FI57418B - FOERFARANDE FOER FRAMSTAELLNING AV ANTIBIOTIKUMET 6-ACYLAMIDO-6-SUBSTITUERAD-PENICILLANSYRA - Google Patents

Description

I55FH [B] (11) EXTENSION PUBLICATION 5 7418 JKSTa W [J1J UTLäGGN | N6Sskii, FT or ** I o ^ v ^ (51) K ».ik? / I« .a.3 c 07 D 499/44 ENGLISH I —-FI N LAN D pi) ρημ «ιι» ι ^ - ^ ιμιμ · μιιι 167U / 71 (22) HakemispUvt — An »5knlnpd« | 15.06.71 ^ ^ (23) AlkuptM — GHtlgh «tadi | 15.06.71 (41) Tullut | ulktMk * l - Bllvtt offwttllg ^ 7 12 71

National Board of Patents and Registration / 44) Niht * v »lptnoi> ji Date of issue. -

Patent · och registerstyrelsan Amttkan utlagd och utl.skrifMfl publfcand 30. OU. 80 (32) (33) (31) Gear Front — Begird Priority χ6.q6.70 30.0U.71 England-England (GB) 29157/70, 29157/70 Proven-Styrkt (71) Merck & Co., Inc. , Rahway, New Jersey, USA (72) Burton Grant Christensen, Scotch Plains, New Jersey, Lovji Dadi Cama, Edison, New Jersey, USA (7U) Oy Roister Ab (5U) Method of antibiotic 6-acylamido For the preparation of -6-substituted-penicillanic acid - Förfarande for framställning av antibiotikumet 6-acylamido-6-substituerad-penicillansyra

The present invention relates to a process for the preparation of a novel antibiotic 6-acyl-amido-6-substituted-penicillanic acid of formula H R1

R »-N - ^ S

J-N - C00H

o 'wherein R' is acetyl, phenylacetyl, o'-aminophenylacetyl, phenoxyacetyl or phenylmalonyl and R 1 is methoxy or ethoxy, and a salt thereof.

The invention of penicillin, which was found to be an important and effective antibiotic, sparked great interest in this field. Subsequently, various other antibiotics were found, such as streptomycin, tetracyclines, novobiocin, and the like, which greatly increased the medical options available to physicians to treat infections caused by various pathogens. Unfortunately, the use of these antibiotics has resulted in strains of pathogens that are resistant to these known antibiotics. In addition, known antibiotics have the disadvantage that they are effective only against certain species of microorganisms and are not effective against a wide range of pathogens. Thus, the search for other antibiotics has continued.

However, several penicillin compounds are relatively unstable because they can degrade the enzyme β-lactamase, i. penicillanase.

The novel 6-aminopenicillanic acid derivatives prepared according to the invention have good antibiotic activity and improved resistance to β-lactamase. It has been shown that the 6? -Alkoxy group, in particular the 6? - methoxy group, confers a surprisingly high resistance to degradation by β-lactamase.

The novel 6-aminopenicillanic acid derivatives prepared according to the invention are compounds in which the penam backbone, namely the thioazolidine ring, is fused to a β-lactam containing a substituent in the 6-position.

The novel penicillanic acid derivatives can also be used as antibiotics in the form of a metal salt, e.g. the sodium, potassium or ammonium salt, or in the form of an amine salt, e.g. the procaine or N, N-dibenzylethylenediamine salt.

The process according to the invention is characterized in that the corresponding ester of 6-amino-6-R-substituted penicillanic acid of the formula fΎ JN - COORp in which R1 is as defined above and Rg is an ester group, preferably benzyl, is treated with an acylating agent of formula The carboxylic acid according to R'-OH or a reactive derivative thereof, and the ester group (Rg) is cleaved from the penicillanic acid ester obtained thereafter, and that, if desired, the penicillanic acid is converted into its salt. This process can be described by the following reaction scheme, which also illustrates the preparation of the starting material.

3 57418 »2 = τ-γΝ <J · - N -! - COORg

II

sl ·

Hai "3-i— N COORg

V

xl / f3 si — r'6 ^ ^ -N --- 1 - COORg

VI

R1 H R1 S / (f1 ~ acylation R'-N - x..K_L ^ 0 * ^ COORg J * - N-— COORg

VII IX

cleavage N / H R.

1 S..

R * -N - ^ S

/) - N - * COOH

O

k The acylation of the 6-amino group is carried out in a manner known per se, such as by reaction with an acyl halide or anhydride in a solvent such as dioxane, methylene chloride or pyridine. The starting 6-amino-penicillanic acid compound is prepared by reduction of the corresponding 6-azido-penicillanic acid compound, and the reaction mixture can be used for the subsequent acylation, i. by isolating the amino compound.

The cleavage of the ester to the free acid also takes place in a manner known per se. Thus, the benzyl ester can be cleaved by reacting in the presence of a reducing agent such as hydrogen and a noble metal catalyst. Acidic or basic hydrolysis can also be used for cleavage. In the latter case, a salt is formed, e.g. an alkali metal salt.

The starting material can be prepared from 6-diazopenicillanic acid (Formula II in the Reaction Scheme) which is converted to 6-azido-6-halopenicillanate (V) in which the halogen atom is replaced by an alkoxy group, preferably a methoxy group (compound of formula VI). The azido group is then hydrogenated to the amino group (Compound Formula VII).

To demonstrate the stability of the compounds of the invention, a comparative test was performed between sodium H 2 -methoxy-δ-phenylacetamido-penicillanate (6-methmethoxy-penicillin G) and sodium 6-phenylacetamido-penicillanate (penicillin G). Both compounds were mixed at a concentration of 1000 with penicillasease preparations. The mixtures were kept at 37 ° C. Samples were taken at regular intervals up to 10 minutes for penicillin Gl and up to 2 hours for o (, - methoxypipenicillin Gl. The residual concentration of antibiotic was determined iodometrically. The inactivation rate V obtained is shown in nicuCS · Table 1.

Table 1.

Compound V (millimoles / ml / min / mg protein)

Penicillin G 3750 6of-methoxy-penicillin G 0.2

The table shows that 6-Hethoxy-penicillin G, in contrast to penicillin G, is virtually completely stable against degradation in the presence of β-lactamase.

Table 2 shows the in vivo test results of 6β-methoxy-6-phenylmaloneacetamidopenicillanic acid (6-methoxycarbenicillin) prepared according to the invention and Table 3 shows the in vitro test results of this compound compared to penicillin G, ampicillin and carbenicillin (6-phenylmalonacamidamidacetamidine).

5,57418

Table 2

In vivo test results (subcutaneous administration) EE> 50 pg / dose

Test organism 6-methoxycarbenisil- Ampicillin Carbenicillin line

Proteus mirabilis 331 + 3 1330> 10,000> 10,000

Proteus morganii 3376 1 + 000> 10,000> 10,000

Escherichia coli 3296 1 + 000 1250 2960

Klebsiella pneumoniae 3083> 8000> 8000> 8000

Aerobacter aerogenes 311 + 8;

Test 1. <125 9 * + about 625 _ Aerobacter aerogenes 3lU8 j

Test 2 62.5 700 j 331 ί ____ 1-

Table 3

In vitro test results

Minimum inhibitory strength (MIC) ^ μg / ml -; - “Ti TT |

The test organism Carbeni- ‘PenicilliniiAmpicillinJ 6-methoxysillin ί G * j carbenicillin -! --- j-

Klebsiella pneumoniae 3083 (> 100)! 100 10 ° 6.25 C-17> 100 ί> 100> 100 50

Escherichia coli 2017 3.12 j 50 3.12 6.25 331 + 9> 100> 100> 100 25

Aerobacter aerogenes 311 + 8 | 50 50 50 12.5

Aerobacter cloacae 261 + 6 I 100> 100> 100 50 I i

Proteus vulgaris 1880 j 0,78 50 12,5 | 6.25

Proteus mirabilis 331 + 3! > 100 j> 100> 100 j 12.5 "Proteus morganii 3376> 100> 100> 100 J 12.5

Klebsiella 965> 100 '> 100> 100 25

Providence 50> 100> 100> 100 6.25 9I + 6 1.56 100 100 3.12 ί Ϊ

The following examples illustrate the invention.

6 57418

Example 1 6β-Acetamido-6-methoxypenicillanic acid and sodium salt

Step A: Benzyl 6-diazopenicillanate 0.5 g of benzyl 6-aminopenicillanate p-toluenesulfonic acid salt is added to a mixture of 50 ml of methylene chloride, 50 ml of ice and 1.5 g of sodium nitrite and mixed well. A total of 0.2 g of p-toluenesulfonic acid is added to the resulting mixture in three equal portions every 5 minutes and the cold mixture (10 ° C) is stirred again by shaking for 20 minutes. The yellow methylene chloride solution of benzyl 6-diazopenicillanate is then separated, dried over sodium sulfate at 0-10 ° C, filtered and the dried solution is evaporated at room temperature to a volume of about 5 ml.

Step B: Benzyl 6/3-azido-6-bromopenicillanate

To a solution of 2.0 g of benzyl 6-diazopenicillanate in 20 ml of methylene chloride is added 20 ml of nitromethane followed by 20 ml of triethylammonium azide solution. The resulting reaction mixture is cooled to 5 ° C, and to this cooled solution is added 20 ml of bromoazide solution over 15 seconds. To the resulting cooled reaction mixture is added 50 ml of 10-N sodium thiosulfate solution while stirring vigorously. After stirring for a further 2 minutes, give the solution on a negative test on starch iodine paper. Sodium bicarbonate is then added to the solution and the mixture is stirred until CO 2 formation ceases. The organic phase is separated and the aqueous phase is extracted with 20 ml of methylene chloride. The combined organic phase is washed with saturated sodium bicarbonate solution until no more ΟΟ ^ develops. The organic phase is then dried over magnesium sulphate and evaporated to dryness to give 1.3 g of crude benzyl 6-azido-6-bromopenicillanate.

The crude product is purified by absorbing it on 2.5 g of silica gel, placing the absorbent on top of a column of 50 g of silica gel in a mixture of equal amounts of hexane and benzene. At the same time, the column is developed as a solvent, 200 ml of the eluate fraction are collected and a solution of benzyl 6/3-azido-6-bromopenicillanate (0.550 g) is recovered from fractions 5-10. This product has the following values: Rf = 0.60 (CHCl3).

IR: U, 69 p (azido), 5.53 μ (β-lactam structure) and at 5.71 ester) NMR: 2.62 tau (s), (phenyl); U, 7 tau (s), (5H); U, 79 tau (s), (CH 2 Cl 2 H); 5.7 (s), (3H); 8, Ui tau (s); 8.63 tau (s), (gem CH 2) · / f s) denotes a singlet ^.

The triethylammonium azide solution is prepared by dissolving 3.0 g of sodium azide in 10 ml of water, cooling this solution to 0-10 ° C, adding 2 ml of methylene chloride and then 3.0 ml of concentrated sulfuric acid dropwise with rapid stirring, separating the organic phase, by extracting the aqueous phase with 5.0 ml of methylene chloride, drying the combined aqueous phases with calcium chloride and adding triethylanine to the dried solution to pH 7.0.

The bromoazide solution is prepared by cooling a mixture of 5.3 g of sodium azide and 16.0 ml of methylene chloride to 5 ° C by adding 1.28 g of brcm to the cooled mixture followed by 1.0 ml of concentrated hydrochloric acid and allowing the sealed mixture to stand at 0- At 10 ° C for 3 hours. The organic phase is separated from the paste-like inorganic phase. The inorganic phase is washed with 1.0 ml of methylene chloride and the organic phases are combined to give 20 ml of solution.

Step C: Benzyl 6/3-azido-6-methoxypenicillanate To a solution of 0.55 g of benzyl 6 / N-azido-6-bromopenicillanate in 50 ml of methanol is added 0.33 * + g of silver tetrafluoroborate and a mixture of allow to stand at room temperature for 2 1/2 hours. The methanol is then removed under reduced pressure and the residue is taken up in 30 ml of methylene chloride, filtered through diatomaceous earth and washed with a small amount of methylene chloride. The filtrate and washings are immediately combined with 5% sodium bicarbonate solution and then brine, dried and evaporated to give 0.16 g of crude benzyl 6-azido-6-methoxypenicillanate. This product is purified by chromatography on 15 g of silica gel and the column is eluted with 70% hexane / benzene. The eluate is collected in 50 ml fractions and fractions 12-26 are evaporated to give benzyl 6,6-dibromo-penicillanate. The column is then eluted with 70 5i of benzene in hexane and fractions 28-39 are evaporated to give 0.325 g of the desired product benzyl 6S-azido-6-methoxypenicillanate. This product has the values: Rf = 0.1 + 35 (CHCl3); IR, 1+, 70 μ (azido), 5.5 μ μ (β-lactam) and 5.71 (ester); and NMR in CDCl 3 2.62 tau (s), (phenyl); 1 +, 60 tau (s), (5H); 1 +, 78 tau (s), (CH 2 Cl 2); 5.1 + 7 tau (s); (3H); 6.35 tau (s), (OCH 3); and 8.1+ tau (s) and 8.58 tau (s), (gem CH3).

Step D: Benzyl 6H-acetamido-6-methoxypenicillanate To a solution of 0.065 g of benzyl 6-azido-6-methoxypenicillanate in 20 ml of acetic anhydride is added 0.065 g of platinum oxide and the mixture is hydrogenated at atmospheric pressure for 18 hours. The resulting solution is concentrated under reduced pressure at a temperature below 1 + 0 ° C to remove acetic anhydride. The residue obtained is taken up in a mixture of equal volumes of methylene chloride and ethyl ether and filtered through diatomaceous earth to remove the catalyst. The filtrate and washings are evaporated under reduced pressure to give 0.066 g of benzyl 6β-acetamido-6-methoxypenicillanate. The crude product is purified by thin layer chromatography to give 0.03 g of pure product.

5741 8

Rf = 0.52 (2% CH3OH / CHCl3h IR; 5.59 μ (β-lactam) and 5.71 μ (ester) NMR: 2.63 tau (s), (phenyl); U, U tau ( s), (5H), U, 80 tau (s), (CH 2 Cl 2 H 2 O), 5.52 tau (s), (3H), 6.53 tau (s), (OCH 3), 7.9 tau (s), ), (CH3C-), 8.5 tau (s) and 8.60 tau (s), (gem CH3).

Step E: 6 / $ - Acetamido-6-methoxypenicillanic acid and sodium salt

A solution of 0.03 g of benzyl 6β-acetamido-6-methoxypenicillanate in a mixture of 2 ml of dioxane, 1 ml of methanol and 2 ml of water is hydrogenated using 10% palladium on carbon (0.03 g) under pressure. 2.8 kg / cm 1 hour. The catalyst is removed and the solvents are removed in vacuo at room temperature. To the residue of 6,3-acetamido-6-methoxypenicillanic acid is added a solution of 0.03 g of sodium bicarbonate in 10 ml of water. The bicarbonate solution is washed with methylene chloride and the aqueous phase is lyophilized to give sodium 6- (h-acetamido-6-methoxypenicillanate (20% yield). This compound has a barrier against B-subtilis.

Example 2

Benzyl 6,3-acetamido-6-methoxypropane illanate

Step A: Benzyl 6-methoxy-6-aminopenicillanate.

To a solution of 0.05 g of benzyl 6-azido-6-methoxypenicillanate in 8.0 ml of ethyl acetate is added 0.05 g of 10%. palladium on carbon. The mixture is reduced with hydrogen at atmospheric pressure for 20 hours. The catalyst is removed by filtration and the filtrate is evaporated to dryness to give crude benzyl 6-methoxy-6-aminopenicillanate. Thin layer chromatography shows one major spot, R 1. 0.52 (2% methanol, chloroform, silica gel plates). The spot gives a positive ninhydrin test. Upon further purification of this product, the product obtained by thin layer chromatography shows in the infrared spectrum λ-lactam and ester carbonyl at 5.59 and 5.71 μ, respectively, and at N-H 2.90 μ. No azide function is observed.

Step B: Benzyl 6,3-acetamido-6-methoxypenicillanate

Benzyl 6-methoxy-6-aminopenicillanate is treated with 2.0 ml of acetic anhydride for 1 hour at room temperature. The acetic anhydride is then evaporated under reduced pressure and the residue is purified by preparative thin layer chromatography to give the product with β-lactam, ester and amide carbonyl in the infrared spectrum and R 4 by thin layer chromatography substantially identical to benzyl-6-acetamido with the values of penicillanate obtained by the latter compound in step D of Example 1.

Example 3 6,3-Phenylacetamido-6-methoxyphenicillanic acid and sodium salt

Step A: Benzyl 6-phenylacetamido-6-methoxy penicillanate

To a solution of 0.5 g of benzyl 6-azido-6-methoxypenicillanate, 2.5 g of phenylacetic anhydride and 20 ml of dioxane are added 0.25 g of platinum-957418 oxide, and the resulting mixture is hydrogenated at atmospheric pressure for 20 hours. The dioxane is removed under reduced pressure and the residue is chromatographed on 30 g of silica gel. The column is developed to remove benzene-unreacted phenylacetic anhydride and any unreacted azide. The column is eluted with chloroform and five 50 ml fractions are evaporated to give a crude product containing phenylacetic acid. The crude product is dissolved in 30 ml of methylene chloride, washed once with 5% sodium bicarbonate solution, dried over magnesium sulphate and evaporated to give 0.175 g of benzyl ester of 6-methoxybenzylpenicillin. This product is chromatographed on silica gel using a methylene chloride / ethyl acetate gradient to give 0.06 g of pure benzyl 6,3-phenylacetamido-6-methoxypenicillanate. This product has the following values:

Rf = 0tk3h (2% CH 3 OH / CHCl 3) i IR, 5.59 μ (β-lactam), 5.71 μ (ester), 5.95 μ (amide I) and 6.58 μ (amide II); MMR, 2.65 tau (s), (C 9 H 8); i +, ί + Ι tau (s), (5 ~ H); i +, 8l tau (s), (OCH 2 C 6 H 5); 5.59 tau (s), (3H); 6.35 tau (s), (CO 2 Cl 2) ·, 6.60 tau (s), (O-COCH 3); and 8.6k tau (s), (gem CH3).

Step B: 6β-Phenylacetamido-6-methoxypenicillanic acid and sodium salt To a solution of 0.317 g of benzyl 6β-phenylacetamido-6-methoxypenisilanate in a mixture of 9.0 mL of dioxane, 5.0 mL of methanol and 10 ml of water, 0.317 g of 10% palladium on carbon and 0.063 g of sodium bicarbonate are added. The mixture is hydrogenated at 2.8 kg / cm 2 for 2 hours. The catalyst is filtered off, the pH of the filtrate is adjusted to 7.3 and most of the dioxane and methanol are removed under reduced pressure below room temperature. The resulting aqueous solution is washed twice with methylene chloride and the aqueous phase is freeze-dried. The lyophilized material is mixed with anhydrous methanol (20 ml) and the insoluble inorganic material is filtered off. The filtrate is evaporated below room temperature to give 0.208 g of sodium 6/3-phenylacetamido-6-methoxy penicillanate.

IR: 5.65 μ (Λ-lactam), 5.95 μ (amide I) and 6.19 μ (C00-); NMR: 2.58 tau (s), (C 9 H 6); 1 +, U5 tau (s), (5H) and 5.72 tau (s), (3H)> 6.27 tau (s), (COCHgCgHj.); 6, k8 tau (s), (OCH. ^); and 8.55 tau (s) and 8.58 tau (s), (gem CH3).

Example U

6 β-Phenoxyacetamido-6-methoxypenicillanic acid sodium salt

Step A: Benzyl 6,3-phenoxyacetamido-6-methoxypenicillanate

To a solution of 0.075 g of benzyl 6,3-azido-6-methoxypenicillanate in 1.0 ml of dioxane containing 0.13 g of phenoxyacetic anhydride is added 0.075 g of platinum oxide and the mixture is hydrogenated for 18 hours with stirring under hydrogen pressure for 27 hours. The resulting mixture is concentrated in vacuo at room temperature and the residue is absorbed onto a column containing 20 g of silica gel with benzene. The product is eluted from the absorbent with chloroform with phenoxyacetic acid. The combined fractions comprising 0.377 g are dissolved in chloroform and washed three times with 5% sodium bicarbonate solution. The chloroform solution is then concentrated and the residue is rechromatographed using 5 g of silica gel, eluting with 2-3% ethyl acetate / methylene chloride, and the eluate is evaporated to give benzyl 6-phenoxyacetamido-6-methoxypenicillanate.

3R: 3.00 μ, 5.63 μ, 5.7¾ p, 5.91 μ

Step B: 6/3-Phenoxyacetamido-6-methoxy-penicillanic acid and sodium salt

Solution of 0.028 g of Bent cause li-6 / Sr-phenoxyacetamido-6-methoxypenicillanate in 8 ml of solvent (dioxane: methanol: water 1: 2: 1) containing 0.028 g of 10% platinum on carbon, hydrogenated at 2.8 kg / cm 1 for 1 hour. The catalyst is removed by filtration and the orgasmic solvents are removed by evaporation in vacuo at room temperature to give 6-methoxy-6-phenoxyacetamidopenicillanic acid, to which is added a solution of 0.06 g of sodium bicarbonate in 10.0 ml of water. The aqueous solution is then extracted with a small amount of methylene chloride and lyophilized to give sodium β / 3-phenoxyacetamido-6-methoxypenicillanate.

IR: 5.66 μ, 5.91 μ, 6.0 μ.

Example 5

Sodium β / 3-methoxy-6-phenylacetamidopenicillanate

Step A: Benzyl 6β-bromo-6-methoxypenicillanate

To a solution of 2.0 g of benzyl 6-diazopenicillanate in 20 ml of methylene chloride is added a cold solution of 56 g of N-bromoacetamide in 20 ml of methanol. The mixture is kept at room temperature for 30 minutes and then the solvents are rapidly removed under reduced pressure. The gummy residue is dissolved in methylene chloride and washed with aqueous sodium bicarbonate solution. The methylene chloride solution is dried over magnesium sulfate, filtered and evaporated. The residue is chromatographed on a column of 60 g of silica gel, eluting with a 1: 1 mixture of hexane and methylene chloride. The middle fraction comprising 860 mg of benzyl 6/3-bromo-6-methoxy penicillanate obtained is solidified on standing. A sample recrystallized from ether / petroleum ether melts at 90-91 ° C. NMR CDCl 3 requires 1.55 tau (s), (5H); 5.18 tau (s), (3H); 6.36 tau (s), (OCH 2); 8.1 tau (s), 8.62 tau (s), (gem CH 2).

The IR is 5.62 μ {β <-actam) and 5.78 μ (ester) (in nujol).

Elemental Analysis

Lask. C, 18.01; H, 1.53; N, 3.50; Br, 19.96; Found: C, 1 * 7.53; H, 1.30; N, 3.60; Br, 20.65.

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Step B: Benzyl 6-4 (azo-6-methoxypenicillanate)

A solution of 6 mg of benzyl 6/3-bromo-6-methoxypenicillanate and 1 + 35 mg of lithium azide in 5 ml of dimethylformamide is kept at 30-35 ° C for 6 hours. The dimethylformamide is evaporated off under high vacuum and the residue is taken up in a mixture of 50 ml of carbon tetrachloride and 75 ml of water. The aqueous solution is discarded and the organic solution is washed twice with water to remove residual dimethylformamide. The carbon tetrachloride phase is dried and evaporated and the residual oil is chromatographed on a column of 20 g of silica gel. Elution with $ 50 hexane / methylene chloride gives 0.56 g of benzyl 6,6-azido-6-methoxypenicillanate.

NMR in CDCl 3 has 1+, 73 tau (s), (5H->; 5.5 tau (s), (3H); 6.1 + 3 tau (s), (OCH 2); 8.1+ 1 tau (s) and 8.62 tau (s), (gem CH 2). IR is 1 +, TU μ (azide), 5.60 μl (β-lactam) and 5.76 μ (ester) ( film).

Initial analysis i

Lask. : C, 53.03; H, 5.0; N, 15.1 + 6; Found: C, 52.92; H, 5.13; N, 15.69.

Step C: Benzyl 6A-methoxy-6-phenylacetamidopenicillanate A mixture of 150 mg of benzyl-6α-azido-6-methoxypenicillanate, 0.1 ml of diisopropylethylamine and 75 mg of 10β palladium on carbon in 1.5 ml in dry ethyl acetate, hydrogenated at atmospheric pressure and room temperature for 3 hours. To the resulting solution of 6α-amino-6-methoxypenicillanate is added a solution of 200 mg of phenylacetic anhydride in 5 ml of methylene chloride. The mixture is stirred at room temperature for 20 minutes, then the catalyst is filtered off and the solvents are evaporated. The residue is chromatographed on 20 g of silica gel. Methylene chloride is passed through the column until the eluate is free of phenylacetic acid and the product is eluted with 2% ethyl acetate in methylene chloride. 75 mg of benzyl 6,3-methoxy-6-phenylacetamidopenicillanate are obtained.

NMR: 2.63 ΐεφ (s) and 2.68 tau (s), (phenyl); 1 +, 3 tau (s), (5H); 1 +, 8 tau (s), (OCHgCgHj-); 5.53 tau (s), (3H); 6.35 tau (s); (° (C-CH 2 O); 6.51 tau (s), (OCH 3); 8.1 + 3 tau (s) and 8.6 l tau (s), (gem CH 2). IR is 5.65 p (Β-lactam), 5.75 p (ester) and a broad band of 5.92-6.02 p (amide) (film) Step D: Sodium 6β-methoxy-6-phenylacetamidopenicillanate Solution with 75 mg of benzyl 6β-ethoxy-6-phenylacetamidopenicillanate and 2 U mg of sodium bicarbonate in 2.2 ml of dioxane, 2.5 ml of water and 1.2 ml of methanol are hydrogenated in the presence of 75 mg of 10 # palladium on carbon at room temperature at a pressure of 2.1 kg / cm 2 hours The catalyst is filtered off and the filtrate is evaporated to dryness in vacuo, the residue is taken up in water, extracted once with methylene chloride and the aqueous layer is freeze-dried to give 51 mg of 12 6 741 8 containing sodium 6 / S-methoxy. -6-fenyyliasetamidopenisillanaattia.

IR: 5.67 μ (lactam) NMR: (solvent - D 2 O) 2.67 tau (s), (phenyl); U, 53 tau (s), (5H); 5.7? tau (s), (3H); 6.38 tau (s), (O-CH 2); 6.58 tau (s), (OCH 2), 8, ** 5 tau (s), and 8.1 * 9 tau (s), (gem CH 2).

Example 6 6 / 8- (D-ofc-aminophenylacetamido) -6-methoxypenicillanic acid

Step A: D (-) - O-azidophenylacetyl chloride 2.29 g of D (-) - o (-azidophenylacetic acid) are dissolved in benzene and the solution is cooled to 10 [deg.] C. with an ice bath, 11.5 ml of thionyl chloride are added dropwise. then heated to reflux for 1/2 hour The reaction mixture is cooled to room temperature and the solvent is removed in vacuo to give 2.5 g of D (-) -? - azidophenylacetyl chloride as a yellow oil. 1790 (C = 0) max.

Step B: 6S- (D-Aminophenylacetamido) -6-methoxypenicillanic acid 1.0 g of benzyl 6H-azido-6-methoxypenicillanate is dissolved in 80 ml of dry ethyl acetate and 1 * ml of N, N-diisopropylethylamine is added. and 1.0 g of 10> S palladium on carbon. The reaction mixture is stirred overnight under hydrogen at atmospheric pressure. The catalyst is filtered off and the solvent is evaporated. The residue is dissolved in 100 ml of methylene chloride and placed in an ice bath at 10 ° C. 20 ml of pyridine are added and then 1.5 g of D (-) - o-azidophenylacetyl chloride are added in one portion. After stirring for 1 * 5 minutes, the mixture is poured onto ice with 1.0 g of sodium bicarbonate. The two layers are separated and the aqueous layer is washed successively three times with methylene chloride. The combined methylene layers are washed with water, dried over magnesium sulfate, filtered and evaporated to give a dark red oil. This reaction is repeated twice more and the products are combined and applied to a column of silica gel (60 g) in benzene.

Elute with benzene and take 200 ml fractions. Fractions 11-23 contain 0.98 g of product which is dissolved in 100 ml of methanol and 100 ml of water and 1.96 g of $ 10 palladium on carbon is added and the mixture is placed on a Parr shaker for 2 hours. The catalyst is removed by filtration. The organic solvents are removed by distillation. The remaining aqueous solution is washed twice with methylene chloride, the combined methylene chloride solutions are washed once with water.

The combined aqueous layers are freeze-dried to give 0.5 g of 6 / J- (D-o-aminophenylacetamido) -6-methoxypenicillanic acid.

TLC: Rf = 0.65 in butanol, acetic acid and water (3: 1: 1) IR: cm nujol 1765 (β-lactam), 1700 (amide), 1600 (C00-) max.

NMR: (CD 3 OD) 7.1 * (m, phenyl), 5.55 (s, 6h), U, 1 (s, 3H), 3.5 (s, -OCH 3), 1.39 (s, 1H), , 12), (s, 2 CH3's).

13 5741 8

Example 7

Sodium 6-ethoxy-6-fenyyliasetamidopenisillanaatti

Step A; Benzyl-6-azido-6-etoksipenisillanaatti

A mixture of 1.542 g of benzyl 6-azido-6-branipenicillanate and 0.743 g of silver fluoroborate in 150 ml of absolute ethanol is stirred at room temperature, protected from moisture and protected from light, for 3 1/2 hours. The ethanol is removed in vacuo, the residue is taken up in methylene chloride and filtered. The filtrate is washed with sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate and evaporated. The residue is chromatographed on 10 g of silica gel. Elution with benzene / hexane gives 657 mg of benzyl 6-azido-6-ethoxypenicillanate, which solidifies on standing.

[Φ + 157 ° C. In 1% MeOH.

NMR: (t) 8.67 (t) OCH 6 CH 2 J 8.42 and 8.6 gem dimethyl; 6.1 (quartet) OCH2CH3, 5.48 (s) (1H, 4.8 (s) CH2O; 4.6 (s) CH2; 2.62.0).

Initial analysis i

Lask. C, 54.23; H, 5.36; N, 14.88; Found: C, 54.15; H, 5.54; N, 15.03.

Step Bi Benzyl 6-ethoxy-6-phenylacetamidopenicillanate

A mixture of 3.6 g of benzyl 6-azido-6-ethoxypenicillanate, 2.8 ml of diisopropylethylamine and 3.6 g of 10 # Pd / C in 40 ml of ethyl acetate is stirred under hydrogen at room temperature at atmospheric pressure 18 hours. A solution of 4 g of phenylacetic anhydride in 50 ml of methylene chloride is added and the mixture is allowed to stand at room temperature for 1/2 hour. The mixture is filtered and the filtrate is evaporated under reduced pressure. The residue is taken up in ether, washed with phosphate buffer pH 7, dried over anhydrous sodium urea sulphate and evaporated. The residue is chromatographed on 500 g of silica gel. Elute with 2 # ethyl acetate in methylene chloride to give 1.5 g of benzyl 6-ethoxy-6-phenylacetamidopenicillanate.

NMR: (r) 8.75 (f), CH 2 CH 3; 8.7, gem dimethyl; 6.4 (q) CH 2 CH 2 6.4 (s) CH 2 CONH; 5.62 (s) C3H; 4.83 (s) OCH 2 O; 4.43 (s) C 1 H 2, 2.6-2.8 aromatic.

Elemental Analysis

Found: C, 64.08; H, 6.02; N, 14.88; Found: C, 63.43; H, 6.15 »N, 15.03.

Step C: Sodium 6-ethoxy-6-phenylacetamidopenicillanate

A solution of 1 g of benzyl 6-ethoxy-6-phenylacetamidopenicillanate and 180 mg of sodium bicarbonate in 34 ml of water, 16 ml of methanol and 30 ml of dioxane is hydrogenated at a pressure of 2.8 kg / cm in the presence of 1 g of 10 JS Pd / C 3 hours. Fresh catalyst (1 g) is added and hydrogenation is continued for a further 2 hours. The catalyst is filtered off and the filtrate is evaporated to a small volume, diluted with water and freeze-dried to give 680 mg of sodium 6-ethoxy-6-phenylacetamidopenicillanate.

lk 5 741 8 TLC butanol / ethanol / water 1+: 1: 5 Rf -0.66.

NMR: (1r) 8.80 (1 H) CH 2 -CH 2 8.55 and 8.6, gem dimethyl; 6.25 (q.) CH 2 CH 3; 6.3 (s) O-CH 2 -C; 5.71 (s), htk6 (s) C 2.6 aromatic.

Example 8 6-Methoxy-6-phenylmalonamidopenicillanic acid and disodium salt

Step A: Benzylphenylmalonic acid

An ethereal solution of phenyldiazomethane is prepared by adding 87 g (0.31 mol) of N-nitro-N-benzyl-p-toluenesulphonamide portionwise over one hour to a well-stirred mixture of 17 g of sodium methoxide (0.315 mol) in 60 ml of methanol and 360 ml of ether. The thick red slurry is heated at reflux for 20 minutes, then cooled and 300 ml of ice water are added. The organic phase is washed three times with 200 ml of water, dried over sodium sulfate and filtered to give 360 ml of a dark red ether solution of phenyldiazomethane. To a solution of phenylmalonic acid (18.2 g) in 200 ml of ether is added 350 ml of phenyldiazomethane ether solution over 10 minutes while maintaining the temperature at 0-5 ° C (nitrogen evolution). The yellow solution is stirred for 10 minutes, 500 ml of water are added and the pH of the mixture is adjusted to 10 with 2N potassium hydroxide. The layers are separated, the outer layer is extracted with 500 ml of ethyl acetate and the combined organic phases are extracted with 250 ml of water. 500 ml of ethyl acetate are added to the combined aqueous extracts and the pH is adjusted to 2 with 2N hydrochloric acid while stirring and cooling with ice. The aqueous phase is extracted with 200 ml of ethyl acetate, the combined organic phases are dried over sodium sulfate and concentrated to dryness to give 25 g of a yellow oil. TLC (silica gel / 10% MeOH in CH 2 Cl 2) showed a spot, Rf. 0.1 and Rf 0.5. Chromatography on 250 g of silica gel eluting with 2 # methanol in methylene chloride gave 11 g (52%) of pure benzylphenylmalonic acid, m.p. 1 + 5 ~ 50 ° C, single spot with TI ε, Rf 0.5. The IR in morpholine is consistent with the putative structure, the ester band at 5.8 fi wide in the carboxylate band at 6.3

Step B: Benzylphenylmalonic acid chloride 8 g of benzylphenylmalonic acid are dissolved in 8 ml of oxalyl chloride and allowed to stand at room temperature for 1 hour. Excess oxalyl chloride is removed in vacuo (bath temperature 25 ° C) and the residue is rinsed with 10 ml of dry benzene to give about 8 g of benzylphenylmalonic acid chloride as a pale yellow oil which is used immediately in the next step. NMR in CDCl 3 showed a shift in the OCH proton from U, δ to 5.0. The IR is consistent with the putative structure with the acid chloride band at 5.6 μl and the benzyl ester band at 5.7} x.

«57418

Step C: Benzyl 6-amino-6-methoxypenicillanate 8 g of benzyl 6-azido-6-methoxypenicillanate dissolved in a mixture of 200 ml of ethyl acetate and 12 ml of N, N-diisopropylethylamine are hydrogenated using 16 g of 10 #. palladium on carbon at room temperature and pressure 2.8 kg / cm for 50 minutes. The catalyst is filtered off and the filtrate is concentrated in vacuo at room temperature to give 8.0 g of a dark red oil. IR (in dichloromethane) showed a strong lactam band at 5.6 yu, an ester band of equal strength at 5.85, while the acid band of the starting material was completely absent. TLC (silica gel / 2% MeOH in CH 2 Cl 2) showed several spots with the desired amine, R 0.5 * +, giving a salmon red color with a ninhydrin spray. The oil is used

✓ X

without cleaning in the next step.

Step D: Dibenzyl 6-methoxy-6-phenylmalonamidopenicillanate 8 g of benzyl 6-amino-6-methoxypenicillanate are dissolved in 160 ml of dichloromethane and cooled to -5 ° C. U, 0 g of pyridine are added, followed dropwise by 8 g of benzylphenylmalonic acid chloride in 160 ml of dichloromethane over 5 minutes. The pH of the pale yellow solution is neutral. After stirring for 30 minutes at room temperature, the mixture is washed three times with 200 ml of water, the organic layer is dried over sodium sulfate and concentrated to dryness to give 15 g of a yellow oil. TLC (silica gel / 2% MeOH in CH 2 Cl 2) shows several spots with the major components Rf 0.5, 0.6 and 0.7 of the desired amine Rf. 0.6 being predominant. Chromatography on 300 g of silica gel (Baker) eluting with 200 ml portions of dichloromethane gives fractions 11-2 * + containing U, 0.0 g of pure product, showing the main spot at Rf. 0.6 and a few smaller impurities. Another similar chromatography using 100 g of silica gel gave 2.0 g (15% in two steps) of pure dibenzyl 6-methoxy-6-phenylmalonamidopenicillanate, single spot Rf, 0.6. IR is consistent with a proven structure with a lactam band at 5.65 μ, an ester band with equal strength at 5.75 μ, and an amide band with lower intensity at 5.9 μ.

Step E: 6-Methoxy-6-phenylmalonamidopenicillanic acid and disodium salt

A solution of 2.0 g of dibenzyl 6-methoxy-6-phenylmalonamidopenicillanate in 200 ml of a mixture of dioxane, methanol and water (1: 2: 1) is hydrogenated using * + g of 10%. palladium on carbon at room temperature at 2.8 kg / cm for 1 hour. After filtration and concentration to dryness at room temperature, 6-methoxy-6-phenylmalonamidopenicillanic acid is converted into the disodium salt by dissolving in 25 ml of water containing 0.5 g of sodium bicarbonate. The aqueous solution is extracted three times with 25 ml of dichloromethane, then freeze-dried to give 1.2 g of pure 6-methoxy-6-phenylmalonamidopenicillanic acid disodium salt (7%) as a pale yellow powder. The IR is consistent with the structure shown, with the β-lactam band at 5.65 and the amide band at 5.95 J and the broad carboxylate band at 6.3 fx. TLC (silica gel / -n-BuOH-HgO-HQAc, U: 1) shows essentially one spot. Titration by Karl Fisher gave 2.5 μg of HgO (after correction NaHCO 2 -> Η ,, Ο + CO 2 + IfegCO 2) · After determining the presence of k% sodium bicarbonate and 2% water was corrected Elemental analysis: Calc . (corr.): C, 1 + 5.58; H, 1 +, 02; N, 5.82; S, 6.65; Na, 10.65; Found: C, 1 + 5.56; H, hr2k; N, 5.59; S, 6.60; Na, 11.10.

Claims (1)

A process for the preparation of the antibiotic 6-acylamido-6-substituted-penicillanic acid of the formula HR -N - COOH 0 in which R 'is acetyl, phenylacetyl, γ-aminophenylacetyl, phenoxyacetyl or phenylmalonyl and R 1 is methoxy or ethoxy, and for the preparation of a salt thereof, characterized in that the ester of 6-amino-6-R-substituted penicillanic acid of the formula '? -? -? - N - COORg in which R1 is as defined above and Rg is an ester group, preferably benzyl, is treated with an acylating agent which is a carboxylic acid of the formula R'-OH or a reactive derivative thereof, and then the ester group (Rg) is cleaved from the penicillanic acid ester obtained and, if desired, the penicillanic acid is converted into a salt thereof.