NO762372L - - Google Patents

Description

The present invention relates to intermediate products

'for the production of therapeutically applicable penicillanic acid derivatives.

Some semi-synthetic penicillins have been developed and marketed on a large scale during the last two decades, e.g. ampicillin, amoxicillin, sulbenicillin, carbenicillin, dicloxacillin and nafcillin. Although these semi-synthetic penicillins

lines appear to be effective in combating a large number of infectious diseases caused by several microorganisms, there is still a need for additional antibiotics that are effective against some specific microorganisms, such as Pseudomonas and Proteus species. The purpose of the invention is to provide new derivatives of amoxicillin, i.e. 6-[a-amino-(p-hydroxybenzylcarbonamido]penicillanic acid, which has interesting properties against microorganisms, e.g. Pseudomonas and/or Proteus in addition to the already known good activities of amoxicillin.

The new penicillanic acid derivatives according to the invention are compounds with the general formula: where E is a hydrogen atom, a salt-forming cation or an ester-forming residue which has been shown to improve the absorption properties of penicillan compounds after oral administration to humans or animals, and where Y is a group with the formula:

where Z, and Z^ are the same or different and where each is a lower alkoxy group, an optionally substituted phenoxy group, an optionally substituted benzyl or benzyloxy group, an optionally substituted lower alkyl group, an optionally substituted phenyl group, a hydroxy group or a group -OM, where M is a salt-forming cation. The substituents which may optionally be present on a phenyl nucleus or groups covered by the definition of the symbol Y can be selected from halogen atoms and nitro, cyano, lower alkyl and lower alkoxy groups. The term "lower" used for alkoxy or alkyl groups means that the group contains no more than 6 carbon atoms.

Ester groups that come under the definition for the symbol E, which can improve the physical absorption properties of the compounds of formula I, can be chosen from e.g. groups with the formula:

where R is a straight or branched alkyl radical with from-1 to 4 carbon atoms which is optionally substituted with one or more substituents selected from lower alkoxy, lower alkylthio and halo-(lower)alkyl.

The salt-forming cations encompassed by the definitions of E and M are those which form non-toxic, pharmaceutically acceptable salts of the compounds of general formula I, such as sodium, potassium, ammonium or calcium salts, or amine, e.g. tri(lower)alkylamine, procaine or benzylamine salts. Hydrates, of salts of the compounds with the general formula I are covered by the invention.

Examples of groups encompassed by formula II are di-(lower)alkylphosphinylaminocarbonyl, diphenoxyphosphinylamino-carbonyl, diphenylphosphinylaminocarbonyl, di(lower)alkylphosphinylaminocarbonyl, hydroxybenzylphosphinylaminocarbonyl, hydroxy-(lower)alkylphosphinylaminocarbonyl, hydroxypheny1- phosphiny1-aminocarbonyl-, hydroxy- (lower ) alkyl-phosphinylaminocarbonyl 1, (lower) alkyl-benzyloxy-phosphinylaminocarbonyl, phenylbenzyl-oxyphosphinylaminocarbonyl, lower-alkylbenzyloxy-phosphinylaminocarbonyl, dibenzyloxy-phosphinylaminocarbonyl, dihydroxy-phosphinylaminocarbonyl, ( lower) alkoxy-benzyl-phosphinylaminocarbonyl and (lower) alkoxy-phenyl-phosphinylaminocarbonyl.

Typical compounds according to the invention are D-6-{ct-[3-(benzyloxy(ethyl)phosphinyl)ureido]-p-hydroxybenzyl-carbon-amido}penicillanic acid, D-6-{a-[3-(benzyloxy(ethoxy) phosphinyl)-ureido]-p-hydroxybenzylcarbonamido}-penicillanic acid, D-6-{a-[3-(dibenzyloxyphosphinyl)ureido]-p-hydroxybenzylcarbonamido}-penicillanic acid, D-6-{a-[3-(hydroxy(ethyl )phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid, D-6-{a-[3-(hydroxy-(ethoxy)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid, D-6-{a-[3~ (hydroxy(benzyloxy)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid, D-6-{a-[3_(benzyloxy-(methoxy)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid , D-6-{a-[ 3-(hydroxy(methoxy)phosphinyl)ureido]-p-hydroxy-benzylcarbonamido}penicillanic acid, and D-6-{a-[3-(dihydroxy-phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid, D-6-{ α-[3-(Benzyloxy(methyl)phosphinyl)ureido]-p-hydroxybenzyl1-carbonamido}penicillanic acid, D-6-{ α-[-3-(benzyloxy(i-propyl)phosphinyl) ur ei do ]-p- hydroxy-benzylcarbonamido}penicillanic acid, D-6-{a-[3(benzyloxy(t-buty1)phosphinyl)ureido]-p-hydroxybenzyl-carbonamido}penicillanic acid, D-6-{a-[3(benzyloxy (t-butoxy)phosphinyl)ureido]-p-hydroxy-benzylcarbonamido}penicillanic acid, D-6-{a-[3(hydroxy(methyl)phosphinyl)ureido]-p-hydroxybenzyl1-carbonamido}penicillanic acid, D-6 - { a--[ 3 (hydroxy ( i -propy 1) pho s phenyl) ureido ] - p - hydroxy b enzy 1-carbonamido}penicillanic acid, D-6-{a-[3 (hydroxy (t-butoxy) phosphinyl) ureido]-p-hydroxybenzyl-carbonamido}penicillanic acid, D-6-{a-[3(hydroxy(t-buty1)phosphinyl)ureido]-p-hydroxybenzyl-carbonamido}penicillanic acid, D-6-{a-[3- (benzyloxy(phenoxy)phosphinyl)ureido]-p-hydroxybenzyl-carbonamido}penicillanic acid, D-6-{a-[3_(benzyloxy(isopropoxy)phosphinyl)ureido]-p-hydroxy-benzylcarbonamido}penicillanic acid, D- 6 - { a-[ 3_ (Hydroxy (phenoxy) :phosphinyl)ureido]-p-hydroxybenzyl-carbonamido}penicillanic acid, D-6-{a-[3(diphenoxy)phosphinyl)ureido]-p-hydroxybenzylcarbon-amido } penicillanic acid, D-6-{a-[3- (diethoxyphosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid. D-6-{a-[3(hydroxy(i-propoxy)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid and their sodium, potassium and ammonium salts (mono-, di- and tri-valent salts) and pharmaceutically acceptable esters as have ester groups corresponding to formula III, where the compounds of formula I, where Z, and/or Z^ is a hydroxy or a group ™OM are preferred. Especially the compounds where Z^ is a hydroxy group or an OM group and Z^ is a methoxy or ethoxy group have given interesting antimicrobial activities. As usual, the D-modification of these compounds shows the most interesting antimicrobial activities.

The compounds of the general formula I can be prepared by using known methods for the preparation of structurally similar penicillins.

According to another aspect of the invention, the compounds of the general formula I are thus prepared by a method which comprises reacting a compound of the general formula:

(wherein Q is a hydrogen atom or a silicon or phosphorus atom having substituents selected from lower alkyl, halogen-substituted lower alkyl, aryl, aralkyl, lower alkoxy, halogen-substituted alkoxy, lower alkylthio, aralkyl, di(lower)alkyl- amino, lower-alkoxyalkyl and alkylenedioxy groups and halogen atoms (preferably a tri(lower)alkylsilyl, i.e. trimethylsilyl group), and where E' is a group protecting the carboxy radical, preferably a group which, if desired, can be easily removed after the reaction, f .eg by hydrolysis, hydrogenation or a substitution reaction that uses a basic or nucleophilic reagent, and which does not affect the reaction, or

a pharmaceutically acceptable absorption-enhancing ester residue ) with a compound of the formula:

(where Z^ and Z^ are as previously defined) or represent groups which can easily be converted into a group covered by the definitions for Z^ and Z^, in an organic solvent at temperatures in the range from -30 to +30°C, preferably between -5 and + 5°C, and preferably under anhydrous conditions, optionally followed by removal of the protecting group E' and the group or groups Q, when this is a group containing silicon or phosphorus, from the compound provided.

The starting material with the general formula IV can be prepared by using known methods. For example, the compound where Q is hydrogen atoms and E' is hydrogen, i.e. amoxicillin, is produced by the method described in British patent spec. Nos. 873-049, 959-853, 978,178, 1,339,605 and 1,347-979, Belgian spec. Nos. 676,594, 790,466, 737,848 and 751106, Dutch Spec. No. 7,215-359, South African spec. No. 64/695, 66 /1304, 67/5627 and 72/05231 or German OLS

No. 2240422. Silyl derivatives and esters of amoxicillin corresponding to formula IV can be prepared from this by known methods.

An alternative method for preparing compounds of the general formula I consists in reacting acetic acid derivatives of the general formula VI:

where Q is as defined above and where Y' has the same meaning as Y or represents a group which can easily be converted into a group with the definition Y (which can be attacked or influenced under the reaction conditions) after the reaction, with 6-amino-

the penicillanic acid or derivatives thereof, in the presence of a carbodiimide (e.g. dicyclohexylcarbodiimide, 1-ethyl-3(3-dimethylamino-propyl)-carbodiimide optionally mixed with 1-hydroxy-benzotriazole and ■1-cyclohexyl- 3-{2-(N-methylmorpholino)ethyl}carbodiimide) as condensing agent.

Derivatives of 6-aminopenicillanic acid, which can also be used, are those with the general formula:

where E" is a salt-forming residue, a protecting group which can be easily removed after the reaction and which does not affect it or a pharmaceutically acceptable absorption-improving ester residue. Examples of easily removable protective residues which fall under the designations E' and E" in the preceding formula are optionally substituted benzyl (eg p-nitrobenzyl, p-methoxybenzy1), benzhydryl, phenacyl (eg p-halogen substituted phenacyl), 2,2,2-trichloroethy1, trityl, to-butyl, isobornyl or a silicon or phosphorus -atom, which has substituents selected from lower alkyl, halogen-substituted lower alkyl, aryl, aralkyl, lower alkoxy, halogen-substituted alkoxy, lower alkylthio, aralkyloxy, di-(lower)alkylamino, lower alkoxyalkyl and alkylenedioxy groups_ and halogen atoms.

The necessary starting material D(-)-2-amino-2-(p-hydroxyphenyl)acetic acid for the production of the starting acids with formula VI or activated derivatives thereof is known, e.g. from German patent document no. 2355785 and Dutch patent specification. No. 73H012.

The acids of formula VI are new compounds and as such are covered by the invention. They can be prepared by reacting a compound with the formula:

(where Q and E' are as defined above) with a compound of formula V in an inert organic solvent at temperatures in the range from -30 to +30°C (preferably between -5 and +5°C) and preferably under anhydrous conditions , followed by removal of the protecting group E' and all other protecting groups if desired. Preferably, the provided acid of formula VI can be converted directly in situ to a compound of formula I by adding to the provided reaction solution a suitable reactant as mentioned earlier.

The starting materials of formula V can be prepared by methods which are known per se, such as e.g. described in G.I. Derkatsch, Angew. Chem. 81, No. 11, 407-436 (1969), L.I. Samarai et al., Zh. Obsch. Kim. 39, 1511 (1969), G.I. Derkach et al., Zh. Obsch. Kim. 38, No. 8, pp. 1784-1788 (1968), E.S. Gubnitskaya et al., Zh. Obshch. Chem., 40, 1205-1210 (1970),

L. I. Samarai et al., Zh. Obshch. Khim 39., No. 8, 1712-1715 (1969 ), A.V. Narbut et al., Zh. Obshch. Khim, 38, No. 6, pp. 1321-1324 (1968) and G. Tomaschewski et al., Arch. Pharm. 301, p. 520

(1968).

The compounds of the general formula I are preferably used for therapeutic purposes in the form of a non-toxic salt such as a sodium, potassium or calcium salt or ammonium salt. Other salts that can be used include non-toxic, crystallizable salts with organic bases such as amines, e.g. trialkylamines, procaine and dibenzylamine.

When treating bacterial infections, the antibacterial agents according to the invention can be applied topically,

orally or parenterally in accordance with usual procedures for the administration of antibiotics. The preparations are supplied in dosage units containing an effective amount of the active ingredient together with suitable physiologically acceptable carriers or excipients.

The dosage units can be in the form of liquid preparations, such as solutions, suspensions, dispersions or emulsions, or in solid form such as powders, tablets and capsules.

Accordingly, the invention encompasses pharmaceutical preparations containing an effective amount of at least one compound of the general formula I, together with a suitable physiologically acceptable carrier or excipient. Such pharmaceutical preparations may also comprise one or more therapeutic ingredients in addition to the compound according to the invention. The term "effective amount" as used herein in relation to the described compounds means an amount sufficient to destroy or inhibit the growth of susceptible microorganisms when the preparation is administered in the usual manner, in other words, an amount sufficient to control the growth of the bacteria. The magnitude of the effective amount can be easily determined by those skilled in the art, using standard methods for determining the relative activity of antibacterial agents used against susceptible organisms through different routes of administration.

Suitable carriers and excipients may be any physiologically acceptable agent which acts to facilitate delivery of the therapeutically active compound. The carriers may also have other functions, such as acting as a diluent, a taste masking agent, binding agent, a retarding agent or a stabilizing agent. Examples of carriers include water, which may contain gelatin, acacia, alginate, dextran, polyvinylpyrrolidone or sodium carboxymethylcellulose, aqueous ethanol, syrup, isotonic saline solution, isotonic glucose, starch, lactose or other such materials commonly used in the pharmaceutical industry or in the veterinary industry.

Another aspect of the invention comprises a method for preventing bacterial growth by adding an effective amount of the antibacterial compounds described to the bacteria's habitat. For example, the method can be used in the treatment of bacterial infections in animals by supplying the host animal with an effective amount of the antibacterial compound according to the invention.

The new penicillanic acid derivatives of formula I can also be used as growth promoters for ruminants.

They are also applicable for use in vitro as e.g. in liquids for desinfection in concentrations of 0.1 - 1% by weight, dissolved or suspended in a suitable inert carrier for use by washing or spraying.

Some typical members of the class of compounds covered by the invention were tested for antibiotic activity in vitro by means of an agar serum dilution test carried out in the following manner: A stock solution of the antibiotic agent at 2000 µg/ml is prepared in a suitable sterile vessel. Twofold dilution is carried out with sterile 1/20 mol phosphate buffer pH 6.5 (KH2POi|-NaOH). A 1 ml amount of each of the dilutions is added to 19 ml brain/heart infusion agar in sterile Petri dishes. The hard surface is sprayed onto the test organisms and incubated for 24 hours at 37°C. The minimum inhibitory concentration (MIC)(Min

Inhibitor Concentration), i.e. the smallest concentration of the antibiotic agent which completely inhibits the growth of the test organism is expressed in ug/ml.'

In some cases, the MIC values were determined according to a

microserum dilution test performed as follows:

Two drops of a stock solution of the antibiotic to be tested at a known concentration are added to the first hole of a nine numbered well sample plate using a sterile Pasteur pipette. After the pipette has been rinsed three times with a physiological sodium chloride solution, two drops of a stock solution of the test organism in a culture medium are placed in all holes except hole 8. In the first hole, the solution of the test compound is half diluted, then, after the solution in the the first hole is stirred and two drops of this mixture are added to the second hole, etc. until hole 8, dilutions of the test compound in decreasing geometric ratio have been provided. Hole 9 does not contain any antibiotic agent and serves to control the growth of the test organism in a clean medium. The sample plate is incubated at 30°C or 37°C for approx. 18 hours. The MIC values determined according to this last test method are placed in parentheses in the following table. The MIC values of the compounds prepared according to Examples 4, 5 and 7 below were determined.

The ABA test to which reference is made in the following

tables are performed with groups of 6 female mice Swiss SPF of approx. 20 g.

After intraperitoneal and oral administration of the tested compounds in doses of 100 mg/kg in physiological saline solution, blood and urine samples were taken.

The content of the added antimicrobial compounds

in these samples is determined qualitatively according to the method of "Vincent by measuring the diameter of the zone of inhibition around paper discs with a cross-section of 7 mm, which are inserted with the sample and placed in an agar culture in Petri dishes, where the desired microorganism is cultivated. The mean values of two or three independent samples are indicated.

For a quantitative determination of the content of the tested compound in the blood samples, the diameter of the inhibition zones is measured around the paper discs added to the sample and with samples of a series of standard solutions of the compound to be tested, in serum, and the unknown content is determined by means of a standard line e.

The production samples that are reproduced in the following

tables are performed with a group of 10 female mice, Swiss SPF.

The mice in each group are infected intraperitoneally with a selected microorganism. A solution of the antimicrobial compound is administered as indicated and five times a day. The duration of the treatment was 1 day and the duration of the observation of the test animals was 7 days. ED^q values and the potency ratio with respect to the indicated reference compounds were determined according to probit analysis.

The invention is illustrated by the following examples: Example 1

Preparation of sodium D-6-{α-|3-(benzyloxy(ethyl)-phosphinyl)ureido|-p-hydroxybenzylcarbonamido}penicillanate.

Ethyl phosphonium chloride (C^H^POCl^, bp. 78°C/30 mm Hg, was prepared in 66.6% yield from ethyl bromide and aluminum chloride/phosphorus trichloride (AlCiyPCl^) (Houben Weyl, 12<1>, page 397). The dichloride was converted to benzylethylphosphonamidate |C2H5P(0)(CCH2C6H5)<NH>2I in a yield of 43.7% by the usual procedure consisting of subsequent reactions with benzyl alcohol/pyridine and liquid ammonia in diethyl ether. Again using a In the usual procedure, the isolated, very hygroscopic phosphonamidates were converted with phosgene/pyridine in toluene to impure benzylethylphosphonisocyanatidate | C2H^P(0 ) (OCH2CgH,-) - NCOI. One mole of the phosphonamidate was added to a mixture of 1 mole of phosgene and 2, 5 mol of pyridine at -60° C, after which the temperature was allowed to rise slowly to -10° C. After further stirring for about 90 min at room temperature, the reaction mixture was filtered under dry conditions and the filtrate evaporated to dryness in vacuo. The amount of isocyanate in the crude product was determined by infrared spectrum oscopy.

Using anhydrous conditions, 6.5 nil

(approx. 26 mmol) N,O-bis-trimethylsilyl-acetamide (BSA) added at room temperature very quickly to a suspension of 4.75 g (approx. 13 mmol) of pure but not dry D(-)-(6- α-amino-β-hydroxybenzyl-carbonamido)penicillanic acid ("amoxycillin") which was assumed to contain from 1.0 - 1.5 mol of water per mol of penicillin in 25 ml of dichloromethane. The resulting reaction mixture, which became clear after

10 min., was stirred for 90 min. at room temperature. The clear solution was cooled below +5°C using an ice bath, after which a solution of approx. Equimolar amounts of the above-mentioned isocyanate (prepared in crude form from 30 mmol of benzylethylphosphonamidate) in 30 ml of dichloromethane were added dropwise at 0°C - 5°C. During further stirring at approx. At 5°C the conversion of amoxicillin was followed by thin layer chromatography (silica, 5:4:1 mixture of ethyl acetate, acetone and acetic acid., double spot at Rf of about 0.7). After, approx. 30 min. further stirring .had a moderate conversion of the desired penicillin taken place. As the result had not improved after 60 min. stirring, the reaction mixture was subjected to conventional isolation. After adding a small volume of ethyl acetate, the reaction mixture was concentrated in vacuo to a small volume and then poured into a mixture of equal volumes of diethyl ether and ice water at pH 7.0. The layers were separated, the organic layer discarded and the aqueous layer washed several times with a 1:1 mixture of ethyl acetate and diethyl ether. The remaining solution in water was acidified to pH 3.8 and extracted 10 times with equal volumes of ethyl acetate. These extracts were combined, washed 4 times with 20 ml of ice water saturated with sodium chloride, dried over anhydrous magnesium sulfate, filtered and slightly concentrated in vacuo to a volume of 500 ml. A small amount of sediment was removed by filtration. A solution of sodium α-ethyl hexanoate in ethyl acetate was added. The precipitate formed was collected by filtration, washed with dry ethyl acetate and diethyl ether and dried in vacuo. The yield of the title compound was 3.3

g (approx. 40% based on amoxicillin, approx. 18% based on phosphonamide), according to thin layer chromatography, IR and PMR spectra, the purity of the final product was over 90%.

The asymmetrically substituted group containing phosphorus in the product constitutes a chiral center and consequently the compound has two forms. With this particular penicillin, this phenomenon is reflected in the thin-layer chromatogram (po associated spots) and in the PMR spectra.

IR (KBr disk, values in cm<-1>): + 3100-3500 (broad and intense), shoulders at 3080, 3000, 2930 and 2900, 1765, ca. 1690 (sh), 1665, 1600-1620, 1520, 1460, + 1400, 1325, approx. 1260-ll80, + 1020, 915, 850, 745.

PMR (dg-dimethyl sulfoxide (DMSO), 60 Mc, 2,2-dimethyl-silapentane-5-sulfonate (DSS) as reference, 6 values in dpm): a very complicated HH absorption range from ca. 0.7 - 2.2 comprising singlets at 1.45 and 1.57, 3.98 (s, 1H), 4.95 (center of two doublets, 6v ~0.7, J~7.6 eps, 2H), approx.- 5.3 - 5.5 (multiplet, 3H), 6.65 - 7.25 (q) and approx. 7.3 (2 signals) together 9H; 7.7 (d, J -7.5 eps, 0.8H), ca. 8.4 (broad, ca. 0.6H) 8.85 (d, J =8.5 eps, 0.8H).

Example 2

Preparation of sodium D-6-{α-[3-(benzyloxy(ethoxy)~phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanate.

Starting from ethyl phosphorus dichloridate (C2H50P(0)C12), impure benzyloxy(ethoxy)phosphinylamide [(CgH^CH20)(CgH^O)P-(0)-NH2~] was prepared by a common procedure consisting of subsequent transformations with benzyl alcohol/pyridine and excess liquid ammonia in diethyl ether. The impure amide (approx. 30 mmol) was converted according to the method described in example 1 to impure benzyl (ethyl) phosphorisocyanatidate |"(CgH^CH20) (C2H^0)-■P(0)NC0] which contains approx. 15 mmol of isocyanate according to infrared spectroscopy.

As described in Example 1, a suspension of 5.5 g was

(approx. 15 mmol) of amoxicillin in 25 ml of dichloromethane treated at room temperature with 7.5 ml (approx. 30 mmol) of BSA. After 60 min. with further stirring at room temperature, the clear solution was cooled to 0°C. To this solution was added a solution of the above-mentioned phosphorisocyanatidate in 25 ml of dichloromethane at between 0 and 5°C over the course of 5 minutes. A thin layer chromatogram that was taken a few min. after the addition, suggests satisfactory

conversion of amoxicillin to the desired penicillin (Rf approx.

0.65 on silica with a 5:4:1 mixture of ethyl acetate, acetone and acetic acid). The reaction mixture was mixed with ice water of pH

7.0 and a small volume of ethyl acetate. Dichloromethane was removed in vacuo and the aqueous solution of pH 7.0 was washed three times with a 1:1 mixture of ethyl acetate and diethyl ether. Using the same mixture, the penicillin was removed from its solution in water at pH 3.5 - 4.0. The combined acidic extracts were washed several times with small volumes of ice water saturated with sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The final solution in ethyl acetate was treated with a solution of sodium α-ethyl hexanoate in ethyl acetate, etc. The yield of the title compound was 8.0 g (about 80% based on amoxicillin, about 40% based on impure benzyloxy (ethoxy)phosphinylamide). The thin layer chromatogram and PMR spectra suggest a purity of at least 95%.

IR (KBr disc, values in cm<-1>): approx. 3200-3600 (broad and intense), shoulders at 3060 and 2935, 2980, 1768, approx. 1690 (sk), 1670, 1610, 1535 (sk), 1515, 1480, 1400, 1375 (sk), approx. 1220-1260, 1180, 1135, 1040 and 1020, 920, 840, 745, '700.

PMR<*>(ca. 4:1 mixture of dg-DMSO and DC02D, 60 Mc, DSS, 6 values in dpm).: 1.26 (center of two close triplets, J<=>7.5 eps ), 1.46 (s) and 1.58 (s) all 9H; about. 3.85 - 4.35 (multiplet) and 4.27 (s) together 3H; 5.35 - 3.6 (AB-q, J<*>4.1 eps) and 5.46 (s) together 3H, 6.7 - 7.3 (q-like, J = 8.5 eps) and approx. 7.4 (double signal) together 9H.

x The spectrum of the compound in DMSO showed the usual three NH absorptions, two doublets at approx. 7.7 and 8.9 and a broad absorption at approx. 8.7.

Sodium D-6-{a-[3-(benzyloxy(methoxy)phosphinyl)ureido-p-hydroxybenzylcarbon-amido}penicillanate was prepared in a similar manner.

IR (ibidem): + 3300-3500 (broad and intense), + 3050 (Sk), + 2950 (sk), 1765, approx. 1690 (sk), 1660, 1590-1610, 1550 (sk), 1515, 1460, 1400, 1325, approx. 1220-1280, 1180 (sk), 1135, 1045 (intense) with shoulders, 930, 850, 790, 745, 700.

PMR (DMSO, ibidem):' 1.46 and 1.56 (6H), 3.69 (center of two close doublets, 6 v =1.3 eps, J~11.7cps, 3H), 3.98 ( s, 1H), 5.05 (center of two close doublets, 6v =0.5 eps, J =7.5 cps^2H),.ca. 5.25 - 5.6 (multiplet 3H), 6.65 - 7.3 (q) and approx. 7.35 (2 signals) together 9H, 7.8 (d, J =8 eps, 0.8H), approx. 8.4 (very close, 1H), 8.9 (J ~8 cps,0.8H).

Example 3

Preparation of sodium D-6-{α-[3-(dibenzyloxyphosphinyl)ureidyl-p-hydroxybenzylcarbonamido}penicillanate.

As described by O-.H. Friedman et al. (J. Am. Chem. Soc, 76, 916 (1945)) phosphorus trichloride (PCl^) was treated in the presence of pyridine with three equivalents of benzyl alcohol in dry benzene, resulting in the preparation and isolation of dibenzylphosphonate I(CgH5CH20)20( 0)HI with a yield of 78.6%. This product was dissolved in dry carbon tetrachloride and gaseous ammonia was introduced (maximum reaction temperature was 38°C). After recrystallization from carbon tetrachloride, dibenzyl phosphoramidate |(CgH^CHgO)2P(0)-NH2I with m.p. 104-105°C provided with a yield of 84.4%. This compound was converted in the usual way and as described in example 1 to impure dibenzyl phosphorisocyanatidate |(CgH^CH2O)2~P(0)NCOI.

As described in example 1, a suspension of 2.2 g (approx. 6 mmol) of amoxycillin in 10 ml of dichloromethane was treated at room temperature with 3 ml (approx. 12 mmol) of BSA. After 60 min. further stirring, the clear solution was cooled to 0°C and then a solution of approx. 6 mmol of the above-mentioned phosphorus isocyanatidate in 15 ml of dichloromethane added dropwise. The reaction temperature was maintained at 0-5°C. A thin layer chromatogram that was taken a few minutes ago. later indicated satisfactory conversion to the desired penicillin (Rf approx. 0.6 on silica with a 5:4:1 mixture of ethyl acetate, acetone and acetic acid). At pH 7.0, the reaction mixture was poured into a mixture of 100 ml of ice water and 100 ml of diethyl ether. To obtain a clear two-layer system, 300 ml of ice water and some sodium chloride were added. The layers were separated, the organic layer was drained and the aqueous layer was washed several times with diethyl ether at pH 7.0. The remaining aqueous layer was acidified with dilute hydrochloric acid and extracted at pH 3.5~4.0 with ethyl acetate. From these extracts, in the same way as in examples 1 and 2, 3.9 g (approx. 90% based on amoxicillin and approx. 55% based on phosphoramidate) of the essentially pure compound according to the title were obtained.

IR (KBr disc, values in cm<-1>): approx. 3200-3500 (wide

and intense), shoulders at 3050 and 2980, 1780, 1680, 1620, 1560 (sk), 1520, 1465, +1400, 1330, 1230-1270, 1190 (sk), 1140 (sk), 1030 with shoulders at 1050 and 1015, 935, 750, 710.

PMR (dg-DMSO, 60 Mc, DSS, 6 values in dpm): 1.45 and

1.56 (6H), 4.05 (s, 1H), 5.05 (d, J~7.5 eps, 4H), ca. 5.2-5.6

(multiplet, 3H), 6.65-7.3 (q-like, J =8 eps) and 7.35 together 14H, 7.7 (d, J =8 eps, ca. 0.8H), ca. 8.9 (d and wide s, approx. 1.4H).

Example 4

Preparation of the disodium salt of D-6-{a-[3-(hydroxy-(ethyl)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid.

3.06 g (4.8 mmol) of sodium D-6-{a-[3-(benzyloxy-(ethyl)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanate (prepared as described in example 1) was dissolved in a ice-cold mixture of 35 ml of ethanol and 5 ml of water. To the magnetically stirred solution was added 1.5 g of 10% palladium-on-charcoal. With continuous cooling with ice, hydrogen was passed at atmospheric pressure

above the surface of the solution. During the reduction, 410 mg (4.9 mmol) of sodium bicarbonate was added in small portions. As the reduction was not complete after 4 hours, the vessel was stored overnight at 0°C. The next day, a further 1 g of the catalyst and 10 ml of ethanol were added and the hydrogen supply continued until the reduction was complete (a few hours). The mixture was filtered through a filter-aid using a suction pump. The filtrate was evaporated in vacuo, absolute ethanol and benzene were added to the residue and the mixture evaporated in vacuo. The residue was dissolved in a minimal amount of water. The volume of the solution was doubled by the addition of ethanol and acetone was added until a slight cloudiness was obtained. Filter-aid was added with continuous stirring and the mixture was filtered, after which dry acetone was added to the filtrate. The resulting precipitate was collected by filtration, washed with acetone and dried in vacuo. The yield of the title compound was 2.6 g (about 90%). The purity of the final product was 90% or better. On thin-layer chromatograms using silica and a 4:1:1 mixture of n-butanol, acetic acid and water, the compound has an Rf value of approx. 0.1.

IR (KBr disk, values in cm 1): 3100-3600 (broad and intense), approx. '3050 (sk), 2980, 1765, l640-l660 , I6OO-I615, c. 1560 (sk), 1515, 1460, 1400, 1370, + 1325, + 1275, 1240, 1180, 1135 (sk), 1060, 900, 850, approx. 730.

PMR (ca. 4:1 mixture of dg-DMSO and DC02D, 60 Mc, DSS, values in dpm): a very complicated HH absorption range from ca. 0.7 - 2.2 comprising singlets at 1.46 and 1.59; 4.24 (s, 1H), from approx. 5.3-5.6 (AB-q with J~4 eps and a singlet, 3H), 6.7 - 7.3 (q-like, J -8.0 eps, 4H).

Example 5

Preparation of the disodium salt of D-6-{α-|'3~(hydroxy^toxy)phosphinyl)ureidoJ-p-hydroxybenzylcarbonamido}penicillanic acid.

In much the same manner as described in Example 4, hydrogen was introduced at 0°C above the surface of a magnetically stirred mixture comprising 3 g of 10% palladium-on-charcoal and a solution of 6.3 g (9.7 mmol) of sodium -D-6-{α-|j3-(benzyloxy-(ethoxy)phosphinyl)ureido.^)-p-hydroxybenzylcarbonamido}penicillanate (prepared as described in Example 2) in a mixture of 60 ml of ethanol and 10 ml of water . During the catalytic reduction, a solution of 0.82 g (9.7 mmol) of sodium bicarbonate in 3 ml of water was added stepwise. The reduction was completed after 5.5 hours. The reaction mixture was filtered through a filter-aid using a suction pump and the filtrate was evaporated in vacuo. To remove residual water, toluene was added and the evaporation in vacuum was repeated. The residue was stirred with 150 ml of pure, dry acetone. The resulting colorless solid was collected by filtration, washed with dry acetone and dried in vacuo.

The yield of the title compound was 5.54 g

(93%). The end product was clean.

IR (XBr disk, values in cm<-1>): approx. 3200-3600 (broad and intense), '2975, 2930 (sk), 1765, +1650-1670, + 1610, 1550 (sk), 1515, 1460, 1400, 1375 (sk), +1325, approx. 1240 (wide) ll8o, 1135,' 1085, 1050, 955, 900, 770.

PMR (ca. 5:1 mixture of dg-DMSO and DC02D, DSS, 6 values in dpm): 1.25 (center of two close triplets, J -7.5 eps), 1.46 (s) and 1.59 (s) all together 9H; about. 3.75 - 4.1 (multiple,

2H), 4.22 (s, 1H), 5.42 (s) and approx. 5.3 - 5.6 (extended AB-q) together 3H), 6.65 - 7.35 (q-like, J~8.5 eps, 4H).

Similarly, the disodium salt of D-6-{α-[3-(hydroxy(methoxy)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid.

IR (ibidem): approx. 3280-3600 (broad and intense), +2950 (sk), 1760, 1680 (sk), approx. 1645 -1665, + 1600, + 1540, 1500, 1455, 1395, 1370 (sk), 1345 (sk), 1310-1330, 1215-1245, 1180 (sk) 1125, 1080 (intensive), 1045, 895, + 770.

PMR (ca. 5:1 mixture of dg-DMSO and DC02D, 60 Mc, DSS, cf values in dpm) : 1.47 and 1.59 (6H), 3.50 (d, J<=>11 ,6 eps, 3H),

4.27 (s, 1H), 5.44 (s) and approx. 5.35 - 5.6 (extended AB-q), together 3H, 6.7 - 7.35 (q-like, 4H).

Example 6

Preparation of the disodium salt of D-6-{α-|_3-(hydroxy-(benzyloxy)phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanic acid.

345 mg (0.47 mmol) of sodium D-6-{α-[3-(dibenzyloxo-phosphinyl)ureido]-p-hydroxybenzylcarbonamido}penicillanate (prepared as described in Example 3) and 80 mg (0.95 mmol) ) sodium bicarbonate dissolved in 10 ml of water. 0.1 g of 10$ palladium-on-charcoal was added, and then hydrogen was added at 0°C. After 5 hours of stirring, thin-layer chromatography indicates that the starting material of dibenzyl ester has completely disappeared from the reaction mixture, while the degree of release of carbon dioxide has decreased considerably. The pH of the reaction mixture was about 8.0. The mixture was filtered through a filter-aid using a suction pump and the filtrate evaporated in vacuo. The residue was triturated with dry acetone and the resulting solid collected by filtration. Apart from the presence of inorganic salt, it was determined by the thin layer chromatogram and PMR spectra that the final product contained the title penicillin compound in an amount of 85-90%. The impurities were relatively small amounts of degradation product, the twice-reduced (ie di-debenzylated) penicillin and acetone. Since two equivalents of sodium bicarbonate were

added, the end product should also contain somewhat less than 1 mol of sodium bicarbonate per moles of penicillin. On thin-layer chromatograms (silica, 95:5:5 mixture of methanol, acetic acid and water) the compound according to the title appears to have an Rf of around 0.9 (UV-positive), while the di-debenzylated penicillin appears with approx. Rf 0.25.

IR (KBr disc, values in cm<-1>): approx. 3100-3600 (shoulder at + 3050, 2970 and 2935, 1765, 1690 (sk), 1640-1660, 1595-1615, +1550 (sk), 1515, 1455, 1400, 1380 (sk), 1320-1340, 1220 -1260,

1180, 1135, 1090 (intense), 1010-1035, 985, 900, 870, 845, 750, 710.

PMR (ca. 4:1 mixture of dg-DMSO and DC02D, 60 Mc, DSS, 6 values in dpm): 1.48 and 1.60 (6H), 4.2.6 (s, 1H), 4, 86 (d, J ~ 7.0 eps, 2H), 5.45 (s) and 5.35 - 5.60 (AB-q, J<=>4.0 eps) together 3H), 6.65 - 7.3 (q-like, J -8.2 eps) and approx. 7.35 together 9H.

The experiment described above clearly suggests that it. it is possible to provide a mono-debenzylated reduction product in a pure state due to the fact that the second reduction proceeds at a significantly lower rate. To provide the mono-debenzylated product, it is obvious that only one equivalent of sodium bicarbonate should be used, while a further reduction of the competing formation of the double reduction product can be provided either by using a slightly poisoned catalyst and/or by interrupting the reduction just before complete conversion of the starting material, which can be easily removed from the final product by washing with ethanol.

Example 7

Preparation of the trisodium salt of D-6-{α-|_3-(di-hydroxyphosphinyl)ureido]-p-hydroxybenzylcarbonamido}-penicillanic acid.

690 mg (0.94 mmol) of sodium D-6-{a-\ j>-(dibenzyloxy-phosphinyl)ureido]-p-hydroxybenzylcarbonamido}peniciHanate (prepared as described in example 3) and 168 mg (2 mmol) of sodium bicarbonate was dissolved in 15 ml of water, 0.1 g of 10% palladium-on-charcoal was added and hydrogen was added. After 5 hours of stirring at room temperature, a further 0.2 g of 10% palladium-on-charcoal was added and the reduction continued. After a total of 6 hours of stirring, a thin layer chromatogram indicates complete conversion of the starting material to a mixture of the product of Example 6 and

the penicillin compound according to the title. The reduction continued overnight. The next day, 5 nil of water and another 0.5 g of 10$ palladium-on-charcoal were added. The reduction was then continued for 2.5 hours. The release of carbon dioxide ceased and a thin layer chromatogram indicated that the compound from Example 6 had completely disappeared. Acetone was added, the mixture was filtered through a filter-aid and the filtrate evaporated in vacuo. The residue was triturated with absolute ethanol. The resulting solid was collected by filtration, washed with ethanol and dry acetone and dried in vacuo.

The yield of the title compound was 0.5 g (approx. 80%) and according to ■thin layer chromatograms and PMR spectrum the final product was approx. 90% pure.

IR (ibidem): approx. 3100-3600, 2970 (sk), 1760, 1640-1660 (very intense), l600, 1540-1560, 1510, 1450, 1400, 1370 (sk), 1320, approx. 1250 with shoulders, 1180, 1135 (sk), 1110 (intense), 985 (intense), 890, 840, 790.

PMR (D20, 60 Mc, DSS, 6 values in dpm): 1.46 and 1.53 (6H), 4.20 (s, 1H), 5.2 (light wide s, 1H), 5.44 .(s, 2H), 6.8 - 7.45 (q-like, - 4H).

Example 8

Preparation of sodium D-6-{α-Q5-(diphenoxyphosphinyl)-ureido]-p-hydroxybenzylcarbonamido}penicillanate.

70 ml of dichloromethane was added to a suspension of 2.65 g (6.0 mmol) of α-|_3-(diphenoxyphosphinyl)ureido]-4-hydroxy-phenylacetic acid and 1.40 g (7.3 mmol) of 1-ethyl- 3-(3-Dimethylaminopropyl)carbodiimide hydrochloride in 25 ml of tetrahydrofuran. With stirring, 1.45 g (6.6 mmol) of 6-aminopenicillanic acid, which had previously been silylated using 0.97 ml (6.6 mmol) TEA and -0.84 ml (6.6 mmol) TMCS in 15 ml of dichloromethane, added over 7 min.

After two hours, the transformation appears to be approx. 40% according to thin layer chromatogram. (Rf = 0.7 in an acetic acid/ethyl acetate/acetone (1:5:4) mixture). A yellow oil was formed which was separated and discarded. The remaining reaction mixture was poured into water at pH = 7 and extracted with ethyl acetate.

The pH was adjusted to 4.7 followed by extraction with ethyl acetate. After drying over magnesium sulfate, filtration, evaporation of ethyl acetate and addition of approx. 2 mmol sodium hexanoate became the desired product, the structure of which could be confirmed by PMR isolated with a yield of 1.23 g (32%). According to the thin-layer chromatogram, the product provided appears to be fairly pure. The compound provided was probably a mixture of D-L.

Starting compound α-[3-(diphenoxyphosphinyl)ureido]-4-hydroxyphenylacetic acid was characterized by m.p. l60-170°C (decomp.).

IR: 990, approx. 1200, 1650 and 1710 cm"<1>.

PMR (dg-DMSO) 60 Mc, TMS, 6 values in dpm): 5.15 (d 3 CH) 36.6-734 (aromatic H).

Example 9

Preparation of D-6-{ct-[ 3-(benzyloxy(ethoxy)phosphinyl)ureido^-p-hydroxybenzylcarbonamido}penicillanate.

15 ml of dichloromethane was added to a suspension of 1.0 mmol of α-13-(benzyloxy(ethoxy)phosphinyl)-ureido]-4-hydroxyphenylacetic acid and 0.25 g (1.2 mmol) of 1-ethyl-3(3-dimethylaminopropyl )-carbodiimide hydrochloride in 4 ml of tetrahydrofuran. After stirring, within 7 min. 0.25 g (1.1 mmol) of 6-aminopenicillanic acid, which was previously

been silylated using 0.16 ml (1.1 mmol) TEA and 0.14 ml (1.1 mmol) TMCS in 235 ml dichloromethane added.

After approx. 2 hours, the transformation seems to be approx. 50% according to thin layer chromatogram (Rf = 037 in an acetic acid/ethyl acetate/acetone (1:5:4) mixture). The reaction mixture was poured into water with pH = 7 after separation of an oily precipitate and extracted with ethyl acetate.

The pH was adjusted with 437 followed by extraction with ethyl acetate. After drying over magnesium sulfate3 filtration, in part

evaporation of the ethyl acetate and addition of sodium hexanoate,

the desired product was obtained, the structure of which could be confirmed by PMR, isolated with a yield of 46.6% and according to the thin-layer chromatogram, the product was quite pure.

The starting compound α-Q)-(benzyloxy(ethoxy)phosphinyl)-ureidoJ-4-hydroxyphenylacetoacetic acid is characterized by m.p. 122-127°C (during decomposition).

IR 1020, 1220, 1670 and 1720 cm"<1>.

PMR (dg-DMSO, 60 Mc, TMS, 5 values in dpm): 1.2 (t, CH 3 ), 4.1 (m, -C-CH 2 O), 5.1 (m, CgH^CH^O ), 7.0 and 7.4 (aromatic

H).

In the same way, instead of α-[_3-(benzyloxy(ethoxy)-phosphinyl)ureidoJ-4-hydroxyphenylacetic acid, one could use c-|j5-(diethoxyphosphinyl)ureidoJ-4-hydroxyphenylacetic acid, which was characterized by m.p. at l66-l69°C (decomp.).

IR: 1040, 1220, 1640 and 1705 cm<-1>.

PMR (dg-DMSO, 60 Mc, TMS,. 6 values in dpm): 1.3 (m, CHj), 4.1 (m, CH2), 515 (d, CH) , 7.0 (q, aromatic H).

Example 10

In the same way as in the preceding examples 1-9, compounds were prepared in which Z-^ and Z2 had the meaning indicated below:

Example 11

Capsules containing as an active ingredient a penicillin derivative prepared according to any of Examples 1-9 were prepared in a conventional manner.

The contents of each capsule are listed below:

The capsules can be used for oral administration.

Example 12

Tablets containing as an active ingredient a penicillin prepared according to any one of Examples 1-9 were prepared in a conventional manner.

The composition of each tablet is listed below:

Example 15

From a penicillin derivative prepared according to any of Examples 1-9, a dry powder usable for injections was prepared in a conventional manner.

An amount of 2 g and 5 g of the steric sodium salt of the compound mixed with the usual other substances was aseptically added to an ampoule suitable for an injectable preparation under a nitrogen atmosphere. The ampoule was closed by means of a rubber plate, which was fixed with an aluminum ring to eliminate the exchange of gases or the ingress of microorganisms.

Common other substances may consist of glucose (usually in an amount to obtain a final isotonic solution), buffer salts, stabilizing agents (e.g. Na2EDTA), preservatives, wetting agents and antifoaming agents.

Before use, the powder is dissolved in an appropriate amount of sterile and pyrogen-free water.

E xample lH

From the penicillin derivatives prepared according to any of Examples 1-9, syrups were prepared by mixing the following ingredients:

Claims (3)

1. Intermediates for the production of penicillanic acid derivatives, characterized by the general formula: where Q denotes a hydrogen atom or a silicon or phosphorus atom, bearing substituents selected from lower alkyl, halogen-substituted lower alkyl, aryl, aralkyl, lower alkoxy, halogen-substituted alkoxy, lower alkylthio, aralkyloxy, di(lower)alkyl-amino , lower alkoxyalkyl, alkylenedioxy groups and halogen atoms (preferably a tri(lower)alkylsilyl, e.g. trimethylsilyl group) and Y' denotes the group: where Z-j^ and Z£ are the same or different and each denotes a lower alkoxy group, an optionally substituted phenoxy group, an optionally substituted benzyl or benzyloxy group, an optionally lower alkyl group, an optionally substituted phenyl group, a hydroxy group or a group -OM, where M denotes a salt-forming cation, or represents groups which can be converted into a group within the above formula. 2. Process for the production of intermediates according to claim 1 for the production of penicillanic acid derivatives, characterized in that a compound with the formula: where Q has the meaning given above and E' denotes a group which protects the carboxy radical, preferably a group which, if desired, can be easily removed after the reaction, e.g. by hydrolysis, hydrogenation or a substitution reaction using a basic or nucleophilic reagent, and which does not affect the reaction, is reacted with a compound of the formula: where Z£ and Z£ are as defined above, in an organic solvent at temperatures within the range - J>Q°C to +30°C, after which the protective groups are optionally removed from the thus obtained compounds. 3. Method according to claim 2, characterized in that the reaction is carried out under anhydrous conditions and at temperatures between -5°C and +5°C.