7-7.5 (10H, multiplet, phenyl)

7.5-8.7 (7H, pyridinium residue, protons of p-nitrobenzyl group).

UV (water): A _niax 274 (ε 14 150),

319 (ε 9,445) nm.

C. 3- [2- (1- (2,6-Dimethylpyridinium)) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylate

10% Pd / C, h ₂ tetrahydrofuran, ether, buffer

To a solution of 0.80 g (1.07 mmol) of diphenylphosphate p-nitrobenzyl-3- [2- (1- (2,6-dimethylpyridinium)) ethylthio] -6a- [1- (R) -hydroxyethyl] -7- oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 42 mL wet tetrahydrofuran was added 42 mL ether, 21 mL 0.1 M potassium dihydrogen phosphate buffer and sodium hydroxide [pH 7.22] ) and 0.80 grams of 10% palladium on carbon, the resulting mixture was hydrogenated at 23 ° C under 20 psi for 1 hour and then filtered through a pad of diatomaceous earth. The filtrate was separated into layers and the organic layer was extracted three times with 8 ml of buffer each time. The aqueous phases are combined, washed with 50 ml of ether, the residual organic solvents are removed by evaporation in vacuo and the residue is applied to a column (3.0 x 10.2 cm) of μ-bondapak C-18. Elution of the column with a mixture of 5% acetonitrile and 95% water and lyophilization of the appropriate fractions gave 0.246 g (63%) of the title compound as a yellowish powder.

IR (KBr): _niax

700—2 800 (OH),

750 (C-O / J-lactam),

620 pyridinium residue),

585 cm ^-1 (carboxylate).

¹ H-NMR (deuterium oxide, δ in ppm):

1.23 (3H, doublet, J 6.4 Hz, CH ₃ CHOH),

2.5-3.5 (11H, H-4, H-6, CH _; 18, two methyls on the pyridinium residue),

3.8-4.4 (2H, CH 3 CHOH, H-5),

4.5 - 4.9 (CH ₃ N ⁺ , HOD), ' ^- ''......

7.64 and 7.74 (2H, part A of the N-M system, Hm of the pyridinium residue),

8.07, 8.16, 8.18 and 8.27 (1H, BA ₂ B-system, Hp pyridinium group).

UV (water): A 2 at _x 277 (ε 9 733),

300 (ε 8,271) nm.

[aJrr ^! = + 50.7 ° (c = 0.48, water).

Analysis: for C ₁₈ H ₂₂ N ₂ O ₄ S. 1.5 H ₂ O calculated:

% H, 6.47;% N, 7.19.

H, 6.23; N, 6.46.

Example 11

Preparation of (5R, 6S) -3- [2-methylthio-3-methylimidazolio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1,1-azabicyclo [3.2.0] hept -2-ene-2-carboxylate

SMc

A. 2-Methylthio 3-methyl-1- (2-mercaptoethyl ethylidazolium trifluoromethanesulfate)

N SMa + CF ^ SCLH v / \ 3 3 / \ <2SM <2USCt-LCHiN ' ^Λ V = 7

CF ₃ so

5 52 6 5 8

Κ 4.0 g (0.03 mol) of 2-methylthio-1-methylimidazole [prepared according to the procedure of A. Wohl and W. Marckwald, Chem. Ber. 22, 1353 (1889)], 1.38 ml (0.015 mol) of trifluoromethanesulfonic acid is added dropwise under nitrogen at 0 [ ^deg.] C., then 0.9 ml (0.015 mol) of ethylene sulfide is added and the mixture is heated at 55 ^DEG C. for 24 hours under nitrogen. ^C1 C. the reaction mixture was triturated with ether three times and the residue was taken up in acetone, the solution is filtered and the filtrate evaporated. 4.2 g (82%) of product are obtained as a semi-crystalline solid which is used for the next reaction without purification.

IC (film): in _lllax

550 (w, shoulder) cm ^-1 .

NMR-NMR (perdeuteroacetone, δ in ppm):

7.97 (singlet, 2 H),

4.86 (triplet, J = 7 Hz, 2H, methylene), -117 (singlet, 3H, N-Me),

3.20 (doublet of triplets, J = 7 Hz, J = 9 Hz, 2H, methylene),

2.72 (singlet, 3H, S-Me),

2.20 (triplet, J = 9Hz, 1H, —SH).

B. p-Nitrobenzyl- (5R, 6S) -3- (2- (2-methylthio-3-methylimidazolio) ethylthio) -6- [1- (R) -hydroxyethyl] -7-oxo-1 diphenylphosphate -azabicyclo [3.2.0] hept-2-ene-2-carboxylate

To a solution of 1.40 g (4.0 mmol) of p-nitrobenzyl- (5R, 6S) -6- [1- (R) -hydroxyethyl] -3,7-dioxo-1α-bicyclo [3.2.0] of heptane-2-carboxylate in 50 mL of dry acetonitrile was added dropwise at 0 ° C with 0.76 mL (4.4 mmol) of diisopropylethylamine and then 0.91 mL (4.1 mmol) of diphenyl chlorophosphate dropwise under nitrogen at 0 ° C. The mixture was stirred at room temperature for 1 hour, then 0.76 ml (4.4 mmol) of diisopropylethylamine and a solution of 2.0 g (5.9 mmol) of 2-methylthio-3-methyl-1- (2) were added successively. (mercaptoethyl) imidazolium trifluoromethanesulfonate in 5 mL of acetonitrile, allowed to react at room temperature for 1.5 hours and then concentrated in vacuo. The resinous residue was taken up with water and applied to a reverse phase column ( _C18 ).

Successive elution with water, 20% aqueous acetonitrile and finally 30% aqueous acetonitrile, followed by lyophilization of the appropriate fractions gave 0.90 g (30%) of the product as a yellowish solid.

iC (KBr technology): v, _nax 3,380 (wide band, OH),

1770 (N-lactam carbonyl) cm ^-1 .

¹ H-NMR (perdeuteroacetone, S-value in ppm):

8.35 (broad singlet, 1H),

8.24, 7.78 (AB-quartet, J = 8.8 Hz, 4H, aromatic protons),

7.89 (broad singlet, 1H),

7.25-6.91 (multiplet, 10H, diphenylphosphate), 5.50, 5.25 (AB-quartet, J = 1.2 Hz, 2H, benzyl residue),

4.75-4.27 (multiplet, 3H),

4.03 (s, 3H, _N-CH3);

4.15 - 2.75 (multiplet, 8H),

2.59 (s, 3H, _S-CH3);

1.22 (doublet, J = 6.2 Hz, 3H, -CHCH _3).

C. (5R, 6S) -3- [2- (2-Methylthio-3-methylimidazolio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

To a solution of 1.20 g (1.56 mmol) of p-nitrobenzyl- (5R, 6S) -3- [2- (2-methylthio-3-methylimidazolio) ethylthio] -6- [1- (R) -hydroxyethyl] diphenylphosphate ] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 70 ml tetrahydrofuran, 70 ml ether and 31 ml 0.05 M phosphate buffer (pH 7.4) 2 g of 10% palladium-on-carbon and the mixture was hydrogenated at 50 psi for 55 minutes in a Parr apparatus. The reaction mixture was filtered through a pad of diatomaceous earth and the filter cake was washed with water and ether. The aqueous phase was separated, cooled to 0 ° C and adjusted to pH 7.0 with saturated aqueous sodium bicarbonate. After removal of the residual organic solvents by evaporation in vacuo, the aqueous solution is applied to a reverse phase column ( _C18 ). Elution with water and then 8% aqueous acetonitrile followed by lyophilization of the appropriate fractions gave 0.25 g of solid material which, after purification by reverse-phase high performance liquid chromatography, afforded 0.114 g (19%) of the desired product as an off-white solid.

IR (KBr): _{max max} 420 (OH),

750 (β-lactam carbonyl),

{590 - _CO2 -) cm ^1st

1 H-NMR (deuterium oxide, δ ppm) 7.58 (singlet, 2H),

4.52 (triplet, J = 6Hz, 2H),

4.28-3.82 (multiplet, 2H),

3.90 (s, 3H, _N-CH3);

3.40 - 2.87 (multiplet, 5H),

2.40 (s, 3H, _S-CH3);

1.20 (doublet, J = 6.4 Hz, 3H, -CHCH ₃₎

UV (water): A _max 297 (ε 7 572),

262 (ε 6,296),

222 (ε 7,955) nm.

Example 12

Preparation of (5R, 6S) -3- [2- (3-aminopyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate

A. 3-Amino-1- (2-mercaptoethyl) pyridinium chloride

1.50 g (0.016 mol) of 3-aminopyridine are taken up in 15 ml of 1N methanolic hydrochloric acid and the resulting solution is evaporated. To the remaining oily hydrochloride was added 1.32 g (0.015 mol) of 3-aminopyrldine and 0.89 ml (0.015 mol) of ethylene sulfide and the mixture was heated in an oil bath at 60-65 ° C under nitrogen for 2 hours. After the addition of another equivalent of ethylene sulfide (0.89 ml, 0.015 mol), heating at 55-65 ° C was continued for 65 hours, then the reaction mixture was washed with dichloromethane and taken up in 25 ml of water. The aqueous solution was applied to a reverse phase column ( C _i8) and the column was eluted with water. After evaporation of appropriate fractions gave 1.26 g (44%) desired product as a colorless viscous oil.

IC (film). T> max

180 (NH ₂ ) cm ^-1 .

@ ¹ H-NMR (CDCl3, .delta. In ppm):

8.19 - 7.59 (multiplet, 4H, aromatic for tones),

4.59 (triplet, J = 6.2 Hz, 2H, _N-CH2), 3.5 (br s, 2H, - _NH2),

3.20 - 2.77 (multiplet, 3H).

B. p-Nitrobenzyl- (5R, 6S) -3- [2- (3-aminopyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.1] diphenylphosphate O] hept-2-ene-2-carboxylate

OH

OH

CO, PNB (PhO)?

To a solution of 0.696 g (2.0 mmol) of p-nitrobenzyl- (5R, 6S) -6- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2 of carboxylate in 10 ml of dry acetonitrile at 0 ° C under nitrogen first dropwise dropwise 0.382 ml (2.2 mmol) of diisopropylethylamine and then 0.457 ml (2.2 mmol) of diphenyl chlorophosphate. After stirring at 0 ° C for 30 min, a solution of 0.475 g (2.5 mmol) of 3-amino-1- (2-mercaptoethyl) pyridinium chloride in 1 mL of dry dimethylformamide was added followed by an additional 0.435 mL (2.5 mmol) of diisopropylethylamine, the reaction mixture was allowed to react 1.5 hours at 0 ° C and then concentrated in vacuo. Pryskyřlčnatý residue is taken up in a mixture of equal parts acetonitrile and water and applied to a reverse phase column (C _L8). Elution with water and then with 20% aqueous acetonitrile, followed by lyophilization of the appropriate fractions, afforded 0.730 g (50%) of the products as a beige solid.

IR (KBr-technique): in ' _lfix 3 330 (wide band, OH),

180 (wide belt, NH ₂ ),

770 (β-lactam carbonyl),

690 (—COnPNB) cm ^-1 .

NMR-NMR (perdeuterodimethylsulphoxide, values <Š in ppm):

8.29—7.63 (multiplet, 8H, aromatic protons),

7.2- 6.7 (multiplet, 10H, diphenylphosphate), 5.47, 5.18 (AB-quartet, J = 14 Hz, 2H, benzyl residue),

4.73-4.45 (multiplet, 3H)

4.2- 3.8 (multiplet, 1 H),

3.6-2.6 (multiplet, 8H),

1.15 (doublet, J = 6.2 Hz, 3H, CH _3).

C. (5R, 6S) -3- [2- (3-Amino-pyridinio) -ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylate

oh ι ^Ύ //

I ’

WHAT"

To a mixture of p-nitrobenzyl- (5R, 6S) -3- [2- (3-aminopyridinio) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-diphenylphosphate (0.730 g, 1.0 mmol) 1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate and 0.7 g of 10% palladium on carbon in 25 ml of 0.05 M phosphate buffer (pH 7.4) are added with 8 ml of tetrahydrofuran and 20 ml of ether and the resulting mixture was hydrogenated in a Parr apparatus under 1 atm for 1 hour. The reaction mixture was filtered through a pad of diatomaceous earth and the filter cake was washed with water and ether.

The aqueous phase was separated, washed twice with ether, and the volatiles were evaporated in vacuo. The aqueous solution was immediately loaded onto a reverse phase (C | ₈₎ which was eluted with water. Lyophilization of the appropriate fractions gave 0.45 g of off-white solid. This material, after purification by reverse phase high performance liquid chromatography, yields 0.123 g (35%) of the desired product as an ivory solid.

IR (KBr): in _max

340 (wide belt),

750 (wide band, β-lactam carbonyl),

1580 (br, -CO ₂ ^_) cm ^-1.

* H-NMR (deuterium oxide, δ in ppm):

8.07—7.59 (multiplet, 4H, aromatic protons),

4.61 (triplet, J = 5.8 Hz, 2H, _N-CH2)

4.14 (doublet of quartets, J = J ‘= 6.3 Hz, 1H, Η-Γ)

3.97 (doublet of triplets, J = 9.2 Hz, J = 2.6 Hz, 1H, H-5),

3.38 (triplet, J = 5.8 Hz, 2H, S-CH3),

3.24 (doublet of doublets, J = 6.0 Hz, J = 2.6 Hz, 1H, H-6),

3.17-2.57 (multiplet, 2H, H-4),

1.21 (doublet, J = 6.3 Hz, 3H, CH _3).

UV (water): A _max 299 (ε 7,949),

256 (ε 8,822) nm.

τ _1/2 = 18.5 hours (pH 7.4, 36.8 ° C).

Example 1 d

Preparation of (5R, 6S) -3- [1- (S) -methyl-2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.3.1.0 &lt; 2,7 &gt; 2,0] hept-2-ene-2-carboxylate of formula

N.

in

(5R, 6S) -3- [1- (R-Methyl-2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3,2,0] hept-2-ene-2-carboxylate of formula

A. dl-1- (2-mercapto-2-methylethyl) pyridinium methanesulfonate dl-1- (2-mercapto-1-methylethyl) pyridinium methanesulfonate

100 + No, OH

M & 0 ~

N (t

NsO

To 7.83 g (0.097 mol) of cold pyridine was slowly added 1.95 ml (0.030 mol) of methanesulfonic acid, the resulting mixture was stirred at 40 ° C for 15 minutes, then 25.9 ml (0.033 mol) was added. The mixture was stirred at 60 ° C for 90 hours under a nitrogen atmosphere. The pyridine was evaporated in vacuo, the residue was mixed with water and purified by chromatography (HPLC, Bondapak C-18 preparative column). Appropriate fractions were combined and lyophilized to give 1.14 g (15%) of dl-1- (2-mercapto-2-methylethyl) pyridinium methanesulfonate as a colorless syrup.

IR (film): v, _nax

520 (SH)

640 (pyridinium residue),

1180 (s, CH 2 SO 4 -),

040 (CH0SO3-) cm ^1st

H-1-NMR (perdeuterodimethylliulfide oxide, <5 ppm):

1.35 (doublet, J = 6.8 Hz, 3H, CH ₃ CHS)

2.30 (singlet, 3H, CH3 SOO,

2.90 (doublet, J = 8.5 Hz, 1H, SH),

3.2--3.7 (multiplet, CHSH), .52.5 (double doublet, J _arni - 12.9 Hz, J -

----- 8.4 Hz, CHCH ₂ N ⁺ )

8.0-8.4 (multiplet, 2H, pyridyl residue Hm),

8.5-8.8 (multiplet, 1H, Hp of the pyridinium residue),

9.04 (double doublet, J = 1.4 Hz, J = 6.7 Hz, 2H, Ho of the pyridinium residue).

UV (water): A _max

208 (ε 5,267),

259 (ε 3,338) nm.

For C ₈ H ₁₅ NO ₃ S ₂ .2 H ₂ O calculated:

H, 6.71; N, 4.91; S, 22.47. Found:

% C, 37.49;% H, 6.85;% N, 4.86.

As a second product, 0.82 g (11%) of dl-1- (2-mercapto-1-methylethyl) pyridinium methane sulfonate is obtained as a colorless syrupy material.

IR (film): in _max

500 (SH)

628 (pyridinium residue),

1180 (sulphonate),

035 (sulfonate) cm ^1st

1 H-NMR (perdeuterodimethylliulfide oxide, δ values in pmm):

1.69 (doublet, J = 6.8 Hz, 3H, CH ₃ CHN +),

2.31 (s, 3H, CH _{3 SO,} R)

3.0-3.3 (multiplet, 2H, CH ₂ S)

4.2-5.2 (multiplet, 1 H, CHN +),

8.0-8.4 (multiplet, 2H, pyridinium Hm),

8.5-8.8 (multiplet, 1H, Hp of the pyridinium residue),

9.0-9.2 (multiplet, 2H, Ho of the pyridinium residue).

UV (water): λ _inax

209 (ε 4,897),

258 (ε 3,838) nm.

Analysis: for C ₈ H ₁₅ NO ₃ ; S ₂ . 1.5 H ₂ O calculated:

% H, 6.56;% N, 5.07.

H, 5.92; N, 5.20.

B. (5R, 6S) -p-Nitrobenzyl-3- [1- (R, S) -methyl-2- (1-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl] diphenylphosphate - 7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

101

102 «

1) NEt (CPr).

O

To a solution of 0.523 g (1.5 mmol) of (5R, GS) -p-nitrobenzyl-6- [1- (R) -hydroxyethyl) -3,7-dloxo-1-azabicyclo [3.2.0] heptane- Of 2-carboxylate in 6 ml of acetonitrile, cooled to 0 ° C, first add 0.314 ml (1.8 mmol) of diisopropylethylamine and then 0.373 ml (1.8 mmol) of diphenyl chlorophosphate under nitrogen. A solution of 0.539 g (2.16 mmol) of dl-1- (2-mercapto-2-methylethyl) pyridinium methanesulfonate in 2 ml of acetonitrile and 0.314 ml (1.8 mmol) was added and the reaction was stirred for 30 minutes. diisopropylethylamine. The resulting mixture was stirred at 0 ° C for 1 h, then diluted with 24 mL of 0 ° O water and chromatographed on a Bondapak C-18 preparative column (2.5 x 8.5 cm) using acetonitrile (25 mL). up to 50%) and water as eluents. Lyophilization of the appropriate fractions gave 1.07 g (97%) of the title compound as a yellowish powder.

IR (KBr): _ν , _llax

700-300 (OH),

770 ((3-lactam carbonyl)).

695 (p-nitrobenzyl ester carbonyl),

630 (pyridinium residue),

590 (phenyl),

518 (NO ·,)

348 (NO _2),

885 (NO ₂ ) cm ^-1 .

¹ H-NMR (perdeuterodimethylsulfoxide, δ ppm in ppm):

1.33 (doublet, J 6.3 Hz, 311, C11 | CHS),

4.6-5.0 (multiplet, UI-UN '),

5.14 (doublet, J = 5.2 Hz, 1H, OH),

5.37 (center of AB-quartet, J = - 12.4 lis, 2H,

CH.j of p-nitrobenzyl residue),

6.6-7.5 (multiplet, 10H, phenyl phosphate residue),

7.69 (doublet, J = 8.7 Hz, 2H, Ho of the p-nitrobenzyl residue),

0-8.4 (multiplet, 4H, Hm of the p-nitrobenzyl residue, Hm of the pyridine residue),

8.4-8.8 (multiplet, 1H, Hp of the pyridinium residue),

9.08 (doublet, J = 5.6 Hz, 2H, Ho of the pyridinium residue).

UV (water):

263 (ε 13,325),

308 (ε 8,915) nm.

Analysis: for C ^ HH ^NnOOSP. H ₂ O calculated:

57.52% C, 5.10% H, 5.59% N, 4.27% S, found:

% C, 57.76;% H, 4.96;% N, 5.36.

C. [5R, 6S] -3- (1- (R and S) -methyl-2- (1-pyridinium) ethylthio) -6- [1- (R) -hydroxyethyl-7-oxc-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

1.14 (doublet, J = 6.1 Hz, 3H, CH-CHO)

103

104

10% Pd / C, H ₂

OH

tetrahydrofuran, ether, buffer

To a solution of 0.60 g (0.82 mmol) of diphenylphosphate 5 (R, 6S) -p-nitrobenzyl-3- [1- (R, S) -methyl-2- (1-pyridinium) ethylthio] -6- [ 1- (R) -Hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 33 ml of wet tetrabydrofuran is added 33 ml of ether, 17 ml of 0.15N based buffer potassium dihydrogen phosphate and sodium hydroxide (pH 7.22); and 0.60 g of 10% palladium on carbon.

The resulting mixture was hydrogenated at 20 psi at 23 ° C for 1 hour, the layers were separated and the organic layer was extracted three times with 7 mL of water each time. The aqueous layers were combined, filtered through a pad of diatomaceous earth, washed three times with 20 ml of ether each and chromatographed on a bondapak C-18 preparative column (2.5; <9.5 cm), eluting with water. 0.18 g (63%) of a mixture of diastereomers is obtained. These diastereomers were separated by high pressure liquid chromatography (prep.bondapak C-18 column) using water as eluent. As an isomer with a lower retention time, compound "B" was obtained in a yield of 0.068 g (23 why).

IR (KBr): u _mi , _x

770 (β-lactam carbonyl),

633 [pyridinium residue],

593 (carboxylate group) cm -1.

^lH -NMR (deuterium oxide, δ in ppm):

1.20 (doublet, J = 6.3 Hz, 3H, CH.CHO),

1.42 (doublet, J = 6.9 Hz, 3H, CH ₃ CHS)

2.3-3.2 (multiplet, 3H, H-4, H-6)

3.5-3.9 (multiplet, IH, SCH)

3.9-4.2 (m, 2H, H-5, _CH3 CHO),

4.3 - 5.1 (multiplet, CH ₂ N

7.8-8.2 (multiplet, 2H, pyridinium Hm),

8.4 - 8.7 (multiplet, IH, Hp of the pyridinium residue),

8.7-9.0 (multiplet, 2H, Ho of the pyridinium residue).

UV (water): A _max

260 (ε 6,727),

300 (ε 8,245).

[Α] 25 D -39.3 ° (c = 0.56, water).

Ti / 2 = 12.6 hours (measured at ⁴ M in phosphate buffer pH 7.4 at 36.8 ° C).

Compound "A" is obtained as an isomer with a higher retention time in a yield of 0.081 g (28%).

IR (KBr): in _max

755 (β-lactam carbonyl),

630 (pyridinium residue),

590 (carboxylate group) cm ^-1 .

¹ H-NMR (deuterium oxide, tf in ppm):

1.18 (doublet, J = 6.3 Hz, 3H, _CH3 CHO),

1.40 (doublet, J = 7.0 Hz, 3H, CH ₃ CHS)

2.84 (doublet, J 9.3 Hz, 2H, H-4),

3.26 (double doublet, J = 2.7 Hz, J = 5.9 Hz, IH, H-6),

3.4-4.2 (multiplet, 3H, SCH, CH ₃ CHO, H-5),

4.2- 5.1 (m, CH ₂ N ^+),

7.7-8.1 (multiplet, 2H, pyridinium Hm),

8.3 - 8.65 (multiplet, IH, pyridinium lipid),

8.65-8.9 (multiplet, 2H, Ho of the pyridinium residue).

UV (water): A, _{11 (IX}

259 (with 5,694),

296 (ε 6,930).

_{[?] D} 23 = + 96.9 ° (c 0.56, water).

Π / 2 = 15.6 hours (measured in concentration

10 ~ ^Zl M in phosphate buffer pH 7.4 at 36.8 C ^J).

Example 14

Preparation of (5R, 3S) -3- (2 - [(S) - (1-pyridinium)] -1- (S) -cyclohexylthio) -6- [1- (R) -hydroxyethyl] -7-oxo-1 -azabicyclo [3.2.0] hept-2-ene-2-carboxylate of formula

105

106

(5R, 6S) -3- [2- ((R) - (1-pyridinium)] -1- (R) -cyclohexylthio} -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3,2,0] hept-2-ene-2-carboxylate of formula

OH

N ·

COO

A. dl-1- (2-mercapto-1-cyclohexyl) -pyridinium methanesulfonate

MsOH

0.65 ml (0.01 mol) of methanesulfonic acid was added dropwise to 2.42 ml (0.03 mol) of pyridine while cooling. After stirring under nitrogen for 10 minutes, 1.377 g (0.0102 mol) of dl-cyclohexene sulfide (85% purity) was added and the mixture was stirred at 72 ° C for 25 hours. Excess pyridine was evaporated in vacuo and its last traces removed by distillation with water. The distillation residue was mixed with water and chromatographed on a 5 x 13 cm column of prepbondapak C-18 using 0-2% aqueous acetonitrile as eluent. After lyophilization of the appropriate fractions, 1.57 g (53%) of the product were obtained as a colorless syrup.

IR (film): v, _llax

500 (SH)

625 (pyridinium residue),

1190 _(SO,! -) cm ^1st

Ή-NMR (perdeuterodimethylsulfoxide, δ in ppm):

1.2-2.5 (multiplet, 8H, cyclohexyl residue protons),

2.32 (singlet, 3H, CH 3 SO 3 -),

2.82 (doublet, J = 9.8 Hz, SH),

3.0-3.5 (multiplet, 1H, CHSH);

4.2-4.9 (multiplet, 1H, CHN ⁺ ),

8.0-8.3 (multiplet, 2H, Hm of the pyridinium residue),

8.4-8.8 (multiplet, 1H, Hp of the pyridinium residue),

8.9-9.3 (multiplet, 2H, Ho of the pyridinium residue).

UV water): A _max 214 (ε 5,365),

258 (ε 3500).

For CjaHH ^ NONijOo. 1-UO calculated:

% C, 46.88;% H, 6.88;% N, 4.56.

H, 6.46; N, 4.65.

B. (5R, 6S) -p-Nitrobenzyl-3- [2- (R or S) - (1-pyridinium)] - 1- (R or S) -cyclohexylthio] -6- [1- (R) - diphenylphosphate hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2'-ene-2-carboxylate

107

OH

ČOOPNB

1) NEt (iPr) ₂

O

2) ClP (OPh) ₂

4) NEt (iPr) ₂

OH

✓

o (PhOífO ’

To a solution of 1.37 g (3.93 mmol) of (5R, 6S) -p-1 Lrobenzyl-6- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] Heptane-2-carboxylate in 15 ml of acetonitrile cooled to 0 ° C was added, under a nitrogen atmosphere, 0.822 ml (4.7 mmol) of diisopropylethylamine and 0.979 ml (4.7 mmol) of diphenyl chlorophosphate. The solution was stirred for 30 min before a solution of 1.64 g (5.66 mmol) of dl-1- (2-mercapto-1-cyclohexyl) pyridinium methanesulfonate in 4.7 mL acetonitrile was added followed by 0.822 mL (4.7 mmol) of diisopropylethylamine. The reaction mixture was stirred at 0 ° C for 1 h, then diluted with 75 mL of water at 0 ° C and chromatographed on prep-C-18 prepbondapak column using 25-50% aqueous acetonitrile as eluent. Lyophilization of the appropriate fractions gave 1.9 g (53%) of the title compound.

IR (KBr): in _max

700-3000 (OH),

1770 ((S-lactam carbonyl),

1700 (carbonyl p-nitrobenzyl ester residue),

628 (pyridinium residue),

1590 (phenyl)

1515 (NO ₂ ),

345 (NO)

880 (NO ₃₎ cm ^1st

Ή-NMR (deuterium oxide, δ values in ppm):

1.13 (doublet, J = 6.1 Hz, 3H, CH _and CHO),

1.2-2.5 (multiplet, 8H, cyclohexyl residue protons),

2.7-3.5 (multiplet, 4H, H-4, H-6, CHS)

3.5 to 4.4 (m, 2H, _CH3 CHO, H-5)

4.4 - 5.0 (multiplet, 1H, CHN +),

5.30 (center of AB-quartet, J = 12.8 Hz, CH group, p-nitrobenzyl residue),

6.7-7.4 (multiplet, 10H, phenyl), 7.65 (doublet, J = 8.6 Hz, 2H, Ho of the p-nitrobenzyl residue),

7.9-8.4 (multiplet, 4H, Hm of p-nitrobenzyl residue, Hm of pyridinium residue),

8.4- 8.8 (multiplet, 1H, pyridium Hp residue),

9.0-9.4 (multiplet, 2H, Ho of the pyridinium residue).

UV water): A _max

263 (ε 9,038),

309 (ε 6,394) nm.

Analysis for C39H40N3O10SP. H ₂ O calculated:

% C, 59.16;% H, 5.35;% N, 5.31.

H, 5.15; N, 5.57.

C. (5R, 6S) -3- (2 - [(R or S) - (1-pyridinium)] - (R or S) -cyclohexylthio-6- [1- (R) -hydroxyethyl] -7-oxo -1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

OH

109

110

10% Pd / C, H ₂ tetrahydrofuran, ether, buffer

OH

To a solution of (5R, 6S) -p-nitrobenzyl-3- [2- ((R or S) - (1-pyridinium)) - (R or S) -cyclohexylthio diphenylphosphate 1.85 g (2.34 mmol) 96 mL of ether, 50 mL of 0 mL of 6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 96 mL of wet tetrahydrofuran are added. 15M potassium dihydrogen phosphate and sodium hydroxide buffer, and 1.9 g of 10% palladium on carbon.

The resulting mixture was hydrogenated at 50 psi at 23 ° C for 1.25 hours, then the organic layer was separated and washed with water (3 x 20 mL). The aqueous portions are filtered through a pad of diatomaceous earth, washed twice with 60 ml of ether each time, the organic solvents are removed by evaporation in vacuo and the aqueous residue is chromatographed on a 4.5 x 9 cm column of prepbondapak C-18 using 0 to 5% aq. acetonitrile as eluent. Lyophilization of the appropriate fractions gave 0.705 g (76%) of a mixture of diastereoisomers. Separation of these diastereoisomers is accomplished by high pressure liquid chromatography (prepbondapak C-18) using 4% aqueous acetonitrile as eluent. As a diastereoisomer with a lower retention time, 0.29 g (31%) of compound "A" was obtained.

IR (KBr): in _max

750 (d-lactam carbonyl),

620 (shoulder, pyridinium residue),

685 (carboxylate grouping) cm ^-1 .

1 H-NMR (deuterium oxide, α values in ppm):

1.21 (doublet, J 6.3 Hz, 311, CH, CHO),

1.4-2.5 (multiplet, 8H, cyclohexyl residue protons),

2.5 - 3.05 (multiplet, 2H, H-4),

3.05-3.25 (multiplet, 1H, H-6),

3.3-3.7 (multiplet, 1H, CHS),

3.9-4.3 (m, 2H, H-5, _CH3 CHO),

4.3- 4.8 (multiplet, CHN +)

7.8-8.2 (multiplet, 2H, Hm of the pyridinium residue),

8.3- 8.7 (multiplet, III, Hp of the pyridinium residue),

8.8-9.1 (multiplet, 2H, pyridinium Ho residue).

UV water): _{max max} 260 (ε 7123),

300 (ε 8,685).

[α] _D ²³ = + 6.2 ° (c 3 = 0.63, water).

_{Ti / 2} = 3 3 16.6 hours (measured at a concentration of 10 ^-4 M in phosphate buffer pH 7.4 at 36.8 ° C).

Analysis: for C 20 H 24 N 2 O 4 S. 2 H ₂ O calculated:

H, 6.65; N, 6.60; S, 7.55. Found:

H, 6.47; N, 6.59; S, 7.43%.

0.35 g (38%) of (B) is obtained as a diastereoisomer with a longer retention time.

IR (KBr j: v _{m! LX} 1750 (0-lactam carbonyl,

622 (shoulder, pyridinium residue),

588 (carboxylate group) cm ^-1 .

1 H-NMR (deuterium oxide, δ ppm): 1.19 (doublet, J = 6.4 Hz, 3H, CH 3 CHO),

1.3-2.5 (multiplet, 8H, cyclohexyl residue protons),

2.5-3.1 (multiplet, 2H, H-4),

3.1-3.3 (multiplet, 1H, H-6);

111

112

3.3- 3.8 (multiplet, 2H, H-5, CHS)

4.1 (multiplet center, 1H, CH ₃ CHO),

4.25-4.7 (multiplet, 1H, CHN ⁺ ),

7.8-8.1 (multiplet, 2H, Hm of the pyridinium residue),

8.3- 8.7 (multiplet, 1H, Hp of the pyridinium residue),

8.75-9.0 (mulltiplet, 2H, Ho of the pyridinium residue).

UV water): A _lax 259 (ε 5 992),

296 (ε 7,646).

[α] _D ²³ = + 65.3 ° (c = 0.43, water).

τ _1.2 - 20.2 hours (measured at a concentration of 10 ^"4 M in phosphate buffer pH 7.4 at 36.8 ° C).

Example 15

A. (5R) -allyl-3 - [(2-pyridinioethyl) thio] - (6S) - [(1R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- diphenylphosphate en-2-carboxylate

To a solution of 473 mg (1.87 mmol) of (5R) -allyl-3,7-dioxo- (6S) - [(1R) -hydroxyethyl] -1-azabicyclo [3.2.0] heptane- (2R) - of carboxylate in 6 ml of acetonitrile, at a temperature of about -10 ° C under a nitrogen atmosphere, is first added 0.42 ml (2.4 mmol) of diisopropylethylamine and then 0.50 ml (2.4 mmol) of diphenyl chlorophosphate. The mixture was stirred at 0 ° C for 30 minutes, then cooled to -15 ° C and an oil suspension of N- (2-mercaptoethyl) pyridinium chloride (527 mg, 3.00 mmol) in 1 mL acetonitrile was added first. containing 5 drops of dimethylformamide and then 0.42 ml (2.4 mmol) of diisopropylethylamine. The reaction mixture is stirred at -15 ° C for 30 minutes, then diluted with 20 ml of water and purified by chromatography on 12 g of reverse phase silica gel ( _IS PrepPAK) eluting with 200 ml of water, 100 ml of 10% aqueous acetonitrile, 100 ml of 20% aqueous acetonitrile, 100 ml of 30% aqueous acetonitrile, and finally 100 ml of 40% aqueous acetonitrile. The appropriate fractions were collected, the organic solvent was evaporated in vacuo and the residue subjected to lyophilization. This afforded 786 mg (1.26 mmol; yield 67.3%) of the title compound as a brownish powder.

¹ H-NMR (perdeuterodimethylsulfoxide,

CFT — 20, values in ppm):

1.16 (3H, doublet, J = 6 Hz, l'-CH ₃₎

2.6- 3.7 (multiplet),

3.75 to 4.3 (2H, multiplet, H 5-N'-H), 4.65 (2I-I, m, -COOCH ₃ -),

4.87 (2H, triplet, J - 6 Hz, -CH ₂ -N ^+), 5-6,2 (3H, multiplet, olefinic protons)

6.6-7.4 (multiplet, aromatic protons),

8.15 (2H, triplet, J = 7 Hz, aromatic protons in meta-position to nitrogen), 8.63 (1H, triplet, J - 7 Hz, aromatic proton in para-position to nitrogen) and 9 07 (2H, "doublet", J = 7 Hz, aromatic protons in ortho position to nitrogen).

IR (film): at _max 3,400 (OH),

770 (β-lactam),

690 (ester)

625 (pyridinium) cm ^-1 .

B. (5R) -3 - ((2-pyridinioethyl) thio] - (6S) - [(1R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-en-2 -carboxylate

258658

114

113

'3

N,

To a solution of 156 mg (0.25 mmol) of (5R) -allyl-3 - [(2-pyridinioethyl) thio] - (6S) - [(1R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2] diphenylphosphate Of Hept-2-ene-2-carboxylate in 2 ml of acetonitrile at about 22 ° C is gradually added 0.6 ml of a 0.5 M solution of potassium 2-ethylhexanoate in ethyl acetate (0.3 mmol), 15 mg (0.057) mmol) triphenylphosphine and 15 mg (0.013 mmol) tetrakistriphenylphosphine palladium. The mixture was stirred at about 22 ° C under nitrogen for 2 hours. 7 ml of anhydrous ether was added thereto, and the precipitate was filtered off and washed with 7 ml of anhydrous ether. 101 mg of a brownish solid are obtained, which is purified by reverse phase column chromatography (12 g of _C18 PrepPAK), eluting with water. The appropriate fractions (fractions 7-12, 20 ml each) were collected and lyophilized. Yield: 53 mg (0.16 mmol; 64% yield of the title compound) as a yellowish powder, which is contaminated with potassium diphenylphosphate and potassium 2-ethylhexanoate.

¹ H-NMR (deuterium oxide, CFT-20, δ in ppm):

0.80 (triplet, f = 6.4 Hz, ethylhexanoate methyl group),

1.21 (3H, doublet, _J = 6.3 Hz, 1'-CH3),

2.93 (2H, doublet of doublets, J | _ ₃ = 9 Hz, flax = 4 Hz, 1 protons)

3.28 (1H, double doublet, J = ₆ _ ₃ = 6.2 Hz, J, - 5 = 2.5 Hz, 6-H)

3.42 (2H, triplet, J = 6 Hz, -CH ₂ S)

3.98 (1H, triplet of doublets, J _t = ₅ _ 9 Hz, Js-6 - 2.5 Hz, 5-H)

4.15 (1H, quartet, J = 6.2Hz, 1H-H),

4.80 (2H, triplet, J = 6.0 Hz, -CH ₂ N +),

7-7.5 (multiplet, phenyl protons of diphenylphosphate),

8.03 (2H, multiplet, Hm of pyridinium residue),

8.56 (1H, multiplet, Hp of the pyridinium residue);

8.81 (2H, doublet, J = 6.5 Hz, Ho of pyridinium residue).

Example 16

Preparation of 3- (2- (N-methylthiomorpholinium) ethylthio ⁾ -6α- [Γ- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

A. N-Methyl-N- (2-mercaptoethyl) morpholinum methanesulfonate

1) Ms> OH

FOR

¢.

M,> K <: • f · ‘* \

To 5.00 g (42.7 mmol) of N-methylthiomorpholine [see JM Lehn and J. Wagner, Tetrahedron, 28, 4227 (1970)] cooled in ice, was added 1.47 ml (20.5 mmol) of methanesulfonic acid. acid and 1.30 mL (21.4 mmol) of ethylene sulfide. The mixture was heated at 65 ° C for 24 h, then diluted with 25 mL of water, washed twice with 25 mL of diethyl ether twice, freed from traces of organic solvent in vacuo and applied to a reverse phase silica gel column from which

113

116 of the title elutes with water. The appropriate fractions were combined and evaporated. 4.80 g (86% yield) of the desired thiol is obtained as an oil.

IC (film): υ, _ηαχ

550 ^cm-1 (w, SH).

’Ň-NMR (perdeuterodimethylsulfoxide, <5 ppm):

3.25 to 2.95 (6H, multiplet, CH ₂ N ^+),

3.32 (3H, s, CH ₃ N ^+);

3.20 to 2.65 (7H, multiplet, _CH2 S, S) and

2.32 (3H, s, CH ₃ SO _3).

B. p-Nitrobenzyl-3- [2- (N-methyl-thiomorpholinium diphenylphosphate) ethylthio] -6- [1'- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

HS 3)

1) EtN

2) C1PO (OPh), + / - \ N N / y

To a solution of 557 ing (i, 60 mmol) p-nitrobenzyl-6α- [1‘- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo | 3,2,0] Heptane-2-carboxylate in 8 ml of ice-cooled acetonitrile was added dropwise with 0.336 ml (1.92 mmol) of diisopropylethylamine and 0.400 ml (1.92 mmol) of diphenylchlorophosphate. After stirring for 30 minutes, 893 mg (2.29 mmol) of N-methyl-N- (2-mercaptoethyl) thiomorpholinium methanesulfonate in 4 ml of acetonitrile and 0.336 ml (1.92 mmol) of dlisopropyl ethyl amine are added. , and stirring was continued for 30 minutes. The resulting solution was diluted with 20 ml of water and applied to a reverse phase silica gel column eluting with a mixture of equal parts of acetonitrile and water. Appropriate fractions were pooled, evacuated for 2 hours and then freeze-dried. This gave 1.01 g (yield 85%) of the title compound.

IC (nujol): p _n , _ix

760 (s, β-lactam carbonyl),

510 cnm ¹ (s, NO ₂ ).

Ή-NMR (perdeuterodimethylsulfoxide, δ in ppm):

8.25 (2H, doublet, J = 8.8 Hz, aromatic protons),

7.70 (2H, doublet, J = 8.8 Hz, aromatic protons),

7.33-6.84 (10H, multiplet, aromatic protons),

5.37 (2H, center of AB-quartet, J - 14.2 Hz, _CH2),

5.14 (1H, doublet, 1H-4.5Hz, OH), 4.35-3.80 (2H, multiplet, H-Γ and H-5),

3.75-3.45 (6H, multiplet, CH, N ⁺ ],

3.31 (3H, s, CH ₃ N ^+);

3.45 to 2.75 (9H, multiplet, CH ₂ S, H-6 and H-4) and

1.15 (3H, doublet, J = 6.2 Hz, CH _3).

C. 3- (2- (N-Methyl-thiomorpholinium) ethylthio) -6α- [Γ- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2-en-2 -carboxylate

117

118

OH

A solution of 1.31 g (1.76 mmol) of p-nitrobenzyl-3- [2- (N-methyl-thiomorpholinium diphenylphosphate) ethylthio] -6α- [Γ- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3,2,0] hept-2-ene-2-carboxylate in 48.8 ml of 0.1 M phosphate buffer pH 7.4, 20 ml of tetrahydrofuran and 20 ml of diethyl ether in the presence of 1.5 g of 10% palladium The coal was hydrogenated in a Parr apparatus at 50 psi for 1 hour. The reaction mixture is diluted with 40 ml of diethyl ether, the phases are separated and the organic phase is washed twice with 5 ml of water each time. The aqueous phases are combined, filtered, the filtrate is washed twice with 20 ml of diethyl ether each time and the solvent is evaporated off under vacuum. The aqueous solution is applied to a reverse phase silica gel column and the desired carbapenem compound is eluted with 5% aqueous acetonitrile. Appropriate fractions were combined and lyophilized to give 205 mg (31%) of the title compound as an amorphous solid.

IR (nujol): in _max

750 (s, β-lactam carbonyl),

590 ^cm-1 (s, C = O).

1 H-NMR (deuterium oxide, δ in ppm):

4.25 - 3.95 (2H, multiplet, H-1 ‘, H-5),

3.70-3.40 (6H, m, CH ₂ N ^+),

3.35 (1H, doublet, J = 6.1 Hz, J = 2.6 Hz, H-6),

3.08 (3H, s, CH ₃ N +);

3.25-2.75 (8H, CH ₉ S, H-4) a

1.24 (3H, doublet, J = 6.4 Hz, CH _3).

UV (water, c = 0.062): λ _max

299 (ε 10,962) nm.

τ _1/9 - = 17.7 hours (concentration 0.1M, phosphate buffer pH 7, 37 ° C).

Example 17

Preparation of (5R, 6S) -3- (2- (1-methylmorpholino) ethylthio) -6 - [(R) -1-hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylate

QH \ / “A .N o l Ί '7' //

WHAT,

A. 1-Methyl-1- (2-mercaptoethyl) morpholinium trifluoromethanesulfonate

N

I i CF 'SO' H -7 / \ - + / \ · Z_Δ

To 3.29 ml (0.030 mol) of N-methylmorpholine at 1.degree. C., 1.327 ml (0.015 mol of trifluoromethanesulfone O250698) was added dropwise first

119

120 g of acid and then 0.89 ml (0.015 mol) of ethylene sulfide. The resulting tan solution was heated in an oil bath at 50-60 ° C under nitrogen for 18 hours, the volatiles were removed in vacuo and the oily residue taken up in 10 mL of water. The aqueous solution was washed three times with 5 ml of diethyl ether each time, the residual organic solvent was removed in vacuo and the aqueous solution was applied to a C18 reverse phase column. The column is eluted with water, then with 5% aqueous acetonitrile and finally with 10% aqueous acetonitrile. Evaporation of the appropriate fractions gave a white solid which, after drying under vacuum over phosphorus pentoxide, gave 1.92 g (41% yield) of the desired product.

IR (KBr): ν _max 2,560 (—SH) cm ^-1 .

bl-NMR (perdeuteroacetone, δ values in ppm):

4.25-3.6 (multiplet, 8H),

3.49 (s, 3H, _N-CH3);

3.35-2.7 (multiplet, 5H).

B. p-Nitrobenzyl- (5R, 6S) -3- (2- (1-methylmorpholino) ethylthio) -6 - [(R) -1-hydroxyethyl] -7-oxo-1-azabicyclo [3, biphenylphosphate] 2.0) hept-2-en-2-carboxylate

OH

To a solution of 0.348 g (1.0 mmol) of p-nitrobenzyl- (5R, 6S) -6 - [(R) -1-hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane- Of 2-carboxylate in 25 ml of dry acetonitrile, under nitrogen at 0 ° C, was added dropwise 0.191 ml (1.1 mmol) of diisopropylethylamine and then 0.228 ml (1.1 mmol) of diphenyl chlorophosphate. After stirring at 0 ° C for 1 hour, the resulting nol-phosphate was first treated with 0.226 ml (1.3 mmol) of diisopropylethylamine and then with 0.373 g (1.2 mmol) of 1-ethyl-1- (2-mercaptoethyl) morpholinium trifluoromethanesulfonate.

The reaction mixture was stirred at room temperature for 1.5 hours and then concentrated in vacuo. The residue is taken up in water and applied to a C 1 chromatography column ₈ reverse phases. The column is eluted with water, then with 20% aqueous acetonitrile and finally with 30% aqueous acetonitrile. Lyophilization of the appropriate fractions gave 0.360 g (40%) of the desired product as an amorphous solid.

IC (film): v, _nax 3,300 (—OH),

770 (β-lactam carbonyl),

700 (—CO ₂ PNB) cm ^-1 .

¹ H-NMR (perdeuteroacetone, δ values in ppm):

8.25, 7.80 (AB-quartet, J = 8.6 Hz, 4H, aromatic protons),

7.4-6.8 (multiplet, 10H, diphenylphosphate), 5.56, 5.27 (AB-quartet, J = 14.2 Hz, 2H, benzyl residue).

4.42 (doublet of triplets, J = 9.2 Hz, J = 2.7 Hz, 1H, H-5),

4.1 - 2.7 (multiplet, 17H),

3.40 (s, 3H, _N-CH3);

1.22 (doublet, J = 6.2 Hz, 3H, _-CHCH:?).

C. (5R, 6S) -3- [2- (1-Methylmorpholino) ethylthio] -6 - [(R) -1-hydroxyethyl] -7-oxo-1-azabicyclo [3.2.0] hept- 2-ene-2-carboxylate

121

122

OH

To a solution of p-nitrobenzyl- (5R, 6S) -3- [2- (1-methylmorpholino) ethylthio] -6 - [(R) -1-hydroxyethyl] -7-oxo-diphenylphosphate (0.360 g, 0.49 mmol) of 1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 13 ml of 0.05 M phosphate buffer pH 7.4 is added 0.36 g of 10% palladium on carbon, 20 ml of tetrahydrofuran and 20 ml of diethyl ether, the resulting mixture was hydrogenated in a Parr apparatus at 50 psi for 1 hour, then filtered through a pad of diatomaceous earth, and the filter aid was washed with water and diethyl ether. The aqueous phase was separated and adjusted to pH 7.0 by addition of phosphate buffer pH 7.4. After removal of the organic solvent residues under vacuum, the aqueous solution is applied to a reverse phase C column ₍₈₎ . Elution with water and lyophilization of the appropriate fractions yields 0.130 g of an amorphous solid. 34%) of pure product as an amorphous solid.

IR (KBr): ν max

420 (wide belt, OH),

1750 ([beta] -lactam carbones),

590 (—CO <) cm ¹ .

¹ H-NMR (deuterium oxide, δ ppm) 4.35-2.77 (multiplet, 17H),

3.18 (s, 3H, _N-CH3);

1.23 (doublet, J = 6.3 Hz, 3H, CHCH _{: 1} ).

UV (water): λ _max 300 (ε 6 344) nm ,.

τ _{ι / 2} = 18.5 hours (pH 7.4, 36.8 ° C).

Example 18

Preparation of (5R, 6S) -3- (2- (1,4-dimethyl-1-piperazinyl) ethylthio) -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.2.1] O] hept-2-ene-2-carboxylate

A. 1- (2-Acetylthioethyl) -1,4-dimethylpiperazinium bromide

1 /

CHjCS '/ ~ \

Br + —N \ __ 7

In acetone ~ 50 ~

O ll

-> CH.CS '

123

124

A solution of 2.20 g (0.012 mol) of 2-bromoethylthioacetate [see B. Hansen, Acta Chem. Scand. 11, 537-540 (1957)] and 1.95 ml (0.014 mol) 1,4-dimethylpiperazine in 4 ml acetone were stirred at 50 ° C for 65 hours.

After cooling to 25 ° C, the liquid phase is separated by decantation from the resinous residue which is triturated twice with diethyl ether. 3.2 g (90%) of a hygroscopic yellowish powdered product are obtained.

IC (nujol): at _max

685 (C = O thioester residue) cm ^-1 .

¹ H-NMR (deuterium oxide, δ ppm): 2.37, 2.39 (two singlets, 6H, CH ₃ CO,

II o

B. 1,4-dimethyl-1- (2-mercaptoethyl) piperazinium bromide hydrochloride li

CS \ Z "- \

N-, Qr-y

Br, HCl

HCl heat

A solution of 1- (2-acetylthioethyl) -1,4-dimethylpiperazinium bromide (1.1 g, 3.7 mmol) in 4 ml of 6N hydrochloric acid was heated at 80 degrees Celsius under nitrogen for 1 hour and then concentrated under reduced pressure. 0.41 g (38%) of a white powder product is obtained.

1 H-NMR (perdeuterodimethylsulfoxide, δ in ppm):

H /

2.90 (singlet, N +) —f \

TH,

—.5

3.26 (singlet,).

Analysis: for C ₈ H ₂ N ₃ SBrCl. H ₂ O calculated:

H, 7.16; N, 9.05; S, 10.35. Found:

% C, 31.62;% H, 7.46;% N / 9.19;

C. (5R, 6S) -p-Nitrobenzyl-3- [2- (1,4-dimethyl-1-piperazinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1- azabicyclo [3.2.0] hept-2-ene-2-carboxylate

125

250638

126

OH __________ ί /

COOPNB

1) NEt (iPr) ₂

O ií

2) Cl (P (OPh) ₂ )

3)

HS

4) NEt (iPr) ₂

COOPNB

PhOI ^ PO

To a solution of 0.465 g (1.33 mmol) of (5R, 6S) -p-nitrobenzyl-6- [1- (R) -hydroxyethyl] -3,7-dioxo-1-azabicyclo [3.2.0] heptane- 2- (R) -carboxylate in 2 ml of acetonitrile cooled to 0 ° C was added, under a nitrogen atmosphere, 0.278 ml (1.59 mmol) of diisopropylethylamine and 0.33 ml (1.59 mmol) of diphenyl chlorophosphate. The reaction mixture was stirred for 30 minutes, then a suspension of 0.40 g (1.37 mmol) was added.

1,4-dimethyl-1- (2-mercaptoethyl) piperazinium bromide hydrochloride in a mixture of 3 mL of acetonitrile and 1 mL of water, and 0.278 mL (1.59 mmol) of diisopropylethylamine. After stirring at 5 ° C for ₁₈ hours, 15 mL of cold water is added and the resulting solution is chromatographed on a 2.5 x 7.5 centimeter column packed with PrepPak-500 / C ₁₈ , using 25-35% aqueous acetonitrile as the eluent. reagents. After freeze-drying of the appropriate fractions, 0.50 g (50%) of the product is obtained as a yellowish powder.

IR (KBr): _11lax 1765 ([ _beta ] -lactam carbonyl),

690 (G = O of p-nitrobenzyl ester j,

585 (phenyl)

1512 (NO-)

875 _(NO2) cm ^1st

Ή-NMR (perdeuterodimethylsulfoxide, δ in ppm):

1.16, 1.18 (two doublets, J = 6.1 Hz, 3H), CH 2 CHOH),

2.44 (singlet,

3 / 1½ ZsLngle.t,

5.31 (doublet, J = 6Hz, OII),

5.39 (center of AB-quartet,] - 13 Hz, _CH2 P-nitrobenzylic residue)

6.6-7.4 (multiplet, 10H, phenyl phosphate residue),

7.71 (doublet, J = 8.8 Hz, 2H, Ho of the p-nitrobenzyl residue),

8.26 (doublet, J = 8.8 Hz, Hm of p-nitrobenyl residue).

D. (5R, 6S) -3- [2- (1,4-Dimethyl-1-piperazinium) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3.3.1.0 &lt; 2,7 &gt; 2,0] hept-2-ene-2-carboxylate

128

127

10% Pd / C tetrahydrofuran, ether, buffer

To a solution of (5R, 6S) -p-nitrobenzyl-3- [2- (1,4-dimethyl-1-plperazinium) ethylthio] -6- [1- (R 1 -hydroxyethyl) diphenylphosphate 0.47 g (0.623 mmol) -7-oxo 1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate in 25 ml wet tetrahydrofuran was added 25 ml diethyl ether, 13 ml potassium dihydrogen phosphate buffer and sodium hydroxide (pH 7.22) and 0.47 g of 10% palladium-on-carbon, the resulting mixture was hydrogenated at 23 ° C under 50 psi for 1 hour, the layers were separated, and the organic layer was extracted twice with 7 ml of water each time. The mixture was washed through a pad of diatomaceous earth, washed twice with 15 ml of diethyl ether each time and chromatographed on a 2.5 x 9.5 cm column packed with PrepPak-500 / _C18 .

Water is used as eluent. After freeze-drying the appropriate fractions, 0.097 granm (43%) of the desired product is obtained.

IR (KBr): i> _{m 'x}

000—3 700 (OH)

750 (/ Mactam carbonyl),

585 (carboxylate residue) cm ^-1 .

¹ H-NMR (deuterium oxide, δ in ppm):

1.24 (d, J == 6.4 Hz, 3H, CH ₃ CHOH)

2.33 (singlet, 3H,

Tí \ / —χ Ptíx

- c H, l, 1, ts, y / ³ '

4.0-4.5 (multiplet, H-5, CH ₃ CHOH).

UV (water): _lllax 296 (ε 9 476).

[?],? «----- 61.1 ° (c = 0.26, water).

ie / 2-12.4 hours (measured at 10 ⁴ M in phosphate buffer pH 7.4, at 36.8 ° C).

Example 19

Preparation of (5R, 6S) -3- [2- (N-methyl-thiomorpholinium oxide) ethylthio] -6- [1- (R-hydroxyethyl) -7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylate

O

129

OH

i A

L ..... W.

-ry 'S · / \ _7 Ma

139 m-chloroperbenzoic acid

To a solution of 608 mg (1.65 mmol) of (5R, 6S) -3- [2- (N-methyl-thiomorpholinyl) ethylthio) -6- (1- (R) -hydroxyethyl] -7-oxo-1- azabicyclo (3,2,0] hept-2-ene-2-carboxylate in 9 ml of a mixture of equal parts of acetonitrile and water, cooled to -10 ° C, was added in small portions over 1 hour 334.8 mg (1.65) mmol) of m-chloroperbenzoic acid The reaction mixture was diluted with 15 ml of water and washed three times with 15 ml of diethyl ether each time, the aqueous phase was freed of organic solvents in vacuo and subjected to reverse phase silica gel column chromatography. After separation by HPLC (reverse phase), fraction A yielded 52.4 mg (12%) and fraction B yielded 23.6 mg (6 why), which are diastereomers of the title compound.

Fraction A:

IR (nujol): in _max

750 (s, β-lactam carbonyl),

580 cm ^-1 (s, 0 = 0).

¹ H-NMR (deuterium oxide, δ values in ppm):

4.26-2.91 (20H, multiplet, H-4, H-5, H-6, H-1 ', CH ₉ S, CH ₃ S-O, CH ₃ -N + and CH ₂ N ⁺ ),

1.24 (3H, doublet, J = 6.4 Hz, CH _3).

UV (water; c = 0.06): λ _niax 302 (ε 10 425) nm.

τι / o = 12 hours (concentration 0.065M, phosphate buffer pH 7.4, 37% C).

Fraction B:

IR (nujol): i. _Mr , x

750 (s, β-lactam carbonyl)

585 cm @ ^-1 (s, C = O).

1 H-NMR (deuterium oxide, δ values in ppm): 3.86-2.90 (17H, multiplet, H-4, H-5, H-6,

Hl ', CI-US, CH-O, CH ₂ N +),

3.25 (3H, singlet, CHN + j,

1.24 (3H, doublet, J = 6.4 Hz, CH _3).

UV (water, c = 0.05): λ _max 299 (ε 6517) nm.

τι / ₉ = 10.75 hours (concentration 0.065M, pH 7.4 buffer, 37 ° C).

Example 1 a d 2 0

Preparation of (5R, 6S) -3- (2- (1,4,4-trimethyl-1-piperazinium) ethylthio) -6- [1- (R) -hydroxyethyl] -7-oxo-1-azabicyclo [3] chloride , 2,0] hept-2-ene-2-carboxylate

N

C00

131

132

A. 1- (2-Acetylthioethyl) -1,4,4-trimethylpiperazinium bromide iodide

O \ Z— \

Cl-LCS

Mo-Ifr-oH, N-oor, o

u r <f

Br, I

To a suspension of 1- (2-acetylthioethyl) -1,4-diethylpiperazimide bromide (1.48 g, 5.0 mmol) in isopropyl alcohol (10 mL) was added methyl iodide (0.373 mL, 6.0 mmol) and the mixture was heated at 55-50 for 30 h. The solvents were evaporated under reduced pressure, the residue was triturated with hexane and the solid material was filtered off to give 1.85 g of this solid, which was dissolved in 8 ml of hot water and the solution was diluted with acetone (70 ml) until cloudy. Two successive crystallizations yielded 1.5 g (68%) of the title compound, melting at 220-225 ° C with decomposition.

IR (KBr): _νmax 1692 cm ^-1 (G = O).

Ή NMR (deuterium oxide, δ values in ppm):

2.40 (s, 3H, CH ₃ COO)

3.37 (s, _N-CH3);

3.39 (singlet, N-CH2);

3.99 (singlet).

UV (water): _λm 226 (ε 13 144) nm.

Analysis: for C | ₂ H 4 N ₂ OSBrI Calculated:

% C, 30.08;% H, 5.51;% N, 6.38.

% C, 30.48;% H, 5.53;% N, 6.86.

Β. 1- (2-mercaptoethyl) 1,4,4-trimethylethylpiperazinium chloride ^? O 11

CH, CS ·

6N HCl permutite

S — 1 ciHS '

A mixture of 1.84 g (4.19 mmol) of 1- (2-acetylthioethyl) -1,4,4-trimethylpiperazinium bromide iodide and 15 ml of 6N hydrochloric acid was heated to 57 ° C under nitrogen for 2.5 hours. The resulting solution is concentrated to dryness under reduced pressure, the solid residue is suspended in 10 ml of water and permutite S-1 (Cl ^- ) ion exchanger is added to the vigorously stirred suspension until a solution is obtained. This solution was applied to a column (1.2 x 60 cm) of permutite S-1 (Cl ^- ) ion exchanger and the column was eluted with water at a rate of 1.5 ml / min. The appropriate fractions were combined and lyophilized to give 0.93 g (85%) of a white powder, m.p. 190-191 ° C.

NMR-NMR (deuterium oxide, δ ppm): 3.4 (singlet, N — CHJ,

3.45 (s, _N-CH3);

4.07 (singlet).

Found: C _<2 with 2N H ₂ .0,75 SC1 ₂ H2O Calculated:

H, 8.62; N, 10.20; S, 11.67. Found:

H, 8.39; N, 10.55; S, 11.15.

C. (5R, 6S) -p-Nitrobenzyl-3- [2- (1,4,4-trimethyl-1-piperazinium) ethylthio] -6- (1H-hydroxyethyl) -7-oxo-1-azabicyclo [3] , 2,0] hept-2-ene-2-carboxylate-bischloride

IR (nujol): ν _max 2,460 (SH) cm ^-1 .

133

134

1) Net (iPr) ₂

O

2) ClP (OPh) _a

3) NEt (iPr) ₃

permutit

S — 1, Cl4)

to a solution of 0.94 g (2.7 mmol) of (5R, 6S) -p-nitrobenzyl-6- (1R-hydroxyethyl) -3,7-dioxo-1-azabicyclo [3.2.0] heptane-2R carboxylate 0.5 ml (3.2 mmol) of diisopropylethylamine and 0.663 ml (3.2 mmol) of diphenyl chlorophosphate are added under nitrogen to 3 ml of acetonitrile cooled to 5 ^DEG C. The reaction mixture is stirred at 5 DEG C. for 30 minutes, 0.599 ml (3.44 mmol) of diisopropylethylamine and 4 ml of an aqueous solution of 1- (2-mercaptoethyl) -1,4,4-trimethylpiperazinium bischloride (0.90 g, 3.44 mmol) are added. Diisopropylethylamine (0.1 ml, 0.57 mmol) was added to the family and stirring was continued for 2 hours, part of the acetonitrile was removed by evaporation under reduced pressure and the remaining red colored mixture was chromatographed on a PrepPak-500 / Cl8 column using 25- 75% aqueous acetonitrile as eluent After freeze-drying of the appropriate fractions, 1.4 g of a yellowish powder is obtained, which is dissolved in water and the aqueous solution carries permutite S-1 (Cl ^- ) ion exchangers on a column (1.2 x 58 centimeters). The column is eluted with water and the appropriate fractions are lyophilized. 1.17 g of a powdered product is obtained, which is purified by chromatography on a PrepPak-50 ° C / S column. 0.80 g (53%) of a yellowish powder was obtained from the appropriate fractions by lyophilization.

IR (KBr): in _mav

400 (wide belt, OH),

770 (β-lactam carbonyl),

090 (C-O-p-nitrobenzyl ester),

605 (aromatics),

515 (NO),

345 (NO _a ) tin * ¹ .

NMR-NMR (deuterium oxide, δ in ppm):

1.26 (doublet, J = 6.3 Hz, 3H, CH ₃ CHOH), 3.39 (s, NCH _i)

4.00 (singlet),

5.37 (broad singlet, CH ₃ p-nitrobenzyl residue),

7.60 (doublet, J = 8.6 Hz, 2H, Ho of the p-nitrobenzyl residue),

8.20 (doublet, J3 = 8.7 Hz, 2H, 1H of the p-nitrobenzyl residue).

UV (water): A _max 276 (ε 12,094),

306 (ε 10,752) nm.

Analysis: for C, 5H; _lfi N ₄ O ₀ SC12. 3 H, 0 calculated:

C, 46.51; H, 6.56; M, 8.68; S, 4.97; Cl, 10.98.

H, 6.18; N, 8.57; S, 5.36.

11.37% Cl.

D. (5R, 6S) -3- [2- (1,4,4-trimethyl-1-piperazinium) ethylthio] -6- (1R-hydroxyethyl) -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate chloride

135

136

A mixture of 0.40 g (0.68 mmol) of (5R, 6S) -p-nitrobenzyl-3- [2- (1,4,4-trimethyl-1-piperazinium) ethylthio] -6- (1-hydroxyethyl) - 7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate bis chloride, 30 mL of 0.05 M phosphate buffer pH 7.0, 10 mL of tetrahydrofuran, 30 mL of ether and 0.40 g of 10% palladium-on-carbon was hydrogenated at 23 degrees Celsius at 20 psi for 1 hour. The phases are separated. The organic phase is extracted with 10 ml of water, the aqueous phases are filtered through a pad of diatomaceous earth, washed with 10 ml of ether, concentrated in vacuo to a volume of 10 ml of ether, concentrated in vacuo to a volume of 10 ml and chromatographed on a column (2.2 x 11 cm] with a prepPAK-500 / C _J8, using water as the eluent. After lyophilization of the appropriate fractions gave 70 mg (25%) desired product.

IR (KBr): v _{m ix} 3400 (br, OH)

755 (β-lactam carbonyl),

585 (carboxylate group) cm * ¹ .

¹ H-NMR (deuterium oxide, δ ppm): 1.24 (3H, doublet, J = 6.3 Hz, -CH ₃ CHOH), 3.36 (singlet, NCH _S ),

3.98 (singlet).

UV (water): A _max 296 (ε 7,987).

[.Alpha.] D @ ²³ = 35.9 DEG (c = 0.30, water).

Ti / 2 = - 9.8 hours (measured at 10 ^-4 M in phosphate buffer pH 7.4, at 36.8 ° C).

Claims (6)

A process for the preparation of carbapenem derivatives of the general formula I pS ^K ..... R-I 'COOR ² (I) in which: R ^s represents hydrogen and R ¹ represents a radical selected from the group consisting of hydrogen and optionally substituted alkyl, alkenyl and alkynyl groups having up to 10 carbon atoms, cycloalkyl and cycloalkylalkyl with 3 to 6 carbon atoms in the cycloalkyl part and 1 to 6 carbon atoms an alkyl, phenyl, aralkyl, aralkenyl, and aralkynyl moiety wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains up to 6 carbon atoms, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl moieties, wherein the heteroaryl or heterocyclic moiety or moiety is a mono- a bi- or polycyclic radical containing at most 18 ring members, preferably 5 or 6 members, and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, and wherein the alkyl moiety attached to the heterocyclic radical contains 1 to 6 carbon atoms, mentioned suh the substituent or substituents of the above-mentioned radicals are selected from the group consisting of alkyl groups of 1 to 6 carbon atoms, optionally substituted with amino, halogen, hydroxyl or carboxyl groups, halogen atoms, radicals of the formula 137 138 -OR ³ in which O —OCNR ³ R ⁴ O II — CN ³ R ^{4 —} NR ³ R ⁴ NPJ '—SO ₂ NR ³ R ⁴ o —NHCNR ³ R ⁴ O II. R ³ CNR ⁴ - —CO ₂ R ³ O II —OCR ³ —SR ³ O II —SR ⁹ O —SR ⁹ II o -CN, -N ₃ -OSO ₃ R ³ -OSO 2 R ^3, -NR ³ SO 2 R ^4, -NR ³ C = NR ⁴ AND R ³ —NK ³ CO2K ⁵ a - NOa R ³ and R ⁴ are each independently hydrogen, alkyl, alkenyl or alkynyl containing up to 10 carbon atoms, cycloalltyl, cycloalkylalkyl or alkylcycloalkyl having 3 to 6 carbon atoms in the cycloalkyl ring and 1 to 6 carbon atoms in the alkyl a phenyl, aralkyl, aralkenyl or aralkynyl group wherein the aryl moiety is a phenyl moiety and the aliphatic moiety contains up to 6 carbon atoms, a hetercaryl, heteroarylalkyl, heterocyclyl or heterocyclylalkyl moiety, wherein the heteroaryl or heterocyclic moiety or moiety is the corresponding mono-, bi- or a polycyclic residue containing up to 18 ring members, preferably 5 or 6 members, and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, and the alkyl moiety attached to the heterocyclic residue contains 1 to 6 carbon atoms, or R ³ and R ⁴ together with the nitrogen atom to which at least one of these radicals is attached form a five- or six-membered nitrogen containing heterocyclic ring, and R ⁹ is as defined above for R ³ except hydrogen, or R ¹ and R ⁸ together form an alkylidene group having 2 to 10 carbon atoms, optionally substituted by a hydroxyl group, A represents a cyclopentylene group, a cyclohexylene group or an alkylene group having 2 to 6 carbon atoms, optionally substituted by one or two alkyl groups having 1 to 4 carbon atoms, R ² is hydrogen, an anionic charge or a carboxy protecting group selected from the group consisting of optionally substituted aralkyl groups containing up to 20 carbon atoms, benzhydryl, allyl, haloalkyl groups containing up to 6 halogen atoms and up to 6 carbon atoms, a trialkylsilyl group containing each alkyl moiety having from 1 to 4 carbon atoms, phenacyl, acetonyl, 4-pyrldylmethyl, alkyl of 1 to 6 carbon atoms, pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl; when R ² is hydrogen or a protecting group, it is also present equalizing anion, and R ¹⁴ represents a quaternizable nitrogen containing an aromatic or non-aromatic heterocyclic radical attached to radical A via a ring nitrogen atom to form a quaternary ammonium group selected from the group consisting of pyridine, optionally substituted with one or two C 1 -C 4 alkyl, C 1 alkoxy C 1 -C 4 alkyl, C 1 -C 4 alkyl thio, C 1 -C 4 hydroxyalkyl 139 140 (C 4 -C 4) alkyl, (C 1 -C 4) imidazole, (C 1 -C 4) alkyl and / or (C 1 -C 4) alkylthio, piperazine moiety substituted with one, two or three C 1 -C 4 alkyl groups, alkyl substituted morpholine moiety C 1 -C 4 -alkyl, C 1 -C 4 -alkyl-thiomorpholine radical and C 1 -C 4 -alkyl-thiomorpholine-S-oxide radical, and their pharmaceutically acceptable salts, characterized in that the intermediate of the formula IV in which R ¹ and R ⁸ have the abovementioned meaning, R ² 'represents a conventional, easily cleavable carboxyl-protecting group and L represents a conventional cleavable group, reacted with a thiol of formula VII (HS - A - R ¹⁴ X - (VII) in which A and R ¹⁴ are as defined above, and X "represents a balancing anion, in an inert solvent in the presence of a base, to form a carbapenem derivative of the general formula Γ In 9 something in which R @ ¹ , R @ ⁸ , R @ ² ', A, R @ ¹⁴ and X are as defined above, whereupon optionally carboxy protecting group R @ ² ' is cleaved to give the corresponding free compound of formula (I) or a pharmaceutically acceptable salt thereof. 2. The process according to claim 1, wherein the base is a non-nucleophilic tertiary amine or a trialkylamine containing from 1 to 4 carbon atoms in each alkyl moiety. 3. The method of claim 2, wherein the base is a non-nucleophilic tertiary amine. 4. Process according to claim 1, 2 or 3, characterized in that the reaction is carried out at a temperature of from -15 ° C to room temperature. 5. The process of claim 4, wherein the reaction is carried out at a temperature of from -15 ° C to +15 ° C. 6. The process of claim 1, wherein an inert solvent selected from the group consisting of acetonitrile, dimethylformamide, tetrahydrofuran, a mixture of tetrahydrofuran and water, a mixture of acetonitrile and water, and acetone is used.