NO171271B - QUATERNARY AMINTIOL COMPOUNDS - Google Patents

Description

The present invention relates to quaternary amine thiol compounds.

More specifically, the invention relates to quaternary amine thiol compounds which are characterized by having the formula

there

A is ethylene which is optionally substituted with C1_4~alkyl,

or cyclohex-1,3-ylene and

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 C^g- alkyl, hydroxy-C1-6-alkylthio, C1-6-alkyloxy and amino and is attached to A via the quaternary nitrogen atom.

The present invention is separated from NO-PS 166038 which describes a method for the production of carbapenem derivatives with the formula

there

R 8 is hydrogen,

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

A and R<*4> have the meaning indicated above, 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.

Carbapenem derivatives 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<**> means hydrogen and R<*> 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, wherein the aryl moiety is phenyl; aralkyl, aralkenyl and aralkynyl, wherein the aryl moiety is phenyl and the aliphatic moiety has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, in which 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^_^-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> independently is 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, wherein the aryl moiety is phenyl and the aliphatic moiety has 1-6 carbon atoms; and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, in which 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 bound, form a 5- or 6-membered nitrogen-containing heterocyclic ring, R** is as defined for R<3> except that it cannot be hydrogen; or where R<1> and R^ together form the Cg_^Q<-a>alkylidene or C210-alkylidene substituted with hydroxy, A is cyclopentylene, cyclohexylene or C2_^-alkylene, optionally substituted with one or more C^^-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, whereby a quaternary ammonium group is formed; 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<*> 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, in which the aryl part is phenyl and the aliphatic part has 1-6 carbon

atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl, in which 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 the 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 respect 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 have 1-6 carbon atoms or R<3> and R<4> together with the nitrogen atom to which at least one is bound, form a 5- or 6-membered, nitrogen-containing, heterocyclic ring, R 9 is as defined for Rd except that it cannot be hydrogen; or where R<*> and R<8> together form the C 8-Q alkylidene or hydroxy-substituted C 210 alkylidene, R<5> is selected from 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 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 chosen 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, C₁-alkyl, -OR3, -NR3R4, -SO3R<3>, -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 C1-4 alkylene group which is attached to the ring forming a bridged polycyclic group; A is cyclopentylene, cyclohexylene or C 3 -alkylene, which is optionally substituted with one or more C 3 -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 core:

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 product II. This is then reacted with the desired amine 1 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, for example, 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 nucleus on a later stage of the synthesis and (4) can be used for a simpler formation of quaternary amine products with a large number of amines, i.e. amines with steric hindrance and with lower pK^ values.

NO-PS 166038 describes a new and improved method for the production of such carbapenem derivatives.

The present invention provides, as mentioned above, intermediate products of formula (VII) which find use for the production of the carbapenem compounds of formula (I) according to NO-PS 166038, compounds which are powerful antibacterial agents.

The quaternary amine thiol intermediate (VII) according to the invention can be obtained by working at a temperature in the range

-20'C to +100°C, preferably in the range 50 to 70°C.

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 following examples shall illustrate the invention without limiting it.

Example 1

Preparation of 3-f2-(l-pvridinlum)etvltlo1-6ot--81-(R)-hydroxyvetvll-7-oxo-1-azabicyclo(3.2.0)-hept-2-ene-2-carboxvlate

A. 1-(2-Mercaptoethyl)pyridinelumethanesulfonate

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 p-Bondapak C^g eluted with water. Lyophilization of the appropriate fractions gave a colorless syrup, 6.5 g (9156).

IR (film) vmax: 2300-2600 (br. SH), 1635 (pyridinium), 1490,

1200 (sulfonate), 1068, 1060, 1045, 791, 780 cm-<1>;

<1->H-NMR (DMSO-d6) S: 2.32 (3H, s, CH3SO3<e>), 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 pyrldinium), 8.6 (1H, m, HO of pyrldinium), 9.08 (2H, dd , J-6.8 Hz, J-1.4 Hz, HO of pyrldinium);

UV (H20) Xmax: 206 (c5230), 258 (c3760) nm.

Method A

B. 1-(2-mercaptoethyl) pyridine chloride

An aqueous solution of crude 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 Permutitt S-1 C1<G>. The column was eluted with water in an amount 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 for the next step.

1-H-NMK (D2O) S: 3.22 (2H, m, CH2S), 4.88 (m, CH2N<®>), 8.18

(2H, m, Hm of pyrldinium), 8.7 (1H, m, Hp of pyrldinium), 9.0 (2H, m, HO of pyrldinium) ppm.

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 residual 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 for the next step.

C. p-nitrobenzvl-3-r2-(1-pyridinelum)ethvlthio1-6a-ri-(R)-hydroxyethyl"!-7-oxo-1-azabicyclo(3.2.0)-hept-2-en- 2-carboxylate chloride

A solution of p-nitrobenzyl-6oc-[l-(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, cooled to -5°C and treated successively with a solution of crude 1-(2-mercaptoethyl)pyridinelum chloride (4.3 g, 24 mmol) in 1.0 mL of N ,N-dimethylformamide and dropwise diisopropylethylamine (3.65 mL, 21.0 mmol). The reaction mixture was stirred at 0<*>C for 1 hr, cooled to -30°C and stirred for an additional 15 min. The solid was filtered off and washed with cold (-30°C) acetonitrile, 5.77 g (6556).

IR (nujol) vmax: 3300 (OH), 1775 (C-0 of ε-lactam), 1690

(C-0 of pNB ester), 1630 (pyrldinium), 1605 (phenyl of pNB ester), 1515 (NO 2 ), 1335 (NO 2 ) cm-<1>;

^H-NMR (DMS0-d6) S: 1.17 (3H, d, J-6.1 Hz, CH3CH0H), 3.2-3.75 (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, Ja,b"14»2 Hz» CH2 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 pyrldinium), 8.66 (1H, m, Hp of pyrldinium), 9.17 ( 2H, wide, J-5.5 Hz, Ho of pyrldinium).

The filtrate and washings were combined and diluted with 150 ml of ether. The supernatant liquid 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^g. The column was eluted with 150 ml of 1056 acetonitrile - 9056 water and 100 ml of 5056 acetonitrile - 5056 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]-6a-[1-(R)-hydroxyethyl]-7-pxo-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 Permutitt S-1 Cl<e> with water. Lyophilization of the appropriate fractions gave 1.8 g or 2056 of the title compound. D. p- nltrobenzvl- 3- r2-( l- Pvrldlnl\ im) etvltlo1- 6a- ri-( Rl-hvdroksvetvll- 7- oxo- l- azablcvklo( 3. 2. 0) hept- 2- en- 2- carboxyvlate diphenyl phosphate

A solution of p-nitrobenzyl-6oc-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azablcyclo(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 min 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 0<*>C water and poured onto a 1.5 x 6.4 cm column of ji-Bondapak C^g. The column was eluted with a mixture of 25-5096 acetonitrile in 75-5056 water. The appropriate fractions were combined and lyophilized after the acetonitrile was removed under vacuum to give a yellowish powder, 0.33 g (9256).

IR (KBr) vmax: 3600-300 (OH), 1765 (C-0 of 3-lactam), 1690

(C-0 of pNB ester), 1625 (pyrldinium), 1585 (phenyl), 1510 (NO 2 ), 1330 (NO 2 ), 885 (NO 2 ) cm-<1>;

%-NMR (DMS0-d6) S: 1.16 (3H, d, J-6.2 Hz, CH3CH0H), 4.87

(2H, brt, J-6.6 Hz, CH2S), 5.37 (center of ABq, Ja>D-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 pyrldinium), 8 .4-8.8 (1H, Hp of pyrldinium), 9.09 (2H, dd, J-6.7 Hz, J-1.3 Hz, Ho of pyrldinium).

E. 3-f2-(1- pvridlnium)ethvlthio1-6a-ri-(R)-hydroxymethyl-7-oxo-1-azabiclo(3.2.0)hept-2-ene-2-carboxylate

Method A

To a solution of p-nitrobenzyl-3-[2-(l-pyridinium)ethylthio]-6a-[l-(R)-hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0 )hept-2 -ene-2-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 of 105é palladium-on-charcoal. The resulting mixture was hydrogenated under approx. 3.2 kp/cm<2> 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 readings were combined, washed with 2 x 10 ml of ether and poured onto the top of a 1.5 x 6.2 cm column of ji-Bondapak C^g 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 (8456).

IR (KBr) vmax: 3700-3000 (OH), 1755 (C-0 of p<->lactam), 1630

(pyrldinium), 1590 (carboxylate) cm"<1>;

<1->H-NMR (D2O) S: 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 pyrldinium), 8.5 (m, Hp of pyrldinium), 8.82 (dd, J-3.2 Hz, J-1.1 Hz, Ho of pyrldinium);

UV (H 2 O) <X>max: 259 (c5800), 296 (c7030) nm;

ttø= 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)ethyl-thio]-6oc-[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 1096 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 >i-Bondapak C^g, after the organic solvents had been removed under vacuum. Elution of the column with water, followed by a mixture of 10% acetonitrile in water after lyophilization of the appropriate fractions gave 2.48 g or 6596 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-r2-(2-(3.5-dimethylPvrldinlum)ethvlthio-6a-ri-(R)-hydroxyethyl1-7-oxo-l-azablcyclo(3.2.0)hept-2-ene-2- carboxylate

A. 1-(2- mercaptoethyl)-3.5- dimethylpyridlinium methanesulfonate

Ethylene sulfide ( 0.655 mL, 0.011 mol). The resulting mixture is stirred under a nitrogen atmosphere at 55°C for 24 hours, cooled to 23<*>C and diluted 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 added to the top of a 2.5 x 6.0 cm column of jj-Bondapak C^g. The column is eluted with water and lyophilization of the appropriate fractions gave a colorless syrup, 2.4 g (9196).

IR (film) vmax: 2520 (SH), 1628 (pyrldinium), 1600, 1495,

1325, 1305, 1283, 1200 (sulfonate), 1040, 938, 765,, 680 cm -1 ;

^H-NMR (DMS0-d6) S: 2.31 (3H, s, CH3SO3<e>), 2.47 (6H, s, CH3

on pyrldinium), 2.57, 2.66, 2.69, 2.78 (1H, B part of A2B system, SE), 3.06 (2H, m [with D20 added (2H, t, J= 6.5 Hz)], CH2S), 4.65 (2H, t, J-6.5 Hz, CH2N<®>), 8.34 (1H, s, Hp of pyrldinium), 8.79 (2H, s, Ho of pyrldinium);

UV (H 2 O) <X>max: 271 (c4860) nm;

Analysis for Ciq<H>i7N03S2<*>0.5H20:

Calculated: C 44.09 H 6.66 N 5.14 S 23.54

Found: C 44.26 H 6.49 N 5.17 S 24.18

B. p- nltrobenzyl- 3- r2-( l-( 3. 5- dlmethylpyridinlum) letvlthiol- 6a-1" 1-( R)- hydroxyethyl1- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxylate dlphenyl phosphate

To a cold (0°C) solution of p-nitrobenzyl-6α-[l-(R)-hydroxyethyl]-3,7-dioxo-1-azablcyclo(3.2.0 )hept-2-ene-2-carboxylate ( 0.523 g, 1.50 mmol) in 6.0 mL of acetonitrile, maintained 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 jj-Bondapak C^g column. Elution of the column with 25-5056 acetonitrile - 75-5056 water mixture after lyophilization of the appropriate fractions gave 1.01 g or 9056 of the title compound as a yellowish powder.

IR (KBr) vmax: 3700-3100 (OH), 1778 (C-0 of ε-lactam), 1700

(C-0 of pNB ester), 1635 (pyrldinium), 1595 (phenyl), 1521 (NO 2 ), 1335 (NO 2 ), 895 (NO 2 ) cm-<1>;

3H-NMR (DMS0-d6) δ: 1.16 (3H, dm, J-6.1 Hz, CH3CH0H), 2.43

(s, CH3 on pyrldinium), 4.75 (2H, m, CH2N<®>), 5.38 (center of ÅBq, Ja>|j-14.3 Hz, CH2 of pNB), 6.6-7 .5 (10H, m, phenyl), 7.70 (2H, d, J-8.7 Hz, Ho of pNB), 8.0-8.5 (3H, m, Hp of pyrldinium, Hm of pNB) , 8.82 (2H, s, Ho of pyrldinium);

TJV (H 2 O) Xmax: 270 (cll570), 306 (c7343) nm;

Analysis for C37<H>3g<N>30ios<p*>H20:

Calculated: C 58.03 H 5.26 N 5.48 S 4.18 Found: C 57.98 H 5.05 N 5.22 S 4.34 C. 3- r2-( l-( 3. 5- dlmetvlPvridinlum) letvlthiol- 6a- fl-( R)-hydroxylvvll- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2-carboxvlate

To a solution of p-nitrobenzyl-3-[2-(1-(3,5-dimethyl-pyridinium))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) 1 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 10SÉ palladium-on-charcoal. The resulting mixture is hydrogenated under approx. 3.2 kp/cm<2> at 23°C 1 1.25 hours. The organic layer was separated from and extracted with 2 x 5 ml of buffer. The aqueous layers were combined, filtered through a pad of Celite, washed with 40 mL of ether, pumped to remove traces of organic solvents, and poured onto a 2.5 x 10.0 cm p-Bondapak C 2 g column. Elution of the column with water and lyophilization of the appropriate fractions gave 0.186 g corresponding to 6456 as a yellowish powder.

IR (KBr) vmax: 3700-3100 (OH), 1760 (C-0 of ε-lactam), 1595

(carboxylate) cm"<1>;

<i>H-NMR (D 2 O) S; 1.21 (3H, d, J-6.3 Hz, CH3CHOH), 2.45 (6H,

s, CH3 on pyrldinium), 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, CH3CHOH), 4.71 (t, J-6.2 Hz, CH2N<®>), 8.21 (1H, s, Hp of pyrldinium), 8.46 (2H, s, Ho of pyrldinium);

UV (H 2 O) <X>max: 2.79 (c8345), 296 (c7714) nm;

[a]<g3> +40.7- (c 0.53, H2O);

ten£ - 16.9 hours (measured at a concentration of 10~<4> M in phosphate buffer, pH 7.4, at 36.8°C).

Example 3

Preparation of (5R. 6S)-3- ff2-(3-hydroxymethylpyrldinio)-ethvll tlol - 6- 1" ( R )- l- hydroxyethl" l - 7- oxo- l- azablcvclo( 3. 2. 0)- hept-2-ene-2-carboxylate

A. 3-Hydroxymethyl-1-(2-mercaptoethyl)pyridyl-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 collected in 15 mL H 2 O and extracted with 5 x 5 mL CE 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 vacuo over P20s, this product yielded in an amount of 4.50 g equivalent to 9496 as a viscous oil.

IR (film) vmax: 3450 (s, OH), 2560 (w, SH) cm-<1>;

^H-NMR (d6-acetone) S: 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-CH 2 ), 2.34-2.10 (m, 1H, SH).

B. p- nltrobenzvl- f5R. 6S)- 3- r2-( 3- hydroxymethylDvrldinio) ethvlthiol- 6- ri-( R)- hydroxymethyl- 7- oxo- l- azabicclo( 3. 2. 0 ^-hept- 2- ene- 2- carboxylate- diphenyl phosphate

To a solution of p-nitrobenzyl-(5R,6S)-6-[l-(R)-hydroxy-ethyl]-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 N2. 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 C₂g reverse phase column eluted with 15% acetonitrile-B₂O. Lyophilization of the relevant fractions gave 0.305 g corresponding to 81% of the product as a pod-coloured solid.

IR (KBr) vmax: 3420 (br, OH), 1775 (p-lactam CO), 1695

(-CO2PNB) cm-1;

^H-NMR (dfc-acetone) S: 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- r2-( 3- hydroxymetvlIpvrldinlo) etvlthio1- 6- f1-( R)-hydroxyethyl- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2 - carbox vat

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 2 O was added 6.0 ml of phosphate buffer (0.05 M, pH 7.4), 0.145 g 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 H2O 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 C 2 g reverse phase column which was eluted with H 2 O. 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* of pure product as a solid.

IR (KBr) vmax: 3300 (br, OH), 1755 (e-lactam CO), 1590

(-C02<G>) cm-<1>;

^H-NMR (D 2 O) S: 8.78-7.94 (m, 4H, aromatic), 4.83 (t, J-6.0

Hz, 2H, N-CH2), 4.83 (s, 2H, CH2OH), 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 (H 2 O) Xmax: 294 (c7614), 266 (c6936) nm;

t% (pH 7.4, 36.8°C); 14.0 h.

Example 4

Preparation of (5R, 6S)-3-r2-(4-hydroxymethyl, vridinio)ethyl-thiol-6- fl-(R)-hydroxymethyl-7-oxo-1-azabicyclo(3.2.0)hept-2 -en- 2- carboxylate A. 4- hydroxymethyl- 1-( 2- mercaptoethyl) pyridinium- trifluoro-mphyt. anaiil f nn that.

To a solution of 4-pyridinemethanol (1.635 g, 0.015 mol) in 10 ml of CH 2 Cl 2 at 0°C under N 2 was added dropwise trifluoromethanesulfonic acid (1.327 ml, 0.015 mol). 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 H2O, and the aqueous solution was washed with 5 x 5 mL of CH2Cl2. After removing residual organic solvent under vacuum, the aqueous solution was applied to a Cig 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 P 2 O 5 to give 4.64 g or 97% of the product as a colorless oil.

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

1-H-NMR (d6-acetone) S: 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-nitrobenzvl-(5R.6S)-3-r2-(4-hydroxymethylpvrldinlo)-ethyltlo~l-6-["l-(R)-l-hydroxyethyl-7-oxo-l-azabicyclo-(3 . 2. 0) hept- 2- en- 2- carboxyvlate- diphenyl phosphate

To a solution of p-nitrobenzyl-(5R,6S)-6-[l-(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 10 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) vmakg: 3240 (br, OH), 1775 (e-lactam CO), 1695

(-CO2PNB) cm"1;

^■H-NMR (d^-acetone) S: 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- r2-( 4- hydroxymethylpvrldinlo) etvltlo1- b- tl-( R)-l- hydroxysvetlll- 7- oxo- 1- azabicclo( 3. 2. 0) hept- 2- one - 2-carboxylate

A mixture of p-nitrobenzyl-(5R,6S)-3-[2-(4-hydroxymethyl-pyridinio)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 of 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 with ether three times. 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 Cig reverse phase column. Elution with 2* acetonitrile-H2O and subsequent lyophilization gave a yellow-brown solid. This material was rechromatographed (Cig reverse phase/g2°) whereby 0.060 g corresponding to 36* of the desired product was obtained as a pale yellow solid.

IR (KBr) vmax: 3400 (br, 0H), 1755 (e-lactam CO), 1590

(-C02<9>) cm"<1>;

^H-NMR (D 2 O) S: 8.73, 7.96 (ABq, J-6.8 Hz, 4H, aromatic),

4.93 (s, 2H, CH2OH), 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 (H20) <X>max: 295 (c6880), 256 (c5595), 224 (c8111) nm; ,

t% (pH 7.4, 36.8°C): 14.5 h.

Example 5

Preparation of 3- T2-( l-( 2- methylpvridinium)) etvltlo1- 6a- Q-( R)- hvdroxvetl1- 7- oxo- l- azablcvclo( 3. 2. 0) hent- 2- en- 2-carbox <y>lazy

A. 1-(2- mercaptoethyl )- 2- methylpyridnl^ mmift. nnsulfonate

For a suspension of 2-methylpyridinium methanesulfonate in 2-methylpyridine, prepared by the addition of 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 for 21 hours, cooled to 23<*>C and diluted with 5 mL of water. The aqueous solution was washed with 6 x 4 mL of ether, pumped to remove traces of organic solvents and completely on top of a 2.5 x 10.0 cm p-Bondapak C^g column The column was eluted with water and lyophilization of the appropriate fractions yielded 2.13 g corresponding to 85% of the title compound.

IR (film) vmax: 2520 (SH), 1623 (pyrldinium), 1574, 1512,

1485, 1412, 1195 (sulfonate), 1038 cm-<1>;

1-H-NMR (DMS0-d6 + D2O) S: 2.37 (3H, s, CH3SO3<G>), 2.83 (3H,

s, CH3 on pyrldinium), 3.09 (2H, J-6.9 Hz, CH2S), . 4.71 (2H, t, J=6.9 Hz, CH2N<®>), 7.93 (2H, m, Hm of pyrldinium), 8.44 (1H, m, Hp of pyrldinium), 8.89 (1H, m, Ho of pyrldinium);

UV (H20) Xmax: 266 (c3550) nm.

B. p-nitrobenzyl-3- r2-(l-(2-methylPvridlnlum))ethvlthio1-6a-f1-(R)- hydroxyethyl1- 7-oxo-l- azabicyclo(3.2.0)hept-2-ene - 2-carboxylate-diphenylphosphate

To a cold (0°C) solution of p-nitrobenzyl-6α-[l-(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 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 p-Bondapak C2g column. Elution of the column with 25% water gave, after lyophilization of the appropriate fractions, 1.06 g corresponding to 96% of the title compound as a yellowish powder.

IR (KBr) vmax: 3650-3100 (OH), 1700 (C-0 of ε-lactam), 1695

and 1690 (C-0 of pNB ester), 1630 (pyrlidinium), 1595 (phenyl), 1518 (NO 2 ), 1335 (NO 2 ), 890 (NO 2 ) cm-<1>;

3H-NMR (DMS0-d6) δ: 1.15 (3H, d, J-6.1 Hz, CH3CH0H), 2.87

(s, CH3 on pyrldinium), 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 pyrldinium), 8.24 (2H, d, J-8.8 Hz, Hm of pNB), 8.50 (1H, m, Hp of pyrldinium), 8.95 (1H, brd, J- 6.1 Hz, Ho of pyrldinium);

UV (H20) Xmax: 265 (cll990), 314 (c8020) nm.

C. 3- r2-( 1-( 2- metvlpvrldlnlumnetvltio1- 6a- ri-( R1- hvdroxvetl"! - 7- oxo-1- azabicyclo( 3. 2. 0 ) hept- 2-ene- 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 10* palladium-on-charcoal. The resulting 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 pad of Celite, washed with 40 mL of ether, pumped to remove traces of organic solvents, and poured onto a 2.5 x 10 cm ji-Bondapak C^g 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) vmax: 3650-3100 (0H), 1755 (C-0 of ε-lactam), 1630

(pyrrlidinium) 1595 (carboxylate) cm-<1>;

^H-NMR (D 2 O) S: 1.20 (3H, d, J-6.3 Hz, CH 3 CHOH), 2.83 (s,

CH3 on pyrldinium), 2.7-3.1 (5H, H-4, CH3 on pyrldinium), 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 pyrldinium), 8.3 (1H, m, Hp of pyrldinium), 8.65 (1H, m, Ho of pyrldinium);

UV (H 2 O) <X>max: 268 (c9350), 296 (c8840) nm;

[cOg<3> +41- (c 0.5, H 2 O);

ti^ - 15.0 hours (measured at a concentration of 10-<4> M in phosphate buffer, pH 7.4 at 36.8°C).

1 Example 6

Preparation of 3-f2-(1-(4-methylPvrldinium))ethylthio"1-6a-ri-(R)-hydroxyethyl1-7-oxo-1-azablcvclo(3.2.0)hept-2-ene-2 -karboks<y>lat

A. 1-(2-Mercaptoethyl)-4-methylpyrridlinium 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 ether and added to the top of a 2.5 x 10 cm jj—Bondapak C^g column after traces of ether had 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 colorless syrup.

IR (film) vmax: 2500 (SH), 1640 (pyrldinium), 1572, 1520,

1478, 1200 (sulfonate), 1040, 833 and 768 cm-<1>;

1-H-NMR (DMS0-d6) S: 2.31 (3H, s, CH3SO3<e>), 2.62 (s, CH3 on

pyrldinium), 2.2-2.9 (4H, SH, CH3 on pyrldinium),, 3.04 (2H, m, CH2S), 4.68 (2H, t, J-6.4 Hz, CH2N<® >), 8.01 (2H, d, J-6.6 Hz, Hm of pyrldinium), 8.89 (2H, d, J-6.6 Hz, Ho of pyrldinium);

UV (H20) <X>max: 256 (c4100), 221 (c7544) nm.

B. 1-(2-mercaptoethyl)-4-methylpropyldinium-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°C for 24 h. More ethylene sulfide (0.65 mL, 0.011 mol) was added and stirring was 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 and washed with 3 x 8 ml of ether. The traces of organic solvents were removed under vacuum and the compound chromatographed on γ-Bondapak C^g with water as eluent to give 2.94 g or 90% of the title compound as a colored syrup.

IR (film) vmax: 2510 (SH), 1640 (pyrldinium), 1595, 1582,

1475, 1200 (sulfonate), 1031, 1010, 818 cm-<1>.

^H-NMR (DMS0-d6) S: 2.29 (3H, s, CH3 on pyrldinium), 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 pyrldinium), 8.89 (2H, d, J-6.5 Hz, Ho of pyrldinium);

UV (H20) <X>max: 256 (c4315), 222 (cl7045) nm.

C. p- nitrobenzvl- 3- r2-( l-( 4- methylpyridinlum)) ethyltlo1- 6a- f1-( R)- hydroxyvetl1- 7- oxo- l- azablcyclo( 3. 2. 0) hept- 2- one - 2-carboxyl-diphenyl phosphate

To a cold (0<*>C) solution of p-nitrobenzyl-6a-[l-(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 min 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 jj-Bondapak C^g 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, gave, after lyophilization of the appropriate fractions, 0.91 g or 83* of the title compound as a yellowish powder.

IR (KBr) vmax: 3700-2800 (OH), 1770 (C-0 of ε-lactam),

1700 (C-0 of pNB ester), 1640 (pyrlidinium), 1595 (phenyl), 1520 (NO2), 1340 (NO2), 890 (NO2) cm-<1>;

<i>H-NMR (DMS0-d6) S: 1.16 (3H, d, J-6.2 Hz, CH3CH0H), 2.61

(s, CH3 on pyrldinium), 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 pyrldinium), 8.23 (2H, d, J-8.8 Hz, Hm of pNB), 8.92 (2H, d, J-6.5 Hz, Ho of pyrldinium);

UV (H 2 O) Xmax: 262 (cl0835), 311 (c9670) nm;

Analysis for C36H36N3010SP'1.5H20:

Calculated: C 56.84 H 5.17 N 5.52 S 4.21

Found: C 56.89 H 5.13 N 5.19 S 4.41

D. 3- r2-( l-( 4- metvlpvrldlniumnetvltlo1- 6a- ri-( R)- hydroxy-ethyl- 7- oxo- l- azablcyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

To a solution of p-nitrobenzyl-3-[2-(1-(4-methylpyridinium))ethyltlo]-6oc-[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 and extracted with 2 x 6 ml of buffer. The aqueous extracts were combined, filtered through a pad of Celite, washed with 3 x 20 ml of ether, pumped to remove traces of organic solvents and poured onto a 2.5 x 10 cm column of jj-Bondapak C^ g. 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) vmax: 3700-3000 (0H), 1770 (C=0 of ε-lactam), 1642

(pyrrlidinium), 1592 (carboxylate) cm-<1>;

3-NMR (D 2 O) S: 1.19 (3H, s, J-6.3 Hz, CH 3 CHOH), 2.59 (3H,

s, CH3 on pyrldinium), 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-5.6 Hz, Hm of pyrldinium), 8.58 (2H, d, J=6.6 Hz, Ho of pyrldinium);

UV (H2O) Xmax: 256 (c5510), 262 (c5360), 296 (c7050) nm;

[oc]g 3 -20.8' (c 0.48, H 2 O);

ty = 12.8 hours (measured at a concentration of 10~<4> M in a

phosphate buffer, pH 7.4 at 36.8°C).

Example 7

Preparation of (5R)-3-f2-(4-methylthiopvridinioletvlthio-(6S)-fl-(R)-hydroxyethyl1-7-oxo-1-azablcvclo(3.2.0)hept-2-ene-2-carbox <y>lazy

A. 4-Methylthiopyridine-^<1> The preparation of this compound was reported in King & Ware, "J. Chem. Soc", 873 (1939). The procedure described here was followed.

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

^H-NMR (D 2 O) S: 2.70 (3H, s, -SCH 3 ) and 7.65-7.77-8.35-8.48

(4H, A2B2 type, aromatic-Hs) ppm;

IR (nujol) vmax: 1615, 1585 (aromatic) and 780 cm"<1>;

UV (H20) Xmax: 227 (c2.20 x IO<4>) and 298 (cl.64 x 10<4>) nm.1

The hydroiodide (6.33 g, 25.0 mmol) was dissolved in 40 mL H 2 O and the insoluble material removed and washed with 10 mL 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 MgSO4 and evaporated to yield 2.92 g (23.4 mmol, total yield 50*) of the title compound as an oil;

<i>H-NMR (CDCl 3 ) S: 2.48 (3H, s, -SCH 3 ) and 7.03-7.13-8.38-8.48

(4H, A2B2 type, aromatic-Hs) ppm;

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

B. 4-Methylthio-N-(2-mercaptoethyl)pyridinelumethanesulfonate

4-Methylthiopyridine (2.75 g, 22.0 mmol) was added slowly to methanesulfonic acid<2> (0.65 mL, 10.5 mmol) while cooling in an ice bath. To this solid was added ethylene sulfide 2 (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 H2O and washed with 5 x 4 ml Et2O. The cloudy aqueous layer was filtered over Celite and the filtrate purified by reverse phase silica gel column chromatography (Cig-y-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.

<1->H-NMR (DMS0-d6, CFT-20) S: 2.32 (3H, s, MeSO3<e>), 2.72 (3H,

s, -SMe), 2.68 (1H, m, SH), 2.9-3.2 (3H, m, -H2S-), 4.59 (2H, t, J=6.4 Hz, - CH2N<®>), 7.97 (2H, "d", J-7.2 Hz, aromatic-Hs) and 8.72 (2H, "d", J-7.2 Hz, aromatic-Hz) ppm ;

IR (pure) vmax: 1630, 1200 (br, -S03<G>), <7>85 and 770 cm-<1>.

C. (5R)- p- niltrobenzvl- 3- r2-( 4- metvlthiopvridlnio) etvlthio1-( 6S)- f1-( R)- hvdroxvevl1- 7- oxo- l- azabicclo( 3. 2. 0 ) hept-2 - en- 2- carboxyl chloride

<2> These reagents are distilled before use.

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) at 0-5°C under nitrogen was added 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 of CH 3 CN, 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 CE 3 CN to yield 314 mg of a yellowish solid. This was triturated from 5 ml of 10* MeOE in H2O and 341 mg (0.618 mmol, yield 45.4*) of the title compound was obtained in the form of white crystals with a melting point of 118-120°C.

1-H-NMR (DMS0-d6, CFT-20) S: 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, -CH2N<®>), 5.15 (1H, d, J-4.9 Hz, OH), 5.20-5.35-5.40-5.55 (2H, ABq, C02CH2- 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 (2H, "d", J=7.1 Hz, pyridinio-Hs) ppm: IR (nujol) vmax: 3250 ( OH), 1775 (e-lactam), 1700 (ester) and

1625 (pyridinium) cm-<1>;

UV (abs, EtOH) Xmax: 308 (c4.47 x 10) nm;

[α]<g3> +24.8° (c 0.5, MeOH);

Analysis for C24H26N3O6S2CI'B^O:

Calculated: C 50.56 H 4.95 N 7.37

Found: C 50.63 H 4.72 N 6.89

D. ( 5R )- 3- r2-( 4- methylthiopvrldinioletvlt1- f6S )- f( 1R1-hydroxvetvll- 7- oxo- 1- azablcyclo( 3. 2. 0 ) hept- 2- ene- 2-carboxylate

(5R)-p-nitrophenyl-3-[2-(4-methylthiopyridinio)ethylthio]-(6S)-[(IR)-hydroxyethyl]-7-oxo-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 layer was pumped off to remove all organic solvents and purified by reverse phase column chromatography (Cig-jj-Bondapak, 13 g, Waters Associates), eluting with H 2 O. Fractions with a UV absorption at 307 nm were collected (approx. 1

liter) and lyophilized, whereby 127 mg (0.334 mmol, yield 48.5*) of the title compound was obtained as a yellowish powder.

<*>H-NMR (D 2 O, CFT-20) S: 1.20 (3H, d, J-6.4 Hz, 1'-CH 3 ),

2.64 (3H, s, -SCH3), 2.81 (2H, m, -SCH2-), 3.19 (1H, dd, J()_1»=6.1 Hz, J6_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, J5_6-2, 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 (2H, "d", J=7.1 Hz, aromatic-Hs) ppm;

IR (KBr, plate) vmax: 3400 (OH), 1750 (e-lactam), 1630

(pyrrlidinium) and 1590 (carboxylate) cm-<1>;

UV (H2O) Xmax: 231 (c9800) and 307 (c25000) nm;

[a]g<3> +3.14° (c 0.5, H 2 O).

Example 8

Preparation of 3- r2-(3-Methoxv-1-Pvridinlum)ethvlthiol-6a-ri(R)-hydroxyvetvll-7-oxo-l-azablcvclo(3.2.0)hept-2-ene-2-carboxvlate A .1-(2-mercaptoethyl)-3-methoxypyridinium mpt. ansulfonate

To 5°C precooled 3-methoxypyridine (698 g, 6.4 mmol) methanesulfonic acid (0.216 ml, 3.05 mmol) and ethylene sulfide (0.19 ml, 3.2 mmol) were added dropwise. 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 poured onto a Cig 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 76.356).

IR (CH2C12) <v>max: 2550 (w, SH) and 1620, 1600, 1585 (m,

aromatic) cm-<1>.

<i>H-NMR (DMS0-d6) S: 8.90-7.90 (4H, m, aromatic C-H), 4.72

(2H, t, J-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 (3H, s, CH3SO3) ppm.

B. p- niltrobenzyl- 3- r2-( 3- methoxy- l- pyridinium chloride) ethylthlol-6a- r1,-( R)- hydroxyethl1- 7- oxo- l- azablcyclo( 3. 2. 0 ) hept- 2- en- 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 minutes and cooled at -10°C for 30 minutes. The solid which precipitated from the mixture was filtered off, washed with 2 ml of cold acetonitrile and dried to give the title compound (1.32 g, yield 82*).

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

1700, 1695 (m, ester C=0) and 1520 (s, NO 2 ) cm"<1>;

<1>H-NMR (DMS0-d6) S: 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 Hz, CH2-N<®>), 4.35-3.75 (2H, m, H-5 andH-1'), 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 (3H, d, J-6.1 Hz, CH3CHO) ppm.

C. 3- r2-( 3- methoxy-l- pyridinelum) ethyltlo1- 6a- fl'-( R)- hydroxy-etvll- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

A solution of p-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 shaker over 10 * Pd/C (1.1 g) for 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) vmax: 1750 (s, e-lactam C=0) and 1580 (s,

carboxylate) cm-<1>;

<1->H-NMR (D2O) S: 8.55-8.10 (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<e>), 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 (3H,

d, J-6.4 Hz, CH 3 ) ppm;

UV (H 2 O, c 0.05) Xmax: 290 (cl0517), 223 (c6643) nm;

t% (0.1 M phosphate buffer, pH 7.4, 37<*>C): 20 h.

Example 9

Preparation of (5R.6S)-3-ff2-(3-methylthiopvridlniobetylthiol-6-fl-(R)-hydroxymethyl"l-7-oxo-l-azabiclo(3.2.0)hept-2-ene-2 -carboxylate

A. 3-Methylthio-1-(2-mercaptoethyl) pyrldinium chloride To a solution of 3-methylthiopyridine<3> (2.00 g, 0.016 mol) in 10 ml of ether is added 15 ml of 1N HCl and the whole is shaken thoroughly. The aqueous phase is separated off, washed with 10 ml of ether and evaporated. The remaining hydrochloride is then dried in vacuo over P 2 O 5 to give a white solid. To this solid hydrochloride is added 3-methylthiopyridine (1.88 g, <3> Prepared by the method according to J.A. Zoltewiez and C.

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

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 ?2 for 15 hours. This gave a slightly cloudy oil which was taken up in 125 ml H 2 O and washed with CH 2 Cl 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 C₂g 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 (film) vmax: 2410 (br, -SH) cm-<1>;

^H-NMR (DMS0-d6 + D2O) S: 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- nitrobenzvl-( 5R. 6S)- 3- r2-( 3- methylthiopvridinio) etvlthio" 1-6- f1-( R)- hydroxymethyl- 7- oxo- 1- azabicclo( 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 end of the addition, a precipitate separated from the reaction mixture and further cooling by

-10°C for 10 minutes gave a firm, orange colored mass. This solid was then triturated with acetonitrile and the residue collected by filtration. The residue was washed with acetonitrile, then with acetone and finally dried in vacuo 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) vmax: 3345 (br, -GH), 1770 (e-lactam CO), 1680 (-CO2PNB) cm-1; ^H-NMR (DMSO-dfc) S: 8.98-7.96 (m, 4H, pyrldinium 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- r2-( 3- metvlthiopvrldinio) etvltlo1- 6- ri-( R)-hydroxyetvll- 7- oxo- 1- azabicclo( 3. 2. 0) hept- 2- en- 2 -karboks<y>lat

To a mixture of p-nitrobenzyl-(5R,6S)-3-[2-(3-methylthio-pyridinio)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) gave added 5 ml of THF and 25 ml of ether. This mixture was hydrogenated (Parr) at approx. 2.8 kp/cm<2> for 1 hour. The reaction mixture was then filtered through Celite and the filter cake washed with H2O 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 NaHCO3. This solution was immediately applied to a Cig 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) vmax: 3400 (br, -OH), 1755 (e-lactam CO), 1590

(-C02<e>) cm"<1>;

iB-NMR (D20) S: 8.60-7.76 (m, 4H, aromatic), 4.76 (t,

J-5.8 Hz, 2H, N-CH2), 4.13 (d of q, «W-6.3 Hz, 1H, H-l')t 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) <X>max: 296 (c8509), 273 (cl3005), 231 (cll576) nm;

tthaw (pH 7.4, 36.8°C): 20 h.

Example 10

Preparation of 3- f2-( l-( 2. 6- dimethylPvrldinium) etvltlo1- 6a-ri-( R)- hvdroxvetl1- 7- oxo- l- azablcyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

A. 1-(2-mercaptoethyl)-2.6-dlmethylpyridinium 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 min, 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 pad of Celite, washed with 2 x 15 mL of ether, pumped to remove traces of organic solvents, and poured onto a 3.0 x 12 cm jj-Bondapak C^g column. Elution with a mixture of 3% acetonitrile and 97% water after lyophilization of the appropriate fractions gave 2.5 g of the impure title compound as a syrup. It is purified again by means of HPLC (ji-Bondapak C^g), whereby 0.90 g corresponding to 7* of the title compound was obtained.

IR (film) vmax: 2520 (SH), 1640 and 1625 (pyrldinium), 1585,

1490, 1200 (sulfonate) cm-<1>;

<1->H-NMR (DMS0-d6 + D2O) S: 2.36 (3H, s, CH2SO3<e>), 4.62 (2H,

m, CH 2 N<®>), 7.74 (2H, m, Hm of pyrldinium), 8.24 (1H, m, Hp of pyrldinium);

UV (H20) Xmax: 272 (4080) nm.

B. p-nitrobenzvl-3-r2-(l-(2.6-dimethylpyrldlnlum))ethyltlo1-6a-l"l-(R)- hydroxyethyl"l-7- oxo-l- azabicyclo(3.2.0 ) heptane-2-ene-carboxylate-diphenylphosphate

To a cold (0'C) solution of p-nitrobenzyl-6α-[l-(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, which is kept 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 minutes and then treated with a solution of 1-(2-mercaptoethyl)-2,6-dimethylpyridinelumethanesulfonate (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 the top of a 25 x 9.0 cm column of ji-Bondapak C^g. Elution with mixtures of acetonitrile and water and lyophilization of the appropriate fractions yielded 0.92 g corresponding to 655E of the title compound.

IR (KBr) vmax: 3700-3000 (OH), 1765 (C-0 of ε-lactam), 1690

(C-0 of pNB ester), 1620 (pyrldinium), 1590 (phenyl), 1517 (NO 2 ), 1330 (NO 2 ), 880 (NO 2 ) cm"<1>;

<1->H-NMR (DMS0-d6) S: 1.15 (3H, d, J-6.2 Hz, CH3CH0H), 2.7-3.7 (11H, CH2S, 2-CH3 on pyrldinium, 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, pyrldinium, H's of pNB);

UV (H20) <X>max: 274 (cl4150), 319 (c9445) nm.

C. 3- T2-( l-( 2. 6- dlmetvlpvrldinium)) etvltio1- 6a- fl-( R)-hydroxylvetvll- 7- oxo- l- azablcvclo( 3. 2. 0) hept- 2- en- 2 - carbox vat

To a mixture 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 p-Bondapak C 2 g 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) vmax: 3700-2800 (0H), 1750 (C-0 of e-lactam), 1620

(pyrrlidinium), 1585 (carboxylate) cm-<1>;

^H-NMR (D 2 O) S: 1.23 (3H, d, J=6.4 Hz, CH 3 CH 0 H), 2.5-3.5

(11H, H-4, H-6, CH2S, 2CH3 on pyrldinium), 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 pyrldinium), 8.07, 8.16, 8.18 and 8.27 (1H, B part of A2B system, Hp of pyrldinium);

UV (H 2 O) <X>max: 277 (c9733), 300 (c8271) nm;

[α]<g3> +50.7° (c 0.48, H 2 O);

Analysis for Ci<g>H22<N>204S*1.5H20:

Example 11

Preparation of (5R.6S)-3-T2-(2-methyltlo-3-methylmidazolio1-ethylthio1-6-ri-(R)-hydroxymethyl-7-oxo-1-azabicyclo(3.2.01-hept-2 - a- 2- carboxylate

A. 2- Methylthio- 3- methyl- 1-( 2- mercaptoethyl Umldazolium trifluoromethanesulfonate

Trifluoromethanesulfonic acid (1.38 mL, 0.015 mol) was added dropwise to 2-methylthio-1-methylimidazole<4> (4.0 g, 0.03 mol) at 0°C under N 2 . Ethylene sulfide (0.9 mL, 0.015 mol) was then added and the mixture was heated to 55°C under N 2 for 24 h. 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) vmax: 2550 (w, sh) cm"1; <4> Prepared as in A. Wohl and W. Marckwald, "Chem.

Ber.", 22, 1353 (1889).

<1>H-NMR (dfc-acetcm) S: 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).

B. p- nltrobenzvl-( 5R. 6S)- 3- r2-( 2- methyltlo- 3- methyl- imlda-zolio letvltlol - fl- f R l- hvdroxvetvll - 7- oxo- l- azablcyclo-( 3 .

To a solution of p-nitrobenzyl-(5R,6S)-6-[l-(R)-hydroxy-ethyl]-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 N2 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 10 hours and then concentrated in vacuo to obtain a gum. This was collected in 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) vmax: 3380 (br, OH), 1770 (e-lactam CO) cm-<1>;

<1->H-NMR (d6-acetone) δ: 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- f2-( 2- metvlthio- 3- metvllmidazolio) etvltlo1- ri-( R)- hvdroxvetvll- 7- oxo- 1- azabicclo( 3. 2. 0) hept- 2 - a- 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-dlphenylphosphate (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-on-charcoal was added. 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 C₂g 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) <v>max: 3420 (OH), 1750 (e-lactam CO), 1590 (-C02<e>)

cm<-1>; <i>H-NMR (D 2 O) δ: 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 (c7572), 262 (c6259), 222 (c7955) nm.

Example 12

Preparation of (5R.6S)-3-r2-(3-amnolopvridinlo/)ethylthiol-6-f1-(R)-hydroxyethyl1-7-oxo-1-azabicyclo(3.2.0)hept-2-ene - 2-carboxylate

A. S-amnol-l- f 2- mercaptoetvl ) pyridinelnnicl nrl ri

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 in 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 taken up in 25 mL H 2 O. The aqueous solution was applied to a C 2 g reverse phase column, which was eluted with H 2 O. 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) vmax: 3180 (NH2) cm"<1>;

1-H-NMR (DMS0-d6) δ: 8.19-7.59 (m, 4H, aromatic), 4.59 (t,

J-6.2 Hz, 2H, N-CH 2 ), 3.5 (br s, 2H, -NH 2 ), 3.20-2.77 (m, 3H).

B. p-nitrobenzvl-(5R.6S)-3-r2-f3-aminopyridinioletyltlo"!-6-l"l-(R)-hydroxybutyl-7-oxo-l-azabicyclo(3.2.0)hept - 2- en-2- carboxylate diphenyl phosphate

To a solution of p-nitrobenzyl-(5R,6S)-6-[l-(R)-hydroxy-ethyl]-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 was kept at 0°C for VA hour and then concentrated under vacuum. The resulting gum was collected in 1:1 acetonitrile :H 2 <) 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) vmax: 3330 (br, OH), 3180 (br, NH2), 1770 (e-lactam

CO), 1690 (-CO 2 pNB) cm-<1>;

<1->H-NMR (DMS0-d6) S: 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- aminopvridiniolethyl) tlo)- 6- fl-( R )-hydroxyl- 7- oxo- 1- azabicyclo( 3. 2. 0) hept- 2- a- 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) were 8 ml of THF and 20 ml of ether were added. This mixture was hydrogenated (Parr) at approx. 2.8 kp/cm<2> for 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 added to a C₂₂ reverse-phase column eluted with Et₂O. Lyophilization of the relevant fractions gave 0.45 g of an off-white solid. This was purified again 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) vmax: 3340 (br), 1750 (br, e-lactam CO), 1580 (br,

-C02<9>) cm-<1>; <1->H-NMR (D 2 O) S: 8.07-7.59 (m, 4H, aromatic), 4.61 (t, J-5.8 Hz, 2H, N-CH 2 ), 4.14 (d of q, J-J'-6.3 Hz, 2H, 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 (H 2 O) <X>max: 299 (c7949), 256 (c8822) nm; t^ (pH 7.4, 36.8°C): 18.5 h. Example 13 Preparation of

(5R.6S)-3-r1-(S)-methyl-2-(1-Diviridine)ethylthiol-6-r1-(R)-hydroxymethyl-7-oxo-1-azabicyclo(3.2.0 )hept-2-ene-2-carboxylate

and

( 5R . 6S )- 3- ri-( R )- methyl- 2-( 1- Pvridini\un) ethvlthio1- 6- ri-( R )-hydroxymethyl- 7- oxo- 1- azabicyclo( 3. 2 . 0 ) hept-2-ene-2-carboxylate

A. dl- 1-(2-mercapto-2-methylethyl)pyridinemethanesulfonate

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 d£-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^g). The appropriate fractions were combined and lyophilized to give dA-1-(2-mercapto-2-methylethyl)-pyridinium methanesulfonate, 1.14 g (15*) as a colorless syrup.

IR (film) vmax: 2520 (SH), 1640 (pyrldinium), 1180 (s,

C<H>3SO3<e>), 1040 (CH3SO3<e>) cm-1; 1-H-NMR (DMS0-d6) S: 1.35 (d, J-6.8 Hz, 3H, CH3CHS), 2.30 (s, 3H, CH3SO3<e>), 2.90 (d, J- 8.5 Hz, 1H, SH), 3.2-3.7 (m, CHSH), 4.52 (dd, <J>genr12'9 Hz» J-8.4 Hz, CHCH2N°), 4, 87 (dd, Jgem-12.9 Hz, J-6.0 Hz, CHCH2<N>), 8.0-8.4 (m, 2H, Hm of pyrldinium), 8.5-8.8 (m , 1H, Hp of pyrldinium), 9.04 (dd, J-1.4 Hz, J-6.7 Hz, 2H, Ho of pyrldinium);

UV (H 2 O) Xmax: 208 (c5267), 259 (c3338) nm;

Analysis for CgH15N03S2•2H20:

Calculated: C 37.88 H 6.71 N 4.91 S 22.47

Found: C 37.49 H 6.85 N 4.86 S 22.09

and d£-1-(2-mercapto-1-methylethyl)pyridinium methanesulfonate, 0.82 g (11*) as a colorless syrup;

IR (film) vmax: 2500 (SH), 1628 (pyrrldinium), 1180 (sulfonate), 1035 (sulfonate) cm-<1>;

<i>H-NMR (DMS0-d6) δ: 1.69 (d, J-6.8 Hz, 3H, CH3CHN<®>), 2.32

(s, 3H, CH3SO3<e>), 3.0-3.3 (m, 2H, CH2S), 4.2-5.2 (m, 1H, CHN<®>), 8.0-8, 4 (m, 2H, Hm of pyrldinium),

8.5-8.8 (m, 1H, Hp of pyrldinium), 9.0-9.2 (m, 2H, Ho of pyrldinium);

UV (H 2 O) Xmax: 209 (c4987), 158 (c3838) nm;

Analysis for C9H15NO3S2* 1.5H20:

Calculated: C 39.11 H 6.56 N 5.07

Found: C 39.11 H 5.92 N 5.20

B. ( 5R. 6S)- p- nltrobenzvl- 3- r1- fR. S)- metvi- 2-( 1- pvridinlum)-ethylthiol - 6- l" l-( R )- hydroxyethyl" l - 7- oxo- l- azablciclo-( 3. 2. 0 ) hept- 2- en- 2- carboxylate diphenyl phosphate

To a cold (0°C) solution of (5R,6S)-p-nitrobenzyl-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 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 prepared Bondapak C18 column with 25-50% acetonitrile in water as elution solvents during formation of 1.07 g corresponding to 97% of the title compound as a yellowish powder after lyophilization;

IR (KBr) vmax: 3700-3100 (OH), 1770 (C-0 of ε-lactam), 1695

(C-0 of pNB ester), 1630 (pyrldinium), 1590 (phenyl), 1518 (NO2), 1348 (NO2), 885 (NO2) cm-<1>;

1-H-NMR (DMSO-d6) δ: 1.14 (d, J-6.1 Hz, 3H, CH3CH0), 133 (d,

J-6.3 Hz, 3H, CH3CHS), 4.6-5.0 (m, CH2N<8>), 5.14 (d, J=5.2 Hz, 1H, OH), 5.37 ( center of ABq, J-12.4 Hz, 3H, 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 pyrldinium), 8.4-8.8 (m, 1H, Hp of pyrldinium), 9.08 (d, J -5.6 Hz, 2H, Ho of pyrldinium );

UV (H20) <X>max: 263 (cl3325), 308 (c8915) nm.

Analysis for C36<H>36<N>3<0>10SP'H20:

Calculated: C 57.52 H 5.10 N 5.59 S 4.27 Found: C 57.76 H 4.96 N 5.36 S 4.35 C. ( 5R. 6S)- 3- ri-( R and S)-methyl-2-(1-pvridinium)ethylthiol-6-ri- ( R )- hydroxyethyl" 1 - 7- oxo- 1- azabicyclo( 3. 2. 0 ) hept- 2-ene- 2-carboxylate

To a solution of (5R,6S)-p-nitrobenzyl-3-[1-(R,S)-methyl-2-(1-pyridinium )ethylthio]-6-[1-(R)-hydroxyethyl]-7 -oxo-1-azabicyclo(3.2.0 )hept-2-ene-2-carboxylate-diphenylphosphate (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 M, pH 7.22) 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 Celite pad, washed with 3 x 20 ml ether and chromatographed on a 2.5 x 9.5 cm preparative Bondapak C^g column with water as eluent, whereby 0.18 g corresponding to 63* of a mixture of diastereoisomers was obtained. These were separated by means of HPLC (preparative Bondapak C^g) with water as elution solvent;

lower retention isomer, 0.068 g (23*), compound

"B";

IR (KBr) vmax: 1770 (C=0 of p<->lactam), 1633 (pyrldinium),

1593 (carboxylate) cm-<1>;

%-NMR (D 2 O) S: 1.20 (d, J-6.3 Hz, 3H, CH 3 CH 0 ), 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, CH3CHO), 4.3-5.1 (m, CH2N<®>), 7.8-8.2 (m, 2H, Hm of pyrldinium), 8.4-8.7 (m, 1H, Hp of pyrldinium), 8.7-9.0 (m, 2H, Ho of pyrldinium);

UV (H2O) Xmax: 260 (c6727), 300 (c8245) nm;

[a]<g3> -39.3<*> (c, H2O);

t^ - 12.6 hours (measured at a concentration of 10"<4> M i

phosphate buffer, pH 7.4 at 36.8<*>C);

isomer with higher retention time, 0.081 g (28*), compd

"A";

IR (KBr) vmax: 1755 (C-0 of ε-lactam), 1630 (pyrldinium),

1590 (carboxylate) cm-<1>;

<*>H-NMR (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, CH3CHO, H-5), 4.2-5.1 (m, CH2N<®>), 7.7-8.1 (m, 2H, Hm of pyrldinium), 8, 3-8.65 (m, 1H, Hp of pyrldinium), 8.65-8.9 (m, 2H, Ho of pyrldinium);

UV (H 2 O) Xmax: 259 (c5694), 296 (c6936) nm;

[α]2.3 +96.9° (c 0.56, H 2 O);

ttø = 15.6 hours (measured at a concentration of 10~<4> M i

phosphate buffer, pH 7.4 at 36.8'C).

Example 14

Manufacture of

( 5R. 6S)- 3- r2- r( S)-( l- pvrldlniumn- l-( S)- cvclohexvltlol- 6- r-( R )- hydroxyethyl" l - 7- oxo- l- azabicvclo( 3. 2. 0 ) hept- 2- en- 2-carboxvlat

and

( 5R. 6S)- 3- f2- r( R)-( l- pvrldlniumn- l-( R)- cvclohexvltilo1- 6- ri-( R )- hydroxyethyl" l - 7- oxo- l- azablcyclo( 3. 2. 0) hept-2-ene-2-carboxylate

A. dÆ-l-r2-mercaptol-l-cyclohexyl<py>rjdininmethanesulfonate

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£-cyclohexene sulfide 2.377 g (85% pure), 0.0103 mol and stirred at 72°C for 25 hours. The excess 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 preparative Bondapak C^g column with 0-2* acetonitrile in water as eluent, whereby after lyophilization a colorless syrup was formed, 1.57 g (53* );

IR (film) vmax: 2500 (SH), 1625 (pyrrldinium), 1190 (S03<G>)<;>

^H-NMR (DMSO-d6) S: 1.2-2.5 (m, 8H, cyclohexyl H), 2.32 (s,

3H, CH3SO3<e>), 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 pyrldinium), 8.9-9.3 (m , 2H, Ho of pyridinium);

UV (H2O) Xmax: 214 (c5365), 258 (c3500) nm;

Analysis for C12H1gN03S2"H20:

Calculated: C 46.88 H 6.88 N 4.56

Found: C 46.61 H 6.46 N 4.65

B. ( 5R. 6S)- p- nitrobenzvl- 3- f2- f( R or S)-( 1- pvridinium) H-( R or S)- cvclohexvlto1- 6- fl-( R)- hvdroxvevl1- 7- oxol- l- azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxyvlate- diphenyl phosphate

To a cold (0°C) solution of (5R,6S)-p-nitrobenzyl-6-[l-(R)-hydroxyethyl]-3,7-oxo-1-azabicyclo(3.2.0)hept-2- ene-2-carboxylate (1.37 g, 3.93 mmol) in 15 mL of acetonitrile, kept 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 δ-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 at 0<*>C and chromatographed on preparative Bondapak C^g with 25-50* acetonitrile in water as elution solvent, whereby after lyophilization of the appropriate fractions were formed 1.9 g corresponding to 53* of the title compound.

IR (KBr) vmax: 3700-3000 (0H), 1770 (C-0 of 3-lactam), 1700

(C=0 of pNB ester), 1628 (pyridinium), 1590 (phenyl), 1515 (NO 2 ), 1345 (NO 2 ), 880 (NO 2 ) cm-<1>;

<1->H-NMR (D2O) δ: 1.13 (d, J-6.1 Hz, 3H, CH3CH0), 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, CH3CHO, 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, 1H, Hp of pyridinium), 9.0-9.4 (m, 2H, Ho of pyridinium);

UV (H 2 O) <X>max: 263 (c9038), 309 (c6394) nm;

Analysis for C3g<H>40<N>3010SP-H20:

Calculated: C 59.16 H 5.35 N 5.31

Found: C 58.95 H 5.15 N 5.57

C. ( 5R. 6S)- 3- f2-( R or S)-( l- pvrldlniumn- l-( R or Sl- cvclohexvollol- 6- l" l-( R)- hydroxyvetll1- 7- oxo- l- aza-blcyclo( 3. 2. 0 ) hept- 2- en- 2- carboxylate

To a solution of (5R,6S)-p-nitrobenzyl-3-[2-[(R or S)-(l-pyridinium)-1-(R or S)-cyclohexylthio]-6-[l-(R )-hydroxyethyl]-7-oxo-1-azablcyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (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 of 10* palladium-on-charcoal. The resulting mixture was hydrogenated at 23°C under approx. 2.8 kp/cm<2> 1 1.25 hours. The organic layer was separated and washed with 3 x 25 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 Cig 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; lower retention diastereoisomers, compound "A", (0.29 g, 31*);

IR (KBr) vmax: 1750 (C-0 of ε-lactam), 1620 (sh, pyridinium), 1685 (carboxylate) cm-<1>;

^H-NMR (D2O) S: 1.21 (d, J-6.3 Hz, 3H, CH3CH0), 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 (H 2 O) Xmal£S: 260 (c7123), 300 (c8685) nm;

[α]g 3 +6.2' (c 0.63, H 2 O);

ttø - 16.6 hours (measured at a concentration of 10~<4> M i

phosphate buffer, pH 7.4 at 36.8<*>C);

Analysis for C2oH24<N>2<0>4S<<>2H20:

Calculated: C 56.59 H 6.65 N 6.60 S 7.55

Found: C 56.83 H 6.47 N 6.59 S 7.43;

higher retention diastereoisomers, compound "B", (0.35 g, 38*);

IR (KBr) vmax: 1750 (C-0 of ε-lactam), 1622 (sh, pyridinium), 1588 (carboxylate) cm-<1>;

<i>H-NMR (D2O) S: 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, CH3CHO), 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 (H 2 O) Xmax: 259 (c5992), 296 (c7646) nm;

[α]<g3> +65.3° (c 0.43, H 2 O);

ty - 20.2 hours (measured at a concentration of 10~<4> M i

phosphate buffer, pH 7.4 at 36.8<*>C);

Example 15

A. ( 5R)- allvl- 3- rf2- Pvrldinioetvl) tlo1-( 6S)- ri-( R)- hvdroksvetvll- 7- oxo- l- azabicvklo( 3. 2. 0) hept- 2- en- 2- carboxyvlate-diphenyl phosphate

To a solution of (5R)-allyl-3,7-dioxo-(6S)-[1-(R)-hydroxyethyl]-1-azabicyclo(3.2.0)heptane-2-(2R)-carboxylate (473 mg , 1.87 mmol) in CH3CN (6 ml) at approx. -ICC under nitrogen 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 3 CN containing 5 drops of DMF, followed by diisopropylethylamine (0.42 mL, 2.4 mmol) . The mixture was stirred at -15°C for 30 minutes, then diluted with 20 mL of H 2 O. This mixture

was purified directly on a reverse-phase silica gel column (C^g PrepPÅK, 12 g, Waters Associates) and eluted with H2O (100 mL), 30* CH3CN/H2O (100 mL) and finally 40* CH3CN/ H 2 O (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.

■^E-NMR (DMS0-d6, CFT-20) S: 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, -CH2<N®> ), 5-6.2 (3H, m, olefinic protons), 6.6-7.4 (m, aromatic protons), 8.15 (2H, "t", Jx7 Hz, aromatic protons meta to the nitrogen), 8.63 (1H, "t", J27 Hz, aromatic proton para to the nitrogen) and 9.07 (2H, "d", Js:7 Hz, aromatic protons ortho to the nitrogen) ppm;

IR (film) vmax: 3400 (OH), 1770 (e-lactam), 1690 (ester),

1625 (pyridinium)

B. ( 5R)- 3- r( 2- pvridlnloetvl ) thio1-( 6S)- ri-( R )- hvdroxwetvl' 1- 7-oxo- 1- azablcyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

To a solution of (5R)-allyl-3-[(2-pyridinioethyl)thio]-(6S)-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0 )hept-2 -ene-2-carboxylate diphenyl phosphate (156 mg, 0.25 mmol) in CH3CN

(2 ml) it was successively at approx. At 22°C was added 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 the addition of 7 ml of anhydrous Et 2 O, the precipitate was filtered off, washed with 7 ml of anhydrous Et 2 O and dried in vacuo to form 101 mg of brown 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-ethyl hexanoate.

<1->H-NMR (D20, CFT-20) δ: 0.80 (t, J-6.4 Hz, Me from ethyl hexanoate), 1.21 (3H, d, J-6.3 Hz , 1<*->Me), 2.93 (2H, dd, <J>i_4<=>9 Hz, Jgem=4 Hz• 1-Hs), 3.28 (1H, dd, J6_!-6, 2 Hz, J6_5-2.5 Hz, 6-H), 3.42 (2H, t, J= 6 Hz, -CH2S), 3.98 (1H, td, <J>s-i-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 (2H, "d ", Jx6.5 Hz, Ho of pyridinium) ppm.

Example 16

Preparation of 3-r2-(N-metvl-thiomorpholnium)etvltlo1-6a-ri'-(R)-hydroxyethyl1-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carbox< y>lazy

A. N-Methyl-N-(2-mercaptoethyl)-tlomorpholinelummethanesulfonate

Methanesulfonic acid (1.47 ml, 20.5 mmol) and ethylene sulfide (1.30 ml, 21.4 mmol) were added to ice-bath precooled N-methylthiomorpholine<5> (5.00 g, 42.7 mmol). The mixture was heated at 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) vmax: 2550 (w, SH) cm-1;

1-H-NMR (DMS0-d6) S: 3.25-2.95 (6H, m, CH2N<®>), 3.20-2.65 (7H,

m, CH2S, SH) and 2.32 (3H, s, CH3SO3) ppm.

<5> J.M. Lehn & J. Wagner, "Tetrahedron", 26, 4227

(1970).

B. p- nitrobenzyl- 3- r2-( N- methylthiomorpholinelum- dlphenvlphosphate) ethvlthio1- 6a- ri'-( R)- hvdroxvevl1- 7- oxo- l-azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

A cold (ice bath) solution of p-nitrobenzyl-6α-[1'-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (557 mg, 1.60 mmol) 18 mL of CH3CN was treated dropwise with diisopropylethylamine (0.336 mL, 1.92 mmol) and diphenylchlorophosphate (0.400 mL, 1.92 mmol) and stirred for 30 minutes. The reaction mixture was again treated with N-methyl-N-(2-mercaptoethyl)thiomorpholinium methanesulfonate (893 mg, 2.29 mmol) in 4 mL of CH 3 CN 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 (nujol) <v>max: 1760 (s, e-lactam C-0) and 1510 (s, N02)

cm<-1>; <*>H-NMR (DMS0-d6) S: 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-1' 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 (3H, d, J-6.2 Hz, CH3) ppm. C. 3- r2-( N- metvl- thiomorpholinum) etvltlo1- 6a- ri,-( R)- hvdroksv etvll- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en- 2- carboxylate

A solution of p-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 10* palladium-on-charcoal in a Parr shaker for 1 hour at approx. 2.8 kp/cm<2>. 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 carbapenem 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 (nujol) vmax: 1750 (s, ε-lactam C-0) and 1590 (s, C-0) cm<-1>;

<i>H-NMR (D 2 O) S: 4.25-3.95 (2H, m, H-1' , 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 (H 2 O, c 0.062) Xmax: 299 (cl 0962) nm;

t% - 17.7 hours (0.1 M phosphate buffer, pH 7, 37°C).

Example 17

Preparation of (5R.6S)-3-T2-(1-methylmorpholno)ethylthiol-6-f(R)-1-hydroxymethyl-7-oxo-1-azabiclo(3.2.0)hent-2-en- 2-carboxylate

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

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 tan solution was heated in oil bath at 50-60<*>C under N 2 for 18 h. Volatiles were then removed under vacuum and the remaining oil was collected in 10 mL H 2 O. The aqueous solution was washed with 3 x 5 mL diethyl ether and then residual organic solvent removed under vacuum. The resulting aqueous solution was added to a C 10 reverse phase column, it was diluted with H 2 O, 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 under vacuum over P2C<5 to give the product (1.92 g, 41*).

IR (KBr) vmax: 2560 (-SH) cm-<1>;

<i>H-NMR (d6-acetone) S: 4.25-3.6 (m, 8H), 3.49 (s, 3H, N-Me),

3.35-2.7 (m, 5H).

B. p- niltrobenzvl-( 5R. 6S)- 3- f2-( 1- methylmorpholino) ethyllol- 6-f( R)- l- hydroxybutyl1- 7- oxo- l- azabicyclo( 3. 2. 0) hept- 2- en-2- carboxyvlate- diphenyl phosphate

To a solution of p-nitrobenzyl-(5R,6S)-6-[(R)-1-hydroxy-ethyl]-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) was 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 taken up in H 2 O and applied to a C 2 g reverse phase column. Diluting with H 2 O and then with 20* acetonitrile-H 2 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) vmax: 3300 (-0H), 1770 (e-lactam CO), 1700

(-CO 2 pNB) cm-<1>;

^H-NMR (d6-acetone) S: 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- 9.2 Hz, 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- f2-( 1- methylmorpholino) ethylthiol- 6- f( R)- 1- hydroxymethyl- 7- oxo- 1- azabicchloro( 3. 2. 0) hept- 2- en- 2- carboxylate

To a solution of p-nitrobenzyl-(5R,6S)-3-[2-(1-methyl-morpholino)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 added to 0.36 g of 10* palladium -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 applied to a Cig 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) vmax: 3420 (br, OH), 1750 (e-lactam CO), 1590

(-C02<e>) cm"<1>;

<i>H-NMR (D 2 O) S: 4.35-2.77 (m, 17H), 3.18 (s, 3H, N-Me),

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

UV (H2O) Xmax: 300 (c6344) nm;

ty, (pH 7.4, 36.8°C), 18.5 h.

Example 18

Preparation of (5R.6S)-3-T2-(1.4-Dimethyl-1-plperazine)-ethvltlo1-6-ri-(R)-hydroxvetl1-7-oxo-1-azabicvklof 3.2.0)- hept-2-ene-2-carboxylate

A. l-(2-Acetylthioethyl)-l. 4- dimethylplperazinium bromide

A solution of 2-bromomethylthioacetate^ (2.20 g, 0.012 mol) and 1,4-dimethylpiperazine (1.95 mL, 0.014 mol) in 4 mL of 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, and a hygroscopic yellowish powder was obtained, 3.2 g (90*).

IR (nujol) vmaijS: 1685 (C=0 of tloester) cm-<1>;

0

■^H-NMR (D 2 O) S: 2.37, 2.39 (2s, 6H, CH 3 CO,

<6> B. Hansen, "Acta Chem. Scand.", 11, 537-40 (1957).

B. 1. 4-Dimethyl-1-(2-mercaptoethyl)piperazinium bromide.

hydrogen chloride

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<4>C under nitrogen for 1 hour. The solution was concentrated under reduced pressure to give a white powder, 0.41 g (38*); <1->H-NMR (DMS0-d6) S: 2.90 (s 3.26 (s,

Analysis for CgB^o^SBrCl 'B^O:

Calculated: C 31.03 H 7.16 N 9.05 S 10.35 Found: C 31.62 H 7.46 N 9.19 S 10.19 C. ( 5R. 6S)- p- nitrobenzvl- 3- r2-(1.4-dlmethyl-1-plperazine)-ethylthiol-6-fl-(R)-hydroxyethyl1-7-oxo-1-azablcyclo-(3.2.0)hept-2-ene-2-carboxylate - diphenyl phosphate

To a cold (0<*>C) solution of (5R,6S)-p-nitrobenzyl-6-[l-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane- 2-(R)-Carboxylate (0.465 g, 1.33 mmol) 1 To 2 mL of acetonitrile, kept under nitrogen, was added diisopropylethylamine (0.278 mL, 1.59 mmol) and diphenyl chlorophosphate (0.33 mL, 1.59 mmol ). The reaction mixture was stirred for 130 minutes and treated with a suspension of 1,4-dimethyl-1-(2-mercaptoethyl)piperazinum bromide hydrochloride (0.40 g, 1.37 mmol) 1 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/Cig 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) vmax: 1765 (C-0 of ε-lactam), 1690 (C-0 of pNB ester), 1585 (phenyl), 1512 (N02), 875 (N02) cm-<1>;

^H-NMR (DMSO-d6) δ: 1.16, 1.18 (2d, J-6.1 Hz, 3H, CH3CH0H),

2.44 (p

3.14 (s,

5.31 (d,

J-6 Hz, OH), 5.39 (center of ABq, J-13 Hz, CH2 of pNB), 6.6-7.4 (m, 10H, phenyl of phosphate), 7.71 (d, J -8.8 Hz, 2H, Ho of pNB), 8.26 (d, J-8.8 Hz, Hm of pNB).

D. ( 5R. 6Sl- 3- T2-( 1. 4- dimetvl- l- plperazlniiup) etvltlo1- 6- ri-( R)- hydroxyethyl1- 7- oxo- l- azablcvclo( 3. 2. 0) hept- 2- en- 2-carboxylate

To a solution of (5R,6S)-p-nitrobenzyl-3-[2-(1,4-dimethyl-1-piperazinium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1 -azablcyclo(3.2.0)hept-2-ene-2-carboxylate-dlphenylphosphate (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 kp/cm<2> 1 1 hour. The two layers were separated and the organic layer was extracted with 2 x 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/Cig column (Waters Associates) (2.5 x 9.5 cm) with water as eluent, which gave 0.097 g (43*) after lyophilization.

IR (KBr) vmax: 3000-3700 (OH), 1750 (C-0 of ε-lactam),

1585 (carboxylate) cm-<1>;

^H-NMR (D 2 O) S: 1.24 (d, J-6.4 Hz, 3H, CHoCHOH), 2.33 (s,

3.15 (s,

4.0-4.5 (m,

UV (H 2 O) Xmax: 296 (c9476) nm;

[α]<g3> 61.1° (c 0.26, H 2 O);

t^ =• 12.4 hours (measured at a concentration of 10~<4> M 1

phosphate buffer, pH 7.4, at 36.8<*>C).

Example 19

Preparation of (5R.6S)-3-r2-(N-methyl-thiomorpholinelumoxydl-ethvlthiol-6-fl-(R)-hydroxyethyl1-7-oxo-l-azabiclo(3.2.0)-hept-2- a- 2- carboxylate

To a cold (-10'C) solution of (5R,6S)-3-[2-(N-methylthio-morpholnium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1- azabicyclo(3.2.0)hept-2-ene-2-carboxylate (6.08 mg, 1.65 mmol) in 9 ml of a 1:1 mixture of acetonitrile and water was added m-chloroperbenzoic acid (334.8 mg, 1.65 mmol) in a small amount during 1 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 diastereoisomers of the title compound;

fraction A:

IR (nujol) vmax: 1750 (s, ε-lactam C-0) and 1580 (s, C-0) cm-1;

%-NMR (D2O) 5 : 4.26-2.91 (20H, m, H-4, H-5, H-6, E- I', CH2S,

CH2S-O, CH3-N<®> and CH2-N<®>) and 1.24 (3H, d, J-6.4 Hz, CH3) ppm;

UV (H2O, c 0.06) Xmax: ^02 (cl0425) nm;

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

faction B:

IR (nujol) <v>max: 1750 (s, ε-lactam, C-0) and 1585 (s, C-0) cm-*;

<1->H-NMR (D2O) S: 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 (3H, d, J=6.4 Hz, CH3) p<p>m;

UV (H 2 O, c 0.05) Xmax: 2.99 (c6517) nm;

t^: 10.75 hours (0.065 M, pH 7.4, buffer solution, 37°C).

Example 20

Preparation of (5R.6S)-3-T2-(1.4.4-trimethyl-1-piperazinelum)ethylthio"1-6-["1R-hydroxyethyl1-7-oxo-1-azabicyclo-(3. 2. 0) hept- 2- en- 2- carboxyvlate chloride

A. l-(2-Acetyltloethyl)-l. 4. 4-trimethylplperazinium bromide. 1

A suspension of 1-(2-acetylthioethyl)-1,4-dimethyl-pipera-zlnlumbromide (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 1 30 hours. The solvents were evaporated under reduced pressure, the residue triturated with 1 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 of m.p. 220-225°C with decomposition, 68% of the title compound.

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

1-H-NMR (D 2 O) S: 2.40 (s, 3H, CH 3 CO ), 3.37 (s, N-CH 3 ),

3.39 (s, N-CH 3 ), 3.99 (s);

UV (H20) Xmax: 226 (cl3144) nm;

Analysis for C^B^^OSBrl:

Calculated: C 30.08 H 5.51 N 6.38

Found: C 30.48 H 5.53 N 6.86

B. 1-(2-mercaptoetyl)-1. 4. 4-trimethylpiperazinium chloride

A mixture of 1-(2-acetylthioethyl)-1,4,4-trimethylpiperazinium bromide iodide (1.84 g, 4.19 mmol) and 15 ml of 6N hydrochloric acid was heated to 57<*>C under a nitrogen atmosphere for 2Vi h . The solution was concentrated under reduced pressure to dryness. The solid was suspended in 10 ml of water and the well-stirred suspension was treated with Permutitt S-1Cl<e> until dissolution was obtained. The solution was poured onto a 1.2 x 60 cm column of Permutitt S-1 Cl<®>. 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 melting point 190-191°C, 85*.

IR (nujol) vmax: 2460 (SH);

<1->H-NMR (D 2 O) S: 3.4 (s, N-CH 3 ), 3.45 (s, N-CH 3 ), 4.07 (s);

Analysis for CgH22N2SCl2* 0.75H20:

Calculated: C 39.34 H 8.62 N 10.20 S 11.67

Found: C 39.48 H 8.39 N 10.55 S 11.15

C. ( 5R. 6S)- p- nitrobenzvl- 3- r2-( l. 4. 4- trimethvl- l- Dipera-zlnium ) etvltlo1- riR- hvdroksvetvll- 7- oxo- 1- aza-bicvklof 3. 2. 0) hept- 2- en- 2- carboxyl chloride

To a cold (5'C) solution of (5R,6S)-p-nitrobenzyl-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, was 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 hours, diisopropylethylamine (0.1 mL, 0.57 mmol) was added and stirring continued for 2 hours. A portion of the acetonitrile was removed under reduced pressure and the resulting red mixture was chromatographed on a PrepPAK -500/Cig 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 Permutitt 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/Cig column. Lyophilization of the appropriate fractions gave a yellowish powder, 0.80 g (53*).

IR (KBr) vmax: 3400 (br, OH), 1770 (C-0 of β-lactam), 1690

(C-0 of pNB ester), 1605 (aromatic), 1515 (NO2), 1345 (NO2) cm-<1>;

<1->H-NMR (D2O) S: 1.26 (d, J-6.3 Hz, 3H, CH3CH0H), 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 2 O) Xmax: 276 (cl2094), 306 (cl0752) nm;

Analysis for C25H3(,N406SC12<*>3H20:

Calculated: C 46.51 H 6.56 N 8.68 S 4.97 Cl 10.98 Found: C 46.31 H 6.18 N 8.57 S 5.36 Cl 11.37 D. ( 5R. 6S )- 3- T2-( 1. 4. 4- trimethyl- 1- plperazinium) ethylthiol- 6-riR- hydroxybutyl- 7- oxo- 1- azabicyclo( 3. 2. 0 ) hept- 2- ene- 2-carboxvlatechlorld

A mixture of (5R,6S)-p-nitrobenzyl-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 pressure of 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/Cjg column (2.2 x 11 cm) eluting with water to give 70 mg or 25* after lyophilization;

IR (KBr) vmax: 3400 (br, OH), 1755 (C-0 of ε-lactam), 1585

(carboxylate) cm-<*>;

*H-NMR (D 2 O) δ: 1.24 (3H, d, J-6.3 Hz, CH 3 CH 0 H), 3.36 (s, NCH 3 ), 3.98 (s);

UV (H 2 O) Xmax1 296 (c7987) nm;

[a]g<3> 35.9<*> (c 0.30, H 2 O);

tj£ - 9.8 hours (measured at a concentration of 10-<4> M in phosphate buffer, pH 7.4, at 36.8<*>C).

Claims (1)

1. Quaternary amine thiol compounds, characterized in that they have the formula there A is ethylene which is optionally substituted with C 4 -alkyl, or cyclohex-1,3-ylene and 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 C^_3~ alkyl, hydroxy-C1-6-alkylthio, C1-6-alkyloxy and amino and is attached to A via the quaternary nitrogen atom.