NO166038B - PROCEDURE FOR THE PREPARATION OF CARBAPENEM DERIVATIVES. - Google Patents

Description

The present invention relates to a method for the production of carbapenem derivatives with the formula

there

R<8> is hydrogen,

R<1> is 1-(R)-hydroxyethyl,

A is ethylene which is optionally substituted with C1_4-alkylf or cyclohex-1,2-ylene,

R<14> is a quaternized pyridine, imidazoline, N-methylmorpholine, N-methylthiomorpholine, N-methylthiomorpholine s-oxide or 1,4-dimethylpiperazine group which is optionally substituted with one or more groups selected from C1_3-alkyl , hydroxy-C1_3-alkylthio, C1_3~alkyloxy and amino and is bound to A via the quaternary nitrogen atom, and

R<2> is hydrogen, an anionic mixture or a conventional easily cleavable carboxyl protecting group provided that, when R<2> is hydrogen or a protecting group, a counter anion is also present, or

a pharmaceutically acceptable salt thereof.

The carbapenem derivatives produced according to the method of the invention are described by Choung U. Kim and Peter F. Misco in US-SN 366,910, 471,379 and 389,652. US-SN 366,910 and its assigned US-SN 471,379, corresponding to DE-OS 3,312,533, deal with the preparation of carbapenem antibiotics of the formula

where R<8> means hydrogen and R<1> is selected from hydrogen;

substituted and unsubstituted alkyl, alkenyl and alkynyl with from

1-10 carbon atoms; cycloalkyl and cycloalkylalkyl with 3-6 carbon atoms in the cycloalkyl ring. and 1-6 carbon atoms in the alkyl portion; phenyl; aralkyl, aralkenyl and aralkynyl* where the aryl part is phenyl and the aliphatic part has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, where the heteroatom or atoms in the above-mentioned heterocyclic part are selected from 1-4 oxygen, nitrogen or sulfur atoms and the alkyl parts connected to the heterocyclic parts have 1-6 carbon atoms; where the substituent or substituents with regard to the above-mentioned groups are selected from C 1-6 alkyl which is optionally substituted with amino, halogen, hydroxy or carboxyl,

where, with regard to the above-mentioned substituents, the groups R<3> and R<4> are independently selected from hydrogen; alkyl, alkenyl and alkynyl having from 1 to 10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkylcycloalkyl with 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl part; phenyl; aralkyl, aralkenyl and aralkynyl, where the aryl part is phenyl and the aliphatic part has 1-6 carbon atoms; and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, where the heteroatom

or -atoms in the above-mentioned heterocyclic parts are selected from 1-4 oxygen, nitrogen or sulfur atoms and the alkyl parts in connection with these heterocyclic parts hair 1-6 carbon atoms or R<3> and R<4> together with the nitrogen atom to which at least one is bonded, forming a 5- or 6-membered nitrogen-containing heterocyclic ring, R<9> is as defined for R<3 >except that it cannot be hydrogen; or where R<1> and R<8> together form the C2_1Q alkylidene or C2_1Q alkylidene substituted with hydroxy, A is cyclopentylene, cyclohexylene or C2_6~alkylene, optionally substituted with one or more C1_4~alkyl groups; R<2> is hydrogen, an anionic charge or a conventional readily removable carboxyl protecting group with the proviso that, when R<2> is hydrogen or a protecting group, a counter anion is also present; and

means a substituted or unsubstituted mono-, bi- or polycyclic aromatic heterocyclic group containing at least one nitrogen atom in the ring and bonded to A through a ring nitrogen atom, thereby forming a quaternary ammonium group; and pharmaceutically acceptable salts thereof by the method shown in the following reaction scheme:

US-SN 389,652 and the derivative US-SN 499,690 deal with the preparation of carbapenem antibiotics of the formula:

where R<8> means hydrogen and R<1> is selected from hydrogen; substituted and unsubstituted alkyl, alkenyl and alkynyl having from 1 to 10 carbon atoms; cycloalkyl and cycloalkylalkyl with 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl part; phenyl; aralkyl, aralkenyl and aralkynyl, where the aryl part is phenyl and the aliphatic part has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, where the heteroatom or atoms in the above-mentioned heterocyclic part are selected from 1-4 oxygen, nitrogen or sulfur atoms and the alkyl parts connected to the heterocyclic parts have 1-6 carbon atoms, where the substituent or substituents with regard to the above-mentioned groups are independently selected from C^^-alkyl which is optionally substituted with amino, halogen, hydroxy or carboxyl, where with regard to the above-mentioned substituents the groups R<3> and R<4> are independently selected among hydrogen; alkyl, alkenyl and alkynyl having from 1 to 10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkylcycloalkyl with 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl part; phenyl; aralkyl, aralkenyl and aralkynyl, where the aryl part is phenyl and the aliphatic part has 1-6 carbon atoms; and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, where the heteroatom or atoms in the above-mentioned heterocyclic parts are selected from 1-4 oxygen, nitrogen or sulfur atoms and the alkyl parts in connection with these heterocyclic parts have 1-6 carbon atoms or R<3 > and R<4> together with the nitrogen atom to which at least one is attached, form a 5- or 6-membered, nitrogen-containing, heterocyclic ring, R<9> is as defined for R<3 >except that it cannot be hydrogen ; or where R<1> and R<8> together form the C2_1Q<-a>alkylidene or with hydroxy substituted C2_1Q<-a>alkylidene, R<5> is selected from substituted or unsubstituted alkyl, alkenyl and alkynyl with from 1-10 carbon atoms; cycloalkyl and cycloalkylalkyl with 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl, where the aryl part is phenyl and the aliphatic part has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, where the heteroatom or atoms in the above-mentioned heterocyclic parts are selected from 1-4 oxygen, nitrogen or sulfur atoms and the alkyl parts connected to the heterocyclic rings have 1-6 carbon atoms; where the above-mentioned R<5-> groups are optionally substituted with 1-3 substituents selected from: C. -alkyl, which is optionally substituted with amino, fluorine, chlorine, carboxyl, hydroxy or carbamoyl; fluorine, chlorine or bromine; phenyl, which is optionally substituted with 1-3 fluorine, chlorine, bromine, C.j_6-alkyl, -OR<3>, -NR3 R4, -SO3R3 , -CO2R<3> or -CONR<3>R<4> , where R<3>, R<4> and R<9> in these R<5->substituents are as defined above; or R<5> can mean a divalent phenylene or C 12 -alkylene group attached to the ring while forming a bridged polycyclic group; A is cyclopentylene, cyclohexylene or C2-g-alkylene, which is optionally substituted with one or more C1-4-alkyl groups; R<2> is hydrogen, an anionic charge or a conventional easily removable carboxyl protecting group with the proviso that, when R<2> is hydrogen or a protecting group, a counterion is also present; and means a substituted or unsubstituted mono-, bi- or polycyclic, non-aromatic, heterocyclic group containing at least one nitrogen atom in the ring and bonded to A through a ring nitrogen atom, thereby forming a quaternary ammonium group; and pharmaceutically acceptable salts thereof, by the method shown in the following reaction scheme:

More specifically, the starting material III is reacted in an inert organic solvent with diphenylchlorophosphate in the presence of a base to form intermediate IV. This is then reacted with a mercaptan reagent of the formula HS-A-OH in an inert organic solvent and in the presence of a base to form the intermediate V. This is then acylated with methanesulfonyl chloride in an inert organic solvent and in the presence of a base to form the intermediate VI which is reacted with an iodide ion source in an inert organic solvent to form the intermediate II. This is then reacted with the desired amine in an inert organic solvent and in the presence of a silver ion to form the quaternized product I'A or I'B which can then be deblocked to form the corresponding deblocked carbapenem of formula IA or IB.

The method described above has several disadvantages. Thus, the method includes e.g. several steps, the number of which could be advantageously reduced. The overall reaction yield is also relatively low and the quaternization step is performed on the entire carbapenem compound.

It would be desirable to have a new process for the preparation of the compounds of formula IA or IB which (1) involves fewer steps, (2) gives a higher yield, (3) allows the quaternized amine to be formed first and then attached to the carbapenem- the core at a later stage of the synthesis and (4) can be used for a simpler formation of quaternary amine products with a large number of mines, i.e. amines with steric

obstruction and with lower pKjj values.

The present invention aims to seek to eliminate the shortcomings of the known technique and in this connection concerns a method for the production of the initially mentioned carbapenem derivatives and this method is characterized by reacting an intermediate product with the formula

where R<1> and R<8> have the above meaning and R<2>' is a p-nitrobenzyl group, with diphenylchlorophosphate in the presence of diisopropylethylamine, and treating the resulting mixture with a thio compound of the formula where A and R<14 > has the above meaning and X- is a counter anion, in an inert solvent with a temperature from -15 °C to room temperature and in the presence of diisopropylethylamine to prepare a carbapenem product of the formula

where R<1>, R<8>, R<2>', A, R<14> and X~ have the meaning given above, and, if desired, removes the carboxyl protecting group R<2>' on i and for known way for the formation of the corresponding unprotected group of formula (I), or a pharmaceutically acceptable salt thereof.

The carbapenem compounds of formula I are potent anti-bacterial agents or intermediates which are valuable

for the production of such funds.

The above-mentioned compounds of the general formula I contain the carbapenem nucleus

and can therefore be called l-carba-2-penem-3-carboxylic acid derivatives. Alternatively, the compounds can be considered to have the base structure

and are designated as 7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylic acid derivatives. While the present invention encompasses compounds in which the relative stereochemistry of the 5,6 protons is both cis and trans, the preferred compounds have 5R,6S-(trans) stereochemistry as in the case of thienamycin.

The term "conventional readily removable carboxyl protecting group" refers to a known ester group which has been used to block a carboxyl group during the chemical reaction steps described below, and which can be removed if desired by the method which does not result in any significant destruction of the rest of the molecule, e.g. by chemical or enzymatic hydrolysis, treatment with chemical reducing agents under mild conditions, irradiation with ultraviolet light or catalytic hydrogenation. Examples of such ester protecting groups are benzhydryl, p-nitrobenzyl, 2-naphthylmethyl, allyl, benzyl, trichloroethyl, silyl such as trimethylsilyl, phenacyl, p-methoxybenzyl, acetonyl, o-nitrobenzyl, 4-pyridylmethyl and C^g -alkyl, such as methyl, ethyl or t-butyl. Such protecting groups include those that are hydrolysed under physiological conditions, e.g. pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl. Particularly advantageous carboxyl protecting groups are p-nitrobenzyl, which can be easily removed by catalytic hydrogenolysis, and allyl, which can be removed by Pd(PO3)4-catalyzed reaction.

The above-mentioned pharmaceutically acceptable salts are non-toxic acid addition salts, e.g. salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, etc. and salts with organic acids such as maleic acid, acetic acid, citric acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, lactic acid, gluconic acid and malic acid. Compounds of formula I in the form of addition salts can be written as

(R<2> = H or a protecting group) where Xe means the acid anion. The counteracting anion XO may be chosen to provide pharmaceutically acceptable salts for therapeutic administration, but in the case of intermediates of formula I, Xe may also be a toxic anion. In such a case, the ion may subsequently be removed or substituted with a pharmaceutically acceptable anion to form an active end product for therapeutic use. When acidic or basic groups are present in the R^ group or on the quaternized R<14> group, the present invention also includes suitable basic or acid salts of these functional groups, e.g. acid addition salts in the case of a basic group and metal salts (e.g. sodium, potassium, calcium and aluminium), the ammonium salt and salts with non-toxic amines (e.g. trialkyl amines, procaine, dibenzylamine, 1-ephenamine, N -benzyl-3-phenethylamine, N,N'-dibenzylethylenediamine, etc.) in the case of a

sour group.

Reaction of intermediate IV with thiol VII can be carried out within a wide temperature range, e.g. -15'C up to room temperature, but is preferably carried out at a temperature in the area of approx. -15'C to +15'C and preferably at approx. 0°C.

The carbapenem compound produced by reacting the quaternary amine thiol VII with the intermediate IV will have a counter anion associated with it (eg (C6H50)2PO2<e>, Cl<e >or the anion associated with the quaternary thiols), which at this point may be substituted with another counter anion, e.g. one that is more pharmaceutically acceptable, using conventional methods. Alternatively, the counter anion can be removed during the subsequent deblocking step. Where the quaternized carbapenem compound and the counter anion form an insoluble product, the product may crystallize out as it forms and be collected purely by filtration.

After formation of the desired carbapenem product, the carboxyl protecting group R<2>' of compound I' can optionally be removed by means of conventional methods such as solvolysis, chemical reduction or hydrogenation. Where a protecting group such as p-nitrobenzyl, benzyl, benzhydryl or 2-naphthylmethyl is used, which can be removed by catalytic hydrogenation, intermediate I' can be dissolved in a suitable solvent such as dioxane-water-ethanol, tetrahydrofuran-diethyl ether buffer, tetrahydrofuran-aqueous dipotassium-hydrogen-phosphate-isopropanol or the like, is treated under a hydrogen pressure of from 1-4 atmospheres in the presence of a hydrogenation catalyst such as palladium-on-charcoal, palladium-hydroxide, platinum oxide or the like, at a temperature from 0 -50'C for from 0.24 - 4 hours. When R<2>' is a group such as o-nitrobenzyl, photolysis can also be used for deblocking. Protecting groups such as 2,2,2-trichloroethyl can be removed by mild zinc reduction. The allyl protecting group can be removed using a catalyst comprising 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 protecting groups can be removed by methods known to those skilled in the art. Finally, compounds of formula I', where R<2>' is a physiologically hydrolyzable ester such as acetoxymethyl, phthalidyl, indenyl, pivaloyloxymethyl, methoxymethyl, etc. as mentioned above can be administered directly to the host without deblocking, as such esters are hydrolyzed in vivo under physiological conditions .

It will be understood that when the R<1-> and/or R<8->substituent or the quaternized nucleophilic group R<14> bound to substituent A contains a functional group that can interfere with the intended reaction course, such a group be protected by means of a conventional blocking group and then unblocked to restore the desired functional group. Suitable blocking groups and procedures for their introduction and removal are well known to those skilled in the art.

As is the case with other β-lactam antibiotics, compounds of the general formula I can be converted by means of known procedures into pharmaceutically acceptable salts which, for the purposes of the present invention, are essentially equivalent to non-salt-forming compounds. Thus, one can e.g. dissolving a compound of formula I, where R<2> is an anionic charge in a suitable inert solvent and then adding one equivalent of a pharmaceutically acceptable acid. The desired acid addition salt can be recovered by conventional procedures, e.g. solvent precipitation, lyophilization, etc. When other basic or acidic functional groups are present in conjunction with formula I, pharmaceutically acceptable base addition salts and acid addition salts may

correspondingly produced using known methods.

The quaternary aminothiol intermediate will have a counter anion bound to it and that will be determined by the particular acid used. It is of course possible to substitute with another counter anion at this point using conventional methods for use in the subsequent reaction with carbapenem intermediate IV.

The carbapenem derivatives of the general formula I, where R<2>

is hydrogen, an anionic charge or a physiologically hydrolyzable carboxyl protecting group, or the pharmaceutically acceptable salts thereof, are powerful antibiotics, which are active against various gram-positive and gram-negative bacteria and they can be used e.g. as animal feed additives for growth promotion purposes, as a preservative in foodstuffs, as bactericides in industrial applications, e.g. in water-based paint and in the waste water from paper mills to inhibit the growth of harmful bacteria and as a disinfectant to destroy or inhibit the growth of harmful bacteria on medical and dental equipment. However, they are particularly valuable for the treatment of infectious diseases in humans and animals, caused by gram-positive or gram-negative bacteria.

The pharmaceutically active compounds provided according to the method of the invention can be used alone or formulated as pharmaceutical preparations which, in addition to the active carbapenem component, comprise a pharmaceutically acceptable carrier or a diluent. The compounds can be administered by a variety of means, those of primary interest include: oral, topical or parenteral (intravenous or intramuscular injection). The pharmaceutical preparations can be used in solid form as capsules,

tablets, powders, etc. or in liquid form such as solutions, suspensions or emulsions. Preparations for injection, which is the preferred route of administration, can be prepared in unit dose form in ampoules or in multi-dose containers, and can contain formulation agents such as suspending, stabilizing and dispersing agents. The preparations may be in ready-to-use form or in powder form for reconstitution at the time of administration with a suitable vehicle such as sterile water.

The dose to be administered depends largely on the particular compound used, the particular preparation formulated, the route of administration, the type and condition of the host and the particular site and organism being treated. Selection of the particularly preferred dose and route of administration is therefore left to the treating person. Generally, however, the compounds can be administered parenterally or orally to mammalian hosts in an amount from approx.

5-200 mg/kg/day. The administration is usually carried out in divided doses, e.g. 3-4 times daily.

The following examples shall illustrate the invention without limiting it.

Example 1

Preparation of 3-[ 2-( 1- pyridinium) ethylthio]- 6a-[ 1-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0)- hept- 2- en- 2- carboxylate

A. 1-(2-mercaptoethyl)pyridinium methanesulfonate

To a suspension of pyridinium methanesulfonate in pyridine, prepared by dropwise addition of methanesulfonic acid (1.95 mL, 0.03 mol) to pyridine (8.0 mL, 0.099 mol) under cooling was added ethylene sulfide (1.96 mL, 0.033 mol ). The resulting mixture was stirred at 55°C for 16 hours and concentrated under reduced pressure to a thick syrup which was mixed with a few ml of water. The solution was poured onto the top of a column (40 x 16 cm) of u-bondapak C-18 eluted with water. Lyophilization of the appropriate

fractions gave a farewell syrup, 6.5 g (91%).

IR (fil<m>) <u>max: 2300-2600 (br. SH), 1635 (pyridinium), 1490, 1200 (sulfonate), 1068, 1060, 1045, 791, 780 cm"<1>.

<1>HMR (DMSO-dg) 6: 2.32 (3H, s, CH3S03~), 2.61, 2.70, 2.73, 2.82 (1H, B part of A2B system, SH) , 3.07 (2H, m [with D2O, 3.08 (2H, t, J=6.5 Hz)], CH2S), 4.76 (2H, t, J=6.5 Hz, CH2N + , 8.19 (2H, m, Hm of pyridinium), 8.6 (lH, m Ho of pyridinium), 9.08 (2H, dd, J=6.8 Hz, J=1.4 Hz, Ho of pyridinium ).

UV (H20) *max: <2>06 U5230), 258 (e3760) mu.

Method A

B. 1-(2-mercaptoethyl)pyridinium chloride

An aqueous solution of impure 1-(2-mercaptoethyl)pyridinium methanesulfonate (9.4 g, 0.04 mol) was poured onto the top of a column (2.5 x 41 cm) of permutite s-1 Cl". The column was eluted with water at a rate of 0.5 ml per minute and the appropriate fractions were combined and lyophilized to give a yellowish syrup, 7.0 g (100%), which was used as is in the next step.

<1>HMR (D2O) 5 : 3.22 (2H, m, CH2S), 4.88 (m, CH2N<+>), 8.18 (2H, m, Hm of pyridinium), 8.7 (1H , m, Hp of pyridinium), 9.0 ppm (2H, m, Ho of pyridinium).

Method B

To a previously cooled (ice bath) 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 to form a two-phase system. The lighter phase was removed. The remaining oil was washed with 5 x 10 ml of ether and pumped under high vacuum to give 90-100% of the title compound, which was used as such in the next step.

C. Paranitrobenzyl- 3-[ 2 - ( 1- pyridinium) ethylthio] - 6a ~[_ 1-( R) -

hydroxyethyl]- 7-oxol-azabicyclo(3.2.0)hept-2-ene-2-carboxylate chloride

A solution of p-nitrobenzyl-6α-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate (6.09 g, 17, 5 mmol) in 20 ml of acetonitrile was cooled to +5°C under a nitrogen atmosphere and successively treated with diisopropylethylamine (3.65 ml, 21.0 mmol) and diphenylchlorophosphate (4.34

ml, 21.0 mmol). The resulting mixture was stirred for 30 min at 5<C>C, cooled to -5°C and treated successively with a solution of crude 1-(2-mercaptoethyl)pyridinium chloride (4.3 g, 24 mmol) in 1.0 ml of N,M-dimethylformamide and dropwise with diisopropylethylamine (3.65 ml, 21.0 mmol). The reaction mixture was stirred at 0°C for 1 hour, cooled to -30°C and stirred for an additional 15 minutes. The solid was filtered off and washed with cold (-30°C) acetonitrile, 5.77 g (65%).

IR (nujo<l>) <u>max: 3300 (OH), 1775 (C=0 of 6-lactam), 1690 (C=0 of pNB-ester), 1630 (pyridinium), 1605 (phenyl of pNB- ester), 1515 (N02), 1335 cm"<1> (N02).

<1>HMR (DMSO-d ) 6: 1.17 (3H, d, J=6.1 Hz, CH,CHOH), 3.2-3.75 6 J (5H, H-4, H-6 , CH2S), 3.75-4.5 (2H, H-5, CH3CHOH), 4.92 (2H, brt, J=6.5 Hz, CH2N<+>), 5.18 (1H, d, J=4 ,.9 Hz, OH) , 5.37 (center of ABq, J cl / D =14.2 Hz, CH £, • of pNB), 7.69 (2H, d, J=8.7 Hz, HO of pNB), 8.24 (d, J=8.7 Hz, Hm of pNB), 8.0-8.4 (4H, Hm of pNB, Hm of pyridinium), 8.66 (1H, m,

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

The filtrate and washings were combined and diluted with 150 ml of ether. The supernatant was decanted and the gum dissolved in 40 ml of water containing sufficient acetonitrile to hold a solution which was poured onto the top of a 3 x 10 cm column of y-bondapak C-18. The column was eluted with 150 ml of 10% acetonitrile - 90% water and 100 ml of 50% acetonitrile - 50% water. The appropriate fractions were combined and lyophilized after the acetonitrile was removed under vacuum to give a yellowish powder. NMR confirms the presence of the title compound mixed with some p-nitrobenzyl-3-[2-(1-pyridinium) ethylthio]-6<>- [ 1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0 )hept-2-ene-2-carboxylate diphenyl phosphate (2:1). The powder was dissolved in a minimal amount of water and passed through a 1.5 x 21 cm column of permutite S-1 Cl with water. Lyophilization of the appropriate fractions gave 1.8 g or 20% of the title compound.

D. Paranitrobenzyl- 3-[ 2-( 1- pyridinium) ethylthiol- 6a-[ 1-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxylate diphenyl phosphate

A solution of p-nitrobenzyl-6α-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate (0.174 g, 0.50 mmol ) in 2 mL of acetonitrile was cooled to 0°C under a nitrogen atmosphere and treated successively with diisopropylethylamine (0.105 mL, 0.60 mmol) and diphenylchlorophosphate (0.124 mL, 0.60 mmol). The resulting solution was stirred for 30 minutes at 0°C and treated successively with a solution of 1-(2-mercaptoethyl)pyridinium methanesulfonate (0.170 g, 0.72 mmol) in 0.6 mL of acetonitrile and diisopropylethylamine (0.105 ml, 0.60 mmol). The reaction mixture was stirred at 0°C for 15 minutes, diluted with 7 ml of cold water of 0°C

and on top of a 1.5 x 6.4 cm column of u-bondapak C-18. The column was eluted with a mixture of 25-50% acetonitrile in 75-50% water. The appropriate fractions were combined and lyophilized after the acetonitrile was removed under vacuum to give a yellowish powder, 0.33 g (92%).

IR (KBr) u mak, s: <3>600-300 (OH), 1765 (C=0 of B-lactam),

1690 (C=0 of pNB ester), 1625 (pyridinium), 1585 (phenyl), 1510 (NO 2 ), 1330 (NO 2 ), 885 cm"<1> (NO 2 ).

<1>HMR (DMSO-d6) 6: 1.16 (3H, d, J=6.2 Hz, CH3CHOH), 4.87 (2H, brt, J=6.6 Hz, CH-S), 5 .37 (center of ABq, J K=4.3 Hz, CH2 of pNB), 6.7-7.5 (phenyl), 7.68 (d, J=8.8 Hz, Ho of pNB), 8, 23 (d, J=8.8 Hz, Hm of pNB), 8.0-8.3 (m, Hm of pyridinium), 8.4-8.8 (1H, Hp of pyridinium), 9.09 ( 2H, dd, J=6.7 Hz, J=1.3 Hz, Ho of pyridinium).

E. 3-[ 2-( 1- pyridinium) ethylthio]- 6a-[ 1-( R )- hydroxyethyl]- 7-

oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate

Method A

To a solution of p-nitrobenzyl-3-[2-(1-pyridinium)ethylthio]-6a-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)-hept-2- en-carboxylate diphenyl phosphate (0.16 g, 0.22 mmol) in 10 ml of wet tetrahydrofuran was added 10 ml of ether, monobasic potassium phosphate-sodium hydroxide buffer > pH 7.4, (16 ml, 0.05 M) and 0.16 g 10% palladium on charcoal. The resulting mixture was hydrogenated under approx. 3.2 kp/cm<3> for 1 hour at 25°C. The two phases were separated, and the organic phase extracted with 2 x 3 ml of water. The aqueous solutions were combined, washed with 2 x 10 ml of ether and poured onto a 1.5 x 6.2 cm column of u-bondapak C-18 after the traces of organic solvents had been removed under vacuum. Elution of the column with water gave, after lyophilization of the appropriate fractions, a yellowish powder, 0.062 g (84%).

IR (KBr) umax: 3700-3000 (OH), 1755 (C-0 of S-lactam),

1630 (pyridinium), 1590 cm (carboxylate).

"""HMR (D20): 1.22 (3H, d, J=6.4 Hz, CH3CH0H) , 2.92 (d, J=9.1 Hz, H-4), 2.97 (d, J=9.1 Hz, H-4), 3.20 (dd, J=2.5 Hz, J=6.1 Hz, H-6), 3.44 (t, J=6.0 Hz, CH2S), 3.93 (dd, J=9.1 Hz, J=2.5 Hz, H-5), 4.82 (t, J=6.0 Hz, CH2N<+>), 8.04 (m,

Hm of pyridinium), 8.5 (m, Hp of pyridinium), 8.82 (dd,

J= 3.2 Hz, J=l.l Hz, Ho of pyridinium).

UV (H20) xmax= 259 (e5800), 296 (e7030 mu, t^=13.5 hours (measured at a concentration of 10~4 M in a phosphate buffer,

pH 7.4 at 36.8°C).

Method B

To a solution of p-nitrobenzyl-3-[2-(1-pyridinium)ethylthio]-6a-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene -2-carboxylate chloride (5.77 g, 11.4 mmol) in monobasic potassium phosphate-sodium hydroxide buffer (170 ml, 0.2 M, pH 7.22) was added 20 ml of tetrahydrofuran, 30 ml of ether and 5.7 g 10% palladium on charcoal. The resulting mixture was hydrogenated at 22°C under approx. 3.2 kp/cm<2> for 1 hour and filtered on a Celite pad. The pad was washed with 2 x 15 ml of water. The filtrate and washings were combined and diluted with 100 ml of ether. The aqueous phase was separated from, washed with 3 x 100 ml of ether and poured onto the top of a 4.5 x 20 cm column of u-bondapak C-18, after the organic solvents had been removed under vacuum. Elution of the column with water, followed by a mixture of 10% acetonitrile in water gave, after lyophilization of the appropriate fractions, 2.48 g or 65% of the title compound as a yellowish powder.

The analytical data were identical to the data reported for the compound prepared under Method A.

Example 2

Preparation of 3-[ 2-( 2-( 3, 5- dimethylpyridinium)- ethylthio]- 6a-

LI-(R)- hydroxyethyl]- 7-oxo-l-azabicyclo(3.2.0)hept-2-ene-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 methanesulfonic acid (0.65 mL, 0.010 mol) to cold 3,5-lutidine (2.51 mL, 0.022 mol) is added ethyl sulfide ( 0.655 mL, 0.011 mol). The resulting mixture is stirred under a nitrogen atmosphere at 55°C

for 24 hours, cool to 23°C and dilute with 5 ml of water and 5 ml of ether. The organic layers are separated and the aqueous solution is washed with 6 x 4 ml of ether. The traces of ether are removed under vacuum and the solution is added to the top of a 2.5 x 6.0 cm column of u-bondapak C-18. The column is eluted with water and lyophilization of the appropriate fractions gave a colorless syrup, 2.4 g (91%).

IR (film) umax: 2520 (SH), 1628 (pyridinium), 1600,

1495, 1325, 1305, 1283, 1200 (sulfonate), 1040, 938, 765, 680 cm<-1>.

"""HMR (DMSO-dg) 6: 2.31 (3H, s, CH3SC>3~), 2.47 (6H, S, CH3 on pyridinium), 2.57, 266, 269, 2.78 ( 1H, B part of A2B system), SH), 3.06 (2H, m [with D2O added (2H, t, J=6.5 Hz)], CH2S), 4.65 (2H, t, J =6.5 Hz, CH2N+), 8.34 (1H,

s, Hp of pyridinium), 8.79 (2H, s, Ho of pyridinium).

UV (Ho0) X ; 271 (e4860) mu.

2 max

Analysis calculated for cioH17N03S2"0'5H2°:

B. Paranitrobenzyl- 3-[ 2-( 1-( 3, 5- dimethylpyridnium)) ethylthio]-6g-[ 1-( R)- hydroxyethyl]- 7- oxo- azabicyclo( 3. 2. 0) hept- 2 -en- 2- carboxylate diphenyl phosphate

To a cold (0°C) solution of p-nitrobenzyl-6α-(1-(R)-hydroxyethyl)-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate ( 0.523 g, 1.50 mmol) in 6.0 mL of acetonitrile, which is kept under a nitrogen atmosphere, was added diisopropylethylamine (0.314 mL, 1.8 mmol) followed by diphenylchlorophosphate (0.373 mL, 1.8 mmol). The reaction mixture was stirred for 30 min and treated with a solution of 1-(2-mercaptoethyl)-3,5-dimethylpyridinium methanesulfonate (0.493 g, 1.87 mmol) in 1.9 mL of acetonitrile, followed by diisopropylethylamine (0.314 mL, 1, 8 mmol). The reaction mixture was stirred at 0°C for 1 hour, diluted with 26 mL of cold 0°C water, and retained on top of a 7.0 x 3.5 cm u-bondapak C-18 column. Elution of the column with 25-50% acetonitrile - 75-50% water mixture gave, after lyophilization of the appropriate fractions, 1.01 g or 90% of the title compound as a yellowish powder.

IR (KBr) v mak, s: 3700-3100 (OH), 1778 (C=0 of 6-lactam), 1700 (C=0 of pNB ester), 1635 (pyridinium), 1595 (phenyl), 1521 ( N02), 1335 (N02), 895 cm"<1> (N02).

"""HMR (DMSO-d,) 5: 1.16 (3H, dm J=6.1 Hz, CH,CH0H), 2.43 (s, CH^ on pyridinium), 4.75 (2H, m , CH2N ), 5.38 (center of ABq, J , = 14.3 Hz, CH_ of pNB), 6.6-7.5 (10H, m,

cl r D

phenyl), 7.70 (2H, d, J=8.7 Hz, Ho of pNB), 8.0-8.5 (3H,

m Hp of pyridinium, Hm of pNB), 8.82 (2H, s, Ho of pyridinium).

UV (H2<0>) <*>max: <2>70 U11570), 306 (e7343) um. '

Analysis calculated for C37<H>3gN3O10SP-H20:

C. 3-[ 2-( 1-( 3, 5- dimethylpyridinium)) ethylthio]- 6a -[ 1-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2 - one-2-carboxylate

To a solution of p-nitrobenzyl-3-[2-(1-(3,5-dimethylpyridinium))ethylthio]-6a-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0 )hept-2-ene-2-carboxylate diphenyl phosphate (0.600 g, 0.80 mmol) in 36 mL of wet tetrahydrofuran was added ether (36 mL), monobasic potassium phosphate-sodium hydroxide buffer (0.05 M, pH 7.4, 44 ml) as well as 0.60 g of 10% palladium on charcoal. The resulting mixture is hydrogenated under approx. 3.2 kp/cm<2> at 23°C for 1.25 hours. The organic layer was separated from and extracted with 2 x 5 ml buffer. The aqueous layers were combined, filtered through a Celite pad, washed with 40 mL of ether, pumped to remove traces of organic solvents, and poured onto a 2.5 x 10.0 cm u-bondapak C-18 column. Elution of the column with water and lyophilization of the appropriate fractions gave 0.186 g corresponding to 64% as a yellowish powder.

IR (KBr) u , : 3700-3100 (OH), 1760 (C=0 of g-lactam),

^ max

1595 cm (carboxylate).

<1>HMR (D20) 6; 1.21 (3H, d, J=6.3 Hz, CH3CH0H), 2.45 (6H, s, CH3 on pyridinium), 2.81 (d, J=9.2 Hz, H-4), 2 .96 (d, J=9.2 Hz, H-4), 3.22 (dd, J=2.6 Hz, J=6.2 Hz, H-6), 3.40 (t, J= 6.2 Hz, CH2S), 3.84 (dd, J=9.2 Hz, J=2.6 Hz, H-5), 4.15 (m, CH3CH0H)k 4.71 (t, J= 6.2 Hz, CH2N<+>), 8.21 (1H,

s, Hp of pyridinium), 8.46 (2H, s, Ho of pyridinium).

UV (H2<0>) <x>max: 2.79 (e8345), 296 (e7714) m

[a]^<3> + 40.7 (c 0.53, H-O) , t, = 16.9 t (measured at a concentration of 10 M in phosphate buffer, pH 7.4 at 3 6.8°C) .

Example 3

Preparation of (5R, 6S)- 3-[[ 2-( 3- hydroxymethylpyridinio)-ethyl] thio]- 6-[( R)- 1- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0 ]-hept-2-ene-2-carboxylate

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

Trifluoromethanesulfonic acid (1.327 mL, 0.015 mol) is added dropwise to 3-pyridinemethanol (2.91 mL, 0.030 mol) followed by ethylene sulfide (0.89 mL, 0.015 mol). The resulting homogeneous mixture was heated in an oil bath to 50-70°C under N 2 for 20 hours. The reaction mixture was taken up in 15 mL H 2 O and extracted with 5 x 5 mL CH 2 Cl 2 . The aqueous phase was concentrated in vacuo and then added to a C₂g reverse-phase column. Elution with H 2 O followed by evaporation of the relevant fractions gave a pale yellow oil. This material was rechromatographed to give an almost colorless oil. After drying in vacuum over P2°59a this product in an amount of 4.50 g corresponding to 94% as a viscous oil.

IR (film) umax: 3450 (s, OH), 2560 (w, SH) cm"<1>.

"""HMR (d6-acetone) 6: 9.10-8.05 (m, 4H, aromatic), 5.01 (t,

J=5.5 Hz, 2H, N-CH2), 4.93 (s, 2H, -CH2OH), 4.43 (br S, 1H, -OH), 3.43-3.18 (m, 2H , S-CH2), 2.34-2.10 (m, 1H,

SH).

B. p- nitrobenzyl-(5R, 6S)- 3-[ 2-( 3- hydroxymethylpyridinio)-ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo-[ 3. 2 .0] hept-2-ene-2-carboxylate diphenyl phosphate

To a solution of p-nitrobenzyl (5R,6S)-6-[1-(R)-hydroxyethyl ]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.174 g, 0, 50 mmol) in 2 ml of dry acetonitrile was added diisopropylethylamine (0.096 ml, 0.55 mmol) at 0°C under N->. Diphenylchlorophosphate (0.114 mL, 0.55 mmol) was then added dropwise and the reaction mixture stirred at 0°C for 30 minutes. A solution of 3-hydroxymethyl-1-(2-mercaptoethyl)pyridinium trifluoromethanesulfonate (0.223 g, 0.70 mmol) in 0.50 mL of acetonitrile was then added, followed by diisopropylamine (0.122 mL, 0.70 mmol) . After the reaction mixture was kept at 0°C for 30 minutes, it was concentrated in vacuo and the remaining yellow gum collected in H 2 O (sufficient acetonitrile was added to aid dissolution of the gum). This solution was added to a C 2 g reverse-phase column eluted with 15% acetonitrile-H 2 O. Lyophilization of the relevant fractions gave 0.305 g corresponding to 81% of the product as a beige solid.

IR (KBr) u , : 3420 (br, OH), 1775 (B-lactam CO), 1695

rriciKs

(-CO2pNB) cm-1;

<1>HMR (dg-acetone) 5: 9.44-7.72 (m, 8H, aromatic), 7.22-6.91 (m, 10H, diphenyl phosphate), 5.53, 5.27 (ABq , J=14 Hz, 2H, benzylic), 5.04 (t, J=7.4 Hz, 2H, N-CH2), 4.75 (s, 2H, CH2OH), 4.5-3.1 ( m, 8H), 1.21 (d, J=6.3 Hz, 3H, CHMe).

C. ( 5R, 6S)- 3-[ 2-( 3- hydroxymethylpyridinio) ethylthio]- 6-[ 1-( R) - hydroxyethyl ] ;- 7- oxo- l- azabicyclo[ 3. 2. 0] hept- 2- one-2- carboxylate

To a solution of p-nitrobenzyl-(5R,6S)-3-[2-(3-hydroxymethylpyridinio)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0 ]hept-2-ene-2-carboxylate diphenyl phosphate (0.145 g, 0.194 mmol) in 10 ml of THF containing 5 drops of H,,0 was added 6.0 ml of phosphate buffer (0.05 M, pH 7.4), 0.145 g of 10% palladium on charcoal and 10 ml of ether. The mixture was hydrogenated (Parr) at approx. 3.2 kp/cm<2> for 1 hour and then filtered through a pad of Celite. The filter cake was washed with some H 2 O and ether and the aqueous phase separated from and extracted three times with ether. The aqueous solution was then cooled at 0°C and the pH adjusted to 7.0 with pH 7.4 buffer. After removal of residual volatiles in vacuo, the aqueous solution was applied to a C₂₂ reverse phase column eluted with H₂O. Lyophilization of the relevant fractions gave 36 mg corresponding to 51% of the product as a pale yellow solid. Further purification by reverse-phase HPLC gave 31 mg or 41% pure product as a solid.

IR (KBr) umax-* 3300 (br, OH) , 1755 (s-lactam CO), 1590 (-C02~), era"1;

1HNMR (D 2 O) 6 : 8.78-7.94 (m, 4H, aromatic), 4.83 (t, J= 6.0 Hz, 2H, N-CH 2 ), 4.83 (s, 2H, CH 2 OH ), 4.16 (d of q, J=J'=6.2 Hz, 1H, H-l'), 3.98 (d, of t, J=9.1 Hz, J'=2.6 Hz, 1H, H-5), 3.75-3.20 (m, 3H), 3.20-2.65 (m, 2H),

1.22 (d, J=6.4 Hz, 3H, CHMe).

UV (H20) *max: <2>94 (e7614), 266 (e6936) nm;

t^ (pH 7.4, 36.8°C) 14.0 h.

Example 4

Preparation of (5R,6S)-3-[2-(4-hydroxymethylpyridinio)-ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicycloj_3. 2. O]-hept- 2- ene- 2- carboxylate A. 4- hydroxymethyl- 1-( 2- mercaptoethyl) pyridinium- trifluoromethanesulfonate

To a solution of 4-pyridine methanol (1.635 g, 0.015 mol)

in 10 CH 2 Cl 2 at 0°C under N 2 , trifluoromethanesulfonic acid (1.327 ml, 0.015 mol) was added dropwise. A yellow-brown oil quickly separated. An additional equivalent of 4-pyridine methanol (1.635 g, 0.015 mol) was added to this mixture and the solvent was removed under reduced pressure to leave an oil. To this oil was added ethylene sulfide (0.891 ml, 0.015 mol) and the resulting homogeneous mixture was heated in an oil bath at approx. 60°C for 3 hours. Then the reaction mixture was taken up in 15 mL of H 2 O, and the aqueous solution was washed with 5 x 5 mL of CH 2 Cl 2 . After removing residual organic solvent under vacuum, the aqueous solution was applied to a C-2g reverse-phase column. Elution with H 2 O and subsequent evaporation of the relevant fractions gave an oil which was further dried in vacuo over ^ 2^ 5' nvorvecl 4.64 g or 97% of the product was obtained as a colorless oil.

IR (film) u , : 3455 (s, OH), 2565 (w, SH) cm"<1>;

mciK s <1>HNMR (dg-acetone) 6: 9.07, 8.18 (ABq, J=6.8 Hz, 4H, aromatic), 5.03 (s, 2H, CH2OH), 4.96 ( t, J=6.5 Hz, 2H, N-CH2), 4.09 (br S, 1H, -OH), 3.5-3.1 (m, 2H, S-CH2), 2.25 ( brs, 1H, -SH).

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

To a solution of p-nitrobenzyl-(5R,6S)-6-[1-(R)-1-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.348 g , 1.0 mmol) in 5 mL of dry acetonitrile at 0°C under N 2 was added dropwise diisopropylethylamine (0.191 mL, 1.1 mmol), followed by diphenylchlorophosphate (0.228 mL, 1.1 mmol). The resulting golden yellow solution was stirred at 0°C for 40 minutes. To this solution was added a solution of 4-hydroxymethyl-1-(2-mercaptoethyl)pyridinium trifluoromethanesulfonate (0.447 g, 1.4 mmol) in 1 mL of acetonitrile, followed by diisopropylethylamine (0.191 mL, 1.1 mmol). A reddish-black gum separated from the reaction mixture. After 20 minutes at 0°C, the reaction mixture was filtered and concentrated under vacuum. The residue was collected in a minimal volume of (1:1) acetonitrile: H 2 O and applied to a C 2 g reverse-phase column. Elution with 25% acetonitrile-H 2 O and subsequent lyophilization of the relevant fractions gave 0.353 g corresponding to 47% of the product as a cream-colored solid.

IR (KBr) umakg: 3240 (br, OH), 1775 (ε-lactam CO), 1695 (-CO 2 pNB) cm-1;

"""HNMR (dg-acetone) 6 : 9 .24-7 .84 (m, H, aromatic), 7.4-6.9 (m, 10H, diphenyl phosphate), 5.52, 5.24 (ABq , J=14 Hz, 2H, benzylic), 5.15-4.80 (m, 4H), 4.45-3.05 (m, 7H), 1.35 (d, J=6.6 Hz, 3H, CHMe).

C. ( 5R, 6S)- 3-[ 2-( 4- hydroxymethylpyridinio) ethylthio]- 6- [ 1-( R)- 1- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0] hept - 2-ene-2-carboxylate

A mixture of p-nitrobenzyl-(5R,6S)-3-f 2-(4-hydroxymethylpyridinio)ethylthio]-6-[1-(R)-1-hydroxyethyl]-7-oxo-1-azabicyclo-[3.2 .0]hept-2-ene-2-carboxylate diphenyl phosphate (0.348 g, 0.465 mmol) and 10% palladium on charcoal (0.35 g) in 11 ml phosphate buffer (0.05 M, pH 7.4), 5 ml of THF and 10 ml of ether were hydrogenated at approx. 3.2 kp/cm<2> for 1.25 hours. The mixture was then filtered through a Celite pad and the aqueous phase washed three times with ether. The pH of the aqueous solution was then adjusted to 7.0 using additional pH 7.4 buffer. After removing the remaining volatiles under vacuum, the aqueous solution was applied to a C-2g reverse-phase column. Elution with 2% acetonitrile-H 2 O and subsequent lyophilization gave a tan solid. This material was rechromatographed (C^g reverse phase/H^O) whereby 0.060 g corresponding to 36% of the desired product was obtained as a pale yellow solid.

IR (KBr) u__,. : 3400 (br, OH), 1755 (6-lactam CO), 1590

nitijv S

(-C02~) cm-1;

"""HNMR (D 2 O) 6 : 8.73 , 7.96 (ABq, J=6.8 Hz, 4H, aromatic), 4.93 (s, 2H, CH 2 0 H), 4.77 (t, J= 6.0 Hz, 2H, N-CH2),

4.15 (d of q, J=J'=6.3 Hz, 1H, H-l'), 3.96 (d of t, J=

9.2 Hz, J'=2.6 Hz, 1H, H-5), 3.65-3.20 (m, 3H), 3.13-2.62 (m, 2H), 1.21 ( d, J=6.3 Hz, 3H, CHMe);

UV (H2 -O) X max. p: 295 U6880), 256 U5595), 224 U8111) nm;

t^ (pH 7.4, 36.8°C) 14.5 h.

Example 5

Preparation of 3-[ 2-( l-( 2- methylpyridinium)) ethylthio]- 6a-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2-ene - carboxylate

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

To a suspension of 2-methylpyridinium methanesulfonate in 2-methylpyridine, prepared by adding methanesulfonic acid (0.65 mL, 0.010 mol) to cold 2-methylpyridine (2.17 mL,

0.022 mol) was added ethylene sulfide (0.655 ml, 0.011 mol). The reaction mixture was stirred under nitrogen at 55°C i

21 hours, cooled to 23°C and diluted with 5 ml of water.

The aqueous solution was washed with 6 x 4 mL ether, pumped to remove traces of organic solvents and poured onto a 2.5 x 10.0 cm u-bondapak C-18 column. The column was eluted with water and lyophilization of the appropriate fractions gave 2.13 g corresponding to 85% of the title compound.

IR (film) u : 2520 (SH), 1623 (pyridinium), 1574, 1512,

rricixs i

1485, 1412, 1195 (sulfonate), 1038 cm".

<1>HMR (DMSO-d, + D_0) 6: 2.37 (3H, S, CH-.SO,"), 2.83 (3H, s, CH3 on pyridinium), 3.09 (2H, J =6.9 Hz, CH2S), 4.71 (2H,

t, J=6.9 Hz, CH2N<+>), 7.93 (2H, m, Hm of pyridinium), 8.44 (1H, m. Hp of pyridinium), 8.89 (1H, m, Ho of pyridinium);

UV (H c0. 0) X rriciK s: <2>66 (e 3550) mu.

B. Paranitrobenzyl- 3-[ 2-( 1-( 2- methylpyridinium)) ethylthio]— 6a -

[ 1-( R )- hydroxyethyl ]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2-ene-2- carboxylate- diphenyl phosphate

To a cold (0°C) solution of p-nitrobenzyl-6α-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate ( 0.523 g, 1.50 mmol) in 6 mL of acetonitrile, and to which was kept under a nitrogen atmosphere was added diisopropylethylamine (0.314 mL, 1.80 mmol), followed by diphenylchlorophosphate (0.373 mL, 1.80 mmol). The reaction mixture was stirred for 30 min at 0°C and treated with a solution of 1-(2-mercaptoethyl)-2-methylpyridinium methanesulfonate (0.530 g, 2.16 mmol) in 18 mL of acetonitrile, followed by diisopropylethylamine (0.314 mL, 1, 8 mmol). The reaction mixture was stirred at 0°C for 1 hour, diluted with 26 ml of cold 0°C water and poured onto a 3.5 x 7.0 cm u-bondapak C-18 column. Elution of the column with 25% acetonitrile - 75% water and with 50% acetonitrile - 50% water gave, after lyophilization of the appropriate fractions, 1.06 g corresponding to 96% of the title compound as a yellowish powder.

IR (KBr) "max: <3>650-3100 (OH), 1700 (C=0 of ø-lactam), 1695 and 1690 (C=0 of pNB ester), 1630 (pyridinium), 1595 (phenyl) , 1518 (N02), 1335 (N02), 890 cm"<1> (N02);

<1>HMR (DMSO, dg) 6: 1.15 (3H, d, J=6.1 Hz, CH3CHOH), 2.87 (s, CH3 on pyridinium), 3.6-4.4 (2H, m, H-5, CH3CHOH), 4.75 (2H, m, CH2N<+>), 5.37 (center of ABq, J=14 Hz, CH2 of pNB), 6.5-7.4 (10H , m, phenyl), 7.70 (2H, d, J=8.8 Hz, HO of pNB), 8.0 (2H, m, Hm of pyridinium), 8.24 (2H, d, J=8 .8 Hz, Hm of pNB), 8.50 (1H, m, Hp of pyridinium), 8.95 (1H, brd, J=6.1 Hz, Ho of pyridinium);

UV (H20) *max= <2>65 (ell990), 314 (e8020) mu.

C. 3-[ 2-( 1-( 2- methylpyridinium)) ethylthio]- 6a-[ 1-( R)- hydroxyethyl ]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- one - carboxylate

To a solution of p-nitrobenzyl-3-[2-(1-(2-methylpyridinium))-ethylthio]-6a-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0) -hept-2-ene-2-carboxylate diphenyl phosphate (0.66 g, 0.90 mmol) in 34 ml of wet tetrahydrofuran was added 34 ml of ether, monobasic potassium phosphate-sodium hydroxide buffer (0.15 M, 16.5 ml, pH

7.22) as well as 0.66 g of 10% palladium on charcoal. The result-

final mixture was hydrogenated under approx. 3.2 kp/cm<2> at 23°C

for 1.25 hours. The organic layer was separated from and extracted with 2 x 6 ml of buffer. The aqueous layers were combined, filtered through a Celite pad, washed with 40 mL of ether,

pumped to remove traces of organic solvents and poured onto the top of a 2.5 x 10 cm u-bondapak C-18 column. Elution of the column with water and lyophilization of the appropriate fractions gave 0.098 g corresponding to 31% of the title compound as a yellowish powder.

IR (KBr) " ,: 3650-3100 (OH), 1755 (C=0 of 6-lactam),

mciKS ^

1630 (pyridinium), 1595 cm (carboxylate);

<1>HMR (D20) 6: 1.20 (3H, d, J=6.3 Hz, CH3CH0H), 2.83 (s,

CH^ on pyridinium), 2.7-3.1 (5H, H-4, CH^ on pyridinium), 3.1-3.7 (3H, m, CH2S, H-6), 3.90 (dd , J=9.1 Hz, J=2.6 Hz, H-5), 3.1 (m, CH3CHOH), 4.78 (t, J=6.2 Hz, CH2N<+>),

7.8 (2H, m, Hm of pyridinium), 8.3 (1H, m, Hp of pyridinium), 8.65 (1H, m, Ho of pyridinium);

UV (H20) xmax= 268 (e9350), 296 (e8840) mu

[a]2<3> +41° (c 0.5, H2O)

-4

t^ = 15.0 t (milled at a concentration of 10 ml phosphate buffer, pH 7.4 at 36.8°C).

Example 6

Preparation of 3-[ 2-( 1-( 4- methylpyridinium)) ethylthio]- 6a-[ 1( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

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

To a suspension of 4-picolinium methanesulfonate in 4-picoline, prepared by adding methanesulfonic acid (0.65 ml,

0.010 mol) to 4-picoline (2.14 ml, 0.022 mol) under cooling is added ethylene sulfide (0.655 ml, 0.011 mol). The reaction mixture is stirred under nitrogen at 55°C for 24 hours, cooled to 23°C and diluted with 5 ml of water and 10 ml of ether.

The organic layer is separated from and the aqueous layer is washed with 5 x 5 ml of ether and added to the top of a 2.5 x 10 cm u-bondapak C-18 column after traces of ether have been removed under reduced pressure. Elution of the column with a mixture of 15% acetonitrile and 85% water gave, after lyophilization of the appropriate fractions, 2.66 g corresponding to 100% of a colored loose syrup.

IR (film) u ro ci K , s : <2>500 (SH), 1640 (pyridinium), 1572, 1520, 1478, 1200 (sulfonate), 1040, 833 and 768 cm" ;

"""HMR (DMSO-dg) 6: 2.31 (3H, s, CH3SC>3~) , 2.62 (s, CH3 on pyridinium), 2.2-2.9 (4H, SH, CH3 on pyridinium), 3.04 (2H, m, CH2S), 4.68 (2H, t, J=6.4 Hz, CH2N<+>), 8.01 (2H, d, J=6.6 Hz, Hm of pyridinium), 8.89 (2H, d, J=6.6 Hz, Ho of pyridinium);

UV (H2<0>) <x>max: 256 (e4100), 221 (e7544) mu.

B. 1-(2- Mercatoethyl)-4-methylpyridinium-p-toluenesulfonate

To a suspension of p-toluenesulfonic acid (1.72 g, 0.01 mol)

in 6.5 ml of benzene was added 4-picoline (1.17 ml, 0.012 mol). The resulting mixture was stirred under nitrogen at 23°C for 30 min, treated with ethylene sulfide (0.65 mL, 0.011 mol) and stirred at 75° for 24 h. More ethylene sulfide (0.65 mL, 0.011 mol) was added and stirring continued at 75°C for an additional 24 hours. 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 from and washed with 3 x 8 ml of ether. The traces of organic solvents were removed under vacuum and the compound chromatographed on u-bondapak C-18

with water as eluent, whereby 2.94 g or 90% of the title compound was obtained as a colorless syrup.

IR (film) umax: 2510 (SH), 1640 (pyridinium), 1595, 1582, 1475, 1200 (sulfonate), 1031, 1010, 818 cm"<1>.

<1>HMR (DMSO, dg) 6 : 2.29 (3H, s, CH3 on pyridinium), 2.61

(s, CH3, Ph), 2.4-2.8 (4H, SH, CH3Ph), 3.03 (2H, m [addition of D2O gave a t, J=6.4 Hz, at 3.04] , CH2S), 4.68 (2H, t, J=6.4 Hz, CH2N<+>), 7.11, 7.49 (4H, 2d, J=7.9 Hz, phenyl), 8.00 (2H, d, J=6.5 Hz, Hm of pyridinium), 8.89 (2H, d, J=6.5 Hz, Ho of pyridinium);

UV (H2<0>) <x>max: 256 (e4315), 222 (el7045) mu .

C. Paranitrobenzyl- 3-[ 2-( 1-( 4- methylpyridinium)) ethylthio]- 6a-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2 - en-2-carboxylate diphenyl phosphate

To a cold (0°C) solution of p-nitrobenzyl-6α-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene-' carboxylate (0.522 g, 1.5 mmol) in 6 mL of acetonitrile, maintained under a nitrogen atmosphere, was added diisopropylethylamine (0.314 mL, 1.8 mmol) followed by diphenylchlorophosphate (0.373 mL, 1.9 mmol). The reaction mixture was stirred for 45 minutes and treated dropwise with a solution of 1-(2-mercaptoethyl)-4-methylpyridinium methanesulfonate (0.539 g, 2.16 mmol) in 1.8 mL of acetonitrile, followed by diisopropylethylamine (0.314 mL, 1.8 mmol ). The reaction mixture was stirred at 0°C for 1 hour, diluted with 24 mL of cold 0°C water and poured onto a 2.5 x 8.5 cm u-bondapak C-18 column. Elution of the column, first with 100 ml of a mixture of

25% acetonitrile and 75% water and then with 100 ml of a mixture of 50% acetonitrile and 50% water, after lyophilization of the appropriate fractions gave 0.91 g or 83% of the title compound as a yellowish powder.

IR (KBr) umax: 3700-2800 (OH), 1770 (C=0 of B-lactam),

1700 (C=0 of pNB ester), 1640 (pyridinium), 1595 (phenyl), 1520 (NO 2 ), 1340 (NO 2 ), 890 cm"<1> (NO 2 ).

<1>HMR (DMSO, d,) 6: 1.16 (3H, d, J=6.2 Hz, CH-.CH0H) , 2.61 (s, CH3 on pyridinium), 3.1-3, 7 (3H, m, H-6, CH2S), 3.7-4.4 (2H, m, H-5, CH3CHOH), 4.79 (2H, brt, J=6.3 Hz, CH2N<+ >), 5.17 (d, J=4.9 Hz, OH), 5.37 (center of ABq, J=14.1 Hz, CH2 of pNB), 6.7-7.4 (10H, m, phenyl ), 7.69 (2H, d, J=8.8 Hz, Ho of pNB), 8.00 (2H, d, J=6.5 Hz, Hm of pyridinium), 8.23 (2H, d, J=8.8 Hz, Hm of pNB), 8.92 (2H, d, J=6.5 Hz, Ho of pyridinium);

UV (H2<0>) <*>max: 262 (el0835), 311 (e9670) m|i.

Analysis for C-. ,H.,gN30,QSP . 1.5H2O;

D. 3-[ 2-( 1-( 4- methylpyridinium)) ethylthio]- 6a -[ 1-( R)- hydroxyethyl ]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- one - 2- carboxylate To a solution of p-nitrobenzyl-3-[2-(1-(4-methylpyridinium))-ethylthio]-6a-[1(R)-hydroxyethyl -7-oxo-1-azabicyclo-(3.2 .0)hept-2-ene-2-carboxylate diphenyl phosphate (0.587 g, 0.80 mmol) in 30 ml of wet tetrahydrofuran was added 30 ml of ether, monobasic potassium phosphate-sodium hydroxide buffer (0.15 M, 14.7 ml, pH 7.22) and 0.59 g of 10% palladium on charcoal. The resulting mixture was hydrogenated under approx. 32 kp/cm<2> at 23°C for 1.25 hours. The organic layer was separated from and extracted with 2 x 6 ml buffer. The aqueous extracts were combined, filtered through a Celite pad, washed with 3 x 20 ml ether, pumped to remove traces of organic solvents and poured onto a 2.5 x 10 cm column of u-bondapak C-18. Elution of the column with water and lyophilization of the appropriate fractions gave 0.136 g corresponding to 49% of the title compound as a yellowish powder.

IR (KBr) u , : 3700-300 (OH), 1770 (C=0 of B-lactam), 1642

rriciJC p

(pyridinium), 1592 cm (carboxylate);

<1>HMR (D20) «: 1.19 (3H, s, J=6.3 Hz, CH3CHOH), 2.59 (3H, s, CH3 on pyridinium), 2.84 (d, J= 9, 1 Hz, H-4), 2.90 (d, J=9.1 Hz, H-4), 3.0-3.6 (3H, m, CH2S, H-6), 3.86 (dd , J=9.1 Hz, J=2.6 Hz, H-5), 4.12 (m, CH3CHOH), 4.5-4.9 (CH2N<+> masked with HOD), 7.80 ( 2H, d, J=6.6 Hz, Hm of pyridinium), 8.58 (2H, d, J=6.6 Hz, Ho of pyridinium);

UV (<H>2<0>) ^ axis- <2>56 (e5510), 262 (e5360), 296 (e7050) m,

[a]^<3> -20.8° (c 0.48, H2O),

o o -4

= 12.8 t (ground at a concentration of 10 ml of a phosphate buffer, pH 7.4 at 36.8°C).

Example 7

Preparation of ( 5R)- 3-[ 2-( 4- methylthiopyridinio) ethylthio]-( 6S)-[( IR)- hydroxyethyl]- 7- oxol- azabicyclo[ 3. 2. 0] hept- 2-ene - 2-carboxylate

A. 4- methylthiopyridine*

4-Mercaptopyridine (5.55 g, 50.0 mmol; Aldrich) was dissolved in 50 mL of boiling absolute EtOH. The insoluble material

♦Preparation of this compound was reported in King & Ware, J. Chem. Soc., 873 (1939). The procedure described here was followed.

was removed by filtration over Celite. The filtrate was heated to redissolve and after cooling to approx. At 50°C, methyl iodide (3.17 mL, 51.0 mmol; Aldrich) was added at once. The mixture was cooled to crystallization. Filtration of the solid gave 6.77 g (26.7 mmol, yield 53.5%) of the title compound as hydroiodide;

"""HMR (D2O) 6: 2.70 (3H, s, -SCH3) and 7 , 65-7 .77-8 , 35-8 .48 ppm (4H, A2B2 type, aromatic-Hs);

IR (nujo<l>) <u>max: 1615, 1585 (aromatic and 780 cm"<1>;

UV (H2<0>) <x>max: 227 (e2.20 x IO<4>) and 298 nm (el.64 x IO<4>).

The hydroiodide (6.33 g, 25.0 mmol) was dissolved in 40 mL of H 2 O

and the insoluble material removed and washed with 10 ml of H 2 O. 5 g NaOH tablets were added to the filtrate at 0-5°C and extracted with 3 x 25 ml Et2O, the aqueous layer was saturated with NaCl. The combined organic extracts were washed twice with brine, dried over MgSO 4 , and evaporated to yield 2.92 g (23.4 mmol, total yield 50%) of the title compound as an oil;

"""HMR (CDC13) δ; 2.48 (3H, s, -SCH3) and 7.03-7.13-8.38-8.48 ppm (4H, A2B2~type, aromatic-Hs);

IR (film) <u>max: 1580 and 800 cm<-1>.

B. 4-methylthio-N-(2-mercaptoethyl)pyridinium methanesulfonate

4-Methylthiopyridine (2.75 g, 22.0 mmol) was added slowly

methanesulfonic acid<*> (0.65 mL, 10.5 mmol) while cooling in an ice bath. To this solid was added ethylene sulfide<*>

(0.66 mL, 11.0 mmol, Aldrich), and the mixture was heated to 50-60°C for 2 hours. As the reaction progressed, the solid dissolved. After cooling, the reaction mixture was dissolved in 5 ml H 2 O and washed with 5 x 4 ml Et 2 O. The cloudy aqueous layer was filtered over Celite and the filtrate purified by reverse-phase silica gel column chromatography (<C>lg u-bondapak, 10 g) and eluted with H 2 O. Each fraction of 10 ml was pooled. Fractions 2 and 3 were combined and purified again using the reverse-phase column. Fraction 2 gave 1.258 g (4.48 mmol, yield 42.6%) of the title compound as a viscous oil;

^HMR (DMSO-d6, CFT-20) 6: 2.32 (3H, s, MeSC^<9>), 2.72 (3H, s, -SMe), 2.68 (1H,m SH), 2.9-3.2 (3H, m, -CH2S-), 4.59 (2H, t, J=6.4 Hz, -CH2N<ffi>), 7.97 (2H, "d", J =7.2 Hz, aromatic-Hs) and 8.72 ppm (2H, "d", J=7.2 Hz, aromatic-Hz),

IR (pure) u in ciK, . s : 1630, 1200 (br, -SOj,6, 785 and 770 cm<-1>.

<*> These reagents are distilled before use.

C. ( 5R )- p- nitrobenzyl- 3- 12-( 4- methylthiopyridinio) ethylthio]-( 6S)-[( IR)- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0]-hept - 2- en- 2- carboxylate chloride

To a solution of (5R)-p-nitrobenzyl-3,7-dioxo-(6S)-[(IR)-hydroxyethyl]-1-azabicyclo 3.2.0 heptane-(2R)-carboxylate (475 mg, 1.36 mmol) and diisopropylethylamine (0.24 ml,

1.4 mmol) in CH3CN (5 ml) was added at 0-5°C under nitrogen diphenylchlorophosphate (0.29 ml, 1.41 mmol). The mixture was stirred at 0-5°C for 30 minutes. To this mixture was added an oily suspension of 4-methylthio-N-(2-mercaptoethyl)pyridinium methanesulfonate (678 mg, 1.45 mmol;

60% pure) in 1.5 mL CH3CN, followed by diisopropylethylamine (0.24 mL, 1.4 mmol). The mixture was stirred at 0-5°C for 1 hour. Immediately after addition of the base, a yellowish precipitate was formed. This was filtered off and washed with 3 mL of cold CH 2 CN to yield 314 mg of a yellowish solid. This was triturated from 5 ml of 10% MeOH in H 2 O to give 341 mg (0.618 mmol, yield 45.4%)

of the title compound in the form of white crystals, m.p. 118-120°C;

<1>HMR (DMSO-dg, CFT-20) 6: 1.16 (3H, d, J=6.1 Hz, 1'-CH3), 2.72 (3H, s, -CH3), 3, 1-3.7 (5H,m), 3.7-4.3 (2H,m),

4.71 (2H, t, J=6.3 Hz, -C^N®) , 5.15 (1H, d, J=4.9 Hz, OH) , 5.20-5.35-5, 40-5.55 (2H, ABq, CO2CH2-Ar), 7.70 (2H, "d", J=8.8 Hz, nitrophenyl-Hs), 7.97 (2H, "d", J=7 .0 Hz, pyridinium-Hs), 8.25 (2H, "d", J=8.8 Hz, nitrophenyl-Hs), and 8.76 ppm (2H, "d", J=7.1 Hz, pyridinium-Hs);

IR (nujol) «max: 3250 (OH), 1775 (6-lactam), 1700 (ester) and 1625 cm 1 (pyridinium);

UV (abs, EtOH) *max: 308 nm (e4.47 x 10);

[α]^3° + 24.8 (c 0.5, MeOH);

Analysis for C„.H_,N,0,S~C1 .H_0:

J 24 26 3 6 2 2 D. ( 5R)- 3-[ 2-( 4- methylthiopyridinio) ethylthio]-( 6S)-[( 1R)-hydroxyethyl]- 7- oxo- l- azabicyclo 3. 2. 0 hept-2-ene-2-carboxylate

(5R)-p-nitrobenzyl-3-[2-(4-methylthiopyridinio)ethylthio]-(6S)-[(1R)-hydroxyethyl]-7-oxd-1-azabicyclo[3.2.0]hept-2-ene -2-carboxylate chloride (380 mg, 0.688 mmol) was dissolved in 31.5 mL of THF and phosphate buffer, pH 7.4, (31.5 mL, 0.05 M Fisher) and diluted with 31.5 mL of Et 2 O. This solution was mixed with 10% Pd-C (380 mg, Engelhard) and hydrogenated at approx.

2.5 kp/cm<2> on a Parr shaker at room temperature for 1 hour. The aqueous layer was filtered over Celite to remove the catalyst and washed with 2 x 5 mL H 2 O. The filtrate and washing water were combined and washed with 2 x 30 ml Et 2 O. The

aqueous layers were pumped off to remove all organic solvents and purified by reverse-phase column chromatography (Clg u-bondapak, 13 g. Waters Associates), eluting with H 2 O. Fractions with a UV absorbance at 307 nm were collected (ca. 1 liter) and lyophilized to give 127 mg (0.334 mmol, yield 48.5%) of the title compound as a yellowish powder;

"""HMR (D20, CFT-20) 6 : 1.20 (3H, d, J=6.4 Hz, 1'-CH3) ,

2.64 (3H, s, -SCH3), 2.81 (2H,m, -SCH2~), 3.19 (1H, dd, J6_1,<=>6.1 Hz, Jg_5=2.6 Hz, 6-H), 3.32 (2H, dd, J=ll Hz, J=5.5 Hz, 4-Hs), 3.92 (1H, dt, J=9.2 Hz, Jc ,-=2 .6 Hz, 5-H), 4.1 (1H,m, 1'-H), 4.61 (2H, t, J=5.9 Hz, -CH2N ), 7.70 (2H, "d ", J=7.1 Hz, aromatic-Hs) and 8.40 ppm (2H, "d", J=7.1 Hz, aromatic-Hs);

IR (KBr, plate) "max: 3400 (OH), 1750 (B-lactam), 1630 (pyridinium) and 1590 cm (carboxylate;

UV (H2<0>) xmax: <2>31 U9800) and 307 nm (e25000);

[a]"0 + 3.14 (c 0.5, H 2 O) .

Example 8

Preparation of 3-[ 2-( 3- methoxyl- pyridinium) ethylthio]- 6a-[ 1'-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2-carboxylate A. 1-(2-mercaptoethyl)-3-methoxypyridinium methanesulfonate

3-Methoxypyridine (698 g, 6.4 mmol) cooled to 5°C was

methanesulfonic acid (0.216 ml, 3.05 mmol) was added dropwise

and ethylene sulfide (0.19 mL, 3.2 mmol). The mixture was then heated to 60°C for 18 hours, cooled to 20°C, diluted with 10 ml of water and washed with 3 x 10 ml of ether. The aqueous phase was pumped under high vacuum for 15 minutes and heated on a C-^g reverse-phase column. The title compound was eluted with water. The appropriate fractions were combined and evaporated under high vacuum to give the desired thiol (61.6 mg, yield 76.3%);

IR (CH2C12) <u>max<:><2>550 (w, SH)'and 1620, 1600, 1585 cm"<1 >(m, aromatic);

■""HMR (DMSO-d,) 6: 8, 90-7, 90 (4H, m, aromatic C-H) , 4.72 (2H, t, J=6.6 6 Hz, CH2N + ), 4, 01 (3H, s, 0CH3), 3.5-3.0 (m, hidden CH2S), 2.66 (1H, dd, J=9.5 Hz, J=7.5 Hz, SH) and 2, 31 ppm (3H, s, CH3SO3).

B. para- nitrobenzyl- 3[ 2-( 3- methoxy- l- pyridinium chloride) ethylthio ]- 6a-[ 1'-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0)- hept-2-ene-2-carboxylate

A cold (0°C) solution of p-nitrobenzyl-6α-[1'-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (1.04 g , 3 mmol) in 12 mL of acetonitrile was treated dropwise with diisopropylethylamine (0.63 mL, 3.6 mmol) and diphenylchlorophosphate (0.75 mL, 36 mmol) and stirred at 0 °C for 30 min. The resulting enol phosphate was treated with 1-(2-mercaptoethyl)-3-methoxypyridinium methanesulfonate (1.14 g, 4.30 mmol)

in 7 mL of CH 3 CN,diisopropylethylamine (0.63 mL, 4.30 mmol), stirred for 30 min and cooled at -10°C for 30 min. The solid that precipitated from the mixture was filtered off, washed with 2 mL of cold acetonitrile and dried to give the title compound (1.32 g, 82% yield);

IR (nujol) u : 3320 (m, OH), 1780, 1765 (s, B-lactam C=0),

rriciKS i

1700, 1695 (m, ester C=O) and 1520 cm" (s, NO 2 );

<1>HMR (DMSO dg) 6: 9.01 (1H, bs, H-3 aromatic), 8.75 (1H,

bd, J=5.4 Hz, H-6 aromatic), 8.35-7.95 (4H, m, H-aromatic), 7.70 (2H, d, J=7.7 Hz, H-aromatic ), 5.37 (2H, center of ABq, J=13 Hz, CH2pNB), 5.17 (1H, d, J=4.9 Hz,

OH), 4.87 (2H, t, J=6.3, CH2-N<®>), 4.35-3.75 (2H, m, H-5

and H-l'), 4.00 (3H, s, OCH3), 3.56 (part of a t, J=6.3 Hz, CH2S), 3.5-3.20 (3H, m, H -6, H-3) and 1.16 ppm (3H, d,

J=6.1 Hz, CH3CHO).

C. 3-[ 2-( 3- methoxy-l- pyridinium) ethylthio]- 6a-[ 1'-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0)hept-2- ene-2-carboxylate

A solution of para-nitrobenzyl-3[2-(3-methoxy-1-pyridinium chloride)ethylthio]-6a-[1'-(R)-hydroxyethyl]-7-oxo-1-azabicyclo-(3.2.0)hept -2-ene-2-carboxylate (600 mg, 1.12 mmol) in 25 mL THF, 25 mL ether and phosphate buffer, pH 7.4 (0.1 M, 25 mL) was hydrogenated in a Parr -shake over 10% Pd/C (1.1 g) 1 hour at approx. 2.8 kp/cm2. The mixture was diluted with ether and the aqueous phase was filtered through hardened filter paper No. 52. The aqueous layer was washed with 2 x 20 ml of ether, pumped under vacuum and poured onto a silica gel reverse phase column. The title compound was eluted with water containing 2 and 5% acetonitrile. The appropriate fractions were combined and lyophilized to give a yellow solid, which was again purified by HPLC to give the penem carboxylate (150 mg, 38%);

IR (nujol) u maK , s: 1750 (s, /<3->lactam C=0) and 1580 cm<-1> (s, carboxylate);

<1>HMR (D20) 6: 8.55-8.30 (2H,m, H-2, H-6 aromatic), 8.17-7.75 (2H, m, H-3, H-4 aromatic), 4.77 (2H, t, J=5.9 Hz, CH2N<®>), 4.10 (1H, part of 5 lines, J=6.3 Hz, H-l'), 3, 97 (3H, s, OCH3), 3.85, 3.82 (2 lines, part of dt, J=2.6 Hz, part of H-5), 3.42 (2H, t, J=5, 9 Hz, CH2~S), 3.25 (1H, dd,

J=6.1 Hz, J=2.6 Hz, H-6), 2.99-2.60 (2H, 6 lines, part of H-3) and 1.20 ppm (3H, d, J= 6.4 Hz, CH3);

UV (H 2 O), c 0.05) xmax: 290 (e 10517), 223 (£ 6643);

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

Example 9

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

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

To a solution of 3-methylthiopyridine<1> (2.00 g, 0.016 mol)

add 15 ml IN HC1 to 10 ml of ether and shake the whole thing thoroughly. The aqueous phase is separated off, washed with 10 ml of ether and evaporated. The remaining hydrochloride is then dried in vacuo over P2°5 to give a white solid. To this solid hydrochloride was added 3-methylthiopyridine (1.88 g, 0.015 mol) and ethylene sulfide (0.89 mL, 0.015 mol), and the resulting mixture was heated in an oil bath to 55-65°C under

"'"Prepared by the method of J.A. Zoltewiez and C. Nisi,

"J. Org. Chem." 34, 765 (1969).

N2 for 15 hours. This gave a slightly cloudy oil which was collected

in 125 ml of H 2 O and washed with CH 2 C 2 . The aqueous solution was concentrated to approx. 2 ml and then a few drops of acetonitrile were added to make the mixture homogeneous. The resulting aqueous solution was added to a C-3g reverse-phase column. Elution with H 2 O and subsequent evaporation of the relevant fractions gave the product (2.66 g, 80%) as a pale yellow, viscous oil.

IR (fil<m>) u mak, s: 2410 (br, -SH) cm"<1>;

<1>HNMR (db ,DMSO+D i_0) 6: 8.88-7.88 (m, 4H, aromatic), 4.70

(t, J=6.5 Hz, 2H, N-CH2), 3.08 (oblique t, J=6.5 Hz, 2H, S-CH2), 2.64 (s, 3H, S-Me) .

B. p-nitrobenzyl-(5R,6S)-3-[2-(3-methylthiopyridinio)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0 ] hept-2-ene-2-carboxylate chloride

A solution of p-nitrobenzyl-(5R,6S)-6-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.522 g, 1, 50 mmol) in 7 ml of dry acetonitrile is cooled to 0°C and then diisopropylethylamine (0.287 ml, 1.65 mmol) is added dropwise. To the resulting tan solution was added dropwise diphenylchlorophosphate (0.342 mL, 1.65 mmol) and the reaction mixture was kept at 0°C for 30 minutes. Diisopropylethylamine (0.313 mL, 1.80 mmol) was then added, followed by a solution of 3-methylthio-1-(2-mercaptoethyl)pyridinium chloride (0.398 g, 1.80 mmol) in 0.70 mL of dry DMF. About. 1 minute after the addition was complete, a precipitate separated from the reaction mixture and further cooling at -10°C for 10 minutes gave a solid, orange colored mass. This solid was then triturated with acetonitrile and the residue collected by filtration. The residue was washed with acetonitrile, then acetone and finally dried in vacuum and 0.455 g corresponding to 55% of the product was obtained as a cream colored solid. The combined filtrate was evaporated to give a yellow oil which was collected in a minimal volume of acetonitrile and cooled at 0°C for 30 minutes. Filtration of this mixture gave an additional 0.139 g of product as a pale yellow solid. The overall yield was 0.594 g (72%).

IR (KBr) u_ , : 3345 (br, -GH), 1770 (B-lactam CO), 1680 (-CO2pNB) cm"<1>;

<1>HNMR (dg-DMSO) 6: 8.98-7.96 (m, 4H, pyridinium aromatic), 8.20-7.65 (ABq, J=7.0 Hz, 4H, pNB aromatic), 5.53-4.80 (m, 4H), 4.3-3.7 (m, 2H), 3.6-3.25 (m, 6H), 2.66 (s, 3H, S-Me ), 1.16 (d, J=6.0 Hz, 3H, CHMe).

C. ( 5R, 6S)- 3-[ 2-( 3- methylthiopyridinio) ethylthio]- 6-[ 1-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo 3. 2. 0 hept- 2-ene - 2-carboxylate

To a mixture 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 chloride (0.551 g, 1.0 mmol) and 0.55 g of 10% palladium on charcoal in 25 ml of phosphate buffer (0.05 M, pH 7.4) was added 5 ml of THF and 2 5 ml of ether. This mixture was hydrogenated (Parr) at approx. 2.8 kp/cm<2> in

1 hour. The reaction mixture was then filtered through Celite and the filter cake washed with H 2 O and ether. The aqueous phase was separated and washed three more times with ether. After removal of residual organic solvents

in vacuo, the aqueous solution was cooled to 0°C and the pH adjusted to 7.0 with saturated aqueous NaHCO 3 . This solution was immediately applied to a C₂g reverse-phase column. Elution with H 2 O and subsequent lyophilization of the relevant fractions gave 0.25 g of a clear yellow,

solid. This material was purified again by reverse-phase HPLC and 0.210 g corresponding to 55% of the product was obtained as a pale yellow solid.

IR (KBr) w ITlclJ, C S: 3400 (br, -OH), 1755 (B-lactam CO), 1590 (-C02~) cm<-1>;

<1>HNMR (D2O) «: 8.60-7.76 (m, 4H, aromatic), 4.76 (t, J=5.8 Hz, 2H, N-CH2), 4.13 (d of q, J=J'=6.3 Hz, 1H, H-l■), 3.95 (d of t, J=9.0 Hz, J'=2.8 Hz, 1H, H-5), 3, 45-2.75 (m, 5H), 2.59 (s, 3H, S-Me), 1.20 (d, J=6.4 Hz, 3H, CHMe);

UV (H 2 O) xmax-' 296 U8509), 273 (el3005), 231 (ell576) nm;

t^ (pH 7.4, 36.8°C) 20 h.

Example 10

Preparation of 3-[ 2-( 1-( 2, 6- dimethylpyridinium) ethylthio]- 6a -

[ 1-( R )- hydroxyethyl]- 7- oxo- l - azabicyclo( 3. 2. 0 ) heptane- 2-ene- 2-carboxylate

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

A mixture of 2,6-dimethylpyridine (19.2 mL, 0.165 mol) and methanesulfonic acid (3.27 mL, 0.050 mol) was stirred for 15 minutes, treated with ethylene sulfide (4.17 mL, 0.070 mol) and stirred at 100°C for 42 hours under a nitrogen atmosphere. After cooling to 25°C, the reaction mixture was diluted with 45 ml of ether and 30 ml of water. The two layers were separated and the organic layer extracted with 2 x 5 ml of water. The aqueous layers were combined, filtered through a Celite pad, washed with 2 x 15 mL of ether, pumped to remove traces of organic solvents and poured onto a 3.0 x 12 cm u-bondapak C-18 column. Elution with a mixture of 3% acetonitrile and 97% water gave, after lyophilization of the appropriate fractions, 2.5 g of the crude title compound as a syrup. It is purified again by means of HPLC (u-bondapak C-18), whereby 0.90 g corresponding to 7% of the title compound was obtained.

IR (film) u , : 2520 (SH), 1640 and 1625 (pyridinium),

1585, 1490, 1200 cm (sulfonate);

<1>HMR (DMSO-dg + D2O) 6: 2.36 (3H, s, CH2SC>3~), 4.62 (2H, m, CH2N<+>), 7.74 (2H, m, Hm of pyridinium), 8.24 (1H, m, Hp of pyridinium);

UV (H20) <x>max: 272 (4080 mu.

B. Para- nitrobenzyl- 3-[ 2-( l-( 2, 6- dimethylpyridinium)) ethylthio ]- 6a- [ 1-( R)- hydroxyethyl]- 6- oxo- l- azabicyclo( 3. 2. 0 )-hept- 2-ene- carboxylate- diphenyl phosphate

To a cold (0°C) solution of p-nitrobenzyl-6α-[1-(R)-hydroxyethyl ]-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate ( 0.658 g, 1.89 mmol) in 6 mL of acetonitrile, maintained under a nitrogen atmosphere, was added diisopropylethylamine (0.394 mL, 2.26 mmol) and diphenylchlorophosphate (0.468 mL, 2.26 mmol). The reaction mixture was stirred for 30 min and then treated with a solution of 1-(2-mercaptoethyl)-2,6-dimethylpyridinium methanesulfonate (0.720 g, 2.73 mmol) in 3 mL of acetonitrile, followed by diisopropylethylamine (0.394 mL, 2.26 mmol). The reaction mixture was stirred at 0°C for 2 hours, diluted with 27 ml of cold 0°C water and poured onto a 25 x 9.0 cm column of u-bondapak C-18. Elution with mixtures of acetonitrile

and water and lyophilization of the appropriate fractions gave 0.92

g corresponding to 65% of the title compound;

IR (KBr) umax= 3700-3000 (OH), 1765 (C=0 of B-lactam), 1690 (C=0 of pNB-ester), 1620 (pyridinium), 1590 (phenyl), 1517 (N02), 1330 (N02), 880 cm"<1> (N02).

<1>HMR (DMSO, dg) 6i 1.15 (3H, d, J=6.2 Hz, CH3CHOH), 2.7-3.7 (11H, CH2S, 2-CH3 on pyridinium, H-4, H-6), 3.7-4.4 (2H, CH3CHOH, H-5), 4.7 (2H,m, CH2N<+>), 5.14 (1H, d, J=4.5 Hz , OH), 5.37 (center of ABq, J=13.2 Hz, CH2 of pNB), 6.7-7.5 (10H, m, phenyl), 7.5-8.7 (7H, pyridinium , H's of pNB);

UV (H20) xmax= 274 (e 14150), 319 (e 9445) mu.

C. 3-[ 2-( l-( 2, 6- dimethylpyridinium)) ethylthio]- 6a-[ 1-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2 - one-2-carboxylate

To a solution of 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 diphenyl phosphate (0.80 g, 1.07 mmol) in 42 ml of wet tetrahydrofuran was added 42 ml of ether, monobasic potassium phosphate-sodium hydroxide buffer (0.15 M, pH 7.22, 21 ml) and 0.80 g of 10% palladium on charcoal. The resulting mixture was hydrogenated for 1 hour under approx. 2.8 kp/cm<2> at 23°C and filtered through a Celite pad. The two layers were separated and the organic layer extracted with 3 x 8 ml of buffer. The aqueous phase was combined, washed with 50 mL of ether, pumped to remove traces of organic solvent and poured onto a 3.0 x 10.2 cm u-bondapak G-18 column. Elution of the column with a mixture of 5% acetonitrile and 95% water and lyophilization of the appropriate fractions gave the title compound in an amount of 0.246 g corresponding to 63% as a yellowish powder;

IR (KBr) u . : 3700-2800 (OH), 1750 (C=0 of B-lactam),

maXs _,

1620 (pyridinium), 1585 cm (carboxylate);

<1>HMR (D20) «: 1.23 (3H, d, J=6.4 Hz, CH3CHOH), 2.5-3.5 (11H, H-4, H-6, CH2S, 2CH3 on pyridinium ), 3.8-4.4 (2H, CH3CHOH, H-5), 4.5-4.9 (CH2N+, HOD), 7.64 and 7.74 (2H,

A part of A2B system, Hm of pyridinium), 8.07, 8.16, 8.18 and 8.27 (1H, B part of A2B system, Hp of pyridinium);

UV (H20) xmax-' 277 (e9733), 300 (£8271) mu.

[*]* 3 +50.7° (C 0.48, H 2 O);

Analysis for C^8H22N2°4S'1'5H2°'

Example 11

Preparation of (5R, 6S)- 3-[ 2-( 2- methylthio- 3- methylimidazolio)-ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0] hept-2- ene- 2- carboxylate A. 2- methylthio- 3- methyl 1- 1-( 2- mercaptoethyl) imidazolium trifluoromethanesulfonate

Trifluoromethanesulfonic acid (1.38 mL, 0.015 mol) was added dropwise to 2-methylthio-1-methylimidazole<1> (4.0 g, 0.03 mol) at 0°C under N 2 . Then ethylene sulfide (0.9 mL, 0.015 mol) was added and the mixture was heated to 55°C under N 2 for 24 hours. The reaction mixture was triturated three times with ether and the residue collected in acetone, filtered and evaporated. This gave the product in an amount of 4.2 g corresponding to 82% as a semi-crystalline solid which was used as such without further purification.

IR (film) u , : 2550 (w, sh) cm"<1>;

TC13.KS

<1>HNMR (dg-acetone) 6: 7.97 (s, 2H), 4.66 (t, J=7 Hz, 2H, methylene), 4.17 (s, 3H, N-Me), 3 .20 (d of t, J=7 Hz, J'=9 Hz, 2H, methylene), 2.72 (s, 3H, S-Me), 2.20 (t, J=9 Hz, 1H,

-SH).

Prepared as in A. Wohl and W. Marckwald, "Chem. Ber."

22, 1353 (1889).

B. p- nitrobenzyl-(5R, 6S)- 3-[ 2-( 2- methylthio- 3- methyl-imidazolio)-ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenyl phosphate.

To a solution of p-nitrobenzyl-(5R,6S)-6-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (1.40 g , 4.0 mmol) in 50 mL of dry acetonitrile at 0°C under N 2 was added dropwise diisopropylethylamine (0.76 mL, 4.4 mmol) followed by diphenylchlorophosphate (0.91 mL, 4.1 mmol). After stirring the reaction mixture at room temperature for 1 hour, diisopropylethylamine (0.76 ml, 4.4 mmol) was added and then a solution of 2-methylthio-3-methyl-1-(2-mercaptoethyl)imidazolium trifluoromethanesulfonate (2 ,0 g,

5.9 mmol) in 5 ml of acetonitrile. The reaction mixture was kept at room temperature for 1% hour and then concentrated in vacuo to obtain a gum. This was collected in H 2 O and added to a C 2 g reverse-phase column. Elution with H 2 O, then with 20% acetonitrile-H 2 O and finally 30% acetonitrile-H 2 O, followed by lyophilization of the appropriate fractions gave the product in an amount of 0.90 g corresponding to 30% as a pale yellow solid.

IR (KBr) u , : 3380 (br, OH), 1770 (B-lactam CO) cm"<1>;

IT13XS

<1>HNMR (dg-acetone) 6: 8.35 (br, s, 1H), 8.24, 7.78 (ABq, J=8.8 Hz, 4H, aromatic), 7.89 (br s , 1H), 7.25-6.91 (m, 10H, diphenyl phosphate), 5.50, 5.25 (ABq, J=12 Hz, 2H, benzylic), 4.75-4.27 (m, 3H ), 4.03 (s, 3H, N-Me), 4.15-2.75 (m, 8H)., 2.53 (s, 3H, S-Me) , 1.22 (d, J= 6.2 Hz, 3H, -CHMe).

C. ( 5R, 6S)- 3-[ 2-( 2- methylthio- 3- methylimidazolio) ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0 ] hept-2-ene-2-carboxylate

To a solution of p-nitrobenzyl-(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 diphenyl phosphate (1.20 g, 1.56 mmol) in a mixture of 70 mL THF, 70 mL ether and 31 mL phosphate buffer (0.05 M, pH 7 ,4) 1.2 g of 10% palladium was placed on charcoal. This mixture was hydrogenated (Parr) at approx. 2.5 kp/cm<2> for 55 minutes. The reaction mixture was then filtered through Celite, and the filter cake washed with H2O and ether. The aqueous phase was separated, cooled at 0°C and the pH adjusted to 7.0 with saturated aqueous NaHCO 3 . After removing residual organic solvents under vacuum, the aqueous solution was added to a C-2g reverse-phase column. Elution with H 2 O and then 8% acetonitrile-H 2 O and subsequent lyophilization of the relevant fractions gave 0.25 g of a solid. This material was purified again by reverse-phase HPLC to give the product in an amount of 0.114 g corresponding to 19% as an off-white solid.

IR(KBr) umax: 3420 (OH), 1750 ( 6-lactam CO) , 1590 (-CO^)

<1>HNMR (D20) 6: 7.58 (s, 2H), 4.52 (t, J=6 Hz, 2H), 4.28-3.82 (m, 2H), 3.90 (s , 3H, n-Me), 3.40-2.87 (m, 5H), 2.40 (s, 3H, S-Me), 1.20 (d, J=6.4 Hz, 3H, - CHMe);

UV (H20) xmax: 297 (e7572), 262 (e6259), 222 (e7955) nm.

Example 12

Preparation of (5R, 6S)- 3-[ 2-( 3- aminopyridinio) ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxol- azabicyclo[ 3. 2. 0] hept- 2 - one-2-carboxylate

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

3-Aminopyridine (1.50 g, 0.16 mmol) was taken up in 15 mL of 1N methanolic HCl and the resulting solution evaporated until the hydrochloride formed as an oil. To this oil was added 3-aminopyridine (1.32 g, 0.015 mmol) and ethylene sulfide (0.89 mL, 0.015 mmol), and the resulting solution was heated in an oil bath at 60-65°C under N2 for 2 hours . An additional equivalent of ethylene sulfide (0.89 mL, 0.015 mmol) was added and heating continued at 55-65°C for 65 hours. The reaction mixture was washed with CH 2 Cl 2 and then pooled in 25 ml H 2 O. The aqueous solution was applied to a C₂₂ reverse-phase column, which was eluted with H₂O. Evaporation of the relevant fractions gave the product in an amount of 1.26 g corresponding to 44% as a colorless, viscous oil.

IR (film) umax: 3180 (NH2) cm"<1>;

<1>HNMR (dg-DMSO) 6: 8.19-7.59 (m, 4H, aromatic), 4.59 (t, J=6.2 Hz, 2H, N-CH2), 3.5 ( br s, 2H, -NH 2 ), 3.20-2.77 (m, 3H).

B. p- nitrobenzyl-( 5R, 6S)- 3-( 2-( 3- aminopyridinio) ethylthio)-6-( l-( R)- hydroxyethyl)- 7- oxo- l- azabicyclo( 3. 2. 0 ) hept-2-ene-2-carboxylate diphenyl phosphate

To a solution of p-nitrobenzyl-(5R,6S)-6-(1-(R)-hydroxyethyl )-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.696 g, 2 .0 mmol) in 10 mL of dry acetonitrile at 0°C under N 2 , diisopropylethylamine (0.382 mL, 2.2 mmol) was added dropwise followed by diphenylchlorophosphate (0.457 mL, 2.2 mmol). After stirring at 0°C for 30 minutes, a solution of 3-amino-1-(2-mercaptoethyl)pyridinium chloride (0.475 g, 2.5 mmol) in 1 mL of dry DMF was added, followed by additional diisopropylethylamine (0.435 ml, 2.5 mmol). The reaction mixture is kept at 0°C for 1% hour and then concentrated in vacuo. The resulting gum was collected in 1:1 acetonitrile:H 2 O and applied to a C 2 g reverse-phase column. Elution with H 2 O followed by 20% acetonitrile-H 2 O and subsequent lyophilization of the relevant fractions gave the product in an amount of 0.730 g, corresponding to 50% as a beige solid.

IR (KBr) umax: 3330 (br, OH), 3180 (br, NH2), 1770 (6-lactam CO), 1690 (-CO2pNB) cm"<1>;

"""HNMR (dg-DMSO) 6 : 8 .29-7.63 (m, 8H, aromatic), 7.2-6.7

(m, 10H, diphenyl phosphate), 5.47, 5.18 (ABq, J=14 Hz, 2H, benzylic), 4.73-4.45 (m, 3H), 4.2-3.8 (m , 1H), 3.6-2.6

(m, 8H), 1.15 (d, J=6.2 Hz, 3H, CHMe).

C. ( 5R, 6s )- 3-(( 2-( 3- aminopyridinio) ethyl) thio)- 6- 1-( R)-hydroxyethyl)- 7- oxo- l- azabicyclo( 3. 2. 0) hept - 2- en-2- carboxylate

To a mixture of p-nitrobenzyl-(5R,6S)-3-(2-(3-aminopyridinio)ethylthio)-6-(1-(R)-hydroxyethyl)-7-oxo-1-azabicyclo-(3.2. 0)hept-2-ene-2-carboxylate diphenyl phosphate (0.730 g,

1.0 mmol) and 0.7 g of 10% palladium on charcoal in 25 ml of phosphate buffer (0.05 M, pH 7.4), 8 ml of THF and 20 ml of ether were added. This mixture was hydrogenated (Parr) at approx. 2.8 kp/cm 2 in

1 hour. The resulting mixture was filtered through a pad of Celite and the filter cake was washed with H 2 O and ether. The aqueous phase was separated, washed twice with ether

and then residual volatiles were removed under vacuum. The aqueous solution was immediately applied to a C^g" reverse-phase column eluted with H^O. Lyophilization of the relevant fractions gave 0.45 g of an off-white solid. This was again purified by reverse -phase HPLC to give the desired product in an amount of 0.123 g corresponding to 35% as an ivory-colored solid.

IR (KBr) u : 3340 (br), 1750 (br, B-lactam CO), 1580

(br, -C02 ) cm ;

<1>HNMR (D2O) 6: 8.07-7.59 (m, 4H, aromatic), 4.61 (t, J=5.8 Hz, 2H, N-CH2), 4.14 (d of q, J=J'=6.3 Hz, 1H,

H-1'), 3.97 (d of t, J=9.2 Hz, J'=2.6 Hz, 1H, H-5),

3.38 (t, J=5.8 Hz, 2H, S-CH2), 3.24 (d of d, J=6.0 Hz, J'=2.6 Hz, 1H, H-6), 3.17-2.57 (m, 2H, H-4), 1.21 (d, J=6.3 Hz, 3H, CHMe);

UV (H20) *max: <2>99 (e7949), 256 (e8822) nm;

(pH 7.4, 36.8°C), 18.5 h.

Example 13

Manufacture of

( 5R, 6S)- 3- [ 1-( S)- methyl1- 2-( 1- pyridinium) ethylthio ]- 6-[ 1-( R)-hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2 .0)hept-2-ene-2-carboxylate and (5R,6S)3-[1-(R)-methyl-2-(1-pyridinium)ethylthio]-6-[1-(R)-hydroxyethyl] - 7-oxo-l-azabiclo(3.2.0)hept-2-ene-2-carboxylate A. d&- 1- ( 2 - mercapto- 2- methylethyl) pyridinium methanesulfonate d&- 1-( 2- mercapto- 1-methylethyl) pyridinium methanesulfonate

Methanesulfonic acid (1.95 mL, 0.030 mol) was slowly added to cold pyridine (7.83 mL, 0.097 mol), and the resulting mixture was stirred at 40°C for 15 min, treated with da-propylene sulfide (2.59 mL , 0.033 mol) and stirred at 60°C under nitrogen for 90 hours. Pyridine was removed under vacuum, the residue was mixed with water and purified by chromatography (preparative HPLC, bondapak C-18). The appropriate fractions were combined and lyophilized to give dn-1-(2-mercapto-2-methylethyl)pyridinium methanesulfonate, 1.14 g (15%) as a colorless syrup;

IR (film) u : 2520 (SH), 1640 (pyridinium), 1180 (s,

IT13.X S -i

CH3SO3~), 1040 (CH3SO3~) cm ;

<1>HMR (DMS0-d6) S: 1.35 (d, J=6.8 Hz, 3H, CH3CHS), 2.30 (s, 3H, CH3SO3~), 2.90 (d, J=8 .5 Hz, 1H, SH), 3.2-3.7 (m, CHSH), 4.52 (dd, Jgem=12.9 Hz, J=8.4 Hz, CHCH2N<+>), 4, 87 (dd, Jgem=12.9 Hz, J=6.0 Hz, CHCH2N<+>), 8.0-8.4 (m, 2H, Hm of pyridinium), 8.5-8.8 (m , 1H, Hp of pyridinium), 9.04 (dd, J=1.4 Hz, J=6.7 Hz, 2H, Ho of pyridinium);

UV (Ho0) X : 208 (e5267), 259 (e3338);

Analysis for CqH,cNO^S,.2H~0:

and dfc-1-(2-mercapto-1-methylethyl)pyridinium methanesulfonate, 0.82 g, (11%) as a colorless syrup;

IR (film) "max: 2500 (SH), 1628 (pyridinium), 1180 (sulfonate), 1035 (sulfonate) cm "*";

"""HMR (DMSO-dt) 6: 1.69 (d, J=6.8 Hz, 3H, CH0CHN+ ), 2.31 (s, 3H, CH3SO3 ), 3.0-3.3 (m, 2H, CH2S), 4.2-5.2 (m, 1H, CHN ), 8.0-8.4 (m, 2H, Hm of pyridinium), 8.5-8.8 (m, 1H, Hp of pyridinium), 9.0-9.2 (m, 2H, Ho of pyridinium);

UV (H20) Xmakg: 209 (e4987), 258 (e3838).

Analysis for CgH,5NO.,S2.1,5H20: B . ( 5R, 6S)- paranitrobenzyl- 3-[ 1-( R, S) methyl- 2-( 1- pyridinium) ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo ( 3. 2. 0) hept- 2- ene- 2- carboxylate diphenyl phosphate To a cold (0°C) solution of (5R,6S)-paranitrobenzyl-6-[1-(R)-hydroxyethyl]-3,7-dioxo -1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate (0.523 g, 1.5 mmol) in 6 mL of acetonitrile, kept under a nitrogen atmosphere, was added diisopropylethylamine (0.314 mL, 1.8 mmol) followed by of diphenylchlorophosphate (0.373 mL, 1.8 mmol). The reaction mixture was stirred for 30 min and treated with a solution of d£-1-(2-mercapto-2-methylethyl)pyridinium methanesulfonate (0.539 g, 2.16 mmol) in 2 mL of acetonitrile and diisopropylethylamine (0.314 mL, 1.8 mmol ). The reaction mixture was stirred at 0°C for 1 hour, diluted with 24 mL of cold 0°C water and chromatographed over a 2.5 x 8.5 cm preparative bondapak C-18 column with 25-50% acetonitrile in water as eluent - solvents to give 1.07 g corresponding to 97% of the title compound as a yellowish powder after lyophilization;

IR (KBr) umax<:><3>700-3100 (OH), 1770 (C=0 of 6-lactam), 1695 (C=0 of pNB ester), 1630 (pyridinium), 1590 (phenyl), 1518 (N02), 1348 (N02), 885 (N02) cm"<1>;

J1=HM6R ,3 (DHMzS, O-3Hd6 ,, ) CH63: CHS1),1, 4 4(d,6, -J5=,0 6,l (mH, zC, H23N H- , )C, H 3 ..C5H,O14 ) , (d13,3 (d,

J=5.2 Hz, 1H, OH), 5.37 (center of ABq, J=12.4 Hz, 2H, CH2 of pNB), 6.6-7.5 (m, 10H, phenyl of phosphate) , 7.69 (d, J=8.7 Hz, 2H, Ho of pNB), 8.0-8.4 (m, 4H, Hm of pNB, Hm of pyridinium), 8.4-8.8 ( m, 1H, Hp of pyridinium), 9.08 (d, J=5.6 Hz, 2H, Ho of pyridinium);

UV (H20) xmax: 263 (el3325), 308 (e8915).

Analysis for <C>36<H>36<N>3<0>i<qSP>'H20:

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

To a solution of (5R,6S)-paranitrobenzyl-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 diphenyl phosphate (0.60 g, 0.82 mmol) in 33 ml of wet tetrahydrofuran was added 33 ml of ether, monobasic potassium phosphate-sodium hydroxide buffer (17 ml , 0.15 N, pH7.22) and 0.60 g of 10% palladium on charcoal. The resulting mixture was hydrogenated for 1 hour under approx. 2.8 kp/cm<2> at 23°C. The two layers were separated and the organic layer extracted with 3 x 7 ml of water. The aqueous layers were combined, filtered through a pad of Celite, washed with 3 x 20 mL of ether and chromatographed on a 2.5 x 9.5 cm preparative bondapak C-18 column with water as eluent to obtain 0.18 g corresponding to 63% of a mixture of diastereoisomers. These were separated by HPLC (preparative bondapak C-18) with water as elution solvent: lower retention isomer, 0.068 g (23%) compound "B";

IR (KBr) u . : 1770 (C=0 of 3-lactam), 1633 (pyridinium), 1593 (carboxylate) cm ;

<1>HMR (D20) 6: 1.20 (d, J=6.3 Hz, 3H, CH3CHO), 1.42 (d, J=6.9 Hz, 3H, CH3CHS), 2.3-3 .2 (m, 3H, H-4, H-6), 3.5-3.9 (m, 1H, SCH), 3.9-4.2 (m, 2H, H-5, CH.. CHO) , 4,3-

+ J

5.1 (m, CH2N ), 7.8-8.2 (m, 2H, Hm of pyridinium),

8.4-8.7 (m, 1H, Hp of pyridinium), 8.7-9.0 (m, 2H, Ho of pyridinium);

UV (H_0) X ; 260 (<£>6727), 300 (e8245);

£. rrict.K p

[a]23 -39.3° (c, H2O),

-4

ti = 12.6 h (milled at a concentration of 10 ml phosphate buffer, pH 7.4 at 36.8°C);

higher retention isomer, 0.081 g (28%), compound

"A" ;

IR (KBr) max = 1755 (C=0 of β-lactam), 1630 (pyridinium), 1590 (carboxylate). cm

<1>HMR (D20) S : 1.18 (d, J=6.3 Hz, 3H, CH3CH0), 1.40 (d, J=7.0 Hz, 3H, CH3CHS), 2.84 (d , J=9.3 Hz, 2H, H-4),

3.26 (dd, J=2.7 Hz, J=5.9 Hz, 1H, H-6), 3.4-4.2 (m, 3H, SCH, CH3CH0, H-5), 4, 2-5.1 (m, CH2N<+>), 7.7-8.1 (m, 2H, Hm of pyridinium), 8.3-8.65 (m, 1H, Hp of pyridinium), 8, 65-8.9 (m, 2H, Ho of pyridinium);

UV (H20) xmax: 259 (e 5694), 296 (e 6936),

[a]^<3> + 96.9° (c 0.56, H 2 O),

tj^ = 15.6 t (milled at a concentration of 10 ml of phosphate buffer, pH 7.4 at 36.8°C).

Example 14

Manufacture of

( 5R , 6S )- 3-[ 2-[( S )-( 1- pyridinium )]- 1-( S )- cyclohexylthio]- 6-[ 1-( R )- hydroxyethyl ]- 7- oxo- l- azabicyclo(3.2.0)hept-2-ene-2-carboxylate and (5R,6S)-3-[2-[(R)-(1-pyridinium)]-1-(R)- cyclohexylthio]- 6- [ 1-( R )- hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2-ene- 2- carboxylate A. dg,- l- [ 2- mercapto- l- cyclohexyl ] pyridinium methanesulfonate

Methanesulfonic acid (0.65 mL, 0.01 mol) was added dropwise to pyridine (2.42 mL, 0.03 mol) with cooling. The mixture was stirred under nitrogen for 10 minutes, treated with d.sub.-cyclohexene sulfide 1.377 g (85% pure), 0.0103 mol and stirred at 72°C for 25 hours. The excess of pyridine was removed under vacuum and the traces co-distilled with water. The residue was mixed with water and chromatographed on a 5 x 13 cm preparation bondapak C-18 column with 0-2% acetonitrile in water as eluent, whereby after lyophilization a colorless syrup was formed, 1.57 g (53%);

IR (film) u ITlclK S : <2>500 (SH), 1625 (pyridinium), 1190 (SOj~);

1HiyiR (DMSO dg) 6: 1.2-2.5 (m, 8H, cyclohexyl H) , 2.32 (s, 3H, CH3SO3~), 2.82 (d, J=9.8 Hz, SH) , 3.0-3.5 (m, 1H, CHSH), 4.2-4.9 (m, 1H, CHN<+>), 8.0-8.3 (m, 2H, Hm of pyridinium) , 8.4-8.8 (m, 1H, Hp of pyridinium), 8.9-9.3 (m, 2H, Ho of pyridinium);

UV (H 2o. 0) X max: 214 (e 5365), 258 (e 3500).

Analysis for C12H19N03S2,H20; B. ( 5R , 6S )- paranitrobenzyl- 3-[ 2-[( R or S )-( 1- pyridinium ) ]- 1-( R or S )- cyclohexylthio]- 6-[ 1-( R )-hydroxyethyl ]- 7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenyl phosphate

To a cold (0°C) solution of (5R,6S)-paranitrobenzyl-6-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)hept-2-ene- 2-Carboxylate (1.37 g, 3.93 mmol) 15 mL of acetonitrile, under nitrogen, was added diisopropylethylamine (0.822 mL, 4.7 mmol) and diphenylchlorophosphate (0.979 mL, 4.7 mmol). The resulting solution was stirred for 30 min and treated with a solution of d -1-(2-mercapto-1-cyclohexyl)pyridinium methanesulfonate (1.64 g, 5.66 mmol) in 4.7 mL of acetonitrile, followed by diisopropylethylamine ( 0.822 mL, 4.7 mmol). The reaction mixture was stirred at 0°C for 1 hour, diluted with 75 ml of cold water of 0°C and chromatographed on preparative bondapak C-18 with 25-50% acetonitrile in water as elution solvent, whereby after lyophilization of the appropriate fractions, the 1.9 g corresponding to 53% of the title compound;

IR (KBr) vj : 3700-3000 (OH), 1770 (C=0 of B-lactam),

rricLK p

1700 (C=0 of pNB ester), 1628 (pyridinium), 1590 (phenyl),

1515 (N02), 1345 (N02), 880 (N02) cm - 1.

<1>HMR (D 2 O) 6: 1.13 (d, J=6.1 Hz, 3H, CH 3 CHO), 1.2-2.5

(m, 8H, cyclohexyl H), 2.7-3.5 (m, 4H, H-4, H-6, CHS), 3.5-4.4 (m, 2H, CH3CH0, H-5) , 4.4-5.0 (m, 1H, CHN<+>), 5.30 (center of ABq, J=12.8 Hz, CH2 of pNB), 6.7-7.4 (m, 10H , phenyl), 7.65 (d, J=8.6 Hz, 2H, Ho of pNB), 7.9-8.4 (m, 4H, Hm of pNB, Hm of pyridinium), 8.4-8 .8 (m, lH,

Hp of pyridinium), 9.0-9.4 (m, 2H, Ho of pyridinium);

UV (H20) xmax: 263 (e 9038), 309 (e 6394).

Analysis for C3gH4nN30,gSP.H20: C. ( 5R, 6S)- 3-[ 2-[( R or S)-( 1- pyridinium)]- 1-( R or S)- cyclohexylthio ]- 6- [ 1 -( R)- hydroxyethyl] - 7- oxo- l -azabicyclo( 3. 2. 0 ) hept- 2- ene- 2- carboxylate To a solution of (5R,6S)-paranitrobenzyl-3-[2-[( R or S)-(1-pyridinium)]-1-(R or S)-cyclohexylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2 -ene-2-carboxylate diphenyl phosphate (1.85 g, 2.34 mmol) in 96 ml of wet tetrahydrofuran was added to 96 ml of ether, monobasic potassium phosphate-sodium hydroxide buffer (0.15 M, pH 7.22, 50 ml) and 1.9 g 10% palladium on charcoal. The resulting mixture was hydrogenated at 23°C under approx. 2.8 kp/cm<2> for 1.2 5 hours. The organic layer was separated and washed with 3 x 20 ml of water. The aqueous solutions were filtered through a Celite pad, washed with 2 x 60 mL ether, pumped to remove traces of organic solvents and chromatographed on a 4.5 x 9 cm preparative bondapak C-18 column with 0-5% acetonitrile in water as eluent, whereby after lyophilization 0.705 g corresponding to 76% of a mixture of diastereoisomers was formed. Separation of these was carried out using HPLC with 4% acetonitrile in water as eluent; the lower retention diastereoisomer, compound "A", (0.29 g, 31%);

IR (KBr) u : 1750 (C=0 of B-lactam), 1620 (sh, pyri-IT13KS ^ ^

dinium), 1685 (carboxylate) cm ;

<1>HMR (D2O) <S: 1.21 (d, J=6.3 Hz, 3H, CH3CHO) , 1.4-2.5 (m, 8H, cyclohexyl H), 2.5-3, 05 (m, 2H, H-4), 3.05-3.25 (m, 1H, H-6), 3.3-3.7 (m, 1H, CHS), 3.9-4.3 (m, 2H, H-5, CH3CHO), 4.3-4.8 (m, CHN<+>), 7.8-8.2 (m, 2H, Hm of pyridinium), 8.3-8 .7 (m, 1H, Hp of pyridinium), 8.8-9.1 (m, 2H, Ho of pyridinium);

UV (H2<0>) <x>max: 260 (e7123), 300 (e8685);

[al^<3> +6.2° (c 0.63, H2O),

= 16.6 h (measured at a concentration of 10 4 M in phosphate buffer, pH 7.4 at 36.8°C);

Analysis four C2n<H>24<N>2<0>4<S.>2H20;

diastereoisomers with higher retention time, compound "B", (0.35 g, 38%);

IR (KBr) "max: 1750 (C=0 of ε-lactam), 1622 (sh, pyridinium), 1588 (carboxylate) cm 1;

<1>HMR (D2O) 6: 1.19 (d, J=6.4 Hz, 3H, CH3CHO), 1.3-2.5 (m, 8H, cyclohexyl H), 2.5-3.1 (m, 2H, H-4), 3.1-3.3 (m, 1H, H-6), 3.3-3.8 (m, 2H, H-5, CHS), 4.1 ( center of m, 1H, CH3CH0), 4.25-4.7 (m, 1H, CHN<+>), 7.8-8.1 (m, 2H, Hm of pyridinium), 8.3-8, 7 (m, 1H, Hp of pyridinium), 8.75-

9.0 (m, 2H, Ho of pyridinium);

UV (H20) xmax: 259 (e5992), 296 (e7646);

[a]p<3>° +65.3° (c 0.43, H2O),

= 20.2 h (measured at a concentration zone of 10 4 M in phosphate buffer, pH 7.4 at 3 6.8°C).

Example 15

A. ( 5R )- allyl- 3-[( 2- pyridinedioethyl) thio]-( 6S )-[( IR )- hydroxyethyl ]- 7- oxo- l- azabicyclo[ 3. 2. 0] hept- 2- one - 2- carboxylate diphenyl phosphate

To a solution of (5R)-allyl-3,7-dioxo-(6S)-[(IR)-hydroxyethyl]-1-azabicyclo[3.2.0]heptane-(2R)-carboxylate (473 mg, 1.87 mmol) in CH3CN (6 ml) at approx. -10°C under nitrogen was added diisopropylethylamine (0.42 mL, 2.4 mmol) followed by diphenylchlorophosphate (0.50 mL, 2.4 mmol). The mixture was stirred at 0°C for 30 minutes and then cooled to -15°C. To this was added an oily suspension of N-(2-mercaptoethyl)-pyridinium chloride (527 mg, 3.00 mmol) in 1 mL of CH-jCN containing 5 drops of DMF, followed by diisopropylethylamine (0.42 mL, 2, 4 mmol). The mixture was stirred at -15°C for 30 minutes, and then diluted with 20 mL of H 2 O. This mixture was purified directly on a reverse-phase silica gel column (C^g PrepPAK, 12 g, Waters Associates) eluting with H 2 O (200 mL), 10% CH 3 CN/H 2 O (100 mL), 20% CH3CN/H2O (100 mL), 30% CH3CN/H2O (100 mL) and finally 40% CH3CN/H2O (100 mL). Appropriate fractions were collected, the organic solvents removed using a vacuum pump and lyophilized to obtain 786 mg (1.26 mmol, yield 67.3%) of the title compound as a brown powder; <1>HMR (DMSO-dg), CFT-20) 6: 1.16 (3H, d, J=6 Hz, 1'-CH3), 2.6-3.7 (m), 3.75- 4.3 (2H, m, 5-H and 1'-H), 4.65 (2H, m, -CH2CH2-), 4.87 (2H, t, J=6 Hz, -CH2N<+>) , 5-6.2 (3H, m, olefinic protons), 6.6-7.4 (m, aromatic protons),

8.15 (2H, "t", J=7 Hz, aromatic protons meta to the nitrogen), 8.63 (1H, "t", J=7 Hz, aromatic proton para to the nitrogen) and 9.07 ppm (2H , "d", J=7 Hz, aromatic protons ortho to the nitrogen);

IR (film) u; 3400 (OH), 1770 (B-lactam), 1690 (ester),

1625 (pyridinium).

B. ( 5R )- 3-[( 2- pyridinedioethyl) thio]-( 6S )-[( IR )- hydroxyethyl]- 7-oxol- l- azabicyclo[ 3. 2. 0] hept- 2- en- 2 - carboxylate

To a solution of (5R)-allyl-3-[(2-pyridinioethyl)thio]-(6S)-[(1R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene -2-carboxylate diphenyl phosphate (156 mg, 0.25 mmol) in CH 2 CN

(2 ml) it was successively at approx. At 22°C, a solution of potassium 2-ethylhexanoate in EtOAc (0.5 M, 0.6 mL, 0.3 mmol), triphenylphosphine (15 mg, 0.057 mmol) and tetrakistriphenylphosphine palladium (15 mg, 0.013 mmol). The mixture was stirred at approx. 22°C under nitrogen for 2 hours. After adding 7 ml of anhydrous Et 2 O, the precipitate was filtered off, washed with

7 ml of anhydrous Et.,0 and dried in vacuo to give 101 mg of brownish solid. This was purified by reverse-phase column chromatography (C-^g PrepPAK, 12 g, Waters Associates), eluting with H 2 O. Appropriate fractions (namely fractions 7-12, 20 mL each) were pooled and lyophilized to obtain 53 mg (0.16 mmol, yield 64%) of the title compound as a yellowish powder. This material was

contaminated with potassium diphenyl phosphate and potassium 2-ethylhexa-

nothing;

<1>HMR (D20, CFT-20) 6: 0.80 (t, J=6.4 Hz, Me from ethylhexa-

noat) , 1.21 (3H, d, J=6.3 Hz, l'-Me), 2.93 (2H, dd, J],_4 =

9 Hz, J =4 Hz, 1-Hs), 3.28 (1H, dd, J, ,=6.2 Hz, J, c=

gem o—± o—d

2.5 Hz, 6-H), 3.42 (2H, t, J=6 Hz, -CH2S), 3.98 (1H, td,

<J>5_1<=>9 Hz, J5_6=2.5 Hz, 5-H), 4.15 (1H, q, J=6.2 Hz, l'-H),

4.80 (2H, t, J=6.0 Hz, -CH2N<+>), 7-7.5 (m, phenyl protons from diphenyl phosphate), 8.03 (2H, m, Hm of pyridinium), 8, 56 (1H,

m, Hp of pyridinium) and 8.81 ppm (2H, "d", J-6.5 Hz, Ho of pyridinium).

Example 16

Preparation of 3-[ 2-( N- methyl- thiomorpholinium) ethylthio]- 6a-[ 1'-( R)- hydroxyethyl]- 7- oxol- azabicyclo[ 3. 2. 0] hept- 2-en- 2- carboxylate

A. N-methyl-N-(2-mercaptoethyl)-thiomorpholinium methanesulfonate

*

To ice-bath precooled N-methylthiomorpholine (5.00 g, 42.7 mmol) was added methanesulfonic acid (1.47 ml, 20.5 mmol) and ethylene sulfide (1.30 ml, 21.4 mmol). The mixture was heated at

<*>J.M. Lehn & J. Wagner, "Tetrahedron", 26, 4227 (1970)

65°C for 24 hours and diluted with 25 ml of water. The aqueous solution was washed with 3 x 25 mL of diethyl ether, pumped under vacuum and poured over a silica gel reverse phase column, eluting the title compound with water. The appropriate fractions were combined and evaporated to give the thiol as an oil (4.80 g, yield 86%);

IR (film) u . : 2550 cm"<1> (w, SH);

2T13JC P

<1>HMR (DMSO-d,) fi: 3.25-2.95 (6H, m, CH_N<+>), 3.32 (3H, s,

+

CH3N ), 3.20-2.65 (7H, m, CH2S, SH) and 2.32 ppm (3H, s, CH3SO3).

B. para- nitrobenzyl- 3-[ 2-( N- methyl- thiomorpholinium- diphenylphosphate) ethylthio]- 6a-[ 1'-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0] hept-2-ene-2-carboxylate

A cold (ice bath) solution of para-nitrobenzyl-6α-[1'-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (557 mg, 1.60 mmol) in 8 mL CH3CN was treated dropwise with diisopropylethylamine (0.336 mL, 1.92 mmol) and diphenylchlorophosphate (0.400 mL, 1.92 mmol) and stirred for 30 min. The reaction mixture was again treated with N-methyl-N-(2-mercaptoethyl)thiomorpholinium methanesulfonate (893 mg, 2.29 mmol) in 4 mL of CH2CN and diisopropylethylamine (0.336 mL, 1.92 mmol) and stirred for 30 minutes. The solution was diluted with 20 ml of water and poured over a silica gel reverse-phase column. The desired compound was diluted with a 50% acetonitrile-water mixture. The appropriate fractions were combined, pumped under vacuum for 2 hours and lyophilized to give the title compound (1.01 g, yield 85%);

IR (nujo<l>) vmax-- <1>760 (s, B-lactam C=0) and 1510 cm"<1> (s, N02);

<1>HMR (DMSO-d^) 6: 8.25 (2H, d, J=8.8 Hz, H-aromatic), 7.70 (2H, d, J=8.8 Hz, H-aromatic ), 7.33-6.84 (10H, m, H-aromatic), 5.37 (2H, center of ABq, J=14.2 Hz, CH2), 5.14

(1H, d, J=4.5 Hz, OH), 4.35-3.80 (2H, m, H-l<*> and H-5), 3.75-3.45 (6H, m, CH2N <+>), 3.31 (3H, s, CH3N<+>), 3.45-2.75 (9H, m, CH2S, H-6 and H-4) and 1.15 ppm (3H, d , J=6.2 Hz,

CH3).

C. 3-[ 2-( N- methyl- thiomorpholinium) ethylthio3- 6a-[ 1'-( R)- hydroxyethyl ] 1- 7- oxo- l- azabicyclo[ 3. 2. 0] hept- 2- en- 2- carboxylate

A solution of para-nitrobenzyl-3-[2-(N-methyl-thiomorpholinium-diphenylphosphate)ethylthio]-6a-[1'-(R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept -2-ene-2-carboxylate (1.31 g, 1.76 mmol) in 0.1 M phosphate buffer, pH 7.4 (48.8 mL), tetrahydrofuran (20 mL) and diethyl ether (20 mL) was hydrogenated over 1.5 g of 10% palladium on charcoal in a Parr shaker for 1 hour at approx. 2.8 kp/cm2. The reaction mixture was diluted with 40 ml of diethyl ether and the phases separated. The organic phase was washed with 2 x 5 ml of water. The aqueous phases were combined, filtered through filter paper hardened 52 times, washed with 2 x 50 ml of diethyl ether and pumped under vacuum. The aqueous solution was poured onto a silica gel reverse-phase column and the desired kabapenem was diluted with 5% acetonitrile-water. The appropriate fractions were combined and lyophilized to give the title compound as an amorphous solid (205 mg, 31%);

IR (nujo<l>) <v>max: 1750 (s, e-lactam C=0) and 1590 cm"<1> (s, C=0) ;

"""HMR (D-O) 5: 4.25-3.95 (2H, m, H-l1 , H-5) , 3.70-3.40 (6H, m, CH2N ), 3.35 (1H , dd, J=6.1 Hz, J=2.6 Hz, H-6), 3.08 (3H, s, CH3N<+>), 3.25-2.75 (8H, CH2S, H- 4) and 1.24 ppm (3H, d, J=6.4 Hz, CH3);

UV (H20, c 0.062) Xmax<:><2>99 (el0962)

t, 17.7 h (0.1 M phosphate buffer, pH 7, 37°C).

Example 17

Preparation of (5R,6S)-3-[2-(1-methylmorpholino)ethylthio]-6-[(R)-1-hydroxyethyl]-7-oxol-azabicyclo[3.2.0]hept-2 - ene-2-carboxylate A. 1- methyl- 1-(2-mercaptoethyl) morpholinium- trifluoromethane-sulfonate

To N-methylmorpholine (3.29 mL, 0.030 mol) was added dropwise trifluoromethanesulfonic acid (1.327 mL, 0.015 mol) at 10°C, followed by ethylene sulfide (0.89 mL, 0.015 mol), The resulting yellow-brown solution was heated in an oil bath at 50-60°C under N-, for 18 hours. Volatiles were then removed in vacuo, and the remaining oil was collected in 10 mL of H 2 O. The aqueous solution was washed with 3 x 5 ml of diethyl ether

and then residual organic solvent was removed in vacuo. The resulting aqueous solution was added to a C₂g reverse-phase column, it was diluted with H₂O

and then with 5% acetonitrile-H 2 O and finally with 10% acetonitrile-H 2 O. Evaporation of the relevant fractions gave a white solid which was dried in vacuo over P2°5'

which gave the product (1.92 g, 41%).

IR (KBr) v_„,, : 2560 (-SH) cm"<1>;

<1>HNMR (dg-acetone) 6; 4.25-3.6 (m, 8H), 3.49 (s, 3H, N-Me), 3.35-2.7 (m, 5H).

B. p- nitrobenzyl-(5R, 6S)- 3-[ 2-( 1- methylmorpholino) ethylthio]-6-[( R)- 1- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0 ] hept-2-ene-2-carboxylate diphenyl phosphate

To a solution of p-nitrobenzyl-(5R,6S)-6-[(R)-1-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.348 g, 1 .0 mmol) in 25 ml of dry acetonitrile, diisopropylethylamine (0.191 ml, 1.1 mmol) and then diphenylchlorophosphate (0.228 ml, 1.1 mmol) were added dropwise at 0°C

under N2. After stirring at 0°C for 1 hour, diisopropylethylamine (0.226 mL, 1.3 mmol) was added to the resulting enol phosphate, followed by 1-methyl-1-(2-mercaptoethyl)morpholinium trifluoromethanesulfonate (0.373 g, 1 .2 mmol). The reaction mixture was stirred at room temperature for 1.5 hours and then concentrated under vacuum. The remaining material was collected in H 2 O and applied to a C 2 g reverse-phase column. Dilution with H.,0 and then with 20% acetonitrile-H^O and

finally with 30% acetonitrile-H 2 O, followed by lyophilization of the relevant fractions gave the product (0.360 g, 40%) as an amorphous solid.

IR (film) : 3300 (-0H), 1770 (6-lactam CO), 1700 (-CO-pNB)

-1

cm

<1>HNMR (dg-acetone) 6: 8.25, 7.80 (ABq, J=8.6 Hz, 4H, aromatic), 7.4-6.8 (m, 10H, diphenyl phosphate), 5, 56, 5.27 (ABq, J=14.2'Hz, 2H, benzylic), 4.42 (d of t, J=9.2Hz, J'=

2.7 Hz, 1H, H-5), 4.1-2.7 (m, 17H), 3.40 (s, 3H; N-Me),

1.22 (d, J=6.2 Hz, 3H, -CHMe).

C. ( 5R, 6S)- 3-[ 2-( 1- methylmorpholino) ethylthio]- 6-[( R)- 1- hydroxyethyl ]- 7- oxol- azabicyclo[ 3. 2. 0] hept- 2 - a- 2- carboxylate

To a solution of p-nitrobenzyl-(5R,6S)-3-[2-(1-methylmorpholino)ethylthio]-6-[(R)-1-hydroxyethyl]-7-oxo-1-azabicyclo [3.2.0 ]hept-2-ene-2-carboxylate diphenyl phosphate (0.360 g, 0.49 mmol) in 13 ml of phosphate buffer (0.05 M, pH 7.4) was placed 0.36 g of 10% palladium on charcoal, 20 ml of tetrahydrofuran and 20 ml of diethyl ether. This mixture was hydrogenated in a Parr apparatus at 2.24 kp/cm<2> for 1 hour. The mixture was filtered through Celite, and the filter pad washed with H 2 O and diethyl ether. The aqueous phase was separated and the pH adjusted to 7.0 with additional phosphate buffer, pH 7.4.

After removal of residual organic solvents under vacuum, the aqueous solution was added to a C-2g reverse-phase column. Elution with H 2 O and lyophilization of the relevant fractions gave 0.13 g of an amorphous solid. This was purified again by reverse-phase HPLC to give the pure product (0.058 g, 34%) as an amorphous solid.

IR (KBr) <v>max: 3420 (br, OH), 1750 (6-lactam CO), 1590 (-C02~) cm-<1>;

<1>HNMR (D20) S; 4.35-2.77 (m, 17H), 3.18 (s, 3H, N-Me),

1.23 (d, J=6.3 Hz, 3H, CHMe);

UV (<H>2<0>) <x>max<=><3>00 U6344) nm;

t, (pH 7.4, 36.8°C), 18.5 hours.

Example 18

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

A. 1-(2-acetylthioethyl)-1,4-dimethylpiperazinium bromide A solution of 2-bromomethylthioacetate *(2.20 g, 0.012 mol) and 1,4-dimethylpiperazine (1.95 mL, 0.014 mol) 4 mL acetone was stirred at 50°C for 65 hours. After cooling to 25°C, the liquid phase was decanted from the gum which had been triturated twice in diethyl ether to give a hygroscopic yellowish powder, 3.2 g (90%); IR (nuj<ol>) <V>max: 1685 (C=0 of thioester) cm 1; <1>HMR (D20) 6: 2.37, 2.39 (2s, 6H 3.18 (s, 3H,

<*>B. Hansen, "Acta Chem. Scand.", 11, 537-40 (1957)

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

hydrochloride

A solution of 1-(2-acetylthioethyl)-1,4-dimethyl-piperazinium bromide (1.1 g, 3.7 mmol) in 6N hydrochloric acid (4 mL) was heated to 80°C under nitrogen for 1 hour. The solution was concentrated under reduced pressure to give a white powder, 0.41 g (38%); <1>HMR (DMSO, d 6 ) 6 : 2.90

Analysis for C<gH>-<gN-S>BrCl.H_0:

C. (5R,6S)-paranitrobenzyl-3-[2-(1,4-dimethyl-1-piperazinium)-ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicycloJj3. 2. 0]-hept- 2- ene- 2- carboxylate- diphenyl phosphate To a cold (0°C) solution of (5R,6S)-paranitrobenzyl-6-[1-(R)-hydroxyethyl]-3,7- To dioxo-1-azabicyclo[3.2.0]-heptane-2-(R)-carboxylate (0.465 g, 1.33 mmol) in 2 mL of acetonitrile, kept under nitrogen, was added diisopropylethylamine (0.278 mL, 1.59 mmol ) and diphenylchlorophosphate (0.33 mL, 1.59 mmol). The reaction mixture was stirred for 30 min and treated with a suspension of 1,4-dimethyl-1-(2-mercaptoethyl)piperazinium bromide, hydrochloride (0.40 g, 1.37 mmol) in a mixture of 3 mL of acetonitrile and 1 mL of water and diisopropylethylamine (0.278 mL, 1.59 mmol). After stirring for 18 hours at 5°C, 15 ml of cold water was added to the mixture. The resulting solution was chromatographed over a PrepPak -500/C-^g column (Waters Associates) (2.5 x 7.5 cm) eluting with 25-35% acetonitrile in water to give a yellowish powder, 0 .50 g (50%) after lyophilization;

IR (KBr) v : 1765 (C=0 of B-lactam), 1690 (C=0 of pNB-niciK s -i

ester), 1585 (phenyl), 1512 (N02), 875 (N02) cm ;

^HMR (DMSO-dg) 6: 1.16, 1.18 (2d, J=6.1 Hz, 3H, CH3CHOH),

10H, phenyl of phosphate), 7.71 (d, J=8.8 Hz, 2H, Ho of pNB), 8.2 6 (d, J=8.8 Hz, Hm of pNB). D. ( 5R, 6S)- 3- j2-( 1, 4- dimethyl- l- piperazinium) ethylthio]- 6-[ 1-( R)- hydroxyethyl]- 7- oxo- l- azabicyclo[ 3. 2. 0] hept-2-ene-2-carboxylate

To a solution of (5R,6S)-paranitrobenzyl-3-[2-(1,4-dimethyl-1-piperazinium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylate-diphenyl phosphate (0.47 g, 0.623 mmol) in 25 ml of wet tetrahydrofuran was added 25 ml of diethyl ether, potassium phosphate monobasic sodium hydroxide buffer (13 ml, pH 7.22) and 0.47 g of 10% palladium on charcoal. The resulting mixture was hydrogenated at 23°C under approx. 2.8 kgp/cm<2> for 1 hour. The two layers were separated bg the organic layer was extracted with 2x 7 ml of water. The aqueous layers were combined, filtered through a Celite pad, washed with 2 x 15 mL of diethyl ether, and chromatographed on a PrepPak -500/C^g column (Waters Associates) (2.5 x 9.5 cm) with water as eluent, yielding 0.097 g (43%) after lyophilization;

IR (KBr) v , : 3000-3700 (OH), 1750 (C=0 of B-lactam),

ntci.K s ^

1585 (carboxylate) cm ;

<1>HMR (D2O) 6 : 1.24 (d, J=6.4 Hz, 3H, CH3CHOH), 2.33 (s, 3H,

), 3.15 (s,

), 4.0-4.5 (m, H-5, CH3CH0H);

UV (<H>2<0>) <*>max: 296 (e9476);

[α]<23> 61.1° (c 0.26, H 2 O);

t^ = 12.4 hours (measured at a concentration of 10 <4> M in phosphate buffer, ph 7.4, at 3 6.8°C).

Example 19

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

To a cold (-10°C) solution of (5R,6S)-3-[2-(N-methyl-thio-morpholinium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo -1-azabicyclo ( 3 . 2 . 0 ) hept-2-ene-2-carboxylate (6.08 mg, 1.65 mmol)

to 9 ml of a 1:1 mixture of acetonitrile and water was added m-chloroperbenzoic acid (334.8 mg, 1.65 mmol) in small amounts over the course of one hour. The mixture was then diluted with 15 ml of water and washed with 3 x 15 ml of diethyl ether. The aqueous phase was pumped under vacuum and passed through a reverse-phase silica gel (H 2 O) column, yielding a solid consisting of a mixture of compounds. This mixture was separated by reverse-phase HPLC to give fraction A, 52.4 mg (yield 12%) and fraction B, 23.6 mg (yield 6%) as diastereo-

isomers of the title compound;

faction A;

IR (nujol) v_ , : 1750 (s, B-lactam C=0) and 1580 cm<-1>

(s, C=0);

1HMR (D„0) 6 : 4.26-2.91 (20H, m, H-4, H-5, H-6, H-1', CH-S, CH2S-0, CH3-N and CH2 ~N ) and 1.24 ppm (3H, d, J=6.4 Hz, CH3);

UV (H2O, c 0.06) *max: 302 (el0425);

t^: 12 hours (0.065 M, pH 7.4, phosphate buffer, 37°C).

faction B:

IR (nujol) ^max: 1750 (s, B-lactam, C=0) and 1585 cm<-1>

(s, C=0).

"""HMR (D2O) 5: 3.86-2.90 (17H, m, H-4, H-5, H-6, H-1", CH2S, CH2S-0, CH2N<+>), 3, 25 (3H, s, CH3N<+>) and 1.24 ppm (3H, d, J=6.4 Hz, CH3);

UV (H20, c 0.05) *max: 2.99 U6517);

t'^: 10.75 hours (0.065 M, pH 7.4, buffer solution, 37°C).

Example 20

Preparation of (5R,6S)-3-[2-(1,4,4-trimethyl-1-piperazinium)-ethylthio]-6-[1R-hydroxyethyl]-7-oxo-l-azabicyclo( 3. 2. 0) hept-2- ene- 2- carboxylate chloride A. 1-( 2- acetylthioethyl)- 1, 4, 4- trimethylpiperazinium bromide iodide

A suspension of 1-(2-acetylthioethyl)-1,4-dimethyl-piperazinium bromide (1.48 g, 5.0 mmol) in 10 mL of isopropyl alcohol was treated with methyl iodide (0.373 mL, 6.0 mmol) and heated to 55-60°C for 30 hours. The solvents were evaporated under reduced pressure, the residue triturated in hexane and the solid filtered, yielding 1.85 g. The solid was dissolved in 8 ml of hot water and the solution diluted with acetone to cloudiness (70-80 ml). Two subsequent crystallizations gave 1.5 g m.p. 220-225°C with decomposition, 68% of the title compound;

IR (KBr) v : 1692 cm"<1> (C=0);

max

<1>HMR (D 2 O 6 : 2.40 (s, 3H, CH^COO) , 3.37 (s, N-CH" 3 ), 3.39 (s, N-CH 3 ), 3.99 (s );

UV (Ho0) X , : 226 (el3144).

2 max

Analysis for C,,H24N2OSBrI:

B. 1-(2-mercaptoethyl)-1,4,4-trimethylpiperazinium bichloride

A mixture of 1-(2-acetylthioethyl)-1,4,4-trimethyl-piperazinium bromide iodide (1.84 g, 4.19 mmol) and 15 mL of 6N hydrochloric acid was heated to 57°C under a nitrogen atmosphere in

2 hours. The solution was concentrated under reduced stirring to dryness. The solid was suspended in 10 ml of water and the well-stirred suspension was treated with permutite S-1 Cl until dissolution was obtained. The solution was poured onto a 1.2 x 60 cm column of permutite S-1Cl. The column was diluted with water (1.5 ml/min). The appropriate fractions were combined and lyophilized to give a white powder in the amount of 0.93 g and m.p. 190-191°C, 85%;

IR (nujol) umax: 2460 (SH);

"""HMR (D 2 O) 6 : 3.4 (s, N-CH 3 ), 3.45 (s, N-CH 3 ), 4.07 (s).

Analysis for C9H22N2SC12.0,75H-0:

C. ( 5R, 6S)- paranitrobenzyl- 3-[ 2-( 1, 4, 4- trimethyl- l- piperazinium) ethylthio ]- 6-[ 1R- hydroxyethyl]- 7- oxo- l- azabicyclo-( 3. 2. 0) hept-2-ene-2-carboxylate bichloride

To a cold (5°C) solution of (5R,6S)-paranitrobenzyl-6-[1R-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2R-carboxylate (0.94 g , 2.7 mmol) in 3 mL of acetonitrile, kept under nitrogen, were added diisopropylethylamine (0.557 mL, 3.2 mmol) and diphenylchlorophosphate (0.663 mL, 3.2 mmol). The reaction mixture was stirred for 30 minutes at 5°C and treated with diisopropylethylamine (0.599 mL, 3.44 mmol) and 4 mL of an aqueous solution of 1-(2-mercaptoethyl)-1,4,4-trimethylpiperazinium bichloride (0.90 g, 3.44 mmol). After 1.25 h, diisopropylethylamine (0.1 mL, 0.57 mmol) was added and stirring continued for 2 h. A portion of the acetonitrile was removed under reduced pressure and the resulting red mixture was chromatographed on a PrepPak -500/C^g column (Waters Associates) eluting with 25-75% acetonitrile in water to give 1.4 g of a yellowish powder after lyophilization. This was dissolved in water and the solution poured onto a 1.2 x 58 cm column of permutite S-1 Cl using water as eluent. Lyophilization of the appropriate fractions gave 1.17 g of a powder which was purified again on a PrepPak -500/C 2 g column. Lyophilization of the appropriate fractions gave a yellowish powder, 0.80 g (53%);

IR (KBr) v . : 3400 (br, OH), 1770 (C=0 of B-lactam),

max

1690 (C=0 of pNB ester), 1605 (aromatic), 1515 (NO2),

1345 (N02) cm"<1>;

<1>HMR (D20) 6: 1.26 (d, J=6.3 Hz, 3H, CH3CHOH), 3.39 (s, NCH3), 4.00 (s), 5.37 (Br, s , CH2 of pNB), 7.60 (d,

J=8.6 Hz, 2H, Ho of pNB), 8.20 (d, J=8.7 Hz, 2H, HM of pNB);

UV (H-<O>) X , : 276 (el2094), 306 (el0752).

2 max

Analysis for C25<H3>6N4<OgS>Cl2.3H20

D. ( 5R, 6S)- 3-[ 2-( 1, 4, 4-trimethyl- 1- piperazinium) ethylthio]-6-[ 1R- hydroxyethyl]- 7- oxo- l- azabicyclo( 3. 2. 0 )- hept- 2r en- 2- carboxylate chloride

A mixture of (5R,6S)-paranitrobenzyl-3-[2-(1,4,4-trimethyl-1-piperazinium)ethylthio]-6-[1R, hydroxyethyl]-7-oxo-1-azabicyclo ( 3 . 2 . 0 ) hept-2-ene-2-carboxylate bichloride (0.40 g,

0.68 mmol), phosphate buffer (30 mL, 0.05 M, pH 7.0), tetrahydrofuran (10 mL), ether (30 mL) and 0.40 g of 10% palladium on charcoal were hydrogenated at 23°C under a press on

2.45 kp/cm<2> for 1 hour. The two phases were separated. The organic phase was extracted with 10 ml of water. The aqueous phases were filtered on a Celite pad, washed with 10 mL of ether, concentrated under vacuum to 10 mL, and chromatographed on a PrepPak -500/C^g column (2.2 x 11 cm) with water as eluent, some which gave 70 mg or 25% after lyophilization;

IR (KBr) vmakg: 3400 (br, OH), 1755 (C=0 of B-lactam),

1585 (carboxylate) cm <1>;

HMR (D 2 O) 6 : 1.24 (3H, d, J=6.3 Hz, CH 3 CHOH), 3.36 (s, NCH 3 ), 3.98 (s);

uv <H2<0>) <X>max<:><2>96 (e7987);

[a]<23> 35.9° (c, 0.30, H 2 O) ,

-4

t^ = 9.8 hours (measured at a concentration of 10 ml of phosphate buffer, pH 7.4, at 36.8°C.

Claims (1)

1. Process for the preparation of a carbapenem derivative of the formula there R<8> is hydrogen, R<1> is 1-(R)-hydroxyethyl, A is ethylene which is optionally substituted with C1_4-alkyl, or cyclohex-1,2-ylene, R<14> is a quaternized pyridine, imidazoline, N-methylmorpholine, N-methylthiomorpholine, N-methylthiomorpholine s-oxide or 1,4-dimethylpiperazine group which is optionally substituted with one or more groups selected from C1_3-alkyl , hydroxy- C1_3-alkylthio, -alkyloxy and amino and is bound to A via the quaternary nitrogen atom, and R<2> is hydrogen, an anionic mixture or a conventional easily cleavable carboxyl protecting group provided that, when R<2> is hydrogen or a protecting group, a counter anion is also present, or a pharmaceutically acceptable salt thereof, characterized in that an intermediate product with the formula is reacted where R<1> and R<8> have the above meaning and R<2>' is a p-nitrobenzyl group, with diphenylchlorophosphate in the presence of diisopropylethylamine, and treating the resulting mixture with a thio compound of the formula where A and R<14> have the meaning given above and X" is a counter anion, in an inert solvent with a temperature from -15"C to room temperature and in the presence of diisopropylethylamine for the preparation of a carbapenem product of the formula where R<1>, R<8>, R<2>', A, R<14> and X" have the meaning given above, and, if desired, removes the carboxyl protecting group R<2>' on i and for known way for the formation of the corresponding unprotected group of formula (I), or a pharmaceutically acceptable salt thereof.