DE1445419A1 - Process for the preparation of 6 - (? - Aminoacylamino) -penicillic acids - Google Patents

Description

DR. EULE DR. BERG DIPL.-ING. STAPF, _Mr ' PATENT LAWYERS 1 H H O 4 1

β MUNICH 2nd HILBLESTRASSE 20

Df.Owl Dr. Berg Dipl-Infl. Darning. SMOndien 2, Hilblestro8e 20 Our sign - the date

V ™ ¹ * ^68e 24. Mt,) 1968 ''

PU 45 419. 4
New documents

Lawyer file no. 14 688

American Home Products Corp. New York 17 / USA.

"Process for the preparation of 6- (oC-aminoacylamino) -penicillic acids."

The present invention relates generally to the master position of penicillins and more particularly to a new to ' : ■
°. Process for the preparation of 6- (W-amino-acylamino) ~ ^ penicillic acids with an additional substituent m on the cC-carbon atom and their non-toxic salts.

■ O ·· '· ■■■: ... ■

Ntue UiUkriaoen (Aft 711 Ata. 2 No. ι

(Oeil) 'SH 3D η ΐι fATENTtUU MtadMii tonki Boyeriieh «V« r «imbank Munich 453 100 Posticheck: Munich« 33 43

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American Patent 2,985,648 describes penicillic acid derivatives of the formula

- CH - C - m - CH - CH

ι ι ι

M ₉ C N-

R "

.3 O

where R ^, R ₂ , R "are each hydrogen, nitrogen, lower dialkylamino, lower alkanoylamino, lower alkynoyloxy, lower alkyl (including those with straight and branched, saturated, aliphatic groups of 1 to 6 carbon atoms, inclusive ), lower alkoxy, sulfamil, chlorine, iodine, bromine, fluorine and trifluoromethyl radicals and their non-toxic carboxylic acid salts. Examples of such non-toxic metal salts are the sodium, potassium, calcium, aluminum, ammonium and substituted ammonium salts such as _e salts of non-toxic amines such as trialkylamines, including triethylamine, procaine, dibenzylamine, N-Benzylß-phenylethylamine, 1- Ephenamin, N, N ^! -Dibenzylethylenediamine, dehydroabietylamine, NjN'-bis-dehydroabietylethylenediamine, N- (lower) alkylpiperidine, for example ". N-ethylpiperidine and. other amines that were used to form salts with benzyl-penicillin, as well as their 'non-toxic' acid addition salts (i.e. amine salts), such as the hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate,

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and the organic oäureadditionssalze as the maleate, acetate, Gitrat, oxalate, succinate, benzo at, tartrate, fumarate, malate, mandelate, ascorbate u _or the like.

In the above-described penicillic acid derivatives according to the "American patent" mentioned, the α-carbon atom of the acyl group (to which the <jf ~ amino group is attached) is asymmetrical; there are therefore two different optically active isomer forms (namely the D - (-) and L (4) diastereoisomers) and the optically inactive DL-Jt ¹ Orm as a mixture of the two optically active forms,

The compounds mentioned above and described in US Pat. No. 2,985,648 are valuable as Antibiotics, additives to animal feed and agents for the treatment of mastitis in cattle and also as Therapeutics for poultry and mammals including humans where they are used for parenteral or oral treatment of diseases caused by gram-positive or gram-negative bacteria '. In addition, show the compounds have a remarkable resistance to acid degradation.

The 6 ~ (ot-aminoacylamino) -penicillic acids according to the above Formula were based on that in the American patent 2,985,648 processes described. With these

BATH 4 8 0 9 8 0 6.0856

"If"

known processes, a 6-aminopenicillic acid is generally reacted with an oU-aminobenzylic acid / thihalide or anhydride in which the amino group has previously been _{blocked by an acyl group, such as PhCH 2} OCO- or another functionally equivalent group. In order to obtain the desired o-aminobenzylpenicillic acid derivative, the blocking group must be removed by catalytic hydrogenation under conditions so mild that any destruction of the penicillin core is avoided. According to this known process, the following four separate process steps are necessary: 1) the amino group of the amino acid reaction partner must be blocked with an acyl group; 2) the anhydride of this acyl-blocked amino acid must be formed; 3) this blocked anhydride must be reacted with 6-aminopenicillic acid and

de

4) the blocking ^ group must be used in order to obtain the desired Penicillic acid derivative can be removed by catalytic hydrogenation.

As already mentioned, it is known that the aforementioned specific blocking group is replaced by another functionally equivalent group to replace, but in all examples described so far, the blocking The group must then be removed by catalytic hydrogenation in order to obtain the free 6- (oC-Aminoacylamino) -penicillic acid derivative to win, Unfortunately, the sulfur

~ 5 8 0 9 8 0 fi 'Π 8 ζ R

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content of the penicillin molecule is disadvantageous for the application of this known method, since sulfur is known acts as a hydrogenation catalyst poison. This special property of sulfur-containing molecules made extraction difficult of pure 6- (ci-aminoacylamino) -penicillic acids Extraordinary loan in high yield by the process mentioned

According to the invention a new method for manufacturing Λ has now position of 6 "(o ^ Aminoaoylamino) -Penicillinsäuren above type provided can be obtained with a high purity product in an effective and economical manner and in high yield ·

According to the process according to the invention, 6- (c (-aminoaoylamino) -penicillic acids can be produced with higher efficiency compared to known processes, wherein the initial introduction of the residue blocking the amino group and its subsequent removal superfluous by catalytic hydrogenation.

An essential feature of the present invention is the elimination of the catalytic hydrogenation stage, the hitherto in the production of 6-fo ^ -aminoaoylamino) -penicillic acids created so many difficulties that the new process was more effective, more productive and more economical than the previously known method works.

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The economy of the process according to the invention Compared to the processes known from Bicher, the commercial delivery of these penicillins is reasonable Prices in prospect. As can be seen from the following, the method according to the invention also enables the easy production of further 6- (O <-aminoacylamino) -penicillic acid derivatives, which are not described in US Pat. No. 2,985,648.

In peptide chemistry it is already known that so-called Coupling Leuehs anhydrides with amino acids or peptides to form a carbonairide bond permit. It was also known to avoid the side reactions involved, but it was unknown that this reaction is carried out in an aqueous, acidic medium at temperatures above 0 ° to obtain technically acceptable levels Could perform yields.

The process according to the invention relates generally to the reaction of a 2,5-oxazolidinedione substituted in the 4-position (also known as N-carboxy amic acid anhydride is known) according to the formula below with 6-aminopenicillic acid. The implementation can can be shown schematically as follows:

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ι ^x o -L- ² IiI ³

R · · Η C · ^ OS CH-COOH

ti ^v 0 O

.0

R "iffi

f /

0 C B

where in the formulas R is hydrogen or lower alkyl, H ^{f is} hydrogen, alkyl, allyl, tosylaminobutyl, methylmercaptoethyl, iJenzylmerkaptomethyl, nitrophenylmethyl, aoetoxymethyl, oi-carboxyethyl, 2- (lower alkoxycarbonyl) ethyl, benzyloxymethyl, mono- or di-acetoxyphen Benaylimidazolylmethyl, Indolylmethyl, Cycloalkyl, Phenyl or one to three times by di (lower) alkylamino, (lower) alkanoyloxy, (lower) alkoxy, oulfamyl, halogen or trifluoromethyl substituted phenyl and R and R * to form a cycloalkyl ring with 5 to 7 carbon atoms can be linked, R "denotes hydrogen or R * can be linked to form a pyrrolidine, acetoxypyrrolidine or tosyloxypyrrolidine ring and the reaction can be carried out in an exclusively aqueous-acidic medium with a

8 0 9 8 0 6/0 8 5

pH of about 3 »8 to 6.2 and at a temperature of about just above the freezing point of the aqueous mixture to about 37 ^° C.

The classic processes for the preparation of 4-3-substituted 2,5-uxazolidinediones consist of a) the alkoxy process, b; the azide rearrangement process and o) the phosgenation process. The processes, of which phosgenation is preferred, have the advantageous property that they do not cause any change in the steric configuration in the presence of an asymmetric carbon atom. The process for obtaining the 4-substituted 2,5-xazolidinediones by hydrogenation according to process c) generally consists in the reaction of a suitable amino acid with phosgene according to the following general formula:

I /, ι.

RG ü " Oh. ^ --— G ü

R "NH Gl R" N C

Preferably in these Ihosgenierungsverfahren the Amino acid dissolved or suspended in dioxane, introduced into the reaction mixture and the resulting

_M 9 *

Anhydride by adding, for example, benzene to Brought crystallization. The following are examples of amino acids to be used: Glycine, alanine, üjorleucin, o (-aminoisobutyric acid, valine, Isoleucine, leucine, C-allylglycine, ε -tosyllysine, methionine, s-benzyloysteine, p ~ nitrophenyl ~ alanine, methyl and ethyl glutamate, ο-acetylserine, ß-methylaspartic acid, o-benzylserine, o-aoetyltyrosine, 2,5- and 3,4-diaoetoxyphenylalanine, 1-benzylhistidine, tryptophan, 1-aminocyclo- (pentane * hot and heptane) carboxylic acids, proline, o-acetylhydroxyproline, ο-To sylhydroxy-proline.

Since there are many processes in the art for the production of 6-amino-penicillic acid, including that in the American No. 2,985,648 are known, a more detailed description is superfluous here.

According to a preferred embodiment of the process according to the invention, the 2,5-oxazolidinedione substituted in the 4-position is mixed with 6-aminopenioillinic acid in approximately equimolar amounts in a cold aqueous solution at a pH of approximately 3 »8 to approximately 6.2 and preferably 4, 7 to 5t7 · The mixture is reacted for several hours at a temperature between just above the freezing point of the aqueous mixture and about 37 ⁰ C, preferably in the range

- 10 809806/0856

between 0 ° and 10 ⁰ C, stirred. Although not required, it is advantageous to add a buffer with an ionic strength of about 0.02, and preferably about 0.3, in order to keep the reaction mixture within the required pH range. Buffers suitable for this purpose can consist of any mixtures of organic or inorganic, water-soluble acids, bases or salts, such as sodium acetate-acetic acid, calcium acetate-acetic acid, pyridine-acetic acid, formic acid-ammonia or the like. Has a value below 4, can also be kept in the desired pH range by carefully adding a base such as NaOH or the like.

The process according to the invention is all the more surprising since 2,5-oxazolidinediones were previously known to the person skilled in the art because of their tendency to hydrolysis and polymerization when they are reacted with amino acids for the purpose of peptide synthesis. This unanimous prejudice of the professional world clearly results from the assessment of Greenstein and Winitz in "Chemistry of the Amino Acida", 1961, John Wiley & Sons, Inc., New York, HY, where it is stated in Volume II, page 876

¹¹ Assessment of the procedure. The generally pronounced lability of N-carboxyamino acid anhydrides in the presence of traces of moisture as well as the too

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their regulated condensation with a suitably constituted amine necessary precautionary measures have so far from the general use of these substances for the gradual construction of low peptides deterred. "

Considering the special characteristics of 6-amino-acid penicillin, the result of the inventive process ^ so is even more surprising. 6-aminopenicillic acid polymerizes under certain conditions. In addition, the sensitivity of the lactam bond in its molecular backbone is well known. These properties therefore lead the person skilled in the art to assume that no reaction of any appreciable extent with 2,5-oxazolidinediones can take place without decomposition, hydrolysis, polymerization and other side reactions.In contrast, under the regulated pH conditions of the process according to the invention, high yields of relatively pure product are obtained possible. "

The following examples serve to explain the present invention in more detail without restricting it.

example 1

500 mg D »4-phenyl-1,5-oxazolidinedione, 400 mg 6-aminopenicillic acid and 50 ml of calcium acetate-acetic acid buffer (having an ionic strength of 0.25) became a mixture

with a pH of 5.02 and place in an open vessel 3

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80 980 6/0 8 56 ^r ^ ^; -

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Stirred at O G for hours. Samples of the filtrate showed a 857 "ige conversion into D ~ o £" ami ^ benzylpenicillin. The i'iltrat was by 15 minutes while stirring with 17 ml Amberlite MB-3 resin is deionized. The solution then became freeze-dried, with 550 mg of ß-lactam in 83% purity were obtained. The product obtained in this way can be further purified in the following ways:

a) 1> 75 g of D-oC-aminobenzylpenicillin were suspended in 30 ml of a 0.05 iv.ol sodium acetate-acetic acid buffer solution with a pH of 3.9, stirred, filtered, readjusted to a pH of 7.5, diluted to 50 ml and brought to a sodium acetate concentration of 0.1 i.iol. This solution was adsorbed in the sulfate form on a 2 × 37 cm column of polystyrene trimethylbenzylammonium anion exchange resin (Dowex 1-X 10). After adsorption, the column was washed with 50 ml of a 0.1 mol sodium acetate solution and then with 650 ml of water. Then it was eluted with GO2 -saturated Y / water and all hydroxate-nate-positive otoff was collected and freeze-dried. The purity of this product, as monohydrate, based on the hydroxamate per be of the β-lactam and using 6-aminopenioillinic acid as the standard substance, was 99.3 / ". The purity, based on the effectiveness against E.coli , was 88 / 0 and to the opposite Staph.aureus 100>.

- 13 -

ο η η r> η c. η r,

b) 300 mg of D-f & aminobenzylpenicillin were mixed with 3 ml of water

stirred at room temperature, and the mixture was centrifuged. The filtrate was saturated ammonium sulfate solution up to a total saturation of 6.55 * » admitted. The system was treated for 1 hour at room temperature and for 2 hours at 0 °. Of the .

Precipitate was filtered off and dried in vacuo, yielding 290 penicillin C units per mg (vs. % Staph.aureus ). The purity according to the hydroxamate sample was 755 *> and according to the effectiveness against E. coli 60 ^. The crystalline precipitate which separated out from the mother liquor after 18 hours at 0 ° was separated off and dried. This substance showed 85% purity after the hydroxamate test and 100% purity after the test against E. coli,

Example 2

To a mixture of 400 mg of 6-aminopenicillic acid with ml sodium acetate-acetic acid buffer (with an ionic strength of 0.1) were at a temperature of 1 C and a pH of 4.65 in each case 100 mg DL-4-phenyl ~ 2,5-oxazolidinedione added at intervals of 30 minutes up to a total of 500 mg. Before every new addition Samples taken showed gradual conversions of - related to the deficient constituent 94 >, 60? Ί, 63 #. 66 # and finally a total of 86 ^.

-14 809806/0856

- 1-1- -

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Example 3

2 g of DL-4 ~ phenyl-2,5-oxazolidinedione, 1.6 g of 6-aminopenicillic acid and 200 ml of calcium acetate-acetic acid buffer (with an ionic strength of 0.30) in the form of a mixture with a pH of 5.15 were Stirred at 1 ⁰ C for 3 hours. The filtrate of the reaction mixture revealed when examined against Staph aureus _o when using penicillin G 289 units per mg as the reference system 86 a / o conversion into DL "C <-amino benzylpenicillin. After deionization with 99 ml Amberlite MB-3, the mixture was freeze-dried and in this way 1.40 g of a solid substance of 7% purity according to the deijb lactam determination and 697% purity according to the antimicrobial effect was obtained.

Example 4

A mixture of 2 g of BL-4 ~ phenyl-2,5-oxazolidinedione, 1.6 g of 6-aminopenicillic acid and 200 ml of calcium acetate-acetic acid (pH 5.18; u 0.30) was stirred in an ice bath for 3 hours and then filtered. The conversion to DL-Ot-aminobenzylpenicillin was a total of 82% if the filtrate potency of 3.075 penicillin G units per ecm is used as a basis for testing against Staph.aureaus . 198 ml of the solution were adjusted to a pH of 7.6 with KOH and chromatographed in a 35 × 2 cm Dowex 1 ~ X10 column (sulfate form). After 222 ml had passed through, 0.1 mol of sodium acetate solution (pH 7.2) was used to elute

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- 15 -ORIGINAL WSPECTEO

began. After passing an additional 250 ml, the column was washed with water. After going through a total amount the elution with CO_-saturated water was started from 792 ml. A bulk of hydroxamate positive Substance was eluted between 930-2000 ml. The fraction eluted between 930 and 1200 ml (pH range 4.5 - 3.6), which let out the tail material was freeze-dried and sexual addiction. Melting point 197 - 202 with decomposition ^

SB ² I - 21 '°

Analysis: for C ₁₆ H ₁₉ N ₃ O ₄ S. H ₃ O ₅ C 52.3; H 5.8; N 11.4; S 8.7 Found: G 53.95; H 5.30; N 11.78; S 8.2.

The following antibacterial test was carried out: 265 penicillin G units / mg of 90% purity according to the Staph. aureus sample and 83> iger purity according to the E. coll sample were subjected to electrophoresis; the electrojhoresis paper had only one component |

Example 5

1 g of D-4-benzyl-2,5-oxazolidinedione, 0.8 g of 6-aminopenicillic acid and 100 ml calcium acetate-acetic acid buffer (with a pH value of 5.17 and an ionic strength of 0.30) were stirred for 3 hours in an open vessel at 0. The filtrate yielded 2,590 penicillin G-injections per ml

- 16 -

'809806/0 856

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against otaph. aareus. After treatment with Amberlite MB-3, the result was 2.320 units per ml. The filtrate of the conversion mixture showed an activity towards E.coli which corresponds to that of 1.460 micrograms of D-CkAreinobenzylpenicillin per ml.

Example 6

If "L-4-benzyl-2,5-oxazolidinedione" is used under the same conditions as in Example 5, the filtrates before and after the treatment result in 735 units per liter and units per ml E.coIi an activity corresponding to 91C ikiikrogramm D ^ -Aniiuobenzylpenicillin per ml.

Example 7

ülach the procedure described in Example 1 can the corresponding 6- (oC-arr.inoacylamino) -penicillin derivative by reacting 6- ^ miriopenicillic acid with the by ihosgenating glycine and 1-aminocyclopentanecartonic acid prepared 2,5-oxazolidinediones. In the case of I-Amir.ocyclopentanartonsäure the D-, L- or DL-Forai are produced,

The lenicillin products obtained and their analysis results are listed in the following table:

- 17 cnaonc / nor-r ORlGl ^ ⁵ - INSPECTED

" ₁₇ - 1445418

Amino acid obtained penicillin product glycine Analysis for C ₁₀ H ₁ ^ ₄ N ₅ S 2 H ₃ O Calculated: C 38.8, H 6.2, N 13.6
Found: C 38.7, H 5.4, N 12.6

1-aminocyclopentane analysis for ^c 14 ^H pi ⁰ 4 ^N 3 ^S * ^H 2 °

Calculated: C 48.7, H 6.7, N 12.2. Found: C 49.0, H 6.8, N 12.0, S 9.2

carboxylic acid Calculated: C 48.7, H 6.7, N 12.2, S 9.3

Example 8

A mixture of 500 mg of D-phenylglycine-N-carboxylic acid anhydride, 400 mg of 6-aminopenicillic acid and 50 ml of water with an initial pH of 3.5 was ^{stirred at 2 °} C. and 0.3 ml of 1 mol of NaOH was added brought to a pH of 5.1. During the three hours of stirring, the pH was maintained between 5.0 and 5.2. The mixture was filtered. A sample of the filtrate when tested against Staph. aureus 209 P 5.130 penicillin G units per ml, which corresponds to an 88% conversion into D-Ot-aminobenzylpenicillin.

Example 9

A mixture of 500 mg of N-carboxyanhydride of 1-aminocyclopentanecarboxylic acid, 400 mg of 6-aminopenicillic acid and 50 ml of a calcium acetate-acetic acid buffer solution of pH 5.1 and ionic strength 0.3 is used for 200 minutes

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1 G stirred. The resulting clear reaction mixture is desalted by contact with 248 mg of ion exchange resin of the Amberlite MB «3 type and. after filtering off the resin in an amount of 44 ml freeze-dried, whereby 640 mg of the desired 'ö-ouniriocyclopentanecarboxamido) penicillic acid, V. 158-164 with decomposition in the specified range, optical rotation for 260 in 1 hour aqueous solution, are obtained. The compound shows antimicrobial activity against Staph . aureus and E.coli as well as other gram-positive and gram-negative organisms including those resistant to benzylpenicillin.

Example 10

A mixture of 2.5 g of 6-aminopenicillic acid and 50 ml of water is first adjusted to pH 7.0 at room temperature, then 1.35 g of the N-carboxyanhydride of I-aminocyclohexanecarboxylic acid are added while stirring adjusted to 6.2, stirred further at room temperature for 60 minutes and now adjusted to pH 4.0, no precipitate of unreacted starting penicillic acid occurring. After the pH has been re-adjusted to 5.1, the solution is lyophilized and now yields 5.5 g of saline solid which, according to the analysis, has ΊΨ / * purity. 4.5 g of it are extracted with 100 ml of methylene chloride and 1 ml of triethylamine. Of the

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The extract is acidified with 0.6 ml of glacial acetic acid, then concentrated to 20 ml and treated with 100 ml of ethyl ether. When this mixture is refrigerated, 1.4 g of 6- (AminoeyeIohexanecarboxainido) penicillic acid, F.156-158, precipitate with decomposition in the specified range. The compound has an optical rotation of <f264 in aqueous solution and is therapeutically highly effective in VI / arm blood infections with Streptococcus pyopenes G 2Q "5, Staphylococcus aureus Smith (penicillin-sensitive) and Staphylococcus aureus CHP (penicillin-resistant).

Example 11

A mixture of 1 g N-carboxyanhydride of 1 -AminoeyeIoheptancarbonsäure, 0.8 6-aminopenicillanic acid and 100 ml of ice-cold water is adjusted by means of 1ON -NaOH to pH 5.0 and stirred for 60 minutes at 1 ⁰ C. After filtering off and freeze-drying, 6-U ~ aminoheptancarboxamido) penicillic acid is obtained, 153-157 C with decomposition in the specified range. The product has a broad spectrum of antimicrobial activity, as serial dilution tests on agar show.

Claims

ORIGINAL INSPECTED 809806/0856

Claims (1)

Patent claims: 1. Process for the preparation of 6- (O (-aminoacylamino) Penicillic acid compounds of the general formula R t 0 C NH CH CH (J - CH, I ll | ' R "NH C N CH COOH in which R is hydrogen or lower alkyl, R ^{f is} hydrogen, alkyl, allyl, tosylaminobutyl, methylmercaptoethyl, .benzylmercaptomethyl, nitrophenylmethyl, acetoxymethyl, o6-carboxyethyl, β- (lower alkoxycarbonyl) ethyl, joenzyloxymethyl, mono- or di-acetoxyphenylmethyl, -imidazol.ylmethyl, indolylmethyl, cycloalkyl, phenyl or a phenyl substituted one to three times by di- (lower) alkyl I amino, (lower) alkanoyloxy, (lower) alkoxy, iäulfamyl, halogen or trifluoromethyl, and u and R ¹ can be linked to form a cycloalkyl ring with 5 to 7 carbon atoms, R "denotes hydrogen or can be linked to R ¹ to form a pyrrolidine, acetoxypyrrolidine or l'osyloxypyrrolidine ring, characterized in that 6-aminopenicillic acid is combined with a 2, 5-oxazolidindione of the formula - 21 809806/0856 New documents i «rt. / g l Ht> s. 2 ur. 1 sow 3 of the amendment. v. 4.9. Γ. INSPECTED in which R, R ¹ and R "have the above meaning, in an exclusively aqueous-acidic medium at a pH of about 3» 8 to about 6.2 and at a temperature of about just above the freezing point of the aqueous mixture until, about 37 C converts. 2 · New 6-WJJt-Aininoacylamino j «penicillic acid derivatives of the general formula - CH - COOH in ieicheir Ü and R * are connected to a cycloalkyl ring with 5 ßis j iLbhienstöffäibMen. 06/0856