NO159659B - PROCEDURE FOR THE PREPARATION OF 1,1-DIOXOPENICILLANOYLOXYMETHYL-6- (2-AMINO-2-PHENYLACETAMIDO) PENICILLANATE ANTIBIOTICS. - Google Patents

Description

This invention relates to a process for the production of 1,1-dioxopenicillanoyloxymethyl-6-(2-amino-2-phenylacetamido)penicillanate antibiotics with formula (II)

or a pharmaceutically acceptable acid addition salt thereof.

US Patent 4,234,579 describes penicillanic acid 1,1-dioxide

and esters thereof which are readily hydrolyzable in vivo, their use as antibacterial agents and to increase the effectiveness of β-lactam antibiotics against many β-lactamase-producing bacteria.

US patent 4,244,951 describes new antibacterial agents

with formula (VIII) where penicillanic acid-1,1-dioxide is bound to known penicillin antibiotics via a methylenedioxo group,

i.e.

birth

where R is the acyl group in a natural or semi-synthetic penicillin. Particularly preferred meanings for R include 2-amino-2-phenylacetyl and 2-amino-2-(p-hydroxyphenyl)-acetyl. The compounds of formula (VIII) are prepared, e.g. by reacting a carboxylate salt of the penicillin such as the sodium or potassium salt or a tertiary amine salt with a halomethyl ester (or related ester) of penicillanic acid 1,1-dioxide. The halomethyl ester intermediates are produced by esterification

of penicillanic acid 1,1-dioxide.

Harrison et al, Journal of the Chemical Society (London), Perkin I, 1772 (1976) describe: (a) oxidation of 6,6-dibromopenicillanic acid with m-chloroperbenzoic acid, to form a mixture of the corresponding a- and /3 -sulfoxides; (b) oxidation of methyl 6,6-dibromopenicillanate with m-chloroperbenzoic acid to form a methyl 6,6-dibromopenicillanate 1,1-dioxide; (c) oxidation of methyl 6-α-chloropenicillanate with m-chloro-perbenzoic acid to form a mixture of the corresponding α- and β-sulfoxides; and (d) oxidation of methyl 6-bromopenicillanate with m-chloroperbenzoic acid, to form a mixture of the corresponding α- and β-sulfoxides.

Clayton, Journal of the Chemical Society (London) (C),

2123 (1969) describes: (a) preparation of 6,6-dibromo- and 6,6-diiodopenicillanic acid; (b) oxidation of 6,6-dibromopenicillanic acid with sodium periodate to form a mixture of the corresponding sulfoxides; (c) hydrogenolysis of methyl 6,6-dibromopenicillanate to form methyl 6-α-bromopenicillanate; (d) hydrogenolysis of 6,6-dibromopenicillanic acid and its methyl ester to form penicillanic acid and its methyl ester, respectively; and (e) hydrogenolysis of a mixture of methyl 6,6-diiodopenicillanate and methyl 6α-iodopenicillanate to form pure methyl 6-α-iodopenicillanate.

Belgian patent 882,028 describes a process for the preparation of penicillanic acid 1,1-dioxide and its in vivo readily hydrolyzable esters by oxidation of 6-halopenicillanate or a 6,6-dihalopenicillanate to the corresponding 1,1-dioxide, and then dehalogenation to form the desired penicillanate 1,1-dioxide.

US Patent 3,293,242 describes 6-(2-azido-2-phenylacetamido)-penicillanic acid and salts thereof.

Japanese Kokai 78-37,691; Chem. Abstr., 89, 109466v (1978) describes 6-[2-azido-2-(p-hydroxyphenyl)acetamido]penicillanic acid and its reduction with hydrogen and palladium-on-charcoal to form 6-[2-amino-2 --(p-Hydroxy enyl) acetamido] penicillanic acid.

The characteristic of the present invention is that a compound of formula

where R<1> is as defined above, Q is N3 or NHC02CH2C6H4R<4>

where R<4> is H, Cl, Br, NO2, CH3 or OCH3; Y and Z are each Cl,

Br or I, or Y is H and Z is Cl, Br or I, is contacted with hydrogen in the presence of a noble metal catalyst, at least two molar equivalents of acid scavenging agent, or when Y

is H, at least one molar equivalent of acid scavenging agent, and a reactive solvent.

The method of the present invention is in contrast to the contents of Baltzer et al., J. Antibiotics, 33, 1183(1980) and British Patent 2,044,255. Baltzer et al. describes the instability of the 1,1-dioxymethylene ester bond between the beta-lactamase inhibitor and the penicillin antibiotic portions of the conjugate ester compounds, such as those of formula (II) of the present invention under neutral or alkaline conditions. In British patent 2,044,255, the hydrogenation exemplified there is carried out under conditions which either maintain an acidic pH by adding acidic reagents or by forming acids during the course of the reaction. In contrast to these literature references, the method according to the present invention can be carried out in the presence of an acid-binding agent which leads to neutral to alkaline reaction conditions.

The compounds with the formulas (I) and (II) are here referred to as derivatives of penicillanoyloxymethyl penicillanate. The compound of formula (I) where R is hydrogen and Y and Z are each Br, and Q is N^, is designated as 1,1-dioxo-6,6-dibromo-penicillanoyloxymethyl-6-(2-azido-2- phenylacetamido)-penicillanate.

The intermediate product of formula (I) and preparation thereof

is described in more detail in Norwegian patent application 82.0898.

In compounds where Y is H and Z is Cl, Br or I, the Z substituent may be in the α-configuration, Ø-configuration, or may be a mixture of the two isomers. All such compounds are covered by the invention.

Methods for the preparation of penicillanoyloxymethyl penicillanates of formula (II) by esterification as illustrated below are described in US patent 4,244,951 and Dutch patent application 8,000,775 published on August 15, 1980, corresponding

to British Patent Application 2,044,255:

In the above formulas, one of A and B is -CH„X , and the other 11 is M , where X is a good leaving group, e.g. Cl, Br, I,

CH 3 SO 2 O or toluenesulfonyloxy; and M^" is a carboxylate salt-forming cation, e.g., sodium, potassium, triethylammonium, or tetrabutylammonium; and Q<1> is a common amino-protecting group, e.g., benzyloxycarbonyl. The first product obtained is the amino-protected deriv. of (II) which gives the desired antibiotic compound by removal of the protecting group Q by standard methods.

The above-mentioned Dutch and the corresponding British application also describe a process for the preparation of compounds of formula (II) where R is hydrogen, by coupling chloromethyl-6-(2-azido-2-phenylacetamido)penicillanate and a salt of penicillanic acid-1, 1-dioxide, and hydrogenation of the intermediate 1,1-dioxopenicillanoyloxymethyl-6-(2-azido-2-phenyl-acetamido) penicillanate.

In the previously known methods, the 1,1-dioxopenicillanic acid starting material of formula (IX), A = H, is obtained, e.g. by dehalogenation of the corresponding 6-halo- or 6,6-dihalo-penicillanic acid sulfone. The ester (IX), where A is Cf^X<1>, is obtained from the acid by certain methods of esterification.

Reaction scheme A shows the preparation of the compounds of formula (I) and their conversion to antibiotic compounds of formula (II) by simultaneous hydrogenolysis of the halogen groups Y and Z and reduction of the azido or NHC02CH2C6H4R<4-> group to NH2 by reaction of compound (I ) with hydrogen in the presence of a catalyst.

The halopenicillanic acid starting materials of formula (III) where M is hydrogen are prepared, e.g. from 6-amino-penicillanic acid or the corresponding sulfoxide or sulfone by reaction with nitric acid and treatment of the resulting 6-diazo compound with a halogen or hydrogen halide of the formula Y-Z, where Y and Z are as indicated above, by known methods. See e.g. Clayton et al., Journal of the Chemical Society (London) (C), 2123 (1969). of chloromethyl-6-(2-azido-2-phenylacetamido)penicillanate and a salt of penicillanic acid 1,1-dioxide, and hydrogenation of the intermediate 1,1-dioxopenicillanoyloxymethyl-6-(2-azido-2-phenylacetamido) penicillanate.

In the previously known methods, the 1,1-dioxopenicillanic acid starting material of formula (IX), A = H, is obtained, e.g. by dehalogenation of the corresponding 6-halo- or 6,6-dihalo-penicillanic acid sulfone. The ester (IX), where A is CR^ X , is obtained from the acid by certain methods of esterification.

Reaction core A shows the preparation of the compounds of formula (I) and their conversion to antibiotic compounds of formula (II) by simultaneous hydrogenolysis of the halogen groups Y and Z and reduction of the azido or NHC02CH2CgH4R<4-> group to NH2 by reaction of compound (I ) with hydrogen in the presence of a catalyst.

The halopenicillanic acid starting materials of formula (III) where M is hydrogen are prepared, e.g. from 6-amino-penicillanic acid or the corresponding sulfoxide or sulfone by reaction with nitric acid and treatment of the resulting 6-diazo compound with a halogen or hydrogen halide of the formula Y-Z, where Y and Z are as indicated above, by known methods. See e.g. Clayton et al., Journal of the Chemical Society (London) (C), 2123 (1969).

Although different methods can be used for the conversion of intermediates of formula (I) to the antibiotic compounds of formula (II), a particularly suitable and preferred method is simultaneous hydrogenolysis of the halogen groups Y and Z as defined above, and reduction of azido or benzyloxycarbonylamino -group Q, as defined above, to NH2 by means of hydrogen in the presence of a noble metal catalyst. A particularly suitable method for carrying out this simultaneous hydrogenolysis and reduction is to stir or shake a solution of a compound of formula (I) under an atmosphere of hydrogen or hydrogen mixed with an inert diluent such as nitrogen or argon, in the presence of a noble metal hydrogenation catalyst and a reaction inert solvent. Suitable solvents for this hydrogenation reaction are those which essentially dissolve the starting compound of formula (I), but which are not themselves subjected to hydrogenation or hydrogenolysis. Examples of such solvents include lower alkanols such as methanol, ethanol and isopropanol; ethers such as diethyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane; low molecular weight esters such as ethyl acetate and butyl acetate; tertiary amides so

such as N,N-dimethylformamide, N,N-dimethylacetamide and N-methyl-pyrrolidone; methylene chloride and mixtures thereof.

Moreover, it is often desirable to carry out this turnover

in the presence of a sufficient amount of an acid binding agent to bind one or both of the molar equivalents of hydrogen halide formed. Examples of suitable acid-binding agents include sodium bicarbonate, calcium carbonate, trisodium phosphate, potassium citrate and tertiary amines such as triethylamine, N,N-dimethylaniline, N-methylmorpholine, N-methylpiperidine, N,N<1->dimethylpiperazine and the like. Introduction of the hydrogen gas into the reaction medium is usually achieved by carrying out the reaction in a closed vessel containing the compound of formula (I), the solvent, the catalyst and the hydrogen. The pressure inside the reaction vessel can vary from approx. 1 to approx.

100 kg/cm 2. The preferred pressure range, when the atmosphere inside the reaction vessel is almost pure hydrogen, is from approx. 2 to approx.

2

5 kg/cm. The reaction with hydrogen is usually carried out at a temperature of approx. 0 to approx. 60°C, and preferably from approx. 25 to approx. 50°C. Using the preferred temperature and pressure values, hydrogenolysis of the halogens and reduction of the group Q usually takes place within a few hours, e.g.

from approx. 2 to approx. 20 hours. The preferred noble metal catalysts used in this hydrogenation reaction are the type of agents known in the art for this type of conversion, e.g. nickel, palladium, platinum and rhodium.

Palladium is particularly preferred. The catalyst is usually

present in an amount from approx. 0.1 to approx. 25% by weight, and preferably from approx. 1 to approx. 10% by weight, based on the compound of formula (I). It is often appropriate to distribute the catalyst on an inert carrier, and a particularly suitable catalyst is palladium distributed on an inert carrier such as coal. Another suitable catalyst is palladium-on-calcium carbonate where the calcium carbonate serves as a carrier for the precious metal and as an acid binding agent.

When the hydrogenolysis and reduction are almost complete, the desired antibiotic of formula (II) is isolated by standard methods, e.g. the catalyst is removed by filtration, the solvent is evaporated and, if necessary, the product is purified by well-known methods such as crystallization or by chromatography.

Alternatively, the product of formula (II) can be isolated in form

of a pharmaceutically acceptable acid addition salt, e.g. by treating the filtrate obtained by removing the catalyst or a solution of the isolated free base with an equivalent amount of a pharmaceutically acceptable acid and removing the solvent, e.g. by filtration or evaporation. Examples of pharmaceutically acceptable acids which may be used include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, maleic acid, fumaric acid, succinic acid, lactic acid, tartaric acid, citric acid, gluconic acid, saccharin acid and p-toluenesulfonic acid.

It should be emphasized that the hydrochloride, hydrobromide and hydroiodide salt or mixtures thereof of compounds of formula (II) are obtained directly from the hydrogenation mixture if

only one molar equivalent of acid-binding agent is used for the starting materials of formula (I) where each of Y and Z is Cl,

Br or I, or no acid binding agent is used for the starting materials of formula (I) where Y is H and Z is Cl, Br or I.

As mentioned above, the compounds of formula (I) are useful intermediates for the preparation of antibacterial agents of formulas (II) and (VIII) described in US Patent 4,244,951 and British Patent Application 2,044,255.

The compounds of formula (II) and (VIII) have in vivo antibacterial activity in mammals, and this activity can be demonstrated by standard methods for penicillin compounds. E.g. the compound of formula (II) is administered to mice in which acute infections have been induced by intraperitoneal inoculation with a standardized culture of a pathogenic bacterium. The severity of the infection is standardized so that the mice receive one to ten times the LD100 ^LD100: minimum inoculation required to consistently kill 100% of control mice). At the end of the experiment, the activity of the compound is assessed by counting the number of survivors who have been exposed to the bacteria and have also received the compound of formula (II). The compounds of formula (II) can be administered by both oral (p.o.) and subcutaneous (s.c.) administration.

In vivo activity of the antibacterial compounds

(II) and (VIII) make them suitable for combating bacterial infections in mammals, including humans, by both oral and parenteral administration. The compounds are useful for combating infections caused by susceptible b bacteria in humans. In general, it is the substituent R that determines whether a given bacterium will be affected by a given compound of formula (VIII). A compound of formula (VIII) breaks down to the corresponding compound of formula (VII)

(or a salt thereof) and penicillanic acid 1,1-dioxide (XIII) after administration to a mammal either orally or parenterally.

Penicillanic acid 1,1-dioxide then acts as an β-lactamase inhibitor and increases the antibacterial effectiveness of the compound of formula (VII) (or a salt thereof). When R<*3> is D-2-amino-2-phenylacetyl or D-2-amino-2-[4-hydroxyphenyl]-acetyl, the compounds are useful in combating infections caused by susceptible strains of Escherichia coli and Staphylococcus aureus .

When determining whether a particular strain of Staphylococcus aureus or Escherichia coli is sensitive to

a particular compound of formula (II) or (VIII), one can use the previously described in vivo examination. Alternatively, one can measure the minimum inhibitory concentration (MIC)

of a 1:1 mixture of the compound of formula (VII) (or its salt) and the compound of formula (XIII) (or its salt). The MIC values can be measured by the method recommended by the International Collaborative Study on Antibiotic Sensitivity Testing (Ericcson and Sherris, Acta. Pathologica et Microbiologia Scandinav. Supp. 217, section B: 64-68 [1971]), which uses brain-heart infusion - (BHI) agar and inoculum duplicating device. Growth tubes for overnight use are diluted 100-fold for use as standard inoculum (20,000-10,000 cells in approx. 0.002 ml are placed on the agar surface; 20 ml BHI agar/dish). Twelve-fold dilutions of the test compound are used, with an initial concentration of the test compound of 200 Mg/ml.

Individual colonies are disregarded when examining the plates

after 18 hours at 37°C. Susceptibility of the test organism

(MIC) is considered the lowest concentration for a compound

which is capable of producing complete inhibition of growth as judged by the naked eye.

The following examples and presentations will serve

to further illustrate the invention. Infrared spectra (IR)

were measured undiluted, as Nujol slurry or as potassium bromide discs (KBr discs) and diagnostic absorption bands are given in wavenumber (cm . Nuclear magnetic resonance (NMR) spectra were measured at 60 MHz for solutions in deuterated chloroform (CDC13), D20, CD3SOCD3, or deuterated acetone (CD3COCD3), and peak positions are given in parts per million downfield from tetramethylsilane.The following abbreviations for peak shapes

are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, double duplicate.

Example 1 (intermediate)

Chloromethyl- 6, 6- dibromopenicillanate

6,6-Dibromopenicillanic acid (8.0 g, 22 mmol) was stirred with 75 mL of methylene chloride, and 35 mL of water was added. To this solution was added tetrabutylammonium hydroxide to adjust the pH to 8. The organic layer was separated and the aqueous phase was extracted with 30 ml of methylene chloride. The combined organic layers were evaporated to dryness in vacuo to give the tetrabutylammonium salt of 6,6-dibromopenicillanic acid, 14.2 g, as a light brown oil. To this was added 40 ml of chloroiodomethane, and the resulting mixture was stirred under nitrogen for 3 hours at room temperature. The reaction mixture was concentrated in vacuo, the residue was stored at room temperature and purified by chromatography on 300 g of silica gel, elution being carried out with 95:5 (by volume) toluene/ethyl acetate. Fractions containing the least polar material were collected and evaporated to give 5.4 g (59%) of the desired product, m.p. 105-106°C. <1>H-NMR (CDCl 3 ) ppm (6): 1.6 (s, 3H), 1.75 (s, 3H), 4.62

(s, 1H), 5.8 (dd, 2H), 5.82 (s, 1H).

When the above procedure is repeated, except that the chloroiodomethane used there is replaced with an equimolar amount of bromoiodomethane, diiodomethane, di(methylsulfonyloxy)methane, di(isobutylsulfonyloxy)methane, di(n-hexylsulfonyloxy)methane, di(benzenesulfonyloxy)methane or a compound of the formula (R2C6H4S020)2CH2 where R<2> is 4-C1, 2-Br, 4-1, 3-N02, 4-CH3, 3-(CH3)2CH, 4-CH30, 3-C2H50 or 4-n -C3H70, you get respectively: bromomethyl-6,6-dibromopenicillanate,

iodomethyl 1-6,6-dibromopenicillanate,

methylsulfonyloxymethyl-6,6-dibromopenicillanate,

isobutylsulfonyloxymethyl-6,6-dibromopenicillanate, n-hexylsulfonyloxymethyl-6,6-dibromopenicillanate, benzenesulfonyloxymethyl-6,6-dibromopenicillanate, and

R? -CgH^-sulfonyloxymethyl-6,6-dibromopenicillanates, where R 2 is

as indicated above for the di(substituted phenylsulfonyloxy)methane reagent.

Example IA (intermediate product)

Iodomethyl- 6, 6- dibromopenicillanate

4.15 g (10.2 mmol) of chloromethyl-6,6-dibromopenicillanic acid and 7.5 g (50 mmol) of sodium iodide were added to 25 ml of acetone. The mixture was stirred overnight at room temperature, and

the acetone was evaporated to give a dark residue. This was dissolved in 150 ml of ethyl acetate, washed with water (3 x 25 ml),

saturated brine (25 ml), dried (MgSO 4 ), and the solvent was evaporated in vacuo to give a residual oil which was purified by chromatography on 100 g of silica gel, eluting with 1:1 (by volume) ethyl acetate /hexane.

30 ml fractions were collected. The product was eluted in fractions 4-6, which were combined and evaporated to give 5.95 g of colorless oil which crystallized on standing, m.p. 67-68°C. <1>H-NMR (CDCl 3 ) ppm (6): 1.55 (s, 3H), 1.65 (s, 3H), 4.54

(s, 1H), 5.8 (s, 1H), 5.98 (s, 2H).

When using bromomethyl-6,6-dibromopenicillanate, bromomethyl-6,6-dichloropenicillanate, chloromethyl-6,6-dichloropenicillanate, chloromethyl-6-bromo-6-chloropenicillanate, chloromethyl-6-chloro-6-iodo-penicillanate, chloromethyl -6-bromo-6-iodopenicillanate or bromo-methyl-6-bromo-6-iodopenicillanate instead of chloromethyl-6,6-dibromopenicillanate in the above-mentioned method is obtained

in each case the corresponding iodomethyl ester.

Example 2 (intermediate)

Chloromethyl-6,6-dibromopenicillanate-1,1-dioxide

A solution of 7.1 g (17.4 mmol) of chloromethyl-6,6-dibromopenicillanate in 75 ml of ethyl acetate was cooled to 0°C, and 7.3 g (36 mmol) of m-chloro-perbenzoic acid was added. The mixture was stirred under nitrogen at 0°C overnight, diluted to 150 mL with ethyl acetate, and 50 mL of water was added at 0°C. Sufficient sodium bisulfite was added to destroy excess peracid, the mixture was adjusted from pH 2 to pH 7.5 with sodium bicarbonate, and the organic layer was separated and washed with 50 mL saturated sodium bicarbonate, 50 mL water, and 25 mL brine. The washed extracts were dried (MgSO 4 ), concentrated to dryness in vacuo, and the residue was purified by chromatography on 300 g of silica gel, eluting with 9:1 (by volume) toluene/hexane to give 5.0 g ( 65%) of the desired dioxide as a crystalline solid,

sm.p. 95-96°c.

<1>H-NMR (CDCl 3 ) ppm (6): 1.5 (s, 3H), 1.7 (s, 3H), 4.58 (s, 1H), 5.04 (s, 1H), 5.8 (dd, 2H).

Analysis:

Calculated for CgH10N05SBr2Cl: C 24.59, H 2.29, N 3.18 Found: C 24.63, H 2.49, N 3.31.

Another, more polar component was isolated from the chromatography column, 0.8 g. This was identified as a 9:1 mixture of the α- and β-sulfoxides of chloromethyl-6,6-dibromopenicillanate by 1 H-NMR.

Example 2A (intermediate)

Iodomethyl-6,6-dibromopenicillanate-1,1-dioxide

To 40 ml of acetone was added 0.25 g (0.5 mmol) of iodomethyl-6,6-dibromopenicillanate, and the mixture was stirred until a solution was obtained, 10 ml of water was added, followed by sufficient concentrated phosphoric acid to adjust the mixture to pH 4.0. Then 158 mg (1 mmol) of powdered potassium permanganate was added and the mixture was stirred at room temperature for 1.25 hours. Ethyl acetate, 100 ml, and water, 50 ml, were added. The resulting mixture was adjusted to pH 2.0 with 6N hydrochloric acid, and sodium bisulfite was added to consume excess oxidant (pH 2.9). The organic layer was separated, the aqueous phase was extracted with 50 mL of ethyl acetate, and the combined organic layers were washed with saturated saline (3 x 25 mL). After drying over anhydrous sodium sulfate and evaporation of the solvent, 0.29 g of colorless oil was obtained. The oil was purified by chromatography on 25 g of silica gel, eluting with 1:1 ethyl acetate/hexane, and 15 ml fractions were taken. Fractions 4 and 5 were combined and evaporated in vacuo to give 0.27 g (100%) of a colorless oil which crystallized on standing, m.p. 71-73°C.

"""H-NMR (CDCl 3 ) ppm (6): 1.5 (s, 3H) , 1.62 (s, 3H) , 4.49 (s, 1H) , 5.02 (s, 1H), 5.98 (dd, 2H).

Using an equivalent amount of sodium permanganate or calcium permanganate instead of potassium permanganate in the above described process gave the same product in a similar manner.

Attempts to prepare iodomethyl-6,6-dibromopenicillanate-1,1-dioxide from the chloromethyl ester prepared in Example 2, by treatment with sodium iodide in acetone by the method of Example IA, gave iodomethyl-6-a-bromopenicillanate-1,1-dioxide . <1>H-NMR (CDC13) ppm/6: 1.55 (s, 3H), 1.70 (s, 3H), 4.43 (s, 1H), 5.2 (d, 1H), 5.75 (d, 1H), 6.0 (dd, 2H).

Example 3 (intermediate)

Iodomethyl- 6- bromo- 6- chlorpenicillanate- 1, 1- dioxide

A solution of 7.6 g (17.4 mmol) of iodomethyl-6-bromo-6-chloropenicillanate in 75 ml of ethyl acetate was cooled to 0°C, and 7.3 g (36 mmol) of m-chloroperbenzoic acid was added. The mixture was stirred under nitrogen at 0°C overnight, diluted to 150 mL with ethyl acetate, and 50 mL of water was added at 0°C. Sufficient sodium bisulfite was added to destroy excess peracid, the mixture was adjusted from pH 2 to pH 7.5 with sodium bicarbonate, and the organic layer was separated and washed with 50 mL saturated sodium bicarbonate, 50 mL water, and 25 mL brine. The washed extracts were dried (MgSO 4 ), concentrated to dryness in vacuo, and the residue was purified by chromatography on silica gel.

Example 4 (intermediate)

Iodomethyl-6,6-dichloropenicillanate-1,1-dioxide

To a solution of 4.09 g (0.01 mol) iodomethyl-6,6-dichloropenicillanate in 50 ml acetone add 2.5 ml (0.24 mol)

30% hydrogen peroxide and 1 ml of 0.5M aqueous sodium tungstate. The mixture is heated at reflux temperature for 1 hour and stirred overnight at room temperature. Evaporation of the solvent gives the crude product which is purified by chromatography on silica gel.

When aqueous potassium molybdate or zirconium tetrachloride is used instead of sodium tungstate in the method described above, the result is essentially the same.

When methanol, ethanol, isopropanol, methyl ethyl ketone or their mixtures with water are used as solvent instead of acetone and the reaction is carried out at from 25 to 60°C, the title compound is obtained in a similar manner.

Example 5 (intermediate)

Chloromethyl-6-g-bromopenicillanate-1,1-dioxide

2.32 g (8 mmol) of tri- n-butyltin hydride. The resulting mixture is stirred overnight at room temperature, the solvent is evaporated in vacuo, and the residue is purified by column chromatography on silica gel to give the title compound.

Alternatively, the same product is obtained by using chloromethyl-6,6-dibromopenicillanic acid by the method described above and by oxidizing the resulting sulfide, chloromethyl-6-0-bromopenicillanate, to the sulfone by the method according to examples 2, 2A, 3 or 4 .

Example 6 (intermediate)

Iodomethyl-6-g-bromopenicillanate-1,1-dioxide

A solution of 0.12 g (0.33 mmol) of chloromethyl-6-(3-bromopenicillanate-1,1-dioxide) and 0.25 g (1.66 mmol) of sodium iodide

in 5 ml of acetone was stirred for 30 hours at room temperature. The resulting pale yellow suspension was evaporated to dryness, and the residue was taken up in 50 mL of ethyl acetate, washed successively with 2 x 10 mL of water, 10 mL of saturated saline, and dried over anhydrous sodium sulfate. The resulting solution was evaporated under reduced pressure to give the title compound as a solid, 0.14 g.

<1>H-NMR (CDCl 3 ) ppm (6): 1.45 (s, 3H), 1.65 (s, 3H), 4.5 (s, 1H), 4.83 (d, 1H), 5.42 (d, 1H), 6.0 (dd, 2H).

Example 7 (intermediate)

6- g- bromopenicillanic acid- 1, 1- dioxide

To a stirred mixture of 560 ml of water, 300 ml of dichloromethane and 56.0 g of 6-a-bromopenicillanic acid was added 4N sodium hydroxide solution until a stable pH of 7.2 was obtained. This required 55 ml of sodium hydroxide. The mixture was stirred at pH 7.2 in

10 minutes and was then filtered. The teams were separated,

and the organic phase was discarded. The aqueous phase was then poured rapidly with stirring into an oxidizing mixture prepared as follows.

In a 3 liter flask, 63.2 g of potassium permanganate, 1000 ml of water and 48.0 g of acetic acid were mixed. This mixture was stirred for 15 minutes at 20°C and then cooled to 0°C.

After the 6-a-bromopenicillanic acid solution was added to the oxidizing mixture, a cooling bath of -15°C was maintained around the reaction mixture. The internal temperature rose to 15°C and then fell to 5°C over a period of 20 minutes. At this point, 30.0 g of sodium metabisulphite was added with stirring over a 10 minute period at approx. 10°C. After a further 15 minutes, the mixture was filtered, and the filtrate's pH value was lowered to 1.2 by adding 170 ml of 6N hydrochloric acid. The aqueous phase was extracted with chloroform and then with ethyl acetate. Both the chloroform and ethyl acetate extracts were dried using anhydrous magnesium sulfate and then evaporated in vacuo. The chloroform solution gave 10.0 g (16% yield) of the title compound. The ethyl acetate solution gave 57 g of an oil which was triturated under hexane. A white, solid substance was obtained. It was filtered out to

give 41.5 g (66% yield) of the title compound, m.p. 134°C (decomp.).

Analysis: Calculated for CgH^0BrN0^S:

C 30.78, H 3-.23, Br 25.60, N 4.49, S 10.27%,

Found:

C 31.05, H 3.24, Br 25.54, N 4.66, S 10.21%.

Oxidation of 6-α-chloropenicillanic acid and 6-α-iodopenicillanic acid with potassium permanganate according to the method described above yields 6-α-chloropenicillanic acid 1,1-dioxide and 6-α-iodopenicillanic acid 1,1-dioxide, respectively.

Example 8 (intermediate)

6- <3- chlorpenicillanic acid- 1, 1- dioxide

An oxidizing solution was prepared from 185 mg of potassium permanganate, 0.063 ml of 85% phosphoric acid and 5 ml of water. This oxidizing solution was added dropwise to a solution of 150 mg of sodium 6-o-chloropenicillanate in 5 ml of water at 0-5°C until the violet color of the potassium permanganate remained.

About. half of the oxidizing solution was required.

At this point, the potassium permanganate color was removed by addition of solid sodium bisulfite, and then the reaction mixture was filtered. Ethyl acetate was added to the filtrate and the pH was adjusted to 1.8. The layers were separated, and the aqueous layer was further extracted with ethyl acetate. The combined ethyl acetate layers were washed with water, dried and evaporated in vacuo to give 118 mg of the title compound.

The NMR spectrum (in CD3COCD3) showed absorption at 5.82 (d, 1H), 5.24 (d, 1H), 4.53 (s, 1H), 1.62 (s, 3H) and 1.50 ( s, 3H) ppm.

The above product was dissolved in tetrahydrofuran,

and an equal volume of water was added. The pH was adjusted to 6.8 using dilute sodium hydroxide, the tetrahydrofuran was removed by evaporation in vacuo, and the remaining aqueous solution was freeze-dried. This gave the sodium salt of the title compound.

Example 9 (intermediate)

6- <3- bromopenicillanic acid- 1, 1- dioxide

To a solution of 255 mg of sodium 6-Ø-bromopenicillanate

in 5 ml of water, at 0 to 5°C, was placed a solution prepared from 140 mg of potassium permanganate, 0.11 ml of 85% phosphoric acid and 5 ml of water, at 0 to 5°C. The pH value was maintained between 6.0 and 6.4 during the addition. The reaction mixture was stirred at pH 6.3

for 15 minutes, and then the violet solution was covered with ethyl acetate. The pH was adjusted to 1.7 and 330 mg of sodium bisulfite was added. After 5 minutes, the layers were separated, and the aqueous layer was further extracted with ethyl acetate. The combined ethyl acetate solutions were washed with brine, dried (MgSO 4 ) and evaporated in vacuo. This gave 216 mg of the title compound as white crystals. The NMR spectrum (in D 2 O) showed absorptions at 5.78 (d, 1H,

J = 4Hz), 5.25 (d, 1H, J = 4Hz), 4.20 (s, 1H), 1.65 (s, 3H) and 1.4 6 (s, 3H) ppm.

Example 10 (intermediate)

Chlormethyl- 6-a- chlorpenicillanate

A mixture of 2.35 g (0.01 mol) of 6-α-chloropenicillanic acid

and 5.0 ml of water is treated with 5.0 ml of 2N potassium hydroxide. Potassium bicarbonate (6.0 g), tetrabutylammonium hydrogen sulfate (0.34 g, 0.001 mol), dichloromethane (20 mL) and chloromethyl chlorosulfate (1.64 g, 0.011 mol) are added and the resulting mixture is stirred at 25 to 30°C for 2 hours. The reaction mixture is filtered and the layers are separated. The organic phase is dried (Na 2 SO 4 ) and evaporated to dryness to give the title compound.

Example 11 (intermediate)

Chloromethyl-6-a-chlorpenicillanate-1-oxide

A solution of 6.12 g (0.03 mol) 3-chloroperbenzoic acid in 100 ml is added to a stirred solution of 8.49 g (0.03 mol) chloromethyl-6-a-chloropenicillanate in 200 ml chloroform at 0°C chloroform. Stirring is continued for 1.5 hours at 0-5°C.

The reaction mixture is then filtered, washed with sodium bicarbonate solution, water and dried (Na 2 SO 4 ). Evaporation of the solvent in vacuum gives the crude title compound as a mixture of α- and Ø-sulfoxides which can optionally be purified by chromatography on silica gel.

Alternatively, the title compound is prepared by oxidation of 6-α-chloropenicillanic acid by oxidation with an equivalent of 3-chloroperbenzoic acid in tetrahydrofuran at 0-25°C for approx. 1 hour, according to the method of Harrison et al., Jour. Chem. Soc. (London), Perkin I, 1772 (1976). The resulting 6-α-chloropenicillanic acid 1-oxide is then esterified by the method of Example 1 to form the desired chloromethyl ester.

The remaining 6-substituted penicillanate esters and 6,6-dihalopenicillanate esters prepared in examples 1 and 1B are converted to the corresponding 1-oxides by the method described above.

The same compound is obtained by reacting 0.1 mol of chloromethyl-6-a-chloropenicillanate in 150 ml of isopropanol containing 0.8 ml of 0.5M sodium tungstate (Na2W04) or an equivalent amount of potassium molybdate (K-jMoO^) with 0, 1 mol of hydrogen peroxide (30%). The peroxide is slowly added to the other reagents at 60°C, after which the mixture is allowed to cool while it is stirred overnight. The product is isolated as described above.

Example 12 (intermediate)

Chloromethyl-6-α-chlorpenicillanate-1,1-dioxide

To a solution of 2.83 g, 0.01 mol of chloromethyl-6-α-chloropenicillanate in 50 ml of chloroform is added 4.32 g (0.025 mol) of m-chloroperbenzoic acid, and the mixture is stirred under a nitrogen atmosphere for 36 hours at room temperature . The solvent is evaporated in vacuo, the residue is partitioned between ethyl acetate and water at pH 6.0, and sodium bisulphite is added until a test for the presence of peroxides is negative. The pH is adjusted to 8.0, the layers are separated, and the organic phase is washed with brine, dried (Na 2 SO 4 ) and evaporated to dryness in vacuo to give the title compound.

Alternatively, the title compound is obtained by oxidation of the same starting material in methanol or ethanol containing a catalytic amount of sodium tungstate and addition of 2 molar equivalents of hydrogen peroxide at temperatures from 20 to 60°C.

Example 13 (intermediate)

1, l- dioxo- 6, 6- dibromopenicillanoyloxymethyl- 6-( D- 2- azido- 2-phenylacetamido) penicillanate

0.20 g (0.37 mmol) of iodomethyl- 6,6-dibromopenicillanate-1,1-dioxide, and the mixture is stirred at room temperature for 30 minutes. An additional 50 mg of tetrabutylammonium D-(2-azido-2-phenylacetamido)penicillanate was added and stirring was continued for 30 minutes. The reaction mixture was concentrated to dryness and the residue was applied to a column of silica gel (50 g). Elution with 1:1 (v/v) ethyl acetate/hexane was performed, taking 7 mL fractions. Fractions 17-24 were combined and evaporated in vacuo to give 0.14 g (49%) of the desired product as a pale yellow oil.

<1>H NMR (CDCl3 ) ppm (6): 1.4 (s, 3H), 1.5 (s, 3H), 1.59 (s, 3H), 1.62 (s, 3H), 4 .4 (s, 1H), 4.5 (s, 1H), 4.97 (s, 1H) , 5.04

(s, 1H), 5.4-5.70 (m, 2H), 5.85 (s, 2H), 7.05 (d, 1H), 7.35

(p, 5H); Infrared (undiluted) cm<-1>: 1810, 1775.

<*> The tetrabutylammonium salt was prepared as follows:

1 g sodium D-2-azido-2-phenylacetamido-penicillanate,

50 ml of ethyl acetate and 25 ml of water were mixed and adjusted to pH 2.0 (2N HCl). The organic layer was separated, washed with brine (10 mL), and the solvent was evaporated in vacuo. The remaining foam was dissolved in 30 mL of methylene chloride, 15 mL of water was added, and 40% tetrabutylammonium hydroxide solution was added until the aqueous phase reached pH 8.0. The organic layer was separated, the aqueous layer was re-extracted with methylene chloride (2 x 20 mL), and the combined extracts were dried (Na 2 SO 4 ) and concentrated to dryness to give a hard gum. This was triturated with ethyl acetate (2 x 10 ml) and ethyl ether (2 x 10 ml). The resulting whitish solid was air dried to give 1.25 g of the desired tetrabutyl ammonium salt.

the solvent in vacuum gives the crude title compound as a mixture of α- and Ø-sulfoxides which can optionally be purified by chromatography on silica gel.

Alternatively, the title compound is prepared by oxidation of 6-α-chloropenicillanic acid by oxidation with an equivalent of 3-chloroperbenzoic acid in tetrahydrofuran at 0-25°C for approx. 1 hour, according to the method of Harrison et al., Jour. Chem. Soc. (London), Perkin I, 1772 (1976). The resulting 6-α-chloropenicillanic acid 1-oxide is then esterified by the method of Example 1 to form the desired chloromethyl ester.

The remaining 6-substituted penicillanate esters and 6,6-dihalopenicillanate esters prepared in examples 1 and 1B are converted to the corresponding 1-oxides by the method described above.

The same compound is obtained by reacting 0.1 mol of chloromethyl-6-α-chloropenicillanate in 150 ml of isopropanol containing 0.8 ml of 0.5 M sodium tungstate (Na2W04) or an equivalent amount of potassium molybdate (K2Mo04) with 0.1 mol of hydrogen peroxide (30%). The peroxide is slowly added to the other reagents at 60°C, after which the mixture is allowed to cool while it is stirred overnight. The product is isolated as described above.

Example 12 (intermediate)

Chloromethyl-6-α-chlorpenicillanate-1,1-dioxide

To a solution of 2.83 g, 0.01 mol of chloromethyl-6-α-chloropenicillanate in 50 ml of chloroform is added 4.32 g (0.025 mol) of m-chloroperbenzoic acid, and the mixture is stirred under a nitrogen atmosphere for 36 hours at room temperature . The solvent is evaporated in vacuo, the residue is partitioned between ethyl acetate and water at pH 6.0, and sodium bisulphite is added until a test for the presence of peroxides is negative. The pH is adjusted to 8.0, the layers are separated, and the organic phase is washed with brine, dried (Na 2 SO 4 ) and evaporated to dryness in vacuo to give the title compound.

Alternatively, the title compound is obtained by oxidation of the same starting material in methanol or ethanol containing a catalytic amount of sodium tungstate and addition of 2 molar equivalents of hydrogen peroxide at temperatures from 20 to 60°C.

Example 13 (intermediate)

1, l- dioxo- 6, 6- dibromopenicillanoyloxymethyl- 6-( D- 2- azido- 2-phenylacetamido) penicillanate

0.20 g (0.37 mmol) of iodomethyl- 6,6-dibromopenicillanate-1,1-dioxide, and the mixture is stirred at room temperature for 30 minutes. An additional 50 mg of tetrabutylammonium D-(2-azido-2-phenylacetamido)penicillanate was added and stirring was continued for 30 minutes. The reaction mixture was concentrated to dryness and the residue was applied to a column of silica gel (50 g). Elution with 1:1 (v/v) ethyl acetate/hexane was performed, taking 7 mL fractions. Fractions 17-24 were combined and evaporated in vacuo to give 0.14 g (49%) of the desired product as a pale yellow oil.

<1>H NMR (CDCl3 ) ppm (6): 1.4 (s, 3H), 1.5 (s, 3H), 1.59 (s, 3H), 1.62 (s, 3H), 4 .4 (s, 1H) , 4.5 (s, 1H) , 4.97 (s, 1H) , 5.04

(s, 1H), 5.4-5.70 (m, 2H), 5.85 (s, 2H), 7.05 (d, 1H), 7.35 (s, 5H); Infrared (undiluted) cm<-1>: 1810, 1775.

<*> The tetrabutylammonium salt was prepared as follows:

1 g sodium D-2-azido-2-phenylacetamido-penicillanate,

50 ml of ethyl acetate and 25 ml of water were mixed and adjusted to pH 2.0 (2N HCl). The organic layer was separated, washed with brine (10 mL), and the solvent was evaporated in vacuo. The remaining foam was dissolved in 30 mL of methylene chloride, 15 mL of water was added, and 40% tetrabutylammonium hydroxide solution was added until the aqueous phase reached pH 8.0. The organic layer was separated, the aqueous layer was re-extracted with methylene chloride (2 x 20 mL), and the combined extracts were dried (Na 2 SO 4 ) and concentrated to dryness to give a hard gum. This was triturated with ethyl acetate (2 x 10 ml) and ethyl ether (2 x 10 ml). The resulting whitish solid was air dried to give 1.25 g of the desired tetrabutyl ammonium salt.

Example 14 (intermediate)

lyl- dioxo- 6, 6- dibromopenicillanoyloxymethyl- 6-[ D- 2- benzyloxycarbonylamino- 2-( p- hydroxyphenyl) acetamido] penicillanate

To a mixture of 101.3 g (0.25 mol) 6-[D-2-benzyloxycarbonylamino-2-(p-hydroxyphenyl)acetamido]penicillanic acid,

To 250 ml of water, 500 ml of methylene chloride and 84.8 g (0.25 mol) of tetrabutylammonium bisulphate at 5°C, 125 ml of 2N sodium hydroxide are added while the mixture is kept at 5-10°C. The organic

layers are separated, and the aqueous phase is extracted with methylene chloride. The combined organic layers are dried (Na 2 SO 4 ), and the solvent is evaporated in vacuo. The residue is dissolved in 1000 ml of ethyl acetate, evaporated in vacuo to approx. 300 ml and cool overnight. The precipitated tetrabutylammonium 6-[D-2-benzyloxycarbonylamino-2-(p-hydroxyphenyl)acetamido]penicillanate is collected by filtration and dried in a vacuum.

49.8 g (0.10 mol) methylsulfonyl- oxymethyl-6,6-dibromopenicillanate-1,1-dioxide, and the resulting mixture is stirred at 50°C for 1 hour. The solvent is evaporated in vacuo, and the residue is distributed between ethyl acetate and water. The aqueous layer is separated, washed with ethyl acetate,

and the combined organic layers are washed with saline and dried (Na 2 SO 4 ). Evaporation of the ethyl acetate in vacuo gives the title compound.

Example 15 (intermediate)

1, l- dioxo- 6- j3- bromopenicillanoyloxymethyl- 6-( D- 2- azido- 2- phenyl-acetamido) penicillanate

0.14 g (0.25 mmol) of iodomethyl-6-0- bromopenicillanate-1,1-dioxide. The resulting colorless mixture was stirred at room temperature for 30 minutes, the solvent was evaporated in vacuo, and the residue was chromatographed on a column of silica gel (25 g), eluting with 1:1 (v/v) ethyl acetate/hexane. Fractions of 6 ml were taken with approx. 30 second intervals. Fractions 13-17 were combined and concentrated in vacuo to give 0.125 g of the desired product as a foam.

<1>H-NMR (CDCl 3 ) ppm (6): 1.4 (s, 3H), 1.5 (s, 3H), 1.6 (s, 3H), (1.65 (s, 3H) , 4.42 (s, 1H), 4.5 (s, 1H), 4.75 (d, 1H), 5.07

(s, 1H) , 5.3 (d, 1H), 5.4-5.75 (m, 2H), 5.85 (s, wide, 2H),

7.1 (d, 1H), 7.35 (s, 5H);

Infrared (undiluted) cm"<1>: 1800, 1775.

Example 16 (intermediate)

1, l- dioxo- 6- a- bromopenicillanoyloxymethyl- 6-( D- 2- azido- 2- phenyl-acetamido) penicillanate

A mixture of 0.308 g (0.5 mmol) tetrabutylammonium-(D-2-azido-2-phenylacetamido)penicillanate, 0.219 g (0.485 mmol) iodomethyl-6-a-bromopenicillanate-1,1-dioxide and 10 ml of acetone was stirred 30 minutes at room temperature. The solvent was evaporated in vacuo, and the residue was chromatographed on a column of 50 g silica gel, eluting with 1:1 (volume/volume) ethyl acetate/hexane. The product-containing fractions were pooled and the solvent was evaporated in vacuo to give 0.125 g

(38%) of the title compound as an oil.

<1>H-NMR (CDCl 3 ) ppm (6): 1.43 (s, 3H), 1.5 (s, 3H), 1.6 (s, 3H), 1.66 (s, 3H), 4.4 (s, 2H) , 4.64 (d, 1H), 5.05 (s, 1H) , 5.1

(d, 1H), 5.4-5.7 (m, 2H), 5.85 (s, 2H), 7.08 (d, 1H), 7.35

(p, 5H); Infrared (undiluted) cm"<1>: 1795, 1775.

Example 17

1,1-Dioxopenicillanoyloxymethyl-6-(D-2-amino-2-phenylacetamido)-penicillanate

A mixture of 1.2 g of 5% palladium-on-calcium carbonate

and 30 mL of 1:1 (by volume) isopropanol/methylene chloride was hydrogenated at 3.52 kg/cm 2 for 30 minutes. To this mixture was added 0.25 g (0.32 mmol) of 1,1-dioxo-6,6-dibromopenicillanoyloxymethyl-6-(D-2-azidp-2-phenylacetamido)penicillanate dissolved in 3 ml of methylene chloride. The resulting mixture was hydrogenated at 3.52 kg/cm 2 for 1 hour. The catalyst was removed by filtration, and washing was carried out with 30 ml of 1:1 isopropanol/methylene chloride. The filtrate was concentrated in vacuo to give a light brown solid. This was rubbed with 15 ml of ethyl ether,

filtered, washed with 10 ml of ether and air dried to give 0.195 g of product.

<1>H-NMR (CDCl 3 ) ppm (6): 1.5 (d, 6H), 1.6 (d, 6H), 3.55 (d, 2H), 4.45 (s, 1H), 4.55 (s, 1H), 4.6-4.75 (m, 2H), 5.5-5.7 (m, 2H), 5.9 (q, 2H), 7.4 (s, 4H), 8.1 (d, 1H).

Claims (1)

Method for making a compound with the formula or a pharmaceutically acceptable acid addition salt thereof, where R<1> is H or OH, characterized in that a compound of the formula where R<1> is as defined above, Q is N3 or NHC02CH2C6H4R<4 >where R<4> is H, Cl, Br, NO2, CH3 or OCH3; Y and Z are each Cl, Br or I, or Y is H and Z is Cl, Br or I, contacted with hydrogen in the presence of a noble metal catalyst, at least two molar equivalents of acid scavenging agent, or when Y is H , at least one molar equivalent acid binding agent, and a reaction inert solvent.