NO157078B - Electrode baking oven. - Google Patents

Description

The invention relates to a process for the production of aminopenicillins with structural formula

as well as non-toxic salts thereof, where R is a hydrogen atom, phenyl or thienyl and n is an integer from 0 to 4 inclusive, the method being characterized in that a compound with the structural formula

wherein R and n, as defined above, are reacted in water or an aqueous organic solvent with a nucleophilic reagent consisting of iodide, thiocyanate, sulfite, thiosulfate, or dithionite ions or mixtures thereof, and, if desired, transferred formed compound to a non-toxic salt.

The acids produced according to the invention can be used as such or in the form of their non-toxic salts. Such salts include metal salts such as sodium, potassium, calcium, aluminium, ammonium and substituted ammonium salts, e.g. salts of such amines as trialkylamines, procaine, dibenzylamine, N-benzyl-g-phenethylamine, NjN^-dibenzylethylenediamine, dihydroabiethylamine and other amines which have been used to form salts with benzyl or phenoxymethylpenicillin.

If the compounds contain one or more asymmetric centers they may occur in diastereoisomeric forms as pure epimers or epimer mixtures. The epimers are all biologically active. The invention encompasses the production of both the pure epimers and their mixtures.

The compounds produced according to the invention have valuable antimicrobial activity against gram-positive microorganisms as well as against gram-negative ones and they are particularly valuable for the treatment of various infections caused by microorganisms. The compounds can be administered in different preparation forms, e.g. solutions, suspensions, tablets, capsules and. ointments. They can therefore be administered orally by injection or by application to the surface, depending on the nature of the infection treated and those. is suitable for the treatment of both humans and animals.

In the production of aminopenicillins by acylation of 6-aminopenicillanic acid with reactive amino acid derivatives, the amino group in the latter, as in peptide syntheses, must generally be suitably protected in order to participate in the reaction. Thus, e.g. the use of b.enzyloxycarbonyl-amino substituted acyl compounds in the preparation of ~penicillin. The protecting group is then removed by catalytic hydrogenation. However, this is associated with major difficulties as the penicillin's sulfur atom causes catalyst poisoning. Other protective groups used in peptide chemistry which are removed by protolysis or hydrolysis are not applicable as the penicillin's 6-lactam ring is very sensitive to hydrolysis (inactivation of the penicillin).

This fact can be illustrated by a recently published method (Zervas et al. J. Amer. Chem. Soc. 85 (1963) 3660) in which the use of the o-nitrophenylsulfenyl group in peptide synthesis is described.

The nitrophenylsulfenyl group is split off under very weak conditions by the action of acid, preferably HC1

in ether, HB r in ether or HC1 in ethanol. This has been used with success in peptide syntheses, as appears from the Journal of the American Chemical Society, 85 (1963) 3660. However, this cleavage method cannot be applied to nitrophenylsulfenyl

aminopenicillins, as these, due to the g-lactam ring's great sensitivity to electrophilic reagents, decompose very quickly under these conditions.

However, the penicillin molecule is also very sensitive to nucleophilic reagents (F.P. Doyle and J.H.C. Nayler in N.J. Harper and A.B. Simmonds, Advances in Drug Research, Academic Press, 1964, volume 1, page 25) and at least one of the nucleophilic reagents, Sulfite ions used in the present process are known for their ability to rapidly inactivate the penicillin molecules (0. Wintersteiner et al. in H.T. Clarke, J.R. Johnson and R. Robinson", The Chemistry of Penicillin, Princeton University Press, Princeton 19^ 9, page 221).It is therefore very surprising and could not be predicted that the cleavage of the nitrophenylsulfenyl protecting group with the nucleophilic reagents mentioned in the present invention can be carried out without the penicillin molecule being strongly cleaved.

To the extent that the literature suggests something, this should be that the reaction according to the invention for splitting off the nitrophenylsulfenyl group from nitrophenylsulfenylaminopenicillins, if it could at all lead to a splitting off of the group in question, which could not be predicted, should lead to an inactivation -of the penicillin^ .

It has now been shown that with the method according to the invention one can avoid the above-mentioned disadvantages and most often cleave o-nitrophenylsulfenyl groups from aminopenicillins in approximately quantitative yield under particularly mild conditions - and with full retention of the B-lactam structure necessary for antimicrobial activity .

The method according to the invention shall be illustrated in more detail by the following examples.

Example 1-. Cleavage of the o-nitrophenylsulfenyl group from a-(o-nitro-phenyl-sulfenylamino) benzylpenicillin with various reagents.

Stock solutions of ot-(o-nitrophenylsulfenylamino)benzylpenicillin- were prepared by dissolving 2.7 g (5 mmol) of its potassium salt in 50 ml of a dioxane-water mixture (3 •" !)•

Portions of 10.0 ml of this solution (corresponding to 1.0 mmol of penicillin) were withdrawn and the pH adjusted to a value chosen for each solution between 1 and 8.5. The respective solutions were then treated for a specific time (usually 10 minutes) with stirring at room temperature with 2.0 mmol of reagent. By continuously supplying dilute hydrochloric acid during the course of the reaction, the pH was kept constant. (The reagents and reaction conditions used in the experiments are stated in the table below).

After completion of the reaction, the pH was adjusted to 2 and the mixture extracted 3 times with 15 ml of ether, in some cases after the addition of 20 - 25 ml of water to dissolve the formed precipitate. The combined ether phases were washed twice with water and then extracted with 10 ml of 0.5 N potassium bicarbonate solution. The aqueous phase containing remaining α-(o-nitrophenyl-sulfenylamino)benzylpenicillin was separated, neutralized by the addition of dilute hydrochloric acid and diluted to the volume indicated in the table below (solution A). The solution was examined with regard to penicillin content (according to Boxer et al. Anal. Chem. 21 (19^9) 670) and the Rf value of the components was determined in detail by paper chromatography (according to Bachelor et. al. Nature 183 (1959) 257 ).

The acidic, original aqueous phase containing formed α-aminobenzylpenicillin was neutralized and filtered. The solution was then diluted to the specified volume (solution B, see table) and analyzed and chromatographed as described for solution A.

As a comparison and control, trials (no. 1, 2 and 3) were carried out at different degrees of acidity without the addition of a-v special reagent, but otherwise as described above. XI solution B: amount of penicillin in mmol XII solution B: Rf value for antibiotically active substances. Values within () indicate trace quantities.

An authentic sample of α-aminobenzylpenicillin has Rf = 0.39-

Experiments no. 1-3 show the results of the work

in aqueous medium with reagents according to J. Amer. Chem. Soc. and experiments no. 4-19 provide data for the work of the method according to the invention. When working according to the modified known method, the yield of aminopenicillin formed was on average 0.06 mmol, while the yield when working according to the method according to the invention was between 0.41 and 1.0 mmol or, converted into a percentage, the yield when working according to the modified, known method of the order of magnitude 6%, but with the work according to the invention, yields were obtained which in certain cases went up to 100% and in no case did they fall below 41%, i.e. according to the method according to the origin, yields are obtained as

is at least approx. 7 times as large as according to the known method and which can go up to 100% yield.

The potassium salt of α-(o-nitrophenylsulfenylamino)benzylpenicillin used as starting material in the experiments was prepared in a known manner.

Example 2.

Preparation of α-aminobenzylpenicillin.

The potassium salt of α-(o-nitrophenylsulfenylamino)benzylpenicillin (2.7 g, 5.0 mmol) was dissolved in 50 ml of 75% dioxane. The pH was adjusted to 3.0 by the addition of 2.0 N hydrochloric acid after which sodium iodide (1.5 g, 10 mmol) was added and the mixture stirred for 10 minutes at room temperature while the pH was kept constant by the addition of dilute hydrochloric acid. After a few seconds the solution began to take on a strong iodine color and immediately afterwards a precipitate formed. The iodine was decolourised by dropwise addition of 1 molar sodium thiosulphate solution. After 10 minutes, water was added and the mixture acidified to pH 2 and washed well with ether. The majority of the formed precipitate was thereby dissolved. The aqueous phase was neutralized, filtered and analyzed to contain 1.96 g (112%) of α-aminobenzylpenicillin, which was identified by paper chromatography.

The water solution was concentrated in vacuo at 30°C to approx. 25 ml volume, filtered, acidified to pH 4.3 and allowed to stand overnight in a refrigerator. The white precipitate formed was filtered off, washed with ice water and dried to give 0.85 g (-48.5%) of α-aminobenzylpenicillin with a purity of 94.5%

(hydroxylamine determination).

The product formed was identified by paper chromatography and IR spectrographic comparison with an authentic sample of α-aminobenzylpenicillin.

In an analogous manner, a-aminomethylpenicillin and a-amino-3-thenylpenicillin are obtained from the potassium salt of a-(o-nitro-phenylsulfenylamino)-methylpenicillin and a-(o-nitrophenylsulfenyl-amino)-3-thenylpenicillin.

Example 3-

Preparation of α-aminobenzylpenicillin.

Potassium salt of α-(o-nitrophenylsulfenylamino)benzylpenicillin (2.7 g, 5.0 mmol) was dissolved in 50 ml of 75% dioxane. The pH was adjusted to 3>0 by adding 2.0 N hydrochloric acid, after which Na^O-^ . 5 H 2 O (2.5 g, 10 mmol) was added and the mixture was stirred for 10 min at room temperature and constant pH. The mixture was then diluted with water, acidified to pH 2 and washed well with ether. The water phase formed was neutralized, as a colored precipitate slowly formed. After the solution had been left for several hours at room temperature, the precipitate was removed by filtration and washing with ether. The aqueous phase contained 1.4 g (80%) of α-aminobenzylpenicillin, which was identified by paper chromatography.

The water solution was treated as described in example 2, whereby 0.2 g of α-aminobenzylpenicillin with a content of 84% (hydroxylamine determination) was obtained. Example 4.

Preparation of α-amihobenzylpeniciTTin.

Experiment 3 is repeated, however, the thiosulphate is replaced with sodium dithionate Na2S-20Ji( (1.7 gj 10 mmol). During the work-up, as in experiment 3, a slow precipitate is obtained in the neutral water phase. The precipitate is removed by filtration and washing with ether, after which the solution analyzed It was found to contain 1*63 g (93%) of a-aminobenzylpenicillin (identified - paper chromatographically).

At the concentration of the solution as described in example 2, the α-aminobenzylpenicillin could be isolated in the form of a white powder: 0.5 g, with a content of 97% (hydroxylamine determination).

The identity of the product formed was confirmed by a paper chromatographic and IR spectrographic comparison with an authentic sample of α-aminobenzylpenicillin.

Example 5-

Preparation of α-aminobenzylpenicillin.

Experiment 3 was repeated, but instead of sodium thiosulphate, potassium rhodanide (1.0 g, 10 mmol) was used. When working up according to example 2, upon neutralization of the water phase only an insignificant precipitate appeared which was removed by filtration. Analysis showed that the water phase contained 1.74 g (100%) of α-aminobenzylpenicillin which was identified by paper chromatography. By concentrating in vacuum at 30°C, 0.9 g (51%) of α-amino-benzylpenicillin with a content of 87% (hydroxylamine determination) was isolated from the water solution, in the same way as in example 2.

The identity of the product formed was confirmed by a paper chromatographic and IR spectrographic comparison with an authentic sample of α-aminobenzylpenicillin.

Example 6.

Preparation of α-aminobenzylpehicylTin.

To a neutral solution of the potassium salt of α-{o-nitrophenylsulfenylamino)-benzylpenicillin ('2.7 g, 5.0 mmol)

in 50 ml of water, Na<2S20->j was added at room temperature.

5 H 2 O (2.5 g., 10.mmol). The solution was stirred for 20 minutes while the pH was maintained at 7 by the addition of dilute hydrochloric acid. It was then acidified to pH 2 and the formed precipitate removed for the most part by washing with ether (3 x 50 ml).' The water phase was decanted from the remaining precipitate and neutralized, whereby a new precipitate was slowly formed. This too was removed by washing with ether and the clear water solution formed (70 ml) was analyzed and proved to contain 17j9 mg of α-aminobenzylpenicillin per ml, which corresponds to a yield of 1.25 g (71%) of the penicillin, which was identified by paper chromatography.

The aqueous solution was concentrated in vacuo at

30°C to approx. 20 ml volume, acidified to pH 4.3 and allowed to stand

two hours in the fridge. The formed precipitate was filtered off, washed with ice water and dried, thereby yielding 0.2 g of α-amino-benzylpenicillin with a content of 87% (hydroxylamine determination).

The identity of the product formed was confirmed by a paper chromatographic and IR spectrographic comparison with an authentic sample of α-aminobenzylpenicillin.

Example 7-

Preparation of 6-7_ D(-)-α-amino.-.a.-f.eny.l.ac.e.t.ami.d£/.p.e.nic.i.ll.i.ns.yr.e.

The potassium salt of 6-/_ D(-)-α-(o-nitrophenylsulfenyl-amino)-α-phenylacetamido7-penicillanic acid (1.9 g» 0.0035 mol)

was dissolved in 50 ml of 75% dioxane, acidified to pH 2 with 2 N hydrochloric acid and mixed with stirring with sodium iodide (1.5 g,

0.01 mol). Sodium thiosulfate was added dropwise to decolorize the iodine formed, while more hydrochloric acid was added to keep the pH constant at 3. After 10 minutes it was acidified to pH 2 and the reaction mixture was washed with ether and filtered. The water phase was neutralized and analysis showed that

contain 0.94 g of α-aminobenzylpenicillin. The concentration of the water solution in vacuum at 25°C to approx. 15 ml volume, acidification to pH 4.3 and storage in a refrigerator overnight, gave. 0.35 g 6-_/ D(-)-a-amino-a-phenylacetamide £7penicillanic acid , melting point 228°C during decomposition, /_<_>a7^°: .+ 274° (C = 1, citric acid/ disodium phosphate buffer, pH 4-.5) £~Doyle, F.P., Fosker, C.R., Nayler, J.H.C. and Smith H., J. Chem. Soc. 1962, 1440: Melting point approx. 202°C, /"aT^<1>: +281° (C = 1, H2)_)7. Example 8.

Preparation of B-aminoethylpenicillin.

The potassium salt of B-(o-nitrophenylsulfenylamino)ethylpenicillin (5.0 g) was dissolved in 25 ml of 50% dioxane, acidified to pH 3 with 2 N sulfuric acid and mixed with stirring with sodium iodide (3 g)• While stirring was added dropwise M sodium thiosulphate solution to decolorize the formed iodine while 2 N- sulfuric acid was added to keep the pH constant at 3- The reaction mixture was acidified to pH 2 and washed with ether. The water phase was filtered, neutralized and concentrated to a small volume in vacuum at 30°C, after which the pH was adjusted to 4.5 - After 2 hours in a refrigerator, the formed precipitate was filtered off and washed with a little water and dried. This gives 1.0 g of g-aminoethylpenicillin with a content of 72% (hydroxylamine determination with 6-aminopenicillanic acid as standard).

The product formed inhibited the growth of Staph. aureus, Oxford with a concentration of 2.5 mcg/ml.

Example 9 -

Preparation of ε-aminopentylpenic:illin.

A solution of the potassium salt of e-(o-nitrophenyl-sulfenylamino)pentylpenicillin (5.6 g) in 25 ml of 75% dioxane was acidified to pH 3 with dilute sulfuric acid and mixed with stirring dropwise with N sodium iodide solution while more sulfuric acid was added. added to keep the pH constant at 3. After acid consumption had ceased and further addition of sodium iodide did not change the pH, it was acidified to pH 2. and the mixture washed well with chloroform to remove iodine formed, was filtered, neutralized and freeze dried. This resulted in 4.9 g of e-aminopentylpenicillin with a content of 15.4%

(hydroxylamine determination with 6-aminopenicillanic acid as standard) .

The product formed inhibited the growth of Staph.

aureus, Oxford -at a concentration of 1.25 mcg/ml and contained in its IR spectrum a band at 1770 cm"<1> which showed the presence of a 6-onion ring.

Claims (1)

Process for the preparation of aminopenicillins with structural formula as well as non-toxic salts thereof, -where R is a hydrogen atom, phenyl or -thienyl and n is an integer from 0 to 4, characterized in that a compound with the structural formula in which -R and n has the above stated' meaning, is reacted in water or an aqueous organic solvent with a nucleophilic reagent consisting of iodide, thiocyanate, sulfite, thiosulfate or dithionite ions or mixtures thereof, and, if desired, the resulting compound is transferred to a non-toxic salt.