DK162717B - METHOD FOR PREPARING 2BETA-CHLORMETHYL-2ALFA-METHYLPENAM-3ALFA-CARBOXYL ACID SULPHONES AND SALTS AND ESTERS THEREOF - Google Patents

Description

in

DK 162717 B

The present invention relates to a process for the preparation of 2β-chloromethyl-2α-methylpenam-3ft-carboxylic acid sulfone, a pharmaceutically acceptable salt or a readily hydrolyzable ester thereof. The products obtained by the process are valuable as inhibitors of J-lactamases.

The presumed association between the resistance exhibited by certain bacteria to β-lactam antibiotics and the ability of such bacteria to produce β-lactamases has led to an intensive search for β-lac-tamase inhibitors. Clavulanic acid is an example of such a compound, which is currently being studied extensively. Another β-lactamase inhibitor has on the acid form the structure Η H 'P CH, H i, = 3 ί5 γ ^ „3 J - * - 1

COOH

and is disclosed in European Patent Application 2927 published July 11, 20, 1979.

The compound having the structure H H e H_N --2 25 2 ^ CH3 j-Æ-i.

O COOH

are disclosed in United States patents 4,036,847, 4,009,159, 3,993,646, 30 3,989,685 and 3,954,732.

U.S. Patent No. 4,155,912 discloses 2-penem-3-carboxylic acid compounds of formula 35 d "5

0 COOH

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2 and esters and salts, see also Farmdoc Abstracts 82090A, 10336B and 443337B.

The connection (under number CP-45899) to the structure

rw / O

CH3 I-N-

° COOH

10 is an irreversibly acting β-lactamase inhibitor with excellent solution stability. It has weak antibacterial activity and potentiates the in vitro and in vivo activities of ampicillin against / Macta mase producing strains [A.R. English et al., Antimicrobial Agents and Chemotherapy, 14, 414-419 (1978), Aswapokee et al., J. Antibiotics 15 31 (12), 1238-1244 (Dec. 1978), and Derwent's Farmdoc Abstracts 89627A and 73866B].

B. Baltzer et al. have described in Mutual Pro-Drugs of β-Lactam Antibiotics and β-Lactamase Inhibitors, J. Antibiotics, 33 (10), 1183-1192 (1980) that the principle of combining an β-lactam antibiotic with a 20β -lactamase inhibitor in a single molecule acting as pro-drug moiety for the two active components is illustrated by the bound esters 3 and 4, wherein ampicillin and mecillinam are respectively combined with β-lactase inhibitor penicillan. sulfone. These human esters have been shown to be excellently absorbed from the gastrointestinal tract and, after absorption, be hydrolyzed while simultaneously releasing the active components.

As a result, high blood levels and tissue levels of antibiotic and / l-lactamase inhibitor are achieved in a balanced ratio. The advantages of "reciprocal pro-drugs" over simple combinations are discussed.

The esters mentioned 3 and 4 have the structures 30 35

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3

/ y-CH-CO-NHV ^ Y [; -Cli = N \ ^ S

w «» oJZ! _J. KJ jxj 0 0 'CO 0 n Λ 5 c t' 0 0 cc py ^ prsY &

CO-O X -N- I

O // CO-O

10, 5 hours In GB Patent 2044255 published October 15, 1980, it is stated that the present invention relates to novel compounds 15 of the general formula 1:

Η H

R-CII-CO-NH 2 t Sn / * 2 f]

20 /) - N-7X

o J c — O-CH — A

H 11 t η o R 3 1 wherein R 2 represents a phenyl 4-hydroxyphenyl, 1,4-cyclohexadienyl-25 or a 3-thienyl group, R 9 represents a primary amino or a carboxy group, R 3 represents a hydrogen atom or a lower alkyl -, aryl or aral cooling radical, and A represents a radical of a β-lactamase inhibitor containing both a / Mactam ring and a carboxy group and wherein A is bonded via the carboxy group.

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

From DK-B-155,740, Reference Example 1, it is known to hydrolyze 1-benzyl-6,6-dibrompenicillanate-la-oxide to produce penicilan-35-acid-la-oxide. However, on the basis of this prior art, it is not expected that catalytic hydrogenation of a (substituted) benzyl-2β-chloromethyl-2α-methyl-6α-brompenam-3α-carboxylate-1 oxide and subsequent oxidation of the hydrogenated product obtained product produces it

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4 desired 2) 8-Chloromethyl “2a-Niethyl-penam-3a-carboxylic acid sulfone with correct stereochemical configuration. The starting material disclosed in DK-B-155,740 contains a stable CH 2 group at the 2-position, while the starting material of the present invention contains an extremely labile and sensitive -Cl 2 -Cl group at the 2-position, a group which surprisingly and unexpectedly, both hydrogenolysis and oxidation survive.

The advantage of obtaining the compound with the proper stereochemistry is thereby conferring superior molecule / Mactamase inhibitory properties on the molecule.

The process of the present invention for the preparation of 2 / S-chloromethyl-2a-methylpenam-3a-carboxylic acid sulfone of the formula o / # 15-XS \ / CH 2 Cl, f: h3 (i) J-N-1

° COOH

A pharmaceutically acceptable salt of the acid or a readily hydrolyzable ester of the acid is characterized in that in order a) catalytic hydrogenation, e.g. with a precious metal catalyst such as palladium, an ester of the formula 25 A CH_Cl

Sr ", _ Γ 7 // ch3 (II) h N 1

O-CO 2 R

30 υ Z

wherein R * represents benzyl or substituted benzyl, and then b) the hydrogenated product undergoes oxidation to produce the desired acid or salt thereof, and then, if desired, c) esterifies the acid or a salt thereof to form a readily hydrolyzable ester of acid.

The pharmaceutically acceptable salts mentioned above include nontoxic metal salts such as sodium, potassium, calcium and magnesium salts.

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5 toxic amines such as trialkyl amines (eg triethylamine), procaine, dibenzylamine, N-benzyl - [beta] phenethylamine, 1-ephenamine, Ν, Ν'-dibenzylethylene diamine, dehydroabietylamine, N, N'-bis (dehydroabietyl) ethylene diamine, N- (lower) alkylpiperidine (eg, N-ethylpiperidine) and 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 as well as the ammonium salt.

The term "physiologically hydrolyzable esters" herein refers to such pharmaceutically acceptable esters which, it is known, hydrolyze to the free acid form in vivo. Examples of suitable physiological hydrolyses - bare esters include, phenacyl, acetoxymethyl, pialoyloxymethyl, α-acetoxylethyl, α-acetoxybenzyl, α-pivaloyloxyethyl, phthalidyl (3-phthalidyl), indanyl (5-indanyl), methoxymethyl, benzoyloxymethyl -ethylbutyryloxy-methyl, propionyloxymethyl, valeryloxymethyl, isobutyryloxymethyl, 6-15 [(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-hydroxyphenyl-acetamido] -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2 , 0] heptane-2-oxymethyl. The preferred esters are acetoxymethyl, pivaloyloxy-methyl, methoxymethyl, phthalidyl, 5-indanyl, 6 - [(R) -2-amino-2-phenyl-acetamido] -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-thi a -1-azabi cyclo [3,2,0] heptane-2-carbonyloxymethyl.

Many different oxidants known for the oxidation of sulfides to sulfones can be used. However, particularly suitable reagents are all potassium permanganates, e.g. potassium permanganate, and organic peracids, e.g. 3-chloroperbenzoic acid.

Particularly valuable protecting groups for R 1 are the benzyl group and substituted benzyl groups, especially 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 of formula A wherein R * represents benzyl or substituted benzyl 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 an 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, and mixtures of these solvents. However, it is normal to choose conditions under which the starting material

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6 is soluble. The hydrogenation is usually carried out at a temperature in the range of about 0 to approx. 60 ° C and at a pressure in the range of approx.

98 to approx. 98x10 kPa. The catalysts used for this hydrogenation reaction are of the type known in the art of this transformation, and typical examples are precious metals such as nickel, palladium, platinum and rhodium. The catalyst is usually present in an amount of approx. 0.01 to approx. 2.5% by weight, and preferably from ca.

0.1 to approx. 1.0 weight percent based on the compound of formula A. It is often convenient to suspend the catalyst on an inert carrier; In a particularly suitable catalyst, palladium is suspended on an inert carrier such as carbon. Furthermore, it is customary to buffer the reaction mixture to operate at a pH in the range of from

4 to 9, and preferably from 6 to 8. Borate and phosphate buffers are normally used. The reaction typically takes approx. 1 hour.

"Skellysolve B" is a petroleum ether fraction of boiling point 60-68 ° C consisting essentially of n-hexane ("Skellysolve" is a trade name of Skelly Oil Co.).

The methods of the invention are elucidated in the following Examples.

20 5

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7

Example 1

Preparation of Potassium 2g-ChloromethylT-2tt-methylpenam-3a-carboxylate sulfone (BL-P2013)

ISLAND ISLAND

t ^

Br S, Br. ^ V 'I - o N ^ CO2H σ go2-CH2- <g> -NO2 1 2

V

ISLAND

B%, - \ JTch3 - «= - Ϊ _ QJ — n \ o2-cK2- (o) -m2« N ΐο2-αί2- (θ) «ο2 h2 £ 3 Ψ - O o. / 0 • * 4ch0ci * .CH9C1 „Mn0 ^ \ / λ

yS N / 2 KMnO 4 -f '/, / CH

CH3 -> 3

</ - N-ΐο, Η Z V

5 (BL-P2013)

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8 6tt-Bronipenicnianoic acid - $ - sulfoxide (1) 1. 30 g (37.5 mmol) of 6α-brompenicillanic acid, N, N'-dibenzylethylenediamine salt [G. Cignarella et al., J. Org. Chem. 27, 2668 (1962) and E. Evrard, Nature 201, 1124 (1964)] are dissolved in 330 ml of methylene chloride. Stir and cool to 0 ° C.

2. Slowly add 13 ml (156 mmol) of concentrated hydrochloric acid to the methylene chloride solution. Precipitation of the dibenzyl ethylenediamine, HCl salt (DBEDjHCl) takes place over one minute. The slurry is stirred at 0-5 ° C for 10 minutes.

3. The precipitate of DBED, HC1 is filtered through a pre-coated diatomaceous earth filter ("Dicalite"). The cake is washed with 150 ml of methylene chloride. Filtering should be completed as soon as possible. The acidic methylene chloride solution should be stored for a longer period of time. There may be some filtering problems due to the fine nature of the precipitate. Adding filter aid to the slurry may be helpful.

4. Wash the mixture of methylene chloride filtrate and washing liquid with 60 ml of cold water. Stir for 5 minutes and discard the aqueous phase. The pH of the wash liquid is 2.0-2.3.

5. The methylene chloride solution containing 6a-brompenicillanic acid is concentrated under reduced pressure to a volume of 65-80 ml. The solution is cooled and stirred to 5 ° C.

6. With vigorous stirring, carefully add 13 ml (86.9 mmol) of 40% peracetic acid over a period of 30 minutes. The reaction is exothermic. The temperature is kept between 15 and 18 ° C with ice bath cooling. The sulfoxide begins to crystallize after adding 10 ml of peracid. The slurry. gene is cooled and stirred at 0-5 ° C for 2 hours.

7. The snow white cake is filtered and washed with 10 ml of 5 ° C water, then with 10 ml of 0-5 ° C methylene chloride and finally washed with 15 ml of heptane.

8. The cake is dried in an oven at 45 ° C to constant weight, whereby approx.

30 6-10 hours should be sufficient. Excessive warming can develop traces of a pinkish color. The weight of 1 is approx. 16.26 g, yield 73.24%.

9. The reaction mixture and final product can be analyzed by thin layer chromatography using solvent systems consisting of 15 parts of toluene / 4 parts of acetone / 1 part of acetic acid (HAC) or 8 parts of 35 acetone / 8 parts of methanol / 3 parts of toluene / 1 part of HAC. The final product is analyzed by NMR and IR.

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9 p-Nitrobenzyl-6a-brompenicinanate S-Sulfoxide (2)

To a solution of 12 g (0.04 mole) of 6α-brompenicillanic acid S-sulfoxide in 100 ml of acetone was added 7.5 g (0.041 mole) of potassium 2-ethyl-hexanoate.

The salt was collected by filtration, washed with cold acetone and air-dried to yield a total of 10 g. The crystalline potassium salt was dissolved in 75 ml of dimethylacetamide and 7.8 g (0.04 mole) of p-nitrobenzyl bromide was added. The solution was stirred at 23 ° C for 24 hours. The mixture was diluted with 500 ml of water and extracted with ethyl acetate. The ethyl acetate layer was washed four times with water and dried over anhydrous magnesium sulfate. The solvent was evaporated at 35 ° C (15 mm) to an oil which crystallized. The light tan-colored crystals of 2 were slurried with ether and collected by filtration to yield 9 g (70%), m.p. 124-125 ° C dec.

Analysis calculated for C CjHHjBrNNO₂S: C: 41.98, H: 3.05, N: 6.52

Found: C: 42.00, H: 3.48, N: 6.98.

IR (KBr): 1800 (s), 1740 (s), 1610 (w), 1520 (s), 1450 (m), 1350 (s), 1060 (m), 740 (m) cm -1. 1 H-NMR (60 mHz, DMSO): 61.22 (s, 3H), 1.6 (s, 3H), 4.67 (s, 1H), 5.2 (d, J 1H), 5.45 (s, 2H), 5.68 (d, Jl-5 Hz; 1H), 7.5-8.5 (m, 4H).

p-Nitrobenzyl-2β-chloromethyl-2a-methyl-6-brompenam-30: -carboxylate (3)

A solution of 5 g (0.012 mol) of p-nitrobenzyl-6a-brompenicillanate S-sulfoxide (2) in 120 ml of anhydrous dioxane was heated at reflux under nitrogen for 4 hours with 1.5 g (0.012 mol) of quinoline and 1.6 g (0.012 mol) of benzoyl chloride. The solution was diluted with 600 ml of water and extracted with ethyl acetate. The ethyl acetate extract was washed with 5% sodium carbonate solution, 5% phosphoric acid solution and finally with water. The organic layer was dried over anhydrous magnesium sulfate and evaporated to an oil at 35 ° C (15 mm). The oil crystallized and collected, washed with ether and finally with cold toluene to yield 3.5 g (65%), m.p. 130-135 ° C dec.

Analysis calculated for C C CHHgClBrNgOgS: C: 40.06, H: 3.14, N: 6.23, 35 Found: C: 40.19, H: 3.12, N: 6.75.

IR (KBr): 1792 (s), 1740 (s), 1610 (w), 1520 (s), 1353 (s), 1280 (m), 1025 (w), 990 (w), 750 (w) cm NMR (60mHz, DMSO): 61.45 (s, 3H), 3.5-4.3 (m, 2H), 5.05 (s, 1H), 5.42 (s, 2H), 5.5 (d, J-1.5 Hz, 1H), 5.62 (d, J ~ 1.5)

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10

Hz, 1H), 7.5-8.5 (m, 4H).

p-Nitrobenzyl-2g-chloromethyl-2a-methylpenam-6a-carboxylate sulfoxide (4)

A solution of 1 g (0.0022 mol) of p-nitrobenzyl-30-chloromethyl-2a-5-methyl-6a-brompenam-3a-carboxylate (3) dissolved in 50 ml of methylene chloride was stirred with 473 mg (0.0022 mol ) m-chloroperoxide benzoic acid. The solution was stirred at 23 ° C for 3 hours. The methylene chloride was evaporated to 20 ml at 15 mm and 33 ° C and the concentrated solution was diluted with 50 ml of hepatane (Skellysolve B). The solvent was decanted and the residue was slurried with ether and (4) soon crystallized in a yield of 250 mg, 24%, m.p. 136-137 ° C dec.

Analysis calculated for C CH ^BrCl ^O₂S: C: 38.68, H: 3.02, N: 6.02,

Found: C: 39.14, H: 3.13, N: 5.96.

IR (KBr): 1800 (s), 1760 (s), 1520 (s), 1350 (s), 1200 (s), 1050 (m), 830 (w), 740 (w) cm -1. NMR (60 mHz, DMSO): 51.32 (s, 3H), 3.8-4.5 (m, 2H), 4.97 (s, 1H), 5.25 (d, J-1, 5 Hz, 1H), 5.45 (s, 2H), 5.6 (d, Jl, 5 Hz, 1H), 7.8-8.5 (m, 4H).

Potassium-2β-chloromethyl-2α: -methylpenam-3α: -carboxylate sulfone (5) (BL-P2013)

To a solution of 7 g (0.015 mole) of p-nitrobenzyl-2β-chloromethyl-2α-methyl-6o-brompenam-3-o-carboxylate sulfoxide (4) in 150 ml of ethyl acetate was added a suspension of 4 g. % palladium-on-diatomaceous earth ("Celite") and 2.8 g of sodium bicarbonate in 150 ml of water. The mixture was hydrogenated for 3 to 25 hours at 50 psi. The catalyst was separated by filtration and the aqueous layer was separated and treated with 1.5 g of potassium permanganate in 50 ml of water. The mixture was stirred for 1 hour and 250 mg of sodium sulphite was added. The mixture was filtered and the filtrate was adjusted to pH 2 with concentrated hydrochloric acid. The solution was lyophilized to form a white 30 amorphous powder. The solid was extracted with ethyl acetate, evaporated to a volume of 20 ml and diluted with 100 ml of heptane (Skellysolve B). White, hygroscopic solid 2β-chloromethyl-2α-methylpenam-3α-carboxylic acid sulfone was collected. The acid was dissolved in acetone and treated with solid potassium 2-ethyl hexanoate. A crystalline white salt precipitated to form, after filtration, 170 mg of 5, m.p. > 140 ° C dec.

Analysis calculated for C CHyClKNOOSSHgO: C: 28.27, H: 3.24, N: 4.12,

Found: C: 28.27, H: 3.69, N: 3.84.

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11 IR (KBr): 1790 (s), 1770 (m), 1620 (s), 1460 (m), 1370 (s), 1310 (s), 1200 (s), 1140 (s), 955 (m) 40 740 (m) cm "1. 1 H-NMR (100 mHz, D₂O): 51.68 (s, 3H), 3.2-3.9 (m, H ~ 2 Hz, J-4 Hz, J 6 Hz, 2H), 4.0-4.4 (m, 2H), 4.3 (s, 1H), 5.02 (dd, J-4 Hz, J-2 Hz, 1H).

5

Example 2

Pi val oyl oxymethyl -2/1-chloro-methyl-2-methyl-phenam-3-t-carboxy-at-sulfone 2 / J-chloromethyl-2a-methylpenam-3a-carboxylic acid-sulfone in dimethyl formamide was treated with one equivalent of triethylamine and stirred. for 10 application of solution. Bromomethyl pivalate (1 equivalent) in dimethylformamide was then added. The resulting mixture was stirred at room temperature. The mixture was then dried 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 phenyl acyl esters of the same acid were prepared by replacing the bromomethyl pivalate used in the above process with an equimolar weight amount of chloromethyl acetate, chloromethyl methyl ether, chloroacetone and phenacyl bromide, respectively.

20

Example 3

PI valoyloxymethyl-20-chloromethyl-2α-methylpenam-3β-carboxylate sulfone BL-P2024 O and CH3

_ISLAND

I Vch2ci + Nai + h2o + cich2-o-c-c (ch3) 3 acetoIML · »i — H .¾.

o <3 ° 6> co2-K - 30 (BL-P2013)

ISLAND ISLAND

^ CH3 ΓΤ Ίν 35 1 CH_C1 s «s o co2ch2-o-c-c (ch3) 3 (BL-P2024)

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12

To a stirred suspension of 14.6 g (0.0487 mole) of 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 this refluxing suspension was added 14.8 ml (0.1 mole) of redistilled 5 chloromethyl pivalate (bp 34 ° C at 7 mm Hg) at once. The mixture was stirred at reflux for three 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 in vacuo at <22 ° C. The oil was then taken up in 500 ml of ethyl acetate and washed once with water (200 ml) and once with saturated Na 2 SO 4 while stirring with 2 g of decolorizing carbon under cooling (ice bath). After 20 minutes, the mixture was filtered through a suction pad (Dicalite) with suction and the pad was washed with 4 x 100 ml ethyl acetate. The combined filtrates were concentrated in vacuo at 22 ° C to an oil. The oil was then further concentrated at ca. 22 ° C and <1 mm Hg to remove most of the remaining chloromethyl pivalate. The residual oil was then triturated twice with 50 ml portions of n-pentane and left over the weekend in cold storage (about 10 ° C) under n-heptane. The solid crystalline mass was then broken up into a solid powder with 40 ml of a 4: 1 mixture of 20 ether-n-pentane. The product was then collected by filtration, washed with ether-pentane (1: 1), then pentane, then air-dried. After drying in vacuo for four hours over P 93-95 ° C.

Analysis calculated for C CjCINO ^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 2,3-chloromethyl-2H: -methylpenam-3-t-carboxylate sulfone (BL-P2013)

To a mixture of 20 ml of n-butanol and 1 g of BL-P2013 (5) was added water, 1 ml at a time, with shaking in a separatory funnel until a light yellow solution was obtained. The clear solution was filtered through a 35 fold filter and the flask and filter paper washed with ca. 10 ml of 9: 1 n-butanol 20 and the combined filtrates were diluted with an additional 20 ml of n-butanol. The resulting solution was placed in a round bottom flask on "roto-gun" and evaporated under reduced pressure to ca. half of it

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13 original volume. The snow white crystalline product was collected by filtration, washed with 6 x 10 ml acetone and air dried. Yield 810 mg. After vacuum drying for 6 hours over PgOg at <1 mm Hg, 800 mg, m.p. 215 ° C (dec.) (80% yield).

Analysis calculated for C CHgClNOgSK.lHgO: C: 29.67, H: 3.39, N: 4.63, Cl: 10.94, K.F.HgO: 5.56,

Found: C: 29.23, H: 3.38, N: 4.49, Cl: 10.74, K.F.HgO: 5.74.

This recrystallization procedure provides a crystalline monohydrate, different from the starting material, which is substantially anhydrous.

Example 5 CH 2

a) - / O

// \ f / + N-Bromosuccinimide α-azo-isobutyronitrile 20 CCl ^ Δ Ψ CHBr 0Λ * 30 As described in U.S. Pat. N-bromo-succinimide (0.375 mol) for 4.5 hours in the presence of ca. 100 mgm of α-azo-butyronitrile in 1 liter of CCl The mixture was cooled to ca. 15 ° C and filtered to remove succinimide, which itself was washed with ca. 100 ml of 35 CCl 2 and filtered. The combined CCl 4 phases were concentrated in vacuo to ca. 150 ml to give solid 3-bromophthalide, which was collected by filtration, washed with ca. 50 ml of CCl 2 and air-dried to yield 54 g of yield, which weighed 50 g after recrystallization from boiling

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14 cyclohexane, m.p. 84-86 ° C.

O o CHBr CH Cl r <> · '* CM - -N-

Ό C-OK

(IN

5 0

* “DMF

22 ° C. -

15 O O

\ s ^ CH Cl _ / 2 I CH3 X-N -% u C-O-CH-BL-P2056

20 H / I

00 \ c—> * dl

II

To a stirred partial solution and partial suspension of compound 5 (BL-P2013, 2.3 g, 0.0075 mol) in 20 ml of dimethyl formamide (DMF, dried for at least 3 weeks over 3A ° molecular sieves) was added 1, 7 g (0.008 mol of 3-bromophthalide (12)) and the mixture was stirred for 4 hours 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 (the flask being rinsed with a small amount of 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 saturated aqueous 10 SO 2, dried in the cold over Na 2 SO 4, filtered and evaporated to dryness in vacuo "Dissolve Solve B" (b.p. 60-68 ° C, essentially n-hexane) (25 ml) and four times with 25 ml n-hexane to give 2.5 g of compound 13 as an almost white solid after dry -

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15 ring in air. This product was then dried over PgOg at less than 1 mm Hg to give 2.5 g of Compound 13, snip. 104 ° C dec. Its estimated purity was 85-95%.

Analysis calculated for CjgHj ^ClNO₂S: 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 6 10 PI valoyloxymethyl-2ff-chloromethyl-2β-methylpenam-3α-carboxylate sulfone-A mixture of 1 g (0.0031 mol) of potassium-2β-chloromethyl-2α-methyl-penam-3α-carboxylate sulfone, hydrate and 1 g of 3A molecule is stirred in 15 ml of dimethylacetamide for 2 hours at 23 °. To this mixture was added 470 mg (0.0031 mole) of pivaloyloxymethyl 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 ° (15 mm) leaving an oil which was chromatographed on silica using "silicar CC-7" (methylene chloride 8, ethyl acetate 2) which showed one spot at Rf 0.5. The residue obtained was crystallized from heptane ("Skellysol ve B") to yield 100 mg (mp 94-95 °) of valoyloxymethyl-20-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 for the structure.

Example 7

Sodium 2fl-chloromethyl-2tt-methylpenam-3e-carboxyl-at-sulfonate 30 ν ° Λα H + NaEH yC / C * sCi -w * γΥ n J-i-i / ~ N 'V * 35 q rn K ^

CgH C1NO S Na O06) (289.67)

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16

To a stirred solution of 500 mg of BL-P2013 (potassium salt) in 5 ml of HgO 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 NagSO 4, the aqueous layer separated and the organic phase briefly dried in ice over 5 5 SO 2, filtered and treated dropwise with 50 med NaEH (sodium 2-ethylhexanoate) in anhydrous n-butanol to neutral for humid pH paper.

The product did not crystallize by scraping and it was then concentrated in vacuo to an oil which was dissolved in acetone (5 ml), scraped - no crystals, ether added to the cloud point - no crystals. It was concentrated in vacuo on "roto-gun" to an oil dissolved in ethyl acetate added to one drop of H2 O, scraped off - no crystals. It was concentrated in vacuo and the residue triturated with 5 ml of n-butanol, 200 mg of amorphous white powder obtained, washed with ether, air dried and vacuum dried over PgOg for 24 hours. 15 180 mg of final yield of sodium 2/5-chloromethyl-2a-methylpenam-3a-carboxylate sulfone was obtained; dec. point> 100 ° indef.

Analysis calculated for C 6 H 9 ClNO 3 SNa: C: 33.10, H: 3.13, N: 4.89,

Found: C: 33.20, H: 3.69, N: 4.44, K.F.HgO: 4.04-.

20

Example 8

Quay in um-2fl-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2013)

To 10 liters of water, 130 g (1.25 mole) of sodium bicarbonate and 200 g of 10% Pd on BaSO4 was added 272 g (0.565 mole) of p-nitrobenzyl-6a-bromo-2/1 · 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, hydrogen uptake became very slow and 200 g of 10% Pd on BaSO4 was added and the mixture hydrogenated until no further significant hydrogen absorption was observed.

The slurry was filtered through a diatomaceous earth ("Celite") pad, the pad was washed with water and the aqueous phase was washed with 3 liters of ethyl acetate. To the aqueous solution was added 3 liters of ethyl acetate and the pH 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 35 Na 2 SO 4 .10H 2 O and extracted with 2 x 1 liter of ethyl acetate. The combined extracts were dried over magnesium sulfate. The desiccant was removed and 260 ml of 2N potassium-2-ethylhexanoic acid in butanol were added at 0 ° C.

After stirring for 2 hours at 0 ° C, cold was collected in µM-2H-chl ormethyl methyl.

DK 162717 B

17 2-methylpenam-3-carboxylate sulfone (BL-P2013) and dried in vacuo at room temperature.

Yield 134.8 g (about 70%).

Example 9 p-ni trobenzyl-6ft-brompeni ci11 in sodium sulfoxy d

ISLAND

10 S— / NC * TEA + BrCHs - \) - »o2 -

1 (101) '—V

cΓ Xo # <2l6) (296) Ψ 15 o -Τ '1 wsx 20 0 C ° 2CH2- ^ N02 (431.28) 25 Procedure;

To 200 ml of Ν, Ν-dimethylacetamide was added 44 g (0.148 mole) of 6α-bromopicillanic acid sulfoxide followed by 20.5 ml (0.148 mole) of triethylamine and 38.2 g (0.177 mole) of p-nitrobenzyl bromide. Stir at 22 ° for 20 hours.

The reaction mixture was poured into 1 liter of HgO and extracted into 3 x 30 ml of methylene chloride. The combined methyl chloride extracts were washed with 200 ml of 5% aqueous sodium bicarbonate solution and dried over sodium sulfate at 5 ° for half an hour. The solution was filtered and evaporated under vacuum to a residue. The residue was diluted with ether and the solid collected by filtration to yield 54 g of p-nitrobenzyl-6-brompenicillinate sulfoxide after drying. Yield 85%. NMR was consistent for 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.

DK 162717 B

18 (A step that proceeds with a yield of 85-90%).

Example 10

Preparation of p-nitrobenzyl-6α-brompenic manate sulfoxide 5 To 4,375 liters of Ν, Ν-dimethylacetamide was added 873.0 g (2.95 mole) of 6α-brompenicillanic acid (S) sulfoxide and then, with stirring, internal temperature was kept below 35 ° C, 293 g (2.95 mole) of triethylamine followed by 764 g (3.54 mole) of p-nitrobenzyl bromide. The mixture was then stirred at room temperature for 5 hours and left overnight.

The reaction mixture was poured into 20 liters of water and extracted with 3 x 7 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 evaporated to a crystalline residue; 4 liters of diethyl ether were added and the crystals collected to yield, after drying at room temperature, 1171 g (92%) of p-nitrobenzyl-6a-brompenicillanate sulfoxide.

Br 18.48% (calculated 18.53%), (0.25% MeOH) + 162 °.

Example 11

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

To 16 liters of acetic acid was added 364.6 g (0.812 mole) of p-nitrobenzyl-6α-bromo-2β-chloromethyl-2-methylpenam-3-carboxylate. To the thus obtained solution, which was stirred at room temperature, a solution of 282 g (1.78 mole) of KMnO 2 in 26 liters of water was added dropwise over 3 hours.

The mixture was then stirred at room temperature for 1 hour and H 2 O 2 (37%) was added dropwise until a colorless solution was obtained. 30 liters of water were then 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 with 2 x 2 liters of ethanol and dried over vacuum at room temperature.

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

35

DK 162717 B

19

Example 12

Preparation of BL-P2013 Free Acid 5 V rl V CH Cl

<f "// C02eK® g ^" 'C02E

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 dissolved, the mixture was treated dropwise with 50% aqueous phosphoric acid with vigorous shaking until no more material from the aqueous layer was precipitated. The ethyl acetate lid 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 wash liquid is combined with the original filtrate). "Dissolve Solve B" was then added to the ethyl acetate until the cloud point (about 10 ml). The mixture was treated with 500 mg of activated carbon ("Darko KB") and filtered off. The filtrate was diluted with 15 ml of Skellysolve B and then seeded with crystals of BL-P2013 free acid. After about 3 hours at room temperature, the crystalline precipitate of free acid and dried in vacuo (15 minutes) over P 2 O 5 to give 323 mg (46%), mp slow dec over 100 °.

Analysis calculated for C CHHClNOOS: 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.

30 35

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20

Example 13 6-Brompenic in 11 anic acid sulphide 0

Br '"" ^> S \ Br / x -,' rr y

L ° 2l 0 "-bCOgH

10 I- —12 MW 800.64 m 296.14

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

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

750 ml.

This solution was cooled to 5 ° and with vigorous stirring 130 ml of 40% peracetic acid was added dropwise so that the temperature was maintained at 5-12 °. The addition was completely exothermic. At the end of the addition, the slurry was stirred at 5 ° for 2 hours, and the product was collected by filtration and washed with 100 ml cold HgO (5 °) and 100 ml cold methylene chloride (5 °). 126 g (57%) of 6α-brompenicillanic acid sulphoxide were obtained, m.p. 129 °. The IR and NMR spectra were consistent for the desired product.

Analysis calculated for C 8 H 10 BrNO 4 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.

35

DK 162717 B

21

Kaiium-6a-brompenicillanate sulfoxide O

5 BRY-VSY

* '' '"C02R ^ co2k MW 334.24 10

To 3 liters of acetone was added 126 g (0.43 mole) of 6α-brompenicillanic acid sulfoxide and 162 ml of 50% by weight potassium 2-ethylhexanoic acid in n-butanol.

After stirring for 1 hour at 22 °, the product was collected by filtration, washed with 2 x 250 ml of acetone and dried. 127 g (90%) of potassium 6tt brompenicillanate sulfoxide were obtained, m.p. 185 °. The IR and NMR spectra were consistent for the desired structure.

Analysis calculated for C CHHBrKNO ^S: C: 28.75, H: 2.71, N: 4.19,

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

20 p-ni trobenzyl -6tt-brompeni ci 11 anat-sul foxide

0 O

t Rr * 25 ^ N ^

Jr - Wco2K} - ^ oo2CH2 (f) - no2 MW 431.28 To 1 liter of Ν, Ν-dimethylacetamide was added 145 g (0.43 mole) of potassium 6a-brompenicillanate sulfoxide and, with stirring, 115 g (0.53 mol) of p ~ nitrobenzyl bromide at 22 °. The mixture was stirred at 22 ° for 20 hours.

The reaction mixture was poured into 3 liters of 1 0 0 and extracted with 35 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. pressure to a residue to which 1 was added

DK 162717 B

22 liters of diethyl ether, 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-bromopanicillanate sulfoxide, m.p. 111 °. The IR and NMR spectra were consistent for the desired structure.

Analysis calculated for C C CHigBrNgOgS: 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-6-bromo-2/3-chloromethyl-2-methyl penam-3-carboxyl

ISLAND

ϊ Ώ .q j CHpCl

Br a s 2 f Η, _I "" CHj

15 I_N · _ \ f /, —v J N

g NO2

MW 449.7I

To 1 liter of p-dioxane was added 70 g (0.16 mol) of p-nitrobenzyl-6a - brompenicillanate 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 °, poured into 2500 ml H 2 O and extracted into 3 x 800 ml 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 H2 O. The ethyl acetate solution was dried over sodium sulfate for ½ hour and the sodium sulfate removed by filtration. The filtrate was evaporated under reduced pressure to a residue which was redissolved in 1 liter of ethyl acetate and again evaporated under reduced pressure to a residue. Then 1 liter of ethyl ether was added and the product was collected by filtration to yield 41 g (57%) of p-nitrobenzyl-6a-bromo-20-chloromethyl-2-methylpenam-3-carboxylate, m.p. 132 °. The IR and NMR spectra were consistent for the desired structure.

Analysis calculated for C ^ gH ^BrClN₂OgS: 35 C: 40.06, H: 3.14, N: 6.23,

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

DK 162717 B

23 p-Nitrobenzyl-6a-bromo-2,3-chloromethyl-2-methylpenam-3-carboxyl at-sulfoxide 0

Br s yCHgCl ÅyCHSC1 5 YY <1 -> ΓΊ Γ "™ 3 -_ ^ 'iN- ^ 0 ^ ° 2 .N02 CO2Cl ^ \ N02 mw 465.71 10 To 1200 ml of methylene chloride was added 51 g (0.11 mole) of p- nitrobenzyl-6a-bromo-2 / J-chloromethyl-2-methylpenam-3-carboxyl followed by 23 g (0.12 mol) of m-chloroperoxybenzoic acid. The solution was stirred at 22 ° for 2 hours and evaporated under reduced pressure to The residue was stirred with 4 liters of diethyl ether for 1 h and left at 10 ° for 15 h The product crystallized and collected by filtration, washed with 2 x 200 ml diethyl ether and dried to yield 39 g of p-nitrobenzyl -6a-bromo-2 / J-chloromethyl-2-methylpenam-3-carboxylate sulfoxide (75%), mp 132 ° The IR and NMR spectra were consistent for the desired structure.

Calcd for C CgH₂ ^ BrClNgOgS: 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 2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2013)

25 O O

> CH2C1 / SV 2 \ - / Nf -> - \ "" CE-z

30 in NO2 * CO2K

'- BL-P2013 MW 3Ο5.77

To 600 ml of H2 O, 8 g of 30% Pd was added to Celite and 16 g (0.19 mol) of sodium bicarbonate. Then 32 g (0.69 mole) of p-nitrobenzyl-6ar-bromo-2β-chloromethyl-2-methylpenam-3-carboxylate sulfoxide was dissolved in 400 ml of ethyl acetate and added to the aqueous slurry. The mixture was hydrogenated on a Paar apparatus at 50 p.s.i. at 22 ° for 4 hours. Up-

DK 162717 B

The slurry was filtered through a thin "Celite" pad onto a sintered glass funnel, the pad was washed with 2 x 50 ml of HgO, and the aqueous layer of the combined filtrate and the washings were separated. The aqueous layer was washed with 200 ml of diethyl ether, then cooled to 5 °, and, with stirring, a solution of 12 g (0.076 mole) of KMnO 2 in 200 ml of 100 µl was added dropwise over a period of ½ hour. , maintaining the pH between 7.5 and 8.0 by adding 40% H 2 PO 2. When the pink color lasted for 5 minutes, the addition of KMnO4 solution ceased. The reaction mixture was stirred with a small amount (about 50 mg) of sodium bisulfite for 10 hours, then the slurry was filtered through a "Celite" pad. The pad was washed with 2 x 50 ml of 1 O. The combined filtrate and washings were layered with 500 ml of ethyl acetate and, with stirring, the pH was adjusted to 1.5 by the addition of 2N HCl. The layers were separated and the aqueous layer was saturated with sodium sulfate. Extract with 2 x 400 ml of ethyl acetate and the combined ethyl acetate extracts were dried over sodium sulfate for ½ hour at 5 °. 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-ethylhexanoate in n-butanol was added until the solution was neutral for 20 moist pH paper. The potassium salt of BL-P2013 crystallized, collected by filtration, washed with diethyl ether and dried. The yield was 16 g of potassium um-20-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2013) (76%), m.p. 202 °. The IR and NMR spectra were consistent for the desired structure.

Analysis calculated for C CHHHClNO₂S: 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 14 P1 val oyl oxymethyl -2β-chloromethyl-2-methyl penam-3-carboxyl-at-sulfone (BL-P2024)

O O O O

_Js ^ ci ^ ci _ 35 ΓΊ Τ'011? -> rί rCH3 N_ N-1 * J ^ co2K / ^ co2ch2ococ (ch5) 3 BL-P2024 MW 381.83

DK 162717 B

25

To a stirred suspension of 14.6 g (0.0487 mol) of potassium B / J-chloro-methyl-2-methylpenam-3-carboxylate sulfone (BL-P2013) in 200 ml of acetone was added 4 ml of a 10% aqueous solution of sodium iodide, and the mixture is refluxed in a steam bath. To this suspension under reflux was added 14.8 ml (0.1 mole) of redistilled chloromethyl pivalate (kgp. 34 ° C at 7 mm Hg) at once. The mixture was stirred at reflux for three 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 evaporated to an oil under reduced pressure at <22 ° C. The oil was then taken up in 500 ml of ethyl acetate and washed once with water (200 ml) and once with saturated NagSO 4 solution (200 ml). The solution was then briefly dried over Na 2 SO 4 while stirring with 2 g of decolorizing carbon under cooling (ice bath).

After 20 minutes, the mixture was filtered through a "Celite" pad and the pad 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 at ca. 22 ° C and <1 mm Hg to remove most of the remaining chloromethyl pivalate. The remaining oil was then triturated twice with 50 ml portions of n-pentane and left to stand for about 20 weeks at ca. 10 ° C under n-pentane. The resulting solid crystalline mass was then broken up into a powder with 40 ml of a 4: 1 mixture of di ethyl 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 four hours over P2 and 25, 13.37 g of pivaloyloxymethyl-2 / J-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2024) were obtained. (75%), m.p. 93-95 ° C.

Purification of BL-P2024

Ca. 3 g of crude BL-P2024 (obtained as described above) were dissolved in 5 ml of ethyl acetate, placed on a 4.5 x 40 cm column of silica gel ("Mallinckrodt CC-7") and eluted with 4: 1 v / v CH 2 Cl 2. ethyl acetate.

The fractions containing a single spot at 0.84 (TLC on silica gel plates with 4: 1 CH 2 Cl 2 ethyl acetate, I2 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 ("Skellysol ve B") and then stored at room temperature for three days. The crystals that formed were collected by

DK 162717 B

26 filtration, washed with petroleum ether and dried to give 560 mg, m.p. 100-101 °, purified BL-P2024.

Analysis calculated for Cj qCINO ^S: C: 44.03, H: 5.27, N: 3.67, Found: C: 44.11, H: 5.08, N: 3.85.

All temperatures in this application are listed in degrees Celsius.

Example 15

Preparation of the ammonium salt of BL-P2013 1. The free acid of BL-P2013 (250 ml) dissolved in 20 ml of acetone-methanol (1: 1 volume) was filtered to prepare a clear solution.

2. Anhydrous ammonium solution was prepared by adding 1 ml of ammonium hydroxide (30%, reagent grade) to 10 ml of 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 designated "anhydrous ammonium solution".

3. To the filtrate of step 1 was added gradually, ca. 2 ml of "anhydrous ammonium solution" and mixed well.

4. A 100 ml portion of diethyl ether was mixed with the mixture of step 3 to precipitate the ammonium salt of BL-P2013.

5. The white ammonium salt was isolated from the solvent and washed with two portions of 50 ml of diethyl ether each.

6. The isolated powder is 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 16

Preparation of non-hygroscopic sodium salt of BL-P2013 1. 50 mg of the free acid of BL-P2013 was dissolved in 4 ml of acetone-methanol (1: 1 volume) mixture. Filter to obtain a clear solution.

2. Sodium 2-ethylhexanoate solution was prepared by dissolving 40 mg of sodium 2-ethylhexanoate in 10 ml of acetone-methanol (1: 1 volume) mixture.

3. To the filtrate of step 1 was added 10 ml of solution according to step

DK 162717 B

27 2 and mixed well.

4. A 10 ml aliquot of diethyl ether was mixed with the mixture of step 3 to precipitate the sodium salt of BL-P2013.

5. The white salt was immersed in the diethyl ether for 1-2 hours and then solubilized from the solvent and washed with three portions of 5 ml of diethyl ether each.

6. The isolated powder is dried at 30 ° C in a vacuum oven overnight. Example 17 Recrystallization of BL-P2013

00 O O

5 <^^ CHgCl Enclosure Case. ^^ CE ^ Cl

Y * Iron H00-acetone ^ I ^ CBL

In CH 5 2 I * 3 / - CO 2 H 2 O 2 Jr-V, 20 BL-P2013 (400 mg) was dissolved in a minimal amount of acetone-HgO (1: 1) volume and diluted with 10 ml of acetone, filtered, diluted then with acetone to about 25 ml, scraped off and after 30 minutes the crystalline hydrate was collected by filtration, washed well with acetone, air dried and then vacuum dried at <1 mm Hg overnight Yield 280 25 mg.

Analysis calculated for C CHHClNOSK.H₂O: C: 29.67, H: 3.39, N: 4.63, Cl: 10.94, H₂O: 5.55,

Found: C: 29.32, H: 3.32, N: 4.44, Cl: 11.31, H 2 O: 5.90.

30 35

DK 162717 B

28

Example 18 Ν, Ν'-Dibenzylethylenediamine salt of BL-P2013

Acetone-ether / CH 2 Cl 2 / RCH 3 \ N-1 / cO ^ .Cg ^ CHgNH-CH2 J-10 \ 306 mg (0.001 ml) BL-P2013 was dissolved in 7 ml HgO and added to a solution of 180 mg (0.0005 mol) of Ν, Ν'-dibenzylethylenediamine diacetate in 7 ml of HgO. The mixture was stirred and the salt crystallized, and after stirring for ca. For 10-15 minutes, the salt was collected by filtration and air dried to give the N, Ν'-di benzyl ethylenediamine salt of BL-P2013 (300 mg).

The material was recrystallized by dissolving for about -5 min. 10 ml of boiling acetone 20 and dilution with ether to cloud point. 260 mg of air dried and vacuum dried material were obtained.

Analysis calculated: C: 51.69, H: 5.42, N: 7.53, Cl: 9.55,

Found: 25 C: 49.39, H: 5.49, N: 7.05, Cl: 8.96, Η 0: 1.23 (KF).

Example 19

Chloromethyl ester of BL-P2013 30 O O

Wg / -.CHgCl + 01CH2-0-SO2C1 ^ -Uk + 3 KHCO3 (305-7) 2. Cf ^ Clg H2 °

DK 162717 B

29

ISLAND ISLAND

I I CH x 1- N-1 C-OCHoCl

II 2 O

(316.17) 7 To a vigorously stirred mixture of 15.25 g (0.05 mole) of BL-P2013 (5), 15 g (0.15 mole) of KHCO3 and 1.7 g (0.005 mole) of tetrabutylammonium hydrogen sulfate ( Aldrich Chem. Co.) in a mixture of 50 ml of water and 50 ml of CH 2 Cl 2 was added dropwise a solution of 9.5 g (0.0575 mol) of ClCH 2 -O-SO 2 Cl in 40 ml of CH 2 Ig. The temperature rose to 26 ° C and after the addition 15 (which took about 15 minutes), the mixture was stirred for a further 30 minutes.

Because the product crystallized, more CH 2 Cl 2 (about 400 ml) was added to give 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. The mixture was filtered for 30 minutes, concentrated to ca. 50 ml and isopropyl alcohol (150 ml) were added. The rest of CH 2 Cl 2 was then 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-2β-chloromethyl-2-methyl-penam-3-carboxylate sulfone (7), m.p. 116 ° (dec., Darkened above 100 ° C).

Analysis for C 9 H 18 Cl 2 NO 3 S: 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).

30 Estimated purity in the range of 90-95%.

Iodomethyl ester of BL-P2013 in i / [I2C1 \ J / H2C1 '35, -,> Sn / Na I - I' '' CH, -> '' CH, /) "" c-om Cl J ""

o S OCHgLl 0 C-OCH.I

7 0 8 J 2

DK 162717 B

30

To a stirred mixture of 5 g (0.0159 mole) of chloromethyl ester of BL-P2013 (7) in 25 ml of acetone was added 3 g (0.02 mole) of sodium iodide. The resulting slurry was stirred for 17 hours and then cooled to ca.

0 ° C. Two drops of saturated aqueous KHCO 3 were added and the mixture was diluted 5 drops slowly with water over 10 minutes until 50 ml was added. The slurry underwent a sudden change of color from yellow to gray to violet to black, and the crystals were immediately collected by filtration and washed with cold acetone (1: 2) water, then isopropyl alcarbon (3 x 10 ml) to give diethyl ether and finally n. -pentane and air-dry-10 are prepared to give a yield of 5.55 g (91¾ yield) of the iodomethyl - ester of BL-P2013 (8). Mp. 118-119 ° C during decomposition. The purity was estimated at approx. 90%.

Biological data 15 The product of Example 1, compound 5 with the structure \ / °, CH2C1 / V -r '"ch3 2 ° i-N -1

O C02K

will hereinafter be referred to as BL-P2013.

Although BL-P2013 is itself a very weak antibacterial agent at best, it inhibits j8 lactamases and protects ceforanide and. amoxicillin against destruction of / Mactamase-producing bacteria in vitro and in vivo when used in combination with these two agents.

DK 162717 B

31 TABLE Γ

Antibacterial activity of new sulphonate 5 MIC (pg / ml)

Organism BL-P2013 Ampicillin S. pneumoniae A-9585 16 0.004 S. pyogenes A-9604 63 0.004 10 S. aureus A-9537> 125 0.16 S. aureus + 50% serum A-9537> 125 0.06 S. aureus Pen-Res A-9606> 125> 125 S. aureus Meth-Res A15097> 125 125 S. faecal is A20688> 125 0.13 15 E. coli A15119> 125 1 E. coli A20341-1> 125> 125 K pneumoniae Al5130> 125 125 K. pneumoniae A20468> 125> 125 P. mirabilis A-9900> 125 · 0.13.

20 P. vulgaris A21559> 125 125 P. morganii A15153> 125> 125 P. justice A21203> 125 4 S. marcescens A20019> 125 16 E. cloacae A-9659> 125 63 25 E. cloacae A-9656> 125> 125 P. aeruginosa A-9843A> 125> 125 P. aeruginosa A21213> 125> 125

DK 162717 B

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DK 162717 B

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44

Thus, the compounds of this invention are valuable, administered orally and parenterally, to improve the efficacy of / Mactam antibiotics against / Mactamase-producing bacteria. On a weight basis, the dosage is from 1/5 to 5 times, and preferably equal to the dosage of the / Mactam antibiotic. For example, the compounds of the invention, as shown above, used in a 1: 1 ratio markedly enhanced the activity of ceforanide and amoxicillin against / Mactamase-producing strains of anaerobic bacteroids such as B. fragile ice, B. thetaiotaomicron and other species of this genus and also over for resistant Staphylococcus aureus. The compounds of the invention are administered either in admixture with or simultaneously with the β-lactam antibiotic at a dose within the stated ratio, together with the known or usual dose of the antibiotic.

Thus, the ability of the compounds of this invention to increase the efficacy of a / Mactam antibiotic against certain β-lactamase-producing bacteria makes them valuable for co-administration with certain β-lactam antibiotics to treat bacterial infections in humans and mammals. In the treatment of a bacterial infection, a compound of the invention can be admixed with the β-lactam antibiotic and the two agents are administered simultaneously. Alternatively, a compound of the invention may be administered as a separate agent during a course of treatment with a / Mactam antibiotic.

When a compound of the invention or a salt thereof is used to enhance the anti-bacterial activity of a / Mactam antibiotic, it can be administered alone or preferably formulated with standard pharmaceutical carriers and diluents. An compound of the invention which is in acid form or as a pharmaceutically acceptable salt thereof can be administered orally or parenterally. A compound of the invention in the form of an ester which is readily hydrolyzable in vivo is best administered orally. Parenteral administration includes intramuscular, subcutaneous, intraperitoneal and intravenous administration.

When a compound of the invention is used in the presence of a carrier or diluent, the carrier or diluent is selected based on the intended mode of administration. In oral administration, the compound may e.g. used in the form of tablets ", 35 capsules, lozenges, lozenges, powders, syrups, elixirs, aqueous solutions and suspensions, and the like, in accordance with standard pharmaceutical practice. The ratio of active ingredients and carrier will of course depend on the chemical nature of the -

DK 162717 B

45 stability, strength and potency of the active ingredients as well as the intended dose. However, these pharmaceutical compositions are likely to contain from ca. 5% to approx. 80% of carriers. In the case of tablets for oral use, commonly used carriers include lactose, 5 sodium citrate and salts of phosphoric acid. Various disintegrating agents such as starch and lubricants such as magnesium stearate, sodium lauryl sulfate and talc are commonly used in tablets. For oral administration in capsule form, valuable diluents are lactose and high molecular weight polyethylene glycols. For aqueous suspensions for oral use, the active ingredients are combined with emulsifying and suspending agents. If desired, certain sweeteners and / or fragrances can be added. For parenteral administration which includes intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredients are usually prepared and the pH of the solution is suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled so that the preparation remains in sotonic.

Although the prescribing physician ultimately determines the dose to be used for a human subject, the ratio of daily doses between a compound of the invention or a salt thereof and the β-lactam antibiotic will usually be in the range of ca. 1: 5 to 5: 1, and preferably approx. 1: 1. In addition, the daily oral dose of each component will usually be in the range of approx. 10 to approx. 200 mg per and the daily parenteral dose of each component will normally be about 25 kg. 10 to approx. 100 mg per kg body weight. These figures serve to illustrate the invention only, and doses outside these limits may be necessary in some cases.

Claims (4)

46 DK 162717 B A process for the preparation of 2β-chloromethyl-2α-methylpenam-3α-carboxylic acid sulfone of the formula coo S '/ S CH CH / V "cn3 in a XN-J4 O boOH 10 a pharmaceutically acceptable salt of the acid or a physiologically hydrolyzable ester of the acid, CHARACTERIZED by sequentially a) catalytically hydrogenating an ester of the formula 15. wherein R * represents benzyl or substituted benzyl and then b) the hydrogenated product undergoes oxidation to produce the desired acid or salt thereof and then, if desired, c) esterifying the acid or a salt thereof to form a physiologically hydrolysable ester of the acid. 2. A process according to claim 1, characterized in that a pivaloyloxymethyl ester is prepared. 3. A process according to claims 1-2, characterized in that the hydrogenation is carried out with a palladium catalyst. 4. A process according to claims 1-2, characterized in that the oxidation is carried out with an alkali metal permanganate or an organic peracid. 35