CS207371B2 - Method of preparation of ethers of the clavulane acid - Google Patents

Description

The invention relates to a process for the preparation of clavulanic acid ethers.

Belgian Patent No. 827,926 discloses among others rice where elavulanic acid has the structure of formula clavulanic acid and its salts and esters.

Clavulanic acid and its salts and esters are capable of inhibiting the beta-lactamases of bacteria and this property makes it possible to increase the effect of penicillins and cephalosporins against small Gram-positive and Gram-negative bacteria. It has now been found that certain clavulanic acid derivatives also have useful antibacterial and beta-lactamase inhibiting effects.

The present invention relates to a process for the preparation of clavulanic acid ethers of the general formula II,

(II)

207771 where

R is C 1 -C 4 alkyl, C 3 -C 4 alkenyl, benzyl, benzyl substituted with nitro or C 1 -C 4 alkyl, substituted with carboxy in salt form, C 1 -C 4 alkoxycarbonyl, benzyloxycarbonyl, phenylcarbonyl, chlorophenylcarbonyl, cyano, hydroxy, amino or amino substituted by C 1 -C 4 alkyl and / or benzyloxycarbonyl, and

A is an alkali metal cation characterized in that the compound of formula IIa is de-esterified,

wherein B is C 1 -C 4 alkyl, C 3 -C 4 alkenyl, benzyl or benzyl substituted with C 1 -C 4 alkoxy or C 1 -C 4 alkyl substituted with C 1 -C 4 alkoxy, alkylcarbonyloxy C 1-4 alkyl or C 1-4 alkoxycarbonyl, and the free acid obtained is converted by treatment with a base into the corresponding salt.

Certain particularly suitable compounds of the invention, showing particularly good activity against certain beta-lactamase producing strains, including Staphylococcus aureus, are those. wherein R 9 is CH 9 R, wherein R is phenyl or phenyl substituted with a halogen atom, or

R * ® or Or'®, where r'® is alkyl containing up to 3 carbon atoms.

Suitable groups A include a hydrogen atom and salt ions such as lithium, sodium, potassium.

Particularly suitable compounds of the invention include compounds of formula II wherein A is especially sodium or potassium ion, most preferably sodium ion.

The esters of the invention are preferably hydrolyzable in vivo. Suitable esters include those described in Belgian Patent No. 827,926 as being hydrolyzable in vivo when coupled to clavulanic acid. Particularly suitable in vivo hydrolysable esters include acetoxymethyl, alpha-acetoxyethyl, pivaloyloxymethyl, phthalidyl, ethoxycarbonyloxymethyl, ethoxycarboronyloxyethyl and the like.

The benzyl esters of the compounds of formula IIa are particularly suitable hydrogenolyzable esters.

The compounds prepared by the process of the invention may be formulated into pharmaceutical compositions which are characterized in that they comprise the compounds of the invention together with a pharmaceutically acceptable carrier.

Compositions with the compounds of the invention include those in a form adapted for oral, topical or parenteral use and which can be used to treat infections in mammals including humans.

Suitable forms of the compositions of the invention include tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders, and sterile forms suitable for injection and infusion. These forms may contain conventional pharmaceutical suitable materials such as diluents, binders, colorants, flavors, preservatives, disintegrators and the like, in accordance with conventional pharmaceutical practice in the manner commonly used in the treatment of antibiotics.

Compositions used as injections or infusions containing salts of the compounds of formula II are particularly suitable because high tissue levels of the compound of formula II can be achieved after injection or infusion. A preferred composition of the invention is characterized in that it comprises a salt of the compound of formula II in sterile form.

A unit dose of the compositions comprises a compound of Formula II or a salt or ester thereof adapted for oral administration and is a further preferred feature of the composition of the invention.

The compound of formula II or a salt or ester thereof may be present in the mixture either as a single therapeutic agent or together with other therapeutic agents such as beta-lactam antibiotics. Suitable beta-lactam antibiotics for the compositions of the invention are benzylpenicillin, phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcilin, cyclacilin, cefatriazine, pirbencillin, alpha-sulfonyloxybenzylpenicin, cephalilin, cephalil, cephalil, cephalil, cephalil, cephalil, cefamandole naphtha, cefapitin, cefradine, 4-hydroxycephalexin, cefapatol, cephaloglycine and other well-known penicillins and celalosporins, or medicaments prepared therefrom such as hetacillin, metampicillin, 4-acetoxyampicillin, acetoxymethyl, phthivalyciniline methylmethyl, ethoxycarbonyloxymethyl, phoxycarbonyloxymethyl, or cephaloglycine or phenyl, tolyl, indanyl alpha-esters of carbenicillin or ticarcillin and the like. These compounds are often used in the form of salts or hydrates.

Naturally, if penicillin or cephalosporin present in the composition is not suitable for oral administration, then the composition is formulated for parenteral administration.

When a compound of the invention is present in a pharmaceutical composition together with a beta-lactantine antibiotic, then the ratio of the compound of formula II or a salt or ester thereof to the beta-lactam antibiotic may be in a wide range, for example 10: 1 to 1: 3. from 5: 1 to 1: 2, for example from 3: 1 to 1: 1.

The total amount of α L bacterial agents present in a unit dosage form normally ranges from 50 to 1500 mg, and is usually from 100 to 1000 mg.

The compositions of the invention may be used to treat infections, inter alia, to treat airways, urinary tract, soft tissue, and mastitis in cattle.

Normally between 50 and 3000 mg of a compound of the invention is administered each day for treatment, but more usually 100 to 000 mg of a compound of the invention are administered per day, for example in 1 to 6 doses, more usually in 2 to 4 doses.

The penicillin or cephalosporin in the synergistic compositions of the invention may be present in approximately the same amounts as commonly used.

Particularly preferred compositions of the invention may comprise from 150 to 1000 mg of amoxycillin, ampicillin or therapeutics prepared therefrom, and from 50 to 500 mg of a compound of formula II or a salt or in vivo hydrolysable ester thereof, and more preferably mixtures containing from 200 to 500 mg. amoxycillin, ampicillin or medicaments prepared therefrom and from 50 to 250 mg of a compound of formula II or a salt thereof or an in vivo hydrolysable ester.

The substances present in the mixtures may optionally be hydrated, for example ampicillin trihydrate or amoxycillin trihydrate. The weights of the antibiotics in these mixtures are expressed on the basis of the antibiotics theoretically present in the mixtures, and not on the weights of the drugs prepared therefrom.

The starting compounds of formula IIa, as defined above, are prepared by esterifying a clavulanic acid ester and then optionally replacing the ester group with a known method for the carboxylic acid group or a salt or other ester thereof.

207 371

Conveniently, etherification of the reaction is carried out with a clavulanic acid ester that is readily hydrogenolyzable to the parent acid, for example, the benzyl ester or its chemical equivalent.

Esters of formula II may be prepared by reaction of an alcohol of formula X,

R-OH (X) wherein R is as defined in formula II, with the corresponding ester of a compound of formula I in the presence of a Lewis acid catalyst such as boron trifluoride or equivalents thereof such as boron trifluoride etherate, e.g.

This reaction is conveniently carried out in a solvent inert under reaction conditions such as chloroform, dichloromethane, tetrahydrofuran or dioxane at a reduced or elevated temperature, for example -80 ° C to +30 ° C, preferably at a reduced temperature, for example -50 ° C to 0 ° C, preferably at a temperature of about -30 ° C.

An alternative form of this method of preparing esters of formula II is characterized by an alcohol reaction <

formula X,

R-OH (X) wherein R is as defined in formula II, with the corresponding clavulanic acid ester, wherein the hydroxyl group is masked such that the compound has the formula XI,

wherein R is C 1 -C 6 alkyl, optionally inertly substituted with one or more halogen atoms, and CO ₂ A 0 is an ester group in the presence of a Lewis acid.

The conditions for this reaction are similar to those described above for the reaction of a compound of formula X with an ester of a compound of formula I.

Compounds of formula XI can be prepared as described in Belgian Patent No. 834,645, i.e. by reacting a compound of formula

¹¹ O R -C-OH or a reactive acylating derivative thereof with a corresponding ester of clavulanic acid.

The invention is illustrated by the following examples.

He did

Preparation of methylclavulanyl-O-methyl ether

Benzylclavulanate (el), 1.5 g, in methanol (10 mL) is hydrogenated on 10% palladium on carbon (0.4 g, 30 minutes at room temperature and room pressure. The solution is filtered through celite and does not react with the solution. The solution was allowed to stand at 0 ° C overnight, the solvent was evaporated and the oil was chromatographed on silica gel to give the crude compound (e2) (35 mg) as a second eluting product. title compound as a colorless oil (15 mg).

I. fi. (CHCl-1): 1800, 1750, 1695 cm ^-1 ,

NMR (CHCl 3): 3.03 (1H, dd, J 17Hz), J 1Hz, 6P-CH), 3.53 (1H, d, J 17Hz, J 2.5Hz, 6a-CH), 3, 31 (3H, s, OCH ₃ ), 3.79 (3H, s, CO ₂ CH ₃ ), 4.03 (2H, d, J 7Hz, CH ₃ OCH ₃ ,

4.88 (1H, broad t, J 7Hz, olef. H), 5.08 (1H, m, 3-CH), 5,70δ (1H, dd, J 2.5Hz, J 2.5Hz ^s 5 -CH).

₀ ij values inhibioe beta-lactamases in .mu.g / ml for (E2) are the following:

Esoherichia coli JT4 0.9

Klebsiella aerogenes E70 0.25

Staphylococcus aureus Russell 0.5

Esoherichia coli JT410 3.1

P. moig, Cr. 0.68

Pseudomonas aeruginosa 1.9

Citrobacter mantio 0.45

Example 2

Preparation of benzylclavulanyl-O-methyl ether

Benzylclavulanate (e1) (300 mg) was dissolved in anhydrous methylene dichloride (25 mL) and cooled to 0 ° C. Boron trifluoride etherate (5 drops) was added at 0 ° C followed by a solution of diazomethane in ether. The reaction was stirred at 0 ° C for one hour and then washed with 3% sodium bicarbonate solution (2 x 25 mL). The organic phase is dried over magnesium sulphate and evaporated. Chromatographic purification gave the title compound (e3) (66 mg).

I. fi. (CHCl3): 1,800, 1,745, 1,695 cm ’,

N.MR (CDCl3): 3.10 (1H, d, J 17Hz, 6p-CH), 3.35 (3H, s, OCHD, 3.60 (1H, dd, J 17Hz, J '2.5Hz) Δ 6 -CH), 4.12 (2H, d, J 8Hz, CH ₂ OCH ₃ ), 4.94 (1H, t, J 8Hz, = CH-CH ₂ ),

5.24 (lH, broad s, 3-CH), 5.32 (2H, s, C0 ₂ CH ₂ Ph), 5.82 (1H, d, J 2.5Hz, 5-CG) »7.518 (5H , with, COgCHgPhl.

The β ₀ -lactamase inhibition values in µg / ml for (e 3) are as follows:

Escherichia coli JT4 0.1 Klebsiella aerogenes E70 0.05 Staphylococcus aureus Russell 0.05 Proteus mirabilis C889 <0.07 Pseudomonas aeruginosa 1.4 Pséudomonas dalgleish <0.07 Citrobacter mantio 1.1

Example 3

Preparation of clavulanyl-O-methyl ether sodium salt

Ether (e3) (30 mg) was dissolved in tetrahydrofuran (3 mL) and 10% palladium on carbon (10 mg) was added. The solution was hydrogenated at room temperature for 15 minutes under normal pressure, filtered, and sodium bicarbonate (8.4 mg) in 0.5 mL water was added. The solvent was evaporated and, after trituration with ether, the sodium salt (e4) was obtained as an amorphous solid (15 mg).

I. No. (KBr disk): 1,790, 1,690, 1,615 cm -1,

NMR (D ₂ O): 3.07 (1H, d, J 17Hz, 6P-CH), 3.27 (3H, s, OCH 3), 3.54 (1H, dd, J 17Hz, J '2.5Hz) 6a-CH), 4.04 (2H, d, J 8Hz, CH 3 OCH 3), 4.87 (1H, t, J 8Hz, = CH-CH ₂ >, 4.93 (1H, s, 3- CH), 5,698 (1H, d, J 2.5 Hz, 5-CH).

The β0-lactamase inhibition values in µg / ml for (e4) are as follows:

Escherichia coli JT4 0.18 Klebsiella aerogenes E70 0.07 Staphylococcus aureus Sussell 0.05 Proteus mirabilis C889 0.01

Example 4

Preparation of methylclavulanyl-O-benzyl ether

CO ₂ CH ₂ C _e H ₅

(e5)

Benzylclavulanate (el) (300 mg) in methylene chloride (25 mL) was cooled to -30 ° C and treated with boron trifluoride etherate (5 drops) and then a solution of diazotoluene in ether was added. The solution was stirred at -30 ° C for one hour and then washed with 3% sodium bicarbonate solution (3 x 25 mL). The organic phase was dried over magnesium sulfate and chromatographed to give crude benzyl 3- (2-benzyloxyethylidene) -7-oxo-4-oxa-1-azabicyclo (3.2.0) heptane-3-carboxylate (e5) (150 mg). ) in the form of an oil. The oil was dissolved in tetrahydrofuran (5 mL) and hydrogenated at room temperature and normal pressure to 10% Pd on carbon (20 mg) for 20 minutes. The solution was filtered and treated with a solution of diazomethane in ether at 0 ° C. Evaporation of the solvent by chromatography gave the title compound (e5) (30 mg) as a colorless oil.

ID NO. (CHC1 _3): 1800, 1750, 1695 cm ^-1,

NMR (CDCl 3): 3.12 (1H, d, J 17Hz, 6p-CH), 3.62 (1H, dd, J 17Hz, J 2.5Hz, 6a-CH), 3.89 (3H, s) CO ₂ CH ₃ ), 4.25 (2H, d, J 8Hz, = CH-CH ₂ O), 4.61 (2H, s, OCH 2 Ph), 5.02 (1H, broad t, J 8Hz, = CH-CH ₂ ), 5.18 (1H, m, 3-CH), 5.82 (1H, d, J 2.5Hz, 5-CH, 7.49δ (5H, s, OCH _; Ph). [Deg.] = + 15 [deg.] (C = 1.37, MeOH).

Antibacterial effect in vitro (µg / ml)

Staphylococcus aureus Oxford, 5-31

Staphylococcus aureus Russell 8-15

Klebsiella aerogenes A.> 500

I ^ Q values of beta-lactamase inhibition in (µg / ml)

Escherichia coli JT4 0.8

Klebsiella aerogenes E70 0.8

Staphylococcus aureus Russell 0.005 *

Example 5

(e1)

Crude benzyl 3- (2-benzylethylidene) -7-oxo-4-oxa-1-azabicyclo (3.2.0) heptane-2-carboxylate (e1) (150 mg) was dissolved in tetrahydrofuran (5 mL) and hydrogenated at room temperature under normal pressure to 10% Pd on carbon (20 mg) for 20 minutes. The solution was filtered and treated with sodium bicarbonate (15 mg) in 1 mL of water. The solvent was evaporated and the residue was dissolved in water (10 ml) and washed with ethyl acetate (3x, 0 ml). The aqueous phase is evaporated and triturated with ether to give the desired sodium salt (e6) (35 mg) in amorphous form.

I. No. (KBr): 1790, 1690, 1615 cm ^-1

NMR (D ₂ O): 3.09 (1H, d, J, 7Hz, 6P-CH), 3.59 (1H, dd, J 17Hz, J '2.5Hz, 6a-CH), 4.20 (1H 2H, d, J 8Hz, = CH-CH ₂ O), 4.48 (2H, s, OCH ₂ Ph), 4.97 (1H, broad t, J 8Hz, = CH-CH ₂ O), 4, 99 (1H, ra, 3-CH), 5.74 (1H, d, J 2.5Hz, 5-CH), 7.42 δ (5H, s, OCH ₂ Ph), []] β ^θ = + 22.5 ° (c = 1.15, 50% aqueous MeOH).

Antibacterial effect in vitro (µg / ml)

Klebsiella aerogenes A.

Staphylococcus aureus Oxford 4.0

Staphylococcus aureus Russell 8.0

I ^ -lactamase inhibition values _of v (g / ml)

Escherichia coli JT4 0.1

Klebsiella aerogenes E70 0.04

Staphylococcus aureus Russell 0.005

Proteus mirabilis C889 0.02

Pseudomonas dalgleish 0.008

Example 6

Preparation of benzylclavulanyl- (ethoxycarbonylmethyl) ether

Benzylclavulanate (e1) (1.44 g) was dissolved in anhydrous methylene dichloride (100 mL) and boron trifluoride etherate (25 drops, 0.3 mL) was added at -30 ° C. Ethyldiazoacetate (2.90 g, 5 equivalents) in methylene dichloride (10 mL) was added dropwise over half an hour and the reaction mixture was stirred at -30 ° C to -10 ° C for one hour. The solution was washed with 3% sodium bicarbonate solution (3 x 50 mL) and dried over magnesium sulfate. The solvent was evaporated at room temperature and the residue was chromatographed. The solvent was evaporated under reduced pressure to give the title compound (e7) as a colorless oil (600mg).

IC (CHCl-1): 1800, 1745, 1695 cm ^-1 ,

KMR (CDCl 3): 1.29 (3H, t, J 8Hz, -CO ₂ CH ₂ CH 2, 3.00 (1H, d, J 17Hz, ββ-CH), 3.50 (1H, dd, J 17Hz, δ 2.5 Hz, 6a-CH), 3.97 (2H, s, CH 3 COgEt), 3.90-4.50 (4H, complete multiplet, = CH-CH ₂ and CO ₂ CH ₂ CH _3), 4.79 (IH, t, J 8 Hz, = CH-GH ₂₎ 5, l4 (IH, broad s, 3-CH)

5.23 (2H, s, CO ₂ CH ₂ Ph), 5.72 (1H, d, 2.5 Hz, 5-CH), 7.37δ (5H, s, CO ₃ CH ₂ Ph).

M j ¹ = + 35.3 ° (c = 1.29, MeOH).

Ι ^ θ values of beta-lactamase inhibition in ^g / ml for (e7) are as follows:

Escherichia coli JT4 0.14 Klebsiella aerogenes E70 0.30 Staphylococcus aureus Russell 0.12 Proteus mirabilis C889 1.0

Antibacterial effect in vitro (µg / ml)

Klebsiella aerogenes A. 500 Staphylococcus aureus Oxford 125 Staphylococcus aureus Russell 125

Preparation of clavulanyl (ethoxycarbonylmethyl) ether sodium salt

Example 7

(e8) («8)

The ester (e7) (188 mg) was dissolved in anhydrous tetrahydrofuran (5 mL) and 10% added.

Ρά on charcoal (65 mg). The solution is hydrogenated at room temperature θ normal pressure for 15 minutes. The solution was filtered through diatomaceous earth and sodium hydrogencarbonate mg in water (1 ml) was added. The solvent is evaporated and the residue is chromatographed on silica gel, eluting with a 4: 1: 1 mixture of butanol, ethanol and water. Trituration of the product in ether afforded the sodium salt as an amorphous solid (e8) (95 mg).

I.Č. (KBr): 1790, 1740, 1690, 1600 cm ^-1 ,

NMR (D ₂ O): 1.27 (3H, t, J 3Hz, CCl 2 CHCl 3), 3.09 (1H, d, J 17Hz, δβ-CH), 3.57 (1H, da, J 17Hz, J '2.5Hz, 6U-CH), 4.14 (2H, s, OCHgCOgEt), 4.21 (4H, doublet and quartet coupled, J = J' 8Hz, = OH-CHg- and COgCl 2 CH) -p, 4.86 (1H, t, J 8Hz, = CH-CH ₂ -), 4.96 (1H, broad s, 3-CH), 4.71 6 (1H, d, J 2.5Hz, 5-CH3).

[ «Ξ ¹ = 36.5 ° (c = 1.15, 50% aqueous MeOH).

The IgO values of beta-lactamase inhibition in µg / ml for (e8) are as follows:

Escherichia coli JT4 0.16

Klebsiella aerogenes E70 0.034

Staphylococcus aureus Russsel 0.044

Proteus mirabilis C889 0.025

Antibacterial effect in vitro (µg / ml)

Escherichia coli 18418 3i, 0 Klebsiella aerogenes A. 15.0 Proteus mirabilis C977 62.0 Staphylococcus aureus Oxford 15.0 Staphylococcus eureus Russell 15.0

Example 8

Preparation of clavulaethyl (ethoxycarbonylmethyl) ether sodium salt

Ester (e7) (122 mg) was hydrolyzed using 1N sodium hydroxide solution at constant pH (using pH-stat) until complete hydrolysis. Chromatography gave the sodium salt as an amorphous solid for trituration with ether (e8) (15 mg).

Example 9

Preparation of benzylclavulanyl- (benzyloxycarbonylmethyl) ether

Benzylclavulanate (e1) (1.8 g) was dissolved in anhydrous methylene dichloride (100 mL) at -30 ° C and boron trifluoride etherate (25 drops, 0.3 mL;) was added. At -30 ° C benzyldiazoacetate ( 5 g (66% pure material) in methylene chloride (20 mL) and the mixture was stirred at -30 ° C to -10 ° C for one hour, washed with 3% sodium bicarbonate solution (12x50 mL) and dried over magnesium sulfate. The residue was chromatographed on silica gel, eluting with hexane / ethyl acetate to give the somewhat impure oil, which was repeated by chromatography to give pure (e9) as a colorless oil (0.32 g).

IC (film): 1800, 1760, 1750, 1695 cm ^-1 ,

NMR (CDCl ₃ ): 7.29 (10H, aromatic H), 5.59 (1H, d, J 2.5Hz, 5-CH), 5.13 (4H, CH 2 Ph), 5.03 (1H, 3 -CH), 4.79 (1H, t, J 7Hz, = CH ₂ ), 4.16 (2H, d, J 8Hz, = CH-CH ₂ ->, 4.00 (2H, s, = CH- CH ₂ -O-CH ₂ -), 3.40-2.95 δ (2H, dd, J 18 Hz, 6-CH ₂ ).

Benzyldiazoacetate was prepared by reacting benzyl chloroformate and diazomethane at 4 ° C for 3 days in a yield of 66% pure oily product.

Example 10

Preparation of clavulanyl (carboxymethyl) ether disodium salt

The ester (e9) (260 mg) was dissolved in tetrahydrofuran / water (5: 1, 12 mL) and sodium bicarbonate (100 mg) and 10% Pd on carbon (150 mg) were added. The solution was hydrogenated at room temperature and normal pressure for 30 minutes, filtered and evaporated to give the desired disodium salt (E10).

I. (KBr): 1780, 1685, 1600, 1425 cm ^-1 .

1

Example 11

Preparation of benzyl 9- (O-cyanomethyl) clavulanate

+ - + N ₂ - CH - CN

A solution of benzyl clavulanate (e1) (5.18 g in methylene dichloride (50 mL) was dried on 4A molecular sieves and filtered. The solution was cooled to -40 to -30 ° C and 1 mL of boron trifluoride etherate was added. A solution of diazoacetonitrile (prepared from aminoacetonitrile hydrochloride (9.3 g) according to Houben-Weyl vol. 10/4) in methylene dichloride (250 mL) was added dropwise at -40 ° C to -30 ° C and allowed to warm to -10 ° C. After standing at this temperature for one hour, it is washed with 3 portions of dilute sodium bicarbonate solution (200 ml), dried over calcium chloride and evaporated to give a brown gum. The product is purified by column chromatography (silica gel, cyclohexane / ethyl acetate 3: 1). and then 1: 1) to give a slightly contaminated product, and repeated chromatography on the same system afforded the desired product (E1) (1.56 g).

X. No (film): 1 805, 1 750, 1 698 cm '

N. M. R. (CDCl3): 83.02 OH, doublet, J 17Hz), 3.45 (1H, double doublet, J 17Hz and 3Hz),

4.01 (2H, singlet), 4.14 (2H, doublet, J 7Hz), 4.70 (1H, fine coupling triplet, Jtrip 7Hz), 5.06 (1H, singlet), 5. 4 (2H, singlet), 5.66 (1H, singlet), 3.28 (5H, singlet).

Example 12

Preparation of 9- (O-cyanomethyl) clavulanate sodium salt

A mixture of benzyl 9- (O-cyanomethyl) clavulanate (el1) (1.56 g) and sodium bicarbonate (0.399 g) in tetrahydrofuran and water (30 mL of a 5: 1 mixture of tetrahydrofuran and water) was hydrogenated to 10% Pd on carbon (0.5 g) at room temperature and atmospheric pressure until the required amount of hydrogen was consumed. The mixture was then filtered and the catalyst was washed with water. The combined filtrates were freed from the distillation of most of the tetrahydrofuran. The remaining aqueous solution was washed with ethyl acetate and freeze-dried to give the desired product (e12) (913 mg) as a brown solid. A sample (535 mgj) was passed through a short column of silica gel in n-butanol, water and ethanol (4: 1: 1) to give (el2) (213 mg) as a pale yellow solid.

KMR (Ώ ₂ θ): 83.24 (1H, doublet, J 16 Hz), 3.66 OH, double doublet, J 16Hz and 3Hz),

4.30 (2H, doublet, J 7Hz), 4.40 (2H, singlet), 4.91 (1H, triple bond,

J 7Hz), 5.00 OH, singlet), 5.78 (1H, doublet, 3Hz).

X. No. (KBr disc): 1780.1 SiO (Wide) cm ^-1 .

Example 13

Preparation of benzyl 9 - (O- (4-nitrobenzyl)) clavulanate

A solution of benzylclavulanate (el) (4.33 g) in methylene chloride (50 mL) was dried with 4A molecular sieves for 30 minutes. The solution was then filtered and cooled to -30 ° C. Boron trifluoride etherate (0.8 mL) was then added. A solution of 4-nitrophenyldiazomethane (7.34 g) in methylene chloride (150 mL) was added dropwise to the stirred solution at -30 ° C over one hour. The mixture was then warmed to -10 ° C and stirred for one hour. The solution was then washed with sodium bicarbonate solution (3 x 150 mL), dried (MgSO 4) and evaporated. Repeated chromatography (twice with cyclohexane / ethyl acetate 1: 1 and once with methylene chloride as eluent) on silica gel H gave pure ether (el3) (230 mg).

ί. δ. (film): 1800, 1750, 1700, 1520 cm ^-1 .

NMR (CDC1 _3): 2.98 (1H, doublet, J = 17Hz), 3.44 (1H, double doublet, J = 17Hz and 3Hz),

4.09 (2H, doublet, J 7Hz), 3.45 (2H, singlet), 4.8 (1H, fine joining triplet, J 7Hz),

5.05 (1H, singlet), 5.14 (21i, singlet), 5.63 (1H, fine coupling doublet), 7.27 (5H, singlet), 7.38 (2H, doublet, J 9Hz) , 8.11 (2H, doublet, J 9Hz).

Example 14

Preparation of benzyl 8- (O-allyl) clavulanate

i) H-Nitroso-t-allylbenzamide

A solution of nitrogen dioxide (24.1 g) in carbon tetrachloride (150 mL) was added to a stirred mixture of anhydrous sodium acetate (45 g) and carbon tetrachloride (150 mL) at -20 ° C. To the stirred mixture at -5 ° C was added N-allylbenzamid (28.4 g in carbon tetrachloride (150 ml) and the mixture stirred for 30 minutes at 0 DEG C. ^{At the} reaction mixture was washed with aqueous sodium bicarbonate (2x400 mL). dried and evaporated to give N-nitroso-N-allylbenzamid (28.5 g). v max ^{(k ln} P® filJ Ý » '' 7i0, 1650, 1600, 1570, 1355, 1285, i 155, 1040 and 920 cm ^-1 , δ (CDCl-j): 7.83-7.20 (5H, m, ArH), 6.03-4.83 (3H, m, = 4), and 4 33 (2H, broadd, J 4Hz).

ii) 3-Diazo-1-propene

Pyrrolidine (4.52 mL) was added to a solution of N-nitroso-N-allylbenzamide (9.5 g) in methylene chloride (100 mL) at -20 ° C, and the mixture was stirred at -20 ° C for 15 minutes to give solution of 3-diazo-1-propene.

, 3. 207371 max (CH 3 Cl 2): 2030 cm ^-1 . This solution is then stored at 0 ° C until use. Iii) Benzyl 9- (O-sllyl) clavulanate

Boron trifluoride etherate (1 mL) was added to a solution of benzyl clavulanate (2.89 gJ in methylene chloride (150 mL) at -30 ° C. The 3-diazo-1-propene solution prepared above was added to the mixture at -30 ° C and The solution was stirred at -30 ° C to -10 ° C for one hour, washed with aqueous sodium bicarbonate solution (2x200 mL), dried and evaporated to give the crude product as an oil which was chromatographed on silica gel (50 g). Elution with cyclohexane / ethyl acetate gave benzyl 9- (O-allyl) clavulanate (e14) (0.92 g) as a colorless liquid, [α] _D + 45.3 ° (CHCl ₃₎ , 1,0), ν _Μχ (liquid film): 1,800, 1,750,

695, 1 300, 1 175, 1 030, 1 010, 995 cm ^-1 . _H

Δ (CDCl ₃ ): 7.37 (5H, s, Ar-H), 6.05-5.70 (1H, m, 5.60 (1H, d, J, 2.5Hz, 5-CH)),

H

5.30 to 5.10 (2H, m, -CH ₂ -CH = C '), 5.13 (2H, s, CH ₂ Ph), 5.03 H, of t, J 8Hz, 8-CH 4.01 (2H, d, J 8Hz, 9-¾). 3.86 (2H,

3.41 (1H, dd, J 18Hz, J '2.5Hz, 6a-CH), 2.98 (1H, d, J 18Hz, (found: C 65.85, H 5.65, N 4.45) % for C 18 H 16 NO 2 requires C 65.65, H 5.8, N 4.25%).

Example 15

Preparation of benzyl 9-O- (2-benzyloxycarbonylethyl) clavulanate (1H, m, 3-CH), 4.79 (1H, broad d, J 7 Hz, -CH ₂ -CH = CH ₂ ), δβ-CH).

i) Benzyl-N-nitroso-N-benzyloxycarbonyl-beta-alanate

A solution of nitrogen dioxide (6.9 g) in carbon tetrachloride (40 ml) was added to a stirred solution of anhydrous sodium acetate (12.3 g? And carbon tetrachloride (40 ml) at -20 ° C to the stirred mixture at -5 ° C add benzyl-N-benzyloxycarbonyl-beta-alanate 112.95 g) in carbon tetrachloride (50 mL) and stir at 0 ° C for 30 min. The reaction mixture was washed with aqueous sodium bicarbonate solution (2 x 100 mL) and the carbon tetrachloride solution was dried, evaporated to give benzyl N-nitroso-N-benzyloxycarbonyl-beta-alanate (12.5 g) as an oil.

^{max (CC1 4):} 745, 1705, 1520, 1400, 1350, 175: 1140, 1095, 960 cm, 8 (CDC1 _3): 7.30 (10H, s, Ar , 5.46 (2H, s, _CH2 Ph), 4.90 (2H, s, CHgPh), 3.92 (2H, t, J

7Hz). The NMR spectrum indicates that the starting material and this oil are only 73% pure.

(ii) Benzyl-9- (O- (2-benzyloxycarbonylethyl)) clavulanate

To a solution of benzyl N-nitroso-N-benzyloxycarbonyl-beta-alanate (3.24 g) in methylene chloride: (25 mL) was added pyrrolidine (0.58 mL) at -20 ° C and the mixture was stirred for 15 minutes at -20 ° C to give a solution of benzyl 3-diazopropionate which is maintained at 0 ° C for the next step.

To a solution of benzyl clavulanate (1 g) in methylene dichloride 150 mL was added boron trifluoride etherate (0.5 g) at -30 ° C. The benzyl 3-diazopropionate solution prepared above was added to the mixture at -30 ° C and the solution was stirred at -30 ° C to -10 ° C for one hour. The reaction mixture was washed with aqueous sodium bicarbonate solution (2 x 50 mL), dried and evaporated to give the crude product as an oil which was chromatographed on silica gel (30 g). Elution of the column with cyclohexane-ethyl acetate gave benzyl 9- (0- / 2-benzyloxykarbonylethyl /) clavulanate (ei5) (0.2 g) as a colorless liquid, [a] _D 29.3 ° (CHCl ,, c, 1.0), _v (liquid film): 1,805, 1,740, 1,700, 1,240, 1,170, 1,100, 1,040, and 1,020 cms (CDCl-j): 7.28 (10H, s, 1 '). Ar-H), 5.60 (1H, d, J 2.5Hz, 5-CH), 5.14 (2H, s, CH Ph), 5.09 (2H, s, CH 2 Ph), 5.02 ( 1H, m, 3-CH), 4.74 (1H, broad t, J 8 Hz, 8-CH), 4.01 (2H, broad d, J 8 Hz, 9-¾). 3.60 (2H, t, J 7Hz), 3.41 (1H, dd, J 18Hz, J '2.5Hz 6'-CH), 2.99 (1H, d, 1.8Hz, 6P-CH), and 2.57 (2H, t, J 7Hz).

M ⁺ 451.1626, θ25 ^ 25 ^ θ7 ^ν ° 6 ° M ⁺ 451.1631.

Example 16

Preparation of 9- (O- (2-carboxyethyl)) clavulanate disodium salt

CH ₂ -O-CH ₂ - eH ₂ —CO ₃ CH ₃ C ₆ H ₆ CO ₂ CH ₂ C _e H ₅

A mixture of benzyl 9- (O- (2-benzyloxycarbonylethyl)) clavulandte (e15) (0.17 g) and sodium bicarbonate (0.063 g) was hydrogenated in tetrahydrofuran / water 5: 1 (12 mL) in the presence of 10% palladium on coal (0.1 g) at room temperature for 0.5 hours. The mixture was filtered and the filtrate was evaporated to give 9- (O- (2-carboxyethyl)) clavulanate disodium salt (e16) (0.084 g).

ν max (KBr: 1,780, 1,690, 1,620, 1,390, 1,310, 1,195, 1,155, 1,085, 1,040, and 895 cm -1 ⁾ .

Δ (D ₂ O): 5.65 (1H, d, J, 2.5Hz, 5-CH), 4.87 (1H, s, 3-CH), 4.81 (1H, broad t, J 8Hz) 8-CH), 4.06 (2H, d, J 8 Hz, 9-¾). 3.63 (3H, t, J 7Hz), 3.51 OH, dd, J 18Hz, J '2.5Hz, 6 (x-CH), 3.05 OH, d, J 18Hz, 6p-CH), and 2.49 (3H, t, J 7Hz).

Example 17

Preparation of benzyl 9-acetonylclavulanate

(e1)

To a solution of benzyl clavulanate (e1) (1.0 g) and diazoacetone 0.4 g) in methylene dichloride (30 mL) was added boron trifluoride etherate (20 drops) and the solution was stirred at -10 ° C for two hours. The reaction was quenched with dilute sodium bicarbonate solution. The organic extract was washed with brine, dried (MgSO4) and evaporated. Silica gel chromatography (eluting with a mixture of ethyl acetate and cyclohexane) gave the title compound (e17) (190 mg) as a colorless oil.

I. No (CHCl-j): 1 800, 1 730 - 1 760, 1 695 cm ^-1 ,

NMR (CDCl ₃ ): 2.10 (3H, s, -Cg ₃ ), 2.96 (1H, d, J 17Hz, 6P-CH), 3.36 (1H, dd, J 17Hz,

J 1 2.5Hz, 6a-CH), 3.99 (2H, d, J 7 HZ, = CH-CH ₂ ), 4.68 (1H, t, J 17 Hz, = CH-CH ₂ ), 5.03 (1H, broad s, 3-CH), 5.20 (2H, s, -CHgPh), 5.70 (1H, d, J 2.5 Hz, 5-CH), 7.33 (5H, s, COgC) (Ph) M (Mass Spectrometry) 345.

Example 18

Preparation of 9-acetonylclavulanate sodium salt

with

COjCHjCgHg

(e18) (e18)

Clavulanic acid benzyl ester acetone ether (e17) (80 mg) was hydrogenated with a mixture of tetrahydrofuran and water (5: 1) in the presence of sodium bicarbonate (5 mg) and 10% palladium on carbon (30 mg). After 20 minutes, the catalyst was filtered off. The filtrate was diluted with water and extracted with ethyl acetate, then freeze-dried. Obtained as the title compound as an off-white solid.

I. Ď. (KBr): 1780, 1720, 1690, 1661 cm @ 1 NMR (CDCl3): 2.09 (3H, s, --CH2), 3.00 (1H, J '2.5Hz, 6a) -CH), 4.08 (2H, d, J 8 Hz, = CH-CH ₂ ), = CH), 4.90 (1H, s, 3-CH), 5.65 (1H, d, J 2, 5Hz, d, J 17 Hz, 6.beta.-CH), 3.45 (IH, gives, 4.18 (2H, s, CH ₂ C θ), 4.80 (1H, 5-CH).

J 17Hz, t, J 8Hz,

Example 19

Preparation of benzyl 9- (4-chlorophenacyl) clavulanate

(el)

Cl

Benzylclavulanate (el) (500 mg) was dissolved in methylene dichloride (25 ml) and treated with alpha-diazo-p-chloroacetophenone (0.90 g). Boron trifluoride etherate (0.1 mL) was added to the stirred solution at -20 ° C. The solution was stirred at -20 ° C to -10 ° C for two hours, washed with dilute sodium bicarbonate solution (2x), and the organic extract was dried over magnesium sulfate. Evaporation of the solvent and chromatography on silica gel (eluting with chloroform) gave the title compound (e19) (130 mg).

ID NO. (CHC1 _3): 1805, 1750, l 695 cm ^-1,

NMR (CDC1 _3): 3.10 (| H, d, J 17Hz, 6P-CH), 3.60 (1H, dd, J 17Hz, J '2.5 Hz, 6a-CH)

4.36 (2H, s, J 7Hz, = CH-CH ₂ O-), 4.74 (2H, s, -OCH ₂ C-), 5.00 (1H, t, J 7Hz, = CH-CH ₂ ), 5.26 (1H, broad s, 3-CH), 5.37 (2H, s, CO ₂ CH ₂ Ph), 5.82 (1H, d, J 2.5 Hz, 5-CH) 7.48-8.12 (9H, m, aryl).

Example 20

Biological effect

a) When administered to mice, the compounds of the invention do not show a high level of toxicity, for example when administered subcutaneously, the compounds of Examples 5 and 12 have a greater

250 mg / kg.

b) When tested by standard MIC assays in combination with ampicillin, the compounds of the invention increase the effect of penicillin against various gram-positive agramnegative organisms such as Staphylococcus aureus Russell, Klebsiella aerogenes, Proteus mirabilis and Escherichia coli: in the presence of 1 µg / ml of certain compounds of the invention are:

the compound of Example. C. 3 5 9 10 1 1 14 15 16 MIC (µg / ml) 2 0,3 1.6 3 1,25 1,25 1 1 Ampicillin in the absence synergically effectively does not inhibit substances growth tested organis-

at a concentration of 250 µg / ml. none of the synergistically active substances inhibit growth of the test organism at a concentration of 5 µg / ml.

Example 21 <

Preparation of benzyl 9-O- (2 '- N-benzyloxycarbonyl-N-methyl / aminoethyl) clavulanate

i) N-Methyl-N'-nitroso-Ν, Ν'-dibenzyloxycarbonyl-1,2-diaminoethane

To carbon tetrachloride (90 mL), stirred at -5 to -10 ° C, was added anhydrous sodium acetate (27.8 g), followed by a solution of nitrogen dioxide (15.6 g) in carbon tetrachloride (90 mL), such as at a rate such that the temperature does not exceed -5 ° C. A solution of N-methyl-N, Ν'-dibenzyloxycarbonyl-1,2-diaminoethane (29 gI in carbon tetrachloride (90 mL) was then added over 40 minutes to the stirred solution to maintain the temperature at -10 ° C. The mixture was washed with an excess of sodium bicarbonate solution, the aqueous phase was extracted with carbon tetrachloride, and the combined organic phases were dried over magnesium sulfate and evaporated to yield 29.3 g of 80% pure product.

ii) N-Methyl-N, N'-dibenzyloxycarbonyl-1,2-diaminoethane

To a stirred solution of N-methyl-1,2-diaminoethane (7.4 g) and triethylamine (20.2 g) in chloroform (50 mL) at 0 ° C was added benzyl chloroformate (34.1 g) dropwise over 30 minutes.

The mixture was then allowed to warm to room temperature over one hour. The organic solution was then washed with water, citric acid solution, sodium bicarbonate solution and finally water again. The chloroform solution was dried over magnesium sulfate and evaporated to give a yellow oil (29.6 g).

iii) Benzyl 9-O- (2 '- N-benzyloxycarbonyl-N-methyl / aminoethyl) elavulanate.

To a stirred solution of N-methyl-N'-nitroso-Ν, Ν'-dibenzyloxycarbonyl-1,2-diaminoethane (3.71 g) in methylene chloride (10 mL) cooled to -30 ° C was added pyrrolidine (0, 71 g).

, 7

20737,

The mixture was stirred at -25 ° C to -30 ° C for 30 minutes and then used as described below.

A solution of benzylclavulanate (1.0 g) in methylene chloride (10 mL) was cooled to -30 ° C and boron trifluoride etherate (0.5 mL) was added. The diazo compound solution prepared above was then added to the stirred solution at a rate such that the temperature was kept below -25 ° C. The mixture was then stirred for 1 hour and gradually allowed to rise to -10 ° C. An excess of ice-cooled sodium bicarbonate solution is then added and the organic phase is separated. The aqueous phase is extracted with methylene chloride and the combined organic phases are washed with water, dried over magnesium sulphate and evaporated. Repeated chromatography of the residue gives the desired ether of the above formula.

IR (film) (not showing absorption for OH group), 1800, 1745 and 1700 cm -1. The thin layer chromatography properties are consistent with the proposed structure.

Example 22

Preparation of benzyl-9-O- (3'-hydroxy-propyl) -clavulanate

i) N, O-Bis- (benzyloxycarbonyl) -3-aminopropanol

Benzyl chloroformate (47 mL) was added dropwise to a stirred solution of 3-aminopropanol (11.4 mL) in pyridine (150 mL) at 0 ° C. The mixture was then stirred overnight at room temperature.

Pour into ice-water (300 mL) and extract with ethyl acetate (3x200 mL). The organic extract was washed with hydrochloric acid (5M, 3x400 mL), dried over anhydrous sodium carbonate and magnesium sulfate and evaporated to give the title compound as an oil, which was contaminated with a small amount of dimethyl carbonate (30.23 g), ii) N-Nitroso-N O-bis- (benzyloxycarbonyl) -3-aminopropanol

Anhydrous sodium ootane (45 g) was added to carbon tetrachloride (150 mL) at 0 ° C, the mixture was cooled to -20 ° C and a solution of nitrogen dioxide (24.1 g) in carbon tetrachloride (150 mL) was added. A solution of N, O-bis-benzyloxycarbonyl-3-aminopropanol (30.23 g) in carbon tetrachloride (150 mL) was added to the stirred mixture at -5 ° C and stirring was continued at 0 ° C for 30 minutes. The reaction mixture was washed with aqueous sodium bicarbonate solution (2x400 mL), dried and evaporated to give the title compound as an oil (31.23 g).

IR ^max (liquid film): i 750, 1510, 1400, 1260, 1125 and 1035 cm ^-1.

iii) benzyl-9-0- (3 ^of -benzyloxykarbonyloxypropyl) clavulanate

Pyrrolidine (4.94 mL) was added to a solution of N-nitroso-N, O-bis- (benzyloxycarbonyl) -3-aminopropanol (31.22 g, 0.056 mol in the mixture) in methylene chloride (100 mL) at -20 ° C. ° C for 15 minutes to give a solution of 3-diazo-O-benzyloxycarbonylpropanol in (CH ₂ Cl 2) λ rt 030 cm. This solution was stored at 0 C. ^{At the} next step.

To a solution of benzyl clavulanate (3 g) in methylene chloride (150 mL) at -30 ° C was added boron trifluoride etherate (1 mL). To this mixture at -30 ° C was added the 3-diazo-O-benzyloxycarbonylpropanol solution prepared above, and the solution was stirred at -30 ° C to -10 ° C for one hour. Washing with aqueous sodium bicarbonate solution (2x200 mL), drying and evaporation gave an oil which was chromatographed on silica gel (80 g. Eluting the column with cyclohexane / ethyl207371 acetate gave the title compound (1.5 g), δ (CDCl). -p 7.36 (IOH, s, Ar H), 5.70 (IH, d, J = 2.5Hz, 5-CH), 5.18 (4H, s, _CH2 Ph), 5.13 (IH , m, 3-CH), 4.84 (1H, broad t,

J = 8Hz, 8-CH), 4.26 (IH, t, J = 6Hz, CH ₂ CH ₂ OCBz), 4.06 (IH, d, J = 8Hz, _9-CH2), 3.60 - 3.26 (4H, m, O-CH ₂ CH ₂ and 6-CH), 3.02 (IH, d, J = 18 Hz, 6.beta.-CH) and 1.86 (IH, m, CH CH ₂ CH ) Example 23

Preparation of p-methoxybenzyl-9-O-ethoxycarbonylmethyl ethyl acetylate

A solution of p-methoxybenzylclavulanate (9.6 g) in anhydrous dichloromethane (200 mL) was stirred at -30 ° C. Redistilled boron trifluoride etherate (.mu.L) was added followed by ethyldiazoacetate (16 ml). The temperature was slowly allowed to rise to -10 ° C and then stirred at this temperature for 3 hours. A saturated aqueous solution of sodium bicarbonate was added (two portions of 200 ml each), the mixture was shaken, separated and the organic phase obtained was dried over magnesium sulphate, filtered and evaporated. The remaining syrup is chromatographed on a silica gel column and eluted with a 1: 3 to 1: 2 mixture of ethyl acetate and cyclohexane. Fractions containing product (by thin layer chromatography) were combined and evaporated. Yield 5.5 g.

IC (film) 1 805 cm ^-1 (beta-lactam c = 0), 1750 cm ^-1 (ester c = 0), 700 cm -1 (C = C).

Example 24

Preparation of benzyl-9-O-phenacylclavulanate

Benzylclavulanate (0.83 g) and diazoacetophenone (1.45 g) were dissolved in methylene chloride (20 mL) and stirred at -20 ° C. Boron trifluoride etherate (0.15 mL) was added and the solution was stirred at -10 ° C for two hours. The reaction is then quenched by addition of dilute bicarbonate solution. The organic phase was washed with bicarbonate solution, brine and then dried over magnesium sulfate. The product (26 mg) was isolated as a colorless oil which, after double chromatography on silica gel (ethyl acetate-petroleum ether), showed in _max (CHCl 3): δ 805, 1735-1755, 1 705 cm ^-1 ; Δ (CDCl ₃ ), 2.97 (1H, d, J 17 Hz, 6β-CH), 3.47 (1H, dd, J 17 Hz, J 2.5 Hz, δ x-CH), 4.28 (2H, d, J 7Hz, 9-CH ₂ ), 4.72 (2H, s, -OCH 3 CO), 4.93 (1H, t, J 7Hz, 8-CH), 5.17 (1H, s, 3) CH), 5.26 (2H, s, CO ₂ CH ₂ Ph), 5.70 (IH, d, J 2.5Hz, 5-CH), 7.35 to 8.10 (10H, m, aryl ).

Example 25

Preparation of allyl-9-O- (p-chlorophenacyl) clavulanate

Allyl clavulanate (0.55 g) and alpha-diazo-p-chloroacetophenone (0.77 g) were dissolved in methylene chloride (25 mL). Boron trifluoride etherate (25 drops) was added and the solution was stirred at -20 ° C to -10 ° C for two hours. The reaction was quenched by the addition of dilute sodium hydroxide solution and the separated organic phase was washed with water. The solution was dried over sodium sulfate, filtered and evaporated. Chromatography on silica gel (eluting with chloroform) gave the desired product (200 mg). (CHCl 3) 1800, 1745, 1690, 1590 cm ^-1 ; Δ (CDCl3): 2.94 (1H, d, J 17Hz,

ΙΠαΧ jj δβ-CH), 3.42 (2H, dd, J 17Hz, J 2.5Hz, 6o-CH), 4.17 (2H, d, J 7Hz, 9-CHg), 4.72 (2H, s, OCH ₂ CO), 4.57 (2H, d, J 6Hz, CO ₂ CH ₂ ). 4.82 (IH, t, J 7 Hz, 8-CH), 5.02 (IH, s, 3-CH), 5.25 (2H, m, _CH2), 5.61 (1H, d, J 2.5Hz, 5-CH), 5.6 to 6.1 (1H, m, CH ₂ -CH = CH _2), 7.2 to 7.85 (4H, m, aryl).

Example 26 p-Methoxybenzyl-9-O-ethyl clavulanate

CO ₂ CH ₂ C _e H ₄ OCH ₃ 'co ₂ ch ₂ c ₆ h ₄ and ₃

The procedure of Example 2 was repeated using p-methoxybenzylclavulanate instead of benzylclavulanate and diazoethane instead of diazomethane. The ethyl ether obtained in the form of a pale yellow oil exhibited the following properties: IR (liquid film) 1 805 (beta-lactam C = O), 1750 (ester C = O), 1700 cm ^-1 (C = C); NHR (CDCl 3) 1.17 (3H, t, J 7Hz, CH 3 CH 2) 3.79 (3H, s, OCl 3), 2.96 (1H, d, J 17Hz, 6P-CH), 3.41 (2H, q, J = 7Hz, CHjCHg-), 3.47 (1H, dd, J 17 and 3Hz, 6a-CH ^_), 3.79 (3H, s, OCH-j), 4.03 (2H, d, J 7Hz, -CH 2 O), 4.82 OH, t, J 7Hz, C 1 = =, 5.04 OH, s,

3-CH), 5.11 (2H, s, PhCH 8).

Example 27

Preparation of methoxymethyl-O-ethylclavulanate

H i

n

CH ₂ OH

_{R 2} CH ₂ OC Hg

CO ₂ CH ₂ OCH ₃ '' co ₂ ch ₂ and ₃

The procedure of Example 26 was repeated using methoxymethylclavulanate instead of p-methoxybenzylclavulanate.

The product is chromatographed on a silica gel column, eluting with a mixture of ethyl acetate and methylcyclopentane (1: 3) and then ethyl acetate and cyclohexane gradient from 2: 1 to pure ethyl acetate. The desired product is eluted first, followed by a small amount of unreacted starting material.

IR (film): 1 805 (beta-lactam C = O), 1755 (ester C = O), 1700 (C = C).

NMR (CDCl ₃ ) 1.18 (3H, t, J 7Hz, CH ₃ GH ₂ ), 3.02 (1H, d, J 17Hz, 6-P-CH), 3.42 (2H, q,

7Hz, CH ₃ CH ₂ ), 3.44 (1H, dd, J, 17 and 3Hz, 6-7 -CH), 3.45 (3H, s, OCH-j), 4.04 <2H, d, 7Hz, CH ₂ CH =), 4.86 (1H, bt, J 7Hz, CH ₂ -CH =), 5.04 (1H, bs, 3CH ₃ ), 5.23, 5.30 (2H, Aqq, J 5Hz, OCH ₂ O), 5.65 (1H, d, J 3Hz, 5-CH).

The corresponding methoxymethyl O-methylclavulanate can be prepared in the same manner.

(The starting material can be prepared by reacting sodium clavulanate and methyloxymethyl chloride in dimethylformamide in a conventional manner.)

Example 28

Preparation of 9-0- (p-nitrobenzyl) -clavulanic acid sodium salt

A solution of benzyl 9- (O- (4-nitrobenzyl)) clavulanate (110 mg) and sodium hydrogen carbonate (21.7 g) in tetrahydrofuran and water (5: 1, 8 mL) was hydrogenated to 10% palladium on carbon (68). mg) for 5 minutes. The solution was then filtered through celite and the solid was washed with water. The combined filtrates were evaporated to remove most of the tetrahydrofuran and the remaining aqueous solution was washed with ethyl acetate. The aqueous solution was then evaporated and the remaining gummy residue was purified on a column (silica gel H, n-butanol-water-ethanol 4: 1: 1). This afforded the product (39 mg, as a yellow solid (70% purity)).

IR (KBr 1790, 1 605 (broad) cm -1 ⁾

NMR (DMSO) 2.83 (1H doublet, J 17Hz), 3.41 (1H, dd, J 17Hz and 3Hz), 3.88 (2H, d, J 7Hz), 4.4 (2H, s), 4.52 (1H, s), 4.67 (1H, mt), 5.57 (1H, s), 6.46 (2H, d, J 9 Hz), 6.89 (2H, d,

J 9Hz).

Example 29

Preparation of 9-0- (3'-hydroxypropyl) clavulanic acid sodium salt

A mixture of benzyl-9-O- (3-benzyloxycarbonyloxypropyl) clavulanate (0.5 g) and sodium bicarbonate (0.05 g) in tetrahydrofuran / water (5: 1, 12 mL) was hydrogenated at room temperature under normal pressure in presence of 10% palladium on carbon (0.3 g) for 15 minutes. The mixture was filtered and the filtrate evaporated to an oil which was partitioned between water and ethyl acetate. The aqueous fraction was evaporated by freeze-drying to give the title compound, whose NMR was consistent with the structure.

Example 30

9-O- (2 '- [N, N-dimethyl / aminoethyl) clavulanate

co ₂ ch ₂ c ₆ h ₅

A solution of the benzyl ester (90 mg) in tetrahydrofuran (5 mL) and water (1 mL) was hydrogenated on 1056 palladium on carbon (30 mg) for 15 minutes. The catalyst was filtered off and washed with water. The combined filtrates were evaporated to remove tetrahydrofuran and the aqueous solution was extracted twice with ethyl acetate. The aqueous solution was evaporated and the residue was purified by column chromatography. (Whatman cellulose powder CC 31 with butanol: water: ethanol 4: 1: 1). Thus, the desired product is obtained in a yield of 15 mg.

IR (CHCl3) 1790 and 1620 cm ^{@ -1}

NMR (D 6 O) ⁱⁿ accordance with the proposed structure.

Example 31

Preparation of 9-0-ethoxycarbonylmethylclavulanic acid lithium salt

p-Methoxybenzyl O-ethoxycarbonylmethylclavulanate (4 g) in redistilled tetrahydrofuran (40 ml) containing water (0.1 ml) was hydrogenated on 10% palladium on carbon (1.2 g) for 30 minutes at room temperature, where according to thin layer will disappear starting material. The catalyst was then removed by filtration, the filtrate was added to water (50 mL) and filtered to pH 7.0 with 1.0 M lithium hydroxide solution (consumption 7.5 mL, 10 mL theory, slight difference due to solvent in starting material). The solution was evaporated in vacuo, the pale yellow syrup obtained was triturated with acetone (50 mL) and crystallized. The product was filtered off, washed with acetone and ether, air dried to give 1.6 g of an almost colorless crystalline solid.

IR: 1795 (beta-lactam C = O), 1735 (ester C = O), 1695 (C = C), 1620 cm ^-1 (COg) Cnujol).

Example 32

Preparation of lithium salt of 9-O-butylclavulanic acid

A solution of p-methoxybenzyl 9-O-butylolavulanate (80 mg) in tetrahydrofuran (6 mL) containing water (0.02 mL) was hydrogenated on 10% palladium on carbon (40 mg) for 15 minutes when thin layer chromatography was performed. shows the absence of starting material. The catalyst was then removed by filtration to give a solution of clavulanyl butyl ether. This solution was diluted with water (20 mL) and titrated to pH 7.5 with 0.1 U lithium hydroxide solution. The solution was evaporated in vacuo to a semi-solid residue which crystallized by trituration with acetone. The crystalline product is filtered off, washed with ether and air dried. Yield 40 mg of crystalline lithium salt of 9-O- (n-butyl) clavulanic acid.

IR (nujol) 1768 (beta-lactam C = O), 1698 (C = C), 1618 cm ^-1 (CO 2 ^- ),

NMR (D ₂ O) δθ, 73 (3H, t, J 7Hz, (CH ₂ ) ₃ CH ₃ ), 0.95-1.60 (4H, m, CH ₃ CH ₂ CH ₃ , 2.94 (1H, d, J 17Hz) 6-P-CH), 3.36 (2H, t, J 7Hz, OCH ₂ CH ₂ ), 3.43 (1H, dd, J 17 and 3Hz, 6-a-CH), 3.97 (2H δ, J 8Hz, = CHCH ₂ ), 4.76 (1H, t, J 8Hz, CH =), 4.84 (1H, s, 3-CH), 5.99 (1H, d,

J 3Hz, 5-CH 3).

Example 33

Lithium salt of 9-0-ethylclavulanate

N

CH ₂ OCH ₂ CH ₃ 'CO ₂ CH ₂ C _b H ₄ OCH ₃

: H ₂ OCH ₂ ^ch 3 p-Methoxybenzyl-9-O-ethylclavulanate (2.5 g) in tetrahydrofuran (25 ml) containing water (0.1 ml) was hydrogenated to 10% palladium on carbon (0.8 g) . After 2 hours, the absence of starting material was detectable by thin layer ehromatography. The catalyst was filtered through a pad of finely divided silica gel, the filtrate was diluted with an equal volume of water and titrated to pH 7.0 with 1M lithium hydroxide solution. Evaporation of the solvents and trituration of the residue in acetone gave the lithium salt as a light cream crystalline solid (1.05 g).

(The sodium salt was prepared in an identical manner using 1M sodium hydroxide solution, yield 0.85 g.)

The properties of the lithium salt: IR (nujol) 1785 (.beta.-lactam C = 0), 1685 (C = C), 1615 cm ^- '(CO ₂ ^-).

Example 34

Preparation of crystalline lithium salt of 9-O-methylclavulanic acid

Benzyl 9-O-methylclavulanate (0.52 g) and lithium carbonate (0.063 g) in ethanol-water (6: 1, 17 ml) were hydrogenated at room temperature and atmospheric pressure to 10% palladium on carbon (0, 2 g) for 30 minutes. After this time, the mixture was filtered, concentrated in vacuo and freeze-dried to give an amorphous solid. This was dissolved in water (about 1 mL) and acetonitrile (about 30 mL) was added. The mixture was cooled in an ice bath and rubbed until crystalline product formed (about 30 minutes). The colorless solid was filtered and dried to give the desired lithium salt of olavulanyl-O-methyl ether as microcrystals (0.13 g).

Example 35

Preparation of lithium salt of O-ethylclavulanic acid

'COjCHjOCHj' cOjli

Methoxymethyl O-ethylalavulanate (0.5 g) in tetrahydrofuran (5 mL) was added to water (25 mL). The mixture was maintained at pH 8.5-9.0 by adding 1.0 mL of lithium hydroxide solution using a Metrohm pH-stat (1.8 mL consumption). The aqueous solution was then evaporated to dryness in vacuo and triturated with acetone to give a pale yellow crystalline solid which was filtered off, washed with ether and air dried. 0.25 g of the title compound is obtained. This compound exhibits the following properties:

(nujol) 1785 (.beta.-lactam C = 0), 1685 (C = C), 1615 cm ^- '(-COj).

NMR (D ₂ O) 1.11 (3H, t, J 7Hz, CH 3 CH-j), 3.05 (1H, d, J, 17 Hz, 6-β-OΗ), 3.48 (2H, q, J 7Hz, CH ₂ CH ₃ ), 3.50 (1H, dd, J 17Hz and 3Hz, 6-b-CH), 4.04 (2H, d, J, 7Hz, CH ₂ -CH =), 4.82 (1H, t, J 7Hz, CH =), 4.88 (1H, s, 3-CH), 5.65% (1H, d, J 3Hz, 5-CH).

The corresponding lithium salt of O-methyl clavulanate can be obtained in a similar manner.

Example 37

9-0-Allylclavulanic acid sodium salt

A solution of benzyl 9-O-allyl clavulanate (0.5 g) in tetrahydrofuran / water (1: 4, mL) was titrated to constant pH 10 at room temperature with aqueous sodium hydroxide. The reaction mixture was diluted with water (30 mL) and neutral organic material was removed by continuous extraction with ether. The aqueous solution was evaporated by freeze-drying and then chromatographed on silica gel. Elution with butanol-ethanol-water (4: 1: 1.75) gave the title compound (0.01 g).

NMR (D ₂ O): 5.63 (1H, d, J 3Hz, 5-CH), 4.97-5.40 (3H, m, -CH = CH ₂ ), 4.87 (1H, m, 3-CH), 4.83 (1H, broad t, J 8Hz, CH ®), 4.19 (2H, d, J 8Hz, _9-CH2), 3.91 (2H, br d,

J 6Hz, -CH ₂ CH = CH ₂ , 3.49 (1H, dd, J 18Hz, J 3Hz, 6 x x-CH), and 2.97 δ (1H, d, J 18Hz, 6P-CH ).

Claims (4)

A process for the preparation of clavulanic acid ethers of general formula II, wherein R is C 1 -C 4 alkyl, C 3 -C 4 alkenyl, benzyl, benzyl substituted by nitro or C 1 -C 4 alkyl substituted by carboxy in salt form, C 1 -C 4 alkoxycarbonyl, benzyloxycarbonyl, phenylcarbonyl, chlorophenylcarbonyl, cyano, hydroxy, amino or amino substituted with 1 to 4 carbon atoms and / or benzyloxycarbonyl; and A is an alkali metal cation, characterized in that the compound of formula (IIa) is de-esterified (IIa) wherein B is alkyl of 1 to 4 carbon atoms, alkenyl of 3 to 4 carbon atoms, benzyl or benzyl substituted with alkoxy of 1 to 4 carbon atoms or C 1 -C 4 alkyl substituted with C 1 -C 4 alkoxy, C 1 -C 4 alkylcarbonyloxy or C 1 -C 4 alkoxycarbonyl, and the free acid obtained is converted by treatment with a base into the corresponding salt. 2. A process according to claim 1 wherein the deesterification is carried out by hydrogenolysis of a compound of formula (IIa) wherein B is benzyl or benzyl substituted with C1-C4 alkoxy in the presence of a transition metal metal catalyst. 3. The process according to claim 1, wherein the deesterification is carried out by mild basic hydrolysis. 4. A process according to claim 3, wherein the compound of formula IIa is deesterified by mild basic hydrolysis, wherein R is methyl and B is methyl.