NO160298B - PROCEDURE FOR THE PREPARATION OF 2BETA-CHLORAMETHYL-2ALFA-METHYL-PENAM-3ALFA-CARBOXYL ACID SULPHON. - Google Patents

Description

The present invention relates to a process for the production of 26-chloromethyl-2a-methyl-penam-3a-carboxylic acid sulfone with the formula

a pharmaceutically acceptable salt of the acid or a physiologically hydrolyzable ester of the acid. The presumed connection between the resistance shown by certain bacteria to B-lactam antibiotics and the ability of such bacteria to produce B-lactamases has led to an intense search for B-lactamase inhibitors. Clavulanic acid is an example of such a compound that is currently being studied extensively. Another 3-lactamase inhibitor has the acid form structure

and is known from European patent application 2927, published on 11 July 1979.

The connection with the structure

is known from US patents 4,036,847, 4,009,159, 3,993,646, 3,989,685 and 3,954,732. From US patent 4,155,912, 2-penem-3-carboxylic acid compounds with the formula as well as esters and salts are known. See also Farmdoc Abstracts 82090A, 10336B and 443337B. The connection (under the number CP-45899) with the structure

is an irreversibly acting 3-lactamase inhibitor with excellent solution stability. It has weak antibacterial activity and enhances the in vitro and in vivo activities of ampicillin against 3-lactamase-producing strains QA.R. English et al., Antimicrobial Agents and Chemotherapy, vol. 14, 1978, pp. 414-419, Aswapokee et al., J. Antibiotics, vol. 31(12), Dec. 1978, p. 1238-1244 as well as Derwenfs Farmdoc Abstracts 89627A and 73866B]].

B. Baltzer et al have in Mutual Pro-Drugs of B-Lactam Antibiotics and 8-Lactamase Inhibitors, J. Antibiotics, vol. 33(10), 1980, p. 1183-1192 described that the principle of combining an 8-lactam antibiotic with a 3-lactamase inhibitor in a single molecule, which functions as a pro-drug for the two active components, is illustrated by the bound esters 3 and 4, where respectively ampicillin and mecillinam are combined with the 6-lactamase inhibitor penicillin sulphone. It has been shown that these esters in humans are absorbed excellently from the gastrointestinal tract and after absorption are hydrolysed with simultaneous release of the active components. As a result of this, high blood-mirror and tissue values of antibiotic and 8-lactamase inhibitor are achieved in a balanced ratio. The advantages of "mutual pro-drugs" over simple combinations are discussed.

The esters 3 and 4 mentioned there have the structures

GB patent document 2,044,255 relates to hitherto unknown compounds with the general formula 1:

where is a phenyl, 4-hydroxyphenyl, 1,4-cyclohexadienyl or a 3-thienyl group, R 2 is a primary amino group or a carboxy group, R 3 is a hydrogen atom or a lower alkyl, aryl or aralkyl radical, and A is a radical of an 8-lactamase inhibitor that contains both a B-lactam ring and a carboxy group, and where A is bound via the carboxy group.

These new compounds are valuable in the treatment of bacterial infections and are particularly highly active against B-lactamase-producing bacteria. See also Farmdoc Abstracts 60773C and 60776C.

From example 8 - in Norwegian patent document 151,746-is-it-by hydrogenolysis of , 1-benzyl-6,6-dibromopenicillanate-1-a-oxide

known to produce penicillanic acid 1a-oxide. However, on the basis of this known technique, it cannot be expected that catalytic hydrogenation of a

(Substituted)benzyl 1-2-chloromethyl-2-methyl-6-bromopenam-3-carboxylate-1-oxide and subsequent oxidation of the obtained hydrogenated product produces the desired 2-ø-chloromethyl-2a-methyl-penam-3a-carboxylic acid sulfone with the correct stereochemical configuration. The starting material in the patent document contains a stable CH^ group in the 2-position of the ring, while the starting material according to the present invention contains an extremely labile and sensitive -CF^-Cl group in the 2-position, a group which surprisingly and unexpectedly survives both hydrogenolysis and oxidation. The advantage of achieving the compound with the correct stereochemistry is that it gives the molecule superior 6-lactamase inhibitory properties.

The method according to the invention is characterized by

that one in order

a) catalytically hydrogenates an ester with the formula

where R 1 is benzyl or substituted benzyl, and then

b) subjecting the hydrogenated product to oxidation to produce the desired acid or a salt thereof, and

then if desired

c) esterifies the acid or a salt thereof to form a physiologically hydrolysable ester of the acid.

The above pharmaceutically acceptable salts include non-toxic metal salts, such as sodium, potassium, calcium and magnesium salts, the ammonium salt and substituted ammonium salts, e.g. salts of non-toxic amines, such as trialkylamines (e.g. triethylamine), procaine, dibenzylamine, N-benzyl- -phenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, dehydroabiethylamine, N,N'- bis(dehydroabiethyl)ethylenediamine, N-(lower)alkylpiperidine (eg N-ethylpiperidine) as well as other amines which have been used to form pharmaceutically acceptable salts of penicillins and cephalosporins. The most preferred salts are the alkali metal salts, namely the sodium and potassium salts and the ammonium salt.

The term "physiologically hydrolyzable esters" refers here to such pharmaceutically acceptable esters which hydrolyze to the free acid form in vivo. Examples of suitable physiologically hydrolyzable esters are phenacyl, acetoxymethyl, pivaloyloxymethyl, a-acetoxyethyl, o-acetoxybenzyl, a-pivaloyloxyethyl, phthalidyl(3-phthalidyl), indanyl(5-indanyl), methoxymethyl, benzoyloxymethyl, a-ethylbutyryloxymethyl, propionyloxymethyl, valeryloxymethyl , iso-butyryloxymethyl, 6-C(R)-2-amino-2-phenylacetamido-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl and 6- [(R)-2-amino-2-p-hydroxyphenylacetamidoJ-3,3-dimethyl-7-oxo-4-thia-1-azabicycloE3.2.0jheptane-2-carbonyloxymethyl. The preferred esters are acetoxymethyl, pivaloyloxymethyl, methoxymethyl, phthalidyl, 5-indanyl, 6-[(R)-2-amino-2-phenylacetamido-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2 .0]heptane-2-carbonyloxymethyl and 6-[(R)-2-amino-2-p-hydroxyphenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0jheptane -2-carbonyloxymethyl.

Many different oxidizing agents can be used which are known for oxidizing sulfides to sulfones. However, particularly suitable reactants are alkali metal permanganates, e.g. potassium permanganate, and organic peracids, e.g. 3-chlorobenzoic acid.

Particularly useful benzyl groups are 4-nitrobenzyl. Benzyl and substituted benzyl groups can be conveniently removed by catalytic hydrogenation. In this case, a solution in an inert solvent of the compound is stirred or shaken under an atmosphere of hydrogen or hydrogen mixed with an inert diluent, such as nitrogen or argon, in the presence of a catalytic amount of a hydrogenation catalyst. Suitable solvents for this hydrogenation are lower alkanols, such as methanol, ethers, such as tetrahydrofuran and dioxane, low molecular weight esters, such as ethyl acetate and butyl acetate, water, as well as mixtures of these solvents. However, it is common to choose conditions where the starting material is soluble. The hydrogenation is usually carried out at a temperature in the range from 0 to 60° and at a pressure in the range from 1 to 100 kg/cm 2 . The catalysts used for this hydrogenation reaction are of the type known from this type of conversion, and typical examples are noble metals, such as nickel, palladium, platinum and rhodium. The catalyst is usually present in an amount of from 0.01 to 2.5% by weight, preferably from 0.1 to 1% by weight, based on the compound of formula A. It is often convenient to suspend the catalyst on an inert support. A particularly suitable catalyst is palladium suspended on an inert support, such as carbon. It is also common to buffer the reaction mixture in order to work at a pH value in the range from 4 to 9, preferably from 6 to 8. Borate and phosphate buffers are usually used. The reaction typically takes approx. 1 hour.

"Skellysolve B" is a petroleum ether fraction with a boiling point of 60-68°C which mainly consists of n-hexane.

The methods according to the invention will be explained in more detail in the following examples.

Example 1

Preparation of potassium-2-chloromethyl-2-methylpenam-3-carboxylate-sulfone (BL-P2013)

Example 2

Pivaloyloxymethyl- 2B - chloromethyl- 2:i - methylpenam- 3a- carboxylate- sulfone

2ε-Chloromethyl-2α-methylpenam-3α-carboxylate sulfone in dimethylformamide was treated with one equivalent of triethylamine and stirred to effect dissolution. Bromomethyl pivalate (1 equivalent) in dimethylformamide was then added. The resulting mixture was stirred at room temperature. The mixture was then clarified by filtration, and the filtrate was poured into ice water. The separated solid was collected by filtration, washed with water and dried to give the title ester.

The respective acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters of the same acid were prepared by replacing the bromomethyl pivalate used in the above-mentioned method with an equimolar amount of chloromethyl acetate, chloromethyl methyl ether, chloroacetone and phenacyl bromide, respectively.

Example 3

Pivaloyloxymethyl- 2g- chloromethyl- 2a- methylpenam- 3a- carboxylate sulfone BL- P2024

To a stirred suspension of 14.6 g (0.0487 mol) BL-P2013 (5) in 200 ml of acetone was added 4 ml of a 10% aqueous solution of sodium iodide, and the mixture was refluxed on a steam bath. To the suspension under re-cooling, 14.8 ml (0.1 mol) of redistilled chloromethyl pivalate (b.p. 34°C at 7 mm Hg) was added in one go. The mixture was stirred at reflux for 3 hours and then cooled to room temperature (22°C). The crystalline solids were collected by filtration, washed with 3 x 30 mL of acetone, and the combined filtrates were evaporated in an oil under vacuum at below 22°C. The oil was then taken up in 500 ml of ethyl acetate and washed once with 200 ml of water and once with saturated Na 2 SO 4 , while stirring with 2 g of decolorizing carbon while cooling (ice bath). After 20 minutes, the mixture was filtered through a diatomaceous earth ("Dicalite") pad with suction, and the pad was washed with 4 x 100 mL ethyl acetate. The combined filtrates were concentrated under vacuum at 22°C to an oil. The oil was then further concentrated at 22°C and <1 mm Hg to remove most of the remaining chloromethyl pivalate. The remaining oil was then treated twice with 50 ml portions of n-pentane and was stored over the weekend in a cold room (approx. 10°C) under n-heptane. The solid crystalline mass was then broken up to a solid powder with 40 ml of a 4:1 mixture of ether-n-pentane. The product was then collected by filtration, washed with ether-pentane (1:1), then pentane, after which it was air-dried. After drying under vacuum for four hours over P2°5, 13.37 g of pivaloyloxymethyl-28-chloromethyl-2a-methylpenam-3a-carboxylate-sulfone (approx. 75% yield) were obtained, m.p. 93-95°C.

Analysis calculated for C^I^qCINO^S :

C: 44.03, H: 5.27, N: 3.67,

Found: C: 44.11, H: 5.08, N: 3.85.

Example 4

Recrystallization of potassium- 28- chloromethyl- 2a- methylpenam- 3a-carboxylate- sulfone ( BL- P2013)

To a mixture of 20 ml n-butanol and 1 g BL-P2013 (5)

water was added, 1 ml at a time, with shaking in a separatory funnel until a pale yellow solution was obtained. The clear solution was filtered through a folding filter, and the flask and filter paper were washed with approx. 10 ml of 9:1 n-biethanol-H 2 O, and the combined filtrates were diluted with an additional 20 ml of n-butanol. The resulting solution was placed in a round-bottomed flask

a rotary evaporator and evaporated under reduced pressure to approx. half of the original volume. The snow white crystalline product was collected by filtration, washed with 6 x 10 ml of acetone and air dried, yield 810 mg. After vacuum drying for 6 hours over P2°5 at less than 1 mm Hg, 800 mg was obtained,

m.p. 215°C during cleavage (80% yield).

Analysis calculated for CgHgClN05SK.lH20:

C: 29.67, H: 3.39, N: 4.63, Cl: 10.94, K.F.H 2 O: 5.56,

Found:

C: 29.23, H: 3.38, N: 4.49, Cl: 10.74, K.F.H 2 O: 5.74.

This recrystallization process produces a crystalline monohydrate which is different from the starting material and which is largely anhydrous.

Example 5

See Chemical Abstracts, vol 27, 2427<1> and vol 22, 382<8>, as well as GB patent 299,064.

a) A solution of chloromethylchlorosulfate (0.115 mol) in 40 ml of dichloromethane was added dropwise, keeping the reaction temperature below 30°C, to a solution of compound 5 (0.1 mol) and potassium bicarbonate (0.3 mol) and tetrabutylammonium hydrogen- sulfate (0.01 mol) in 200 ml of dichloromethane-water (1:1). At the end of the addition, the mixture was stirred at room temperature for 30 minutes, the organic phase was separated, and the aqueous phase was extracted with 50 ml of dichloromethane. The combined organic phases were dried (Na 2 SO 4 ) and evaporated in vacuo to give a residue which was dissolved in 150 ml of ether. Insoluble material was filtered off after addition of diatomaceous earth, and the filtrate was evaporated under vacuum to form compound 7. b) To a suspension of 1.5 g of compound 5 in 12 ml of dimethylformamide, 1.6 g of bischloromethyl sulfate was added, and the mixture was stirred at room temperature for 45 minutes. After dilution with 50 mL of ethyl acetate, the mixture was washed with water and then aqueous sodium bicarbonate, dried and evaporated in vacuo, leaving compound 7 as an oil. c) To a solution of compound 5 (0.005 mol) in dimethylformamide (7.5 ml) was added triethylamine (0.007 mol) and chloroiodomethane (0.030 mol), and the mixture was stirred at room temperature for 4 hours. After dilution with 30 mL of ethyl acetate, the mixture was washed with 3 x 10 mL of water, followed by 5 mL of saturated aqueous sodium chloride, dried and evaporated in vacuo, leaving compound 7 as an oil. d) To a mixture of compound 5 (0.15 mol), silver nitrate (0.15 mol) and silver oxide (7.5 g) in 750 ml of acetonitrate, 1.5 mol of chloroiodomethane was added. After stirring for 48 hours at room temperature, the silver salts were filtered off, and the filtrate was evaporated to dryness under vacuum. The residue was dissolved in 200 ml of ethyl acetate, and the solution was washed with saturated aqueous sodium chloride, filtered, dried and evaporated under vacuum to give compound 7.

Compound 7 and other intermediates and final products of the present invention were purified, if desired, by column chromatography, as on "Sephadex" LH20 using e.g. chloroform-hexane 65:35 as eluent, or by silica gel chromatography, e.g. using "Mallinckrodt" CC-7 and hexane-ethyl acetate 3:2, or ethyl acetate-petroleum ether 8:2 or 7:3 or 1:9 or 15:85, or ethyl acetate-n-hexane 4:6 or 3:1 , hexane-ethyl acetate 3:1 or 1:1 or 1:4, or cyclohexane-ethyl acetate 1:1.

Thin layer chromatography is also useful. "Sephadex" is cross-linked dextran-2-(diethylamine)-ethyl-2-[[2-(diethylamino)-ethyl]-diethylamino]-ethyl ether chloride-hydrochloride-epichlorohydrin (see Merck Index, Ninth Edition No. 7337).

A solution of compound 7 (0.2 mol) and sodium iodide (0.3 mol) in acetone (150 mL) was stirred at room temperature for 18 h. The resulting suspension was cooled to approx. 0°C and set to approx. pH 7.2 by adding saturated aqueous sodium bicarbonate while stirring. After decolorization by titration with 0.5M aqueous sodium thiosulfate, 150 mL of water was added dropwise to the stirred mixture, whereby compound 8 precipitated and was collected by filtration, washed with acetone-water 1:1

(2 x 20 ml), isopropanol (2 x 20 ml) and ether (2 x 20 ml) and dried.

Ampicillin was converted to compound 9 using methyl acetoacetate according to the methods described in US Patent 3,316,247. To a stirred solution of compound 9 (0.57 mol) in dimethylformamide (1 liter) at 5°C was added 0.5 mol of compound 8. After stirring for 15 minutes at 5°C, the reaction mixture was poured into an ice-cold mixture of ethyl acetate (4 liters) and saturated aqueous calcium chloride (2 liters) with stirring. The organic layer was separated, washed with saturated aqueous calcium chloride (2 x 500 ml), filtered and evaporated to approx. 1 liter under vacuum, whereby a concentrated solution of compound 10 was obtained. To this concentrate was added water (500 ml) and n-butanol (500 ml), and then 4N hydrochloric acid dropwise with stirring, until the amino protecting group was removed , whereby a solution of compound 11 was obtained. After the addition of the acid was complete, ether (1 liter) and water (500 ml) were added to the stirred mixture, the aqueous phase was separated, and the organic phase was extracted with water (800 ml). The combined aqueous extracts were washed with ether (1 L), then sodium chloride (640 g) and dichloromethane (2 L) were added and the mixture was stirred for 15 minutes. The organic phase was separated and the aqueous phase was extracted with dichloromethane

(1 liter), and the combined organic extracts were dried with MgSO 4 and evaporated to approx. 600 ml under reduced pressure,.. whereby

a concentrated solution of compound 11 was obtained. Addition to the concentrate of 200 ml of 2-butanone followed by cooling led to precipitation of solid 6-£(R)-2-amino-2-phenyl-acetamido]-3,3-dimethyl -7-oxo-4-thia-1-azabicyclo£3.2. 0] heptane-2-carbonyloxymethyl-20-chloromethyl-2a-methylpenam-3a-carboxylate sulfone (11), which was collected by filtration.

Example 6

6- C( R)- 2- amino- 2- p- hydroxyphenylacetamido]- 3, 3- dimethyl- 7- oxo-4- thi- l- azabicyclo[ 3. 2. 03heptane- 2- carbonyloxymethyl- 2B- chloromethyl- 2g- methylpenam- 3a- carboxylate- sulfone with the formula

was prepared by replacing the ampicillin used in the method according to example 5 with amoxicillin.

Example 7

As described in US Patent 3,860,579, 50 g (0.375 mol) of recrystallized phthalide and recrystallized N-bromosuccinimide (0.375 mol) were refluxed for 4.5 hours in the presence of approx. 100 mg α-azobutyronitrile in 1 liter CC14- The mixture was cooled to approx. 15°C and filtered to remove succinimide, which itself had been washed with approx. 100 ml of CCl^ and filtered. The combined CCl 2 phases were concentrated under vacuum to ca. 150 ml, which gave solid 3-bromophthalimide, which was collected by filtration, washed with approx. 50 ml of CCl^ and air-dried, whereby 54 g of yield was obtained, which weighed 50 g after recrystallization from boiling cyclohexane, m.p. 84-86°C.

To a stirred partial solution and partial suspension of 2.3 g (0.0075 mol) of compound 5 in 20 ml of dimethylformamide (DMF, dried for at least 3 weeks over 3A° molecular sieves) was added 1.7 g (0.008 mol ) 3-bromophthalimide (12), and the mixture was stirred for 4 h at 22 C. The resulting mixture was poured into a mixture of 200 mL of ice-cold water and 200 mL of ice-cold ethyl acetate (while rinsing the flask with a little ethyl acetate), and the mixture was shaken . Then the organic solvent phase was separated and washed with seven portions of ice-cold water (100 ml). The ethyl acetate phase was washed once with sat. "Skellysolve B" (b.p. 60-68°C, mainly n-hexane) (25 ml)

and four times with 25 ml of n-hexane, whereby 2.5 g of compound 13 was obtained as a next white solid after drying in air. This product was then dried over P^^

below 1 mm Hg, whereby 2.5 g of compound 13 were obtained, m.p. 104°C during cleavage. Its purity was estimated at 85-95%.

Analysis calculated for C1(-H14C1NC>7S:

C: 51.61, H: 3.79, N: 3.77, Cl: 9.53,

Found: C: 52.59, H: 4.67, N: 3.21, Cl: 7.73,

K.F.H 2 O: 0.27.

Example 8

Pivaloyloxymethyl- 2B- chloromethyl- 2a- methylpenam- 3g- carboxylate- sulfone

A mixture of 1 g (0.0031 mol) of potassium 2B-chloromethyl-2a-methylpenam-3a-carboxylate sulfone hydrate and 1 g of 3A molecular sieves was stirred in 15 ml of dimethylacetamide for 2 hours at 23°C. To this mixture was added 470 mg (0.0031 mol) of pivaloyl oxymethyl chloride, and stirring was continued for 18 hours. The molecular sieves were collected and the filtrate was diluted with 100 ml of water and extracted with ethyl acetate. The ethyl acetate was washed nine times with water and dried over anhydrous magnesium sulfate. The solvent was removed at 30°C and 15 mm, leaving an oil which was chromatographed on silica using "silicar CO-7" (methylene chloride 8, ethyl acetate 2), as shown by a spot at Rf 0, 5. The residue obtained was crystallized from heptane ("Skellysolve B"), whereby a yield of 100 mg was obtained

(m.p. 94-95°C) pivaloyloxymethyl-2B-chloromethyl-2a-methylpenam-3a-carboxylate sulfone.

Analysis calculated: C: 44.03, H: 5.27, N: 3.67,

Found: C: 44.20, H: 5.24, N: 3.63.

The NMR and IR spectra were consistent with the structure.

Example 9

Sodium- 2B- chloromethyl- 2g- methylpenam- 3a- carboxylate- sulfone

To a stirred solution of 500 mg of the potassium salt of BL-P2013 in 5 ml of water and 10 ml of ethyl acetate was added

2N HCl, until pH 1 was reached (in an ice bath with vigorous stirring). The mixture was then saturated with Na2SO4, the aqueous layer was separated, and the organic phase was dried briefly in ice over Na2SO4, filtered and treated dropwise with 50% NaEH (sodium 2-ethylhexanoate) in anhydrous n-butanol until neutral too moist pH paper. The product did not crystallize on scraping and was then concentrated under vacuum to an oil, which was dissolved in 5 ml of acetone, scraped - no crystals, ether was added to the cloud point - no crystals. It was concentrated under vacuum on a rotary evaporator to an oil, which was dissolved in ethyl acetate after which one drop of H 2 O was added, scraped - no crystals. It was concentrated under vacuum and the residue treated with 5 ml of n-butanol to give 200 mg of amorphous white powder, which was washed with ether, air dried and vacuum dried over P 2 O 5 for 24 hours. A final yield of 180 mg of sodium 2B-chloromethyl-2a-methylpenam-3a-carboxylate-sulfone was obtained, which decomposed at above 100°C indefinitely.

Analysis calculated for CgHgClNC^SNa:

C: 33.10, H: 3.13, N: 4.89,

Found: C: 33.20, H: 3.69, N: 4.44, K.F.H 2 O: 4.04.

Example 10

Potassium- 2g- chloromethyl- 2- methylpenam- 3- carboxylatesulfon ( BL- P2013).

To 10 liters of water, 130 g (1.25 mol sodium bicarbonate and 200 g 10% Pd on BaSO 4 ) was added 272 g (0.565 mol) p-nitrobenzyl-6a-bromo-2g-chloromethyl-2-methylpenam-3-carboxylate- sulfone dissolved in 5 liters of ethyl acetate. The mixture was hydrogenated at 40°C and a pressure of 1 kg. After 5 hours, the hydrogen uptake became very slow, and 200 g of 10% Pd on BaSO 4 were added, and the mixture was hydrogenated until no further significant hydrogen absorption was observed.

The slurry was filtered through a diatomaceous earth pad ("Celite"), the pad was washed with water, and the aqueous phase was washed with 3 liters of ethyl acetate. 3 liters of ethyl acetate were added to the aqueous solution, and the pH value of the mixture was adjusted to 1.5 with 150 ml of 12N HCl at 10°C. The organic phase was separated, and the aqueous solution was saturated with Na 2 SO 4 10 H 2 O and extracted with 2 x 1 liter of ethyl acetate. The combined extracts were dried with magnesium sulfate. The desiccant was removed,

and 260 ml of 2N potassium 2-ethylhexanoic acid in butanol was added at 0°C.

After stirring for 2 hours at 0°C, potassium 2g-chloromethyl-2-methylpenam-3-carboxylate-sulfone (BL-P2013) was collected and dried under vacuum at room temperature.

Yield: 134.8 g (approx. 70%).

Example 11

p-nitrobenzyl-6a-bromopenicillinate sulfoxide

To 200 ml of N,N-dimethylacetamide was added 44 g (0.148 mol) of 6α-bromopenicillanic acid sulfoxide followed by 20.5 ml (0.148 mol) of triethylamine and 38.2 g (0.177 mol) of p-nitrobenzyl bromide. It was stirred at 22 °C for 20 hours.

The reaction mixture was poured into 1 liter of r 2 O and extracted

in 3 x 300 ml methylene chloride. The combined methyl chloride extracts were washed with 200 ml of 5% aqueous sodium bicarbonate solution and dried over sodium sulphate at 5°C for 1 hour. The solution was filtered and evaporated under vacuum to a residue. The residue was diluted with ether, and the solid was collected by filtration in a yield of 54 g of p-nitrobenzyl-6a-bromopenicillinate sulfoxide after drying. Yield 85%. NMR was consistent with the structure.

The yield in this step was the same as for the K-salt esterification. The advantage was that it was not necessary to prepare the K salt, a step which runs with a yield of 85-90%.

Example 12

Preparation of p-nitrobenzyl-6a-bromopenicillanate sulfoxide

To 4.375 liters of N,N-dimethylacetamide was added 873.0 g (2.95 mol) of 6α-bromopenicillanic acid (S)-sulfoxide, and then, with stirring and while maintaining the internal temperature below 35 C, 293 g (2.95 mol) of triethylamine followed by 764 g (3.54 mol of p-nitrobenzyl bromide. The mixture was then stirred at room temperature for 5 hours and allowed to stand overnight.

The reaction mixture was poured into 20 liters of water and extracted with 3x7 liters of methylene chloride. The combined organic extracts were washed with 5x7 liters of water and then with 7 liters of 5% aqueous sodium bicarbonate solution and dried over magnesium sulfate.

The magnesium sulfate was filtered off, and the solution was evaporated to a crystalline residue. 4 liters of diethyl ether added and the crystal collected, yielding after drying at room temperature 1171 g (92%) of p-nitrobenzyl-6a-bromopenicillanate sulfoxide.

Br: 18.48% (calcd. 18.53%1, aD (0.25% MeOH) + 162°.

Example 13

Preparation of p- nitrobenzyl- 6a- bromo- 2B- chloromethyl- 2- methylpenam-3- carboxylate- sulfone

To 16 liters of acetic acid was added 364.6 g (0.812 mol p-nitrobenzyl-6a-bromo-20-chloromethyl-2-methylpenam-3-carboxylate. To the resulting solution, which was stirred at room temperature, was added a solution of 282 g (1.78 mol) KMnO^ in 26 liters of water dropwise over 3 hours. The mixture was then stirred at room temperature for 1 hour, and I^C^ (37 percent) was added dropwise until a colorless solution Then 30 liters of water was added, the mixture was stirred

for 1 hour at room temperature, and the crystalline precipitate was collected, washed with 3 x 5 liters of water and 2 x 2 liters of ethanol and dried under vacuum at room temperature.

Yield: 297 g (76%). αQ (0.5% CH 2 Cl 2 ) + 75.9°.

Example 14

Preparation of BL-P2013 free acid

To a mixture of 25 ml of ethyl acetate and 10 ml of water was added 800 mg (0.00261 mol) of BL-P2013 potassium salt. After all the solid had dissolved, the mixture was treated dropwise with 50 percent aqueous phosphoric acid with vigorous shaking until no more material precipitated from the aqueous layer. The ethyl acetate layer was separated, then washed with saturated sodium chloride solution and dried over anhydrous magnesium sulfate. The desiccant was removed by filtration and washed with 10 ml of ethyl acetate. The washing liquid was combined with the original filtrate. "Skellysolve B" was then added to the ethyl acetate until the point of cloudiness (about 10 ml). The mixture was treated with 500 mg of activated carbon ("Darko KB") and filtered. The filtrate was diluted with 15 ml "Skellysolve B" and then seeded with crystals of BL-P2013

free acid. After approx. After 3 hours at room temperature, the crystalline precipitate of free acid was collected and dried under vacuum (15 minutes) over P2°5' to give 323 mg (46%), which was slowly decomposed over 100°C.

Analysis calculated for CgH1QClN05S:

C: 35.89, H: 3.77, N: 5.23, Cl: 13.25, Found: C: 35.88, H: 3.91, N: 5.41, Cl: 13.52.

This product was found to be unstable when stored at 23°c for seven days.

Example 15

6ot - bromopenicillanic acid - sulph oxide

To 3 liters of methylene chloride was added 300 g (0.75 mol) 6a-bromopenicillanic acid-N,N'-dibenzylethylenediamine salt, and this suspension was cooled to 5°C. 130 ml of concentrated HCl were then added dropwise over the course of 15 minutes and with good stirring. The slurry was stirred at 5°C for 2 hours. It was then filtered through a pad of diatomaceous earth ("Celite") and the cake was washed with 3 x 250 ml of methylene chloride.

The combined methylene chloride solutions were washed with 2 x 500 mL water and dried over sodium sulfate for 15 minutes. The sodium sulfate was removed by filtration, and the filtrate was evaporated under reduced pressure to approx. 750 ml.

This solution was cooled to 5°C, and with vigorous stirring, 130 ml of 40 percent peracetic acid was added dropwise, so that the temperature was maintained at 5-12°C. The addition was fairly exothermic. After the addition was complete, the slurry was stirred at 5°C for 2 hours, and the product was collected by filtration and washed with 100 mL of cold water (5°C)

and 100 ml of cold methylene chloride (5°C). 126 g were obtained

(57%) 6α-bromopenicillanic acid sulfoxide, m.p. 129°C. The IR and NMR spectra were consistent with the desired product.

Analysis calculated for CgH^gBrNC^S:

C: 32.44, H: 3.40, N: 4.73, Found: C: 32.30, H: 3.35, N: 4.71, H 2 O: 2.18. Callum-6a-bromopeniclllanate-sulphoxide

To 3 liters of acetone were added 126 g (0.43 mol) of 6ct-bromopenicillanic acid sulfoxide and 16 2 ml of 50% by weight potassium-2-ethylhexanoic acid in n-butanol. After stirring for 1 hour at 22°C, the product was collected by filtration, washed with 2 x 250 ml of acetone and dried. 127 g (90%) of potassium 6a-bromopenicillanate sulfoxide were obtained, m.p. 185°C. The IR and NMR spectra were consistent with the desired product.

Analysis calculated for CgHgBrKNO^S:

C: 28.75, H: 2.71, N: 4.19,

Found: C: 29.03, H: 2.78, N: 4.04.

p- nitrobenzy1- 6g- bromopenicillanate- sulfoxide

To 1 liter of N,N-dimethylacetamide, 145 g (0.43 rapi) of potassium 6a-bromopenicillanate sulfoxide was added, and with stirring, 115 g (0.53 mol) of p-nitrobenzyl bromide were added at 22°C. The mixture was stirred at 22°C for 20 hours.

The reaction mixture was poured into 3 liters of water and extracted with 3 x 1500 ml of ethyl acetate. The combined ethyl acetate extracts were washed with 2 x 500 ml of 5% aqueous sodium bicarbonate solution and dried over sodium sulfate for 1 hour. The sodium sulfate was filtered off, and the filtrate was evaporated under reduced pressure to a residue, to which was added 1 liter of diethyl ether, some

which caused crystallization of the product. The crystals were collected by filtration, washed with 2 x 100 ml of diethyl ether and dried to yield 162 g (87%) of p-nitrobenzyl-6a-bromopenicillanate sulfoxide, m.p. 111°C. The IR and NMR spectra were consistent with the structure of the desired product.

Analysis calculated for C-j^H<->^<B>rt^OgS:

C: 41.78, H: 3.51, N: 6.50,

Found: C: 41.66, H: 3.45, N: 6.85, H 2 O: 0.69.

p- nitrobenzyl- 6a- bromo- 2B- chloromethyl- 2- methyl penam- 3- carboxylate

To 1 liter of p-dioxane was added 70 g (0.16 mol) of p-nitrobenzyl-6a-bromopenicillanate sulfoxide, followed by 21.2 ml (0.10 mol) of benzoyl chloride and 21.8 ml (0.19 mol ) quinoline. The reaction mixture was refluxed for 4 hours and then cooled to 22°C, poured into 2500 ml of water and extracted into 3 x 800 ml of ethyl acetate. The combined ethyl acetate extracts were washed with 300 ml of 5% aqueous sodium bicarbonate solution, 300 ml of 5% aqueous phosphoric acid and 300 ml of water. The ethyl acetate solution was dried over sodium sulfate for \ hour,

and the sodium sulfate was removed by filtration. The filtrate was evaporated under reduced pressure to a residue which was dissolved again in 1 liter of ethyl acetate and again evaporated under reduced pressure to a residue. Then 1 liter of diethyl ether was added, and the product was collected by filtration in a yield of 41 g (57%)

p-nitrobenzyl-6a-bromo-2B-chloromethyl-2-methylpenam-3-carboxylate, m.p. 132°C. The IR and NMR spectra were consistent with the desired structure.

Analysis calculated for C-^H^BrClt^O^S:

C: 40.06, H: 3.14, N: 6.23,

Found: C: 40.62, H: 3.11, N: 6.13.

p- nitrobenzyl- 6a- bromo- 2g- chloromethyl- 2- methylpenam- 3- carboxylate sulfoxide

To 1200 ml of methylene chloride was added 51 g (0.11 mol) of p-nitrobenzyl-6a-bromo-2B-chloromethyl-2-methylpenam-3-carboxylate, followed by 23 g (0.12 mol) of m-chloroperoxybenzoic acid. The solution was stirred at 22°C for 2 hours and evaporated under reduced pressure to a wet residue. The residue was stirred with 4 liters of diethyl ether for 1 hour and was left at 10°C for 20 hours. The product crystallized and was collected by filtration, washed with 2 x 200 mL of diethyl ether and dried, yielding 39 g of p-nitro-benzyl-6α-bromo-28-chloromethyl-2-methylpenam-3-carboxylate sulfoxide ( 75%), m.p. 132°c. The IR and NMR spectra were consistent with the desired structure.

Analysis calculated for C15H14BrClN20gS:

C: 38.69, H: 3.03, N: 6.07,

Found: C: 38.98, H: 3.04, N: 5.84, H 2 O: 0.35.

Potassium- 2B- chloromethyl- 2- methylpenam- 3- carboxylate- sulfone ( BL- P2013)

To 600 ml of water was added 8 g of 30% Pd on "Celite"

and 16 g (0.19 mol) of sodium bicarbonate. Then, 32 g (0.69 mol) of p-nitrobenzyl-6a-bromo-2B-chloromethyl-2-methylpenam-3-carboxylate sulfoxide was dissolved in 400 ml of ethyl acetate and added to the aqueous slurry. The mixture was hydrogenated in a Paar apparatus at 3.52 kg/cm<2> at 22°C for 4 hours. The slurry was filtered through a thin "Celite" pad on a sintered glass funnel. The pad was washed with 2 x 50 mL H 2 O, and the aqueous layer of the combined filtrate and washings was separated. The aqueous layer was washed with 200 ml of diethyl ether, then cooled to 5°C, and with stirring a solution of 12 g (0.076 mol) KMnO^ in 200 ml of water was added dropwise over a period of \ hour, the pH -value was kept at between 7.5 and 8.0 by adding 40 percent H3P04. After the light-red color had lasted for 5 minutes, the addition of KMnO 4 solution was stopped. The reaction mixture was stirred with a little (about 50 mg) sodium bisulfite for 1 hour, then the slurry was filtered through a Celite pad. The pad was washed with 2 x 50 ml water. The combined filtrate and washings were partitioned with

500 ml of ethyl acetate, and with stirring the pH value was adjusted to 1.5 by adding 2N HCl. The layers were separated and the aqueous layer was saturated with sodium sulfate. It was extracted again with 2 x 400 ml ethyl acetate, and the combined ethyl acetate extracts were dried over sodium sulfate for 1 hour at 5°C. The sodium sulfate was removed by filtration, and the filtrate was evaporated under reduced pressure to a residue. This residue was dissolved in 160 ml of acetone and 160 ml of diethyl ether, and 50% by weight of potassium 2-ethyl hexanoate in n-butanol was added until the solution was neutral to moist pH paper. The potassium salt of BL-P2013 crystallized out, was collected by filtration, washed with diethyl ether and dried.

The yield was 16 g of potassium 2B-chloromethyl-2-methylpenam-3-carboxylate-sulfone (BL-P2013) (76%), m.p. 202°C. The IR and NMR spectra were consistent with the desired structure.

Analysis calculated for CgHgClKN05S:

C: 31.42, H: 2.97, N: 4.58,

Found: C: 31.18, H: 2.98, N: 4.51, H 2 O: 0.93.

Example 16

Pivaloyloxymethyl- 2g- chloromethyl- 2- methylpenam- 3- carboxylate-sulfone ( BL- P2024)

4 ml of a 10% aqueous solution of sodium iodide, and the mixture was brought to reflux on a steam bath. To this suspension, which was refluxed, 14.8 ml (0.1 mole) of redistilled chloromethyl pivalate (b.p. 34°C at 7 mm Hg) was added in one portion. The mixture was stirred under reflux for 3 hours and then cooled to room temperature (22°C). The crystalline solids were collected by filtration, washed with 3 x 30 mL of acetone, and the combined filtrates were evaporated to an oil under reduced pressure at below 22°C. The oil was then taken up in 500 ml of ethyl acetate and washed once with 200 ml of water and once with 200 ml of saturated Na^O^ solution. The solution was then dried briefly over Na^O^ while stirring with 2 g of decolorizing carbon while cooling (ice bath). After 20 minutes, the mixture was filtered through a Celite pad and the pad was washed with 4 x 100 mL of ethyl acetate. The combined filtrates were concentrated under reduced pressure at 22°C to an oil. The oil was then further concentrated to approx. 22°C and less than 1 mm Hg to remove most of the remaining chloromethyl pivalate. The remaining oil was then treated twice with 50 ml portions of n-pentane and was then stored over the weekend at approx. 10°C under n-pentane. The resulting solid crystalline mass was then broken up to a powder with 40 ml of a 4:1 mixture of diethyl ether-n-pentane. The product was collected by filtration, washed with diethyl ether-n-pentane (1:1), then n-pentane and air-dried. After drying under high vacuum for 4 hours over P2°5, 13.37 g (approx. 75%) of pivaloyloxymethyl-20-chloromethyl-2-methylpenam-3-carboxylate-sulfone (BL-P2024), m.p. 93-95°C.

Purification of BL-P2024

About. 3 g of crude BL-P2024 (obtained as described above) was dissolved in 5 ml of ethyl acetate, applied to a 4.5 x 40 cm column of silica gel ("Mallinckrodt CC-7") and eluted with CH2Cl2-ethyl acetate in a 4:1 volume ratio . The fractions, which contained a single spot at R 0.84 (thin layer chromatography on silica gel plates with 4:1 CH 2 Cl 2 -ethyl acetate, I 2 detection). were combined and concentrated under reduced pressure to 1.38 g of a crystalline solid. A portion of this material (900 mg) was dissolved in 5 ml of ethyl acetate. The resulting solution was filtered, diluted almost to the cloud point with petroleum ether ("Skellysolve B") and then stored at room temperature for 3 days.

The crystals that formed were collected by filtration, washed with petroleum ether and dried, whereby 560 mg were obtained, m.p. 100-101°C, purified BL-P2024.

Analysis calculated for C^<H>^<C>lNO^S:

C: 44.03, H: 5.27, N: 3.67,

Found: C: 44.11, H: 5.08, N: 3.85.

Example 17

Preparation of the ammonium salt of BL-P2013

1. The free acid of BL-P2013 (250 ml) dissolved in 20 ml of acetone-methanol at a ratio of 1:1 by volume was filtered to produce a clear solution.

2. Anhydrous ammonium solution was prepared by addition

of 1 ml of ammonium hydroxide (30 per cent, reagent grade) to

10 ml acetone-methanol (1:1 volume) solvent, and then

1 g of anhydrous magnesium sulfate was added to this solution with gentle stirring, and the mixture was filtered through filter

paper. The filtrate was termed "anhydrous ammonium solution".

3. To the filtrate in step 1, 2 ml of "anhydrous ammonium solution" was gradually added and it was mixed well. 4. A 100 ml portion of diethyl ether was mixed with the mixture from step 3 to precipitate the ammonium salt of BL-P2 013.

5. The white ammonium salt was isolated from the solvent

and washed with 2 portions of 50 ml each of diethyl ether.

6. The isolated powder was dried at 35°C in a vacuum oven overnight.

7. Analysis data were as follows:

Calculated % C: 33.7, H: 4.6, N: 9.8,

Found: C: 33.66, H: 4.63, N: 10.12, dry at KF. Microscopic examination: Crystalline substance.

Example 18

Preparation of non-hygroscopic sodium salt of BL-P2013

1. 50 mg of the free acid of BL-P2013 was dissolved in a mixture of acetone and methanol in the volume ratio 1:1. It was filtered to produce a clear solution. 2. Sodium 2-ethyl hexanoate solution was prepared by dissolving 40 mg of sodium 2-ethyl hexanoate in 10 ml of one mixture of acetone and methanol in the volume ratio 1:1. 3. To the filtrate in step 1, 10 ml of the solution according to step 2 was added, and it was mixed well. 4. A 10 ml portion of diethyl ether was mixed with the mixture of step 3 to produce precipitation of the sodium salt of BL-P2013. 5. The white salt was immersed in the diethyl ether for 1-2 hours and then isolated from the solvent and washed with three portions of 5 ml each of diethyl ether. 6. The isolated powder was dried at 30°C in a vacuum oven overnight.

Example 19

Recrystallization of BL-P2013

400 mg of BL-P2013 was dissolved in a minimal amount of a mixture of acetone and water in the volume ratio 1:1, and diluted with 10 ml of acetone, filtered and then diluted with acetone to approx. 25 ml, and scraped, and after 30 minutes the crystalline hydrate was collected by filtration, washed well with acetone, air dried and then vacuum dried at less than 1 mm Hg overnight. Yield 280 mg.

Analysis calculated for CgHgClNOSK"H20:

C: 29.67, H: 3.39, N: 4.63, Cl: 10.94, H 2 O: 5.55, Found: C: 29.32, H: 3.32, N: 4.44, Cl: 11.31, H 2 O: 5.90.

Example 20

N,N'-dibenzylethylenediamine salt of BL-P2013

BL-P2013 + 1/2 N,N'-dibenzylethylenediamine diacetate

306 mg (0.001 ml) of BL-P2013 was dissolved in 7 ml of water and added to a solution of 180 mg (0.0005 mol) of N,N'-dibenzylethylenediamine diacetate in 7 ml of water. The mixture was stirred, and the salt crystallized, and after stirring for approx. After 10-15 minutes the salt was collected by filtration and air dried to give 300 mg of the N,N'-dibenzylethylenediamine salt of BL-P2013. The material was re-crystallized by solution for approx. 10 ml of boiling acetone and dilution

with ether to the cloud point. 260 mg of air-dried and vacuum-dried material was obtained.

Analysis calculated:

C: 51.69, H. 5.42, N: 7.53, Cl: 9.55,

Found:

C: 49.39, H: 5.49, N: 7.05, Cl: 8.96, H 2 O: 1.23 (KF).

Example 21

Chloromethyl ester of BL- P2 013

To a vigorously stirred mixture of 15.25 g (0.05 mol) BL-P2013 (5), 15 g (0.15 mol) KHCO 3 , and 1.7 g (0.005 mol) tetra-butylammonium hydrogen sulfate in a mixture of 50 ml water and 50

ml CH2C12, a solution of 9.5 g (0.0575 mol) ClCH2-0-SO2Cl in 40 ml CH2C12 was added dropwise. The temperature rose to 26°C,

and after the addition, which took approx. 15 minutes, the mixture was stirred for an additional 30 minutes. Because the product crystallized, more was added, approx. 400 mL, CH 2 Cl 2 to produce a solution. The separated CH 2 Cl 2 layer and a 50 mL CH 2 Cl 2 wash were combined, dried over MgSO 4 with stirring,

and 2 g of decolorizing carbon ("Darco KB") was added. After approx. 30 minutes, the mixture was filtered and concentrated to approx. 50 ml, and 150 ml of isopropyl alcohol was added. Then the rest of the CH2Cl2 was removed under reduced pressure. The resulting crystalline precipitate was collected by filtration, washed well with isopropyl alcohol and air dried. After vacuum drying at less than 1 mm Hg, 8.5 g of chloromethyl-2B-chloromethyl-2-methylpenam-3-carboxylate-sulfone (71, m.p. 116°C during cleavage, darkens above 100°C.

Analysis calculated for CgH^Cl2N05S:

C: 34.18, H: 3.51, N: 4.43, Cl: 22.43,

Found: C: 34.16, H: 3.45, N: 4.47, Cl: 22.46, H 2 O: 0.33 (KF). Estimated purity in the range of 90-95%.

Iodomethyl ester of BL-P2013

To a stirred mixture of 5 g (0.0159 mol) of chloromethyl ester of BL-P2013 (7) in 25 ml of acetone was added 3 g (0.02 mol) of sodium iodide. The resulting slurry was stirred for 17 hours and then cooled to approx. 0°C. Two drops of saturated aqueous KHCO 3 were added and the mixture was slowly diluted dropwise with water over 10 minutes until 50 ml had been added. The slurry underwent a sudden color change from yellow to gray to violet to black, and the crystals were therefore immediately collected by filtration and washed with cold acetone-water (1:2) then with isopropyl alcohol (3 x 10 mL), then diethyl ether and finally with n-pentane, and air-dried, whereby a yield of 5.55 g (91% yield) of the iodomethyl ester of BL-P2013 (8) was obtained. Temp. 118-119°C during cleavage. The purity was estimated at approx. 90%.

6- C( R) - 2- amino- 2- phenylacetamidoJ - 3, 3- dimethyl- 7- oxo- 4- thia- l-azabicyclo[ 3. 2. 0] heptane- 2- carbonyloxymethyl- 2g- chloromethyl- 2a -methylpenam- 3a- carboxylate- sulfone ( 11)

To a stirred mixture cooled in an ice bath of 5.46 g (0.01 mol) of the indicated "Dane" salt of ampicillin (9), which is solvated with one molecule of isopropyl alcohol, in 60 ml of acetone was added 4 .08 g (0.01 mol) of the iodomethyl ester of BL-P2013 (8), and the resulting almost clear solution was stirred for 5 h, the ice bath being removed after 30 min. Then most of the acetone was removed under vacuum on a rotary evaporator, and the resulting concentrated solution was dissolved in 200 mL of cold ethyl acetate, which was then washed with 2 x 50 mL of ice-cold water and 2 x 100 mL of saturated aqueous Na 2 SO 4 . The ethyl acetate solution was then dried over Na 2 SO 4 and filtered, and most of the ethyl acetate was removed under vacuum on the rotary evaporator. The residue was treated with 2 x 200 ml of dry diethyl ether and the resulting solids were collected by filtration to give 5.5 g (10) as a bright red powder. This powder was stirred in a mixture of 50 ml of water, 50 ml of n-butanol and 20 ml of ethyl acetate, while 6N HCl was added dropwise to a pH value of 2.5. Then one or two drops of HCl were added occasionally to maintain the pH value at 2.2-2.5 during 45 minutes. After the pH no longer increased, 100 ml of diethyl ether was added to this mixture with good stirring. The aqueous phase was separated and combined with a another 2 5 ml aqueous extract of the organic layer. The aqueous solution was extracted once with 50 mL of diethyl ether and the ether was discarded.

The aqueous layer was then stirred vigorously under a layer of 100 mL of 2-butanone (methyl ethyl ketone), while Na 2 SO 3 was added to saturate the aqueous layer. The 2-butanone layer was separated, dried over Na 2 SO 4 for 30 min in an ice bath, filtered and concentrated under vacuum to near dryness. The remaining oil was treated to a solid with n-butanol, washed well with ether, then with n-pentane, air-dried and then vacuum-dried over P2^5 at a pressure of less than 1 mm Hg to give a yield of 1.6 g of 6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonylmethyl -23-chloromethyl-2a-methylpenam-3a-carboxylate-sulfone (11) in crude form. The IR and NMR spectra were consistent with structure 11 but not with high purity. It was estimated that this solid product contained at least 40%, perhaps as much as 80% of 6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1 -azabicyclo[3.2.0]heptane-2-carbonylmethyl-28-chloromethyl-2a-methylpenam-3a-carboxylate-sulfone.

Example 22

Improved synthesis of BL-P2013

This method simplifies the preparation of BL-P2013 by eliminating the previous use of catalytic reduction.

(see p. 633 in "Cephalosporins and Penicillins", edited by Edwin H. Flynn, Academic Press, New York, 1972). 30 g (0.1 mol) of 6α-bromopenicillanic acid sulfoxide (1) was dissolved in 1 liter of dry CH 2 Cl 2 followed by the addition of 16.2 ml (0.2 mol) of pyridine and 29.8 (0.2 mol) of trichloroethanol. Then 20 g (0.1 mol) of dicyclohexylcarbodiimide was added, and the mixture was stirred at 22°C for 16 hours. Dicyclohexylurea began to precipitate, at the end it was removed by filtration. The filtrate was washed with 200 ml of 5 percent aqueous sodium bicarbonate, 200 ml of 10 percent phosphoric acid and 100 ml of saturated aqueous sodium sulfate. The organic phase was dried over sodium sulfate at 5°C for 30 minutes, filtered and evaporated to an oil. Diethyl ether was added, and during scraping 27 g (63% yield) of product 2 crystallized. 26.5 g (0.062 mol) of compound 2 was dissolved in 500 mL of p-dioxane, and 8.5 mL (0.078 mol ) benzoyl chloride and 8.75 ml (0.078 mol) quinoline. The solution was refluxed for 4 hours and then poured into 1100 ml of water, and the product 3 was extracted into 2 x 400 ml of ethyl acetate. The ethyl acetate extracts were combined and washed sequentially with 200 ml of 5% aqueous sodium bicarbonate, 200 ml of 5% phosphoric acid and 200 ml of saturated aqueous sodium sulfate, dried over sodium sulfate at 5°C for 30 minutes and evaporated to an oil (3), which was used as such in the next reaction.

Compound 3 obtained in the previous step was resolved in

1 liter of glacial acetic acid, and while stirring at 22°C, a saturated aqueous solution of KMnO^ was added dropwise, until a pink color was stable, i.e. a drop placed on a piece of filter paper gave a pink color. During cooling, 30 percent H 2 O 2 was then added dropwise until a clear solution was obtained. Some white precipitate was present. The solution was poured into 2.5 liters of water, and the product 4 was extracted into 3 x 500 ml of ethyl acetate. The ethyl acetate was washed with 5% aqueous sodium bicarbonate until neutral, i.e. addition no longer caused bubbling, dried over sodium sulfate and evaporated, leaving 4 as a residue. This was left at 10°C for one day and was then treated with "Skellysolve B", whereby a yield of 9.1 g of solid 4 was obtained. The yield was 28% of the theoretical for steps 2 and 3 combined.

(see US Patent 4,164,497).

3.75 g of zinc powder was suspended in 5 ml of glacial acetic acid and cooled to 5°C. To this mixture was added a solution of 3 g (0.005 7 mol) of compound 4 in 15 ml of dimethylformamide, and the resulting slurry was stirred at 5°C for 2.5 hours.

The zinc was then filtered off, and the pale yellow solution was poured into 80 ml of 5 per cent aqueous hydrochloric acid. This mixture was extracted with 3 x 25 ml of ethyl acetate. The combined ethyl acetate extracts were extracted with 3 x 20 ml of 5% aqueous sodium bicarbonate, and the ethyl acetate phase was saved after f raski Helsen.

The bicarbonate extracts were combined, layered with ethyl acetate, adjusted to pH 1.5 by addition of 2N HCl and saturated with sodium sulfate. The ethyl acetate was separated, and the aqueous phase was extracted with 2 x 30 ml of ethyl acetate.

All of the above ethyl acetate phases were combined, dried over sodium sulfate and evaporated to an oil, which was the free acid form of BL-P2013, and which was dissolved in approx. 20 ml of acetone, to which 20 ml of diethyl ether was then added. Then 50 percent potassium 2-ethylhexanoate (KEH) in dry n-butanol was added to a neutral state. The product 5 (BL-P2013) crystallized out. After stirring at 22°C, it was collected by filtration, whereby a yield of 650 mg of compound 5 (37% yield) was obtained.

A 50 mg sample of compound 5 was dissolved in 0.5 ml of water, and 20 mg of N,N'-dibenzylethylenediamine (DBED) diacetate was added. The DBED salt of 5 crystallized, was collected by filtration, washed with water and dried over P2°5 under vacuum to give N,N'-dibenzylethylenediamine-28-chloromethyl-2-methylpenam-3-carboxylate sulfone (the DBED salt of the free acid 5).

Another sample of 5 (450 mg) was dissolved in 3 ml of water to which was added a solution of 270 mg of DBED diacetate in 2 ml of water. During scraping, the DBED salt of 5 crystallized (430 mgl. Recrystallization ion from ca. 5 ml boiling acetate gave a yield of

270 mg.

Example 23

6- [( R )- 2- amino- 2- p- hydroxyphenylacetamido- 3, 3- dimethyl- 7- oxo-4- thi- l- azabicyclo [ 3. 2. 0] heptane- 2- carbonyloxymethyl- 2B- chloromethyl -2g- methylpenam- 3a- carboxylate- sulfone with the formula

was prepared instead of the ampicillin used by the method in Example 21 using the corresponding "Dane" salt of amoxicillin.

Biological data

The product of Example 1, compound 5 with the structure

will hereafter be referred to as BL-P2013.

Although BL-P2013 is at best a very weak antibacterial agent by itself, it inhibits 8-lactamases and protects ceforanid and amoxicillin from destruction by γ-lactamase-producing bacteria in vitro and in vivo when used in combination with these two agents .

The untreated compounds are thus valuable, administered orally and parenterally, for improving the effectiveness of 8-lactam antibiotics against 8-lactamase-producing bacteria. On a weight basis, the dosage is from 1/5 to 5 times, preferably equal to, the dosage of the 6-lactam antibiotic. E.g. the compounds in question as shown above used in a 1:1 ratio markedly improved the activity of ceforanid and amoxicillin against 8-lactamase-producing strains of anaerobic bacteroides, such as B. fragilis, B. thetaiotaomicron and other species of this genus and also against resistant Staphylococcus aureus. The compounds in question are administered either in mixture with or simultaneously with the 8-lactam antibiotic at a dose within the indicated ratio together with the known or usual dose of the antibiotic.

The ability of the compounds to increase the effectiveness of a B-lactam antibiotic against certain ε-lactamase-producing bacteria thus makes them valuable for administration together with certain 8-lactam antibiotics for the treatment of bacterial infections in humans and mammals. In the treatment of a bacterial infection, a compound according to the invention can be mixed with the 8-lactam antibiotic, and the two agents are administered simultaneously. Alternatively, a compound according to the invention can be administered as a separate agent during a course of treatment with a B-lactam antibiotic.

Claims (3)

1. Process for the preparation of 2B-chloromethyl-2a-methylpenam-3<*-carboxylic acid sulfon with the formula a pharmaceutically acceptable salt of the acid or a physiologically hydrolysable ester of the acid, characterized in that in order a) catalytically hydrogenates an ester with the formula where R"*" is benzyl or substituted benzyl, and then b) subjecting the hydrogenated product to oxidation to produce the desired acid or a salt thereof, and then if desired c) esterifying the acid or a salt thereof to form a physiological hydrolyzable ester of the acid. 2. Method in accordance with claim 1, characterized in that the hydrogenation is carried out with a palladium catalyst. 3. Method in accordance with claim 1 or 2, characterized in that the oxidation is carried out with an alkali metal permanganate or an organic peracid.