NO802859L - PHOSPHONIC ACIDS AND MANUFACTURING THEREOF - Google Patents

Description

The present invention relates to new phosphonic acids or peptide derivatives, a method for the production of these as well as pharmaceutical preparations based on this new compound and production thereof. Finally, the present invention relates to the use of the new compounds.

The compound according to the invention concerns peptide derivatives with the general formula

where R is hydrogen or methyl;

2 3

R and R are lower alkyl residues which are characteristic of an α-amino acid not normally occurring in the protein or one of the two substituents is such a lower alkyl residue and the other an alkyl residue which is characteristic of an α-amino acid which normally occurs in proteins,

R<4> is the L-pyroglutamyl residue;

the configuration of the C atom (a) R (provided R"<*>" f H) while the configuration of the C atoms (b) and (c) is L (provided R<2> or R3 f H),

and about their physiologically tolerable salts.

In the present application and the associated claims, the term "lower alkyl" means straight or branched alkyl residues with preferably up to 8 carbon atoms, such as e.g.

ethyl, propyl, butyl, tert-butyl, pentyl and hexyl. At the characteristic residue of a normally occurring in proteins or non-occurring amino acid, it is about the substituent R of a natural α-amino acid with it

general formula

If the amino acid is alanine, R is methyl -

in the case of methionine, R is 2-methylthioethyl, in serine R is hydroxymethyl and in tyrosine R is p-hydroxybenzyl, etc. R can also together with the nitrogen atom of the amino group be cyclized to form a ring as in proline.

When R"'" in the compound of formula I is methyl, the configuration on the C atom indicated by (a) must be R, i.e. the configuration must be that obtained by replacing the carboxyl group of a natural α-amino acid with a PO^H2 group.

Compounds of formula , wherein R"<*>" is methyl, are preferred. Furthermore, compounds are preferred where R 2 and RJ T are lower alkyl residues, which are characteristic of α-amino acids which do not normally occur in proteins.

Examples of compounds according to the invention with formula I are: (IR)-1-(L-pyroglutamyl-L-norvalyl-norvalylamino)-ethylphosphonic acid

(L-pyroglutamyl-L-norvalyl-L-norvalylamino)-methyl-phosphonic acid (IR)-1-[L-pyroglutamyl-L-(2-aminoheptanoyl)-L-norvalylamino)-ethylphosphonic acid and

(IR)-1-(L-pyroglutamyl-L-alanyl-L-norvalylamino)-ethylphosphonic acid.

Compounds of formula I and their physiologically tolerable salts can be prepared according to the invention by reacting a compound of the general formula

wherein R"*" is as defined in claim 1,

20 30 2 3

R and R have the meanings given in claim 1 for R and R, the amino groups being however protected with a hydrogenolytically cleavable protecting group

and the configuration of the C atoms (a), (b) and (c) is as in the compounds of formula I,

with a compound of formula

wherein R 5 is 2,4,5-trichlorophenyl, pentachlorophenyl or succinimidyl,

R is hydrogen or an aralkoxycarbonyl group and the configuration of the C atom (d) is L,

hydrogenolyzes a reaction product containing a protected amino group and/or an aralkylcarbonyl group and, if desired, transfers a compound thus obtained in a physiologically tolerable salt.

Examples of hydrogenolytically cleavable amino protecting groups which can occur in the substituents R and R of a compound II are aralkylcarbonyl groups, such as e.g. benzyloxycarbonyl. An amino group can also be protected with

a nitro group, as in the case of the nitroarginyl group.

The reaction of a compound II with a compound IH can be carried out in a manner known per se, e.g. according to the activated ester method. The turnover can e.g. happen in an organic solvent such as aqueous dimethylformamide at approx. 0°C.

In a preferred embodiment, a compound is condensed

5 of formula II with a compound of formula III, wherein R is the 2,4,5-trichlorophenyl group.

An obtained condensation product containing a protected amino group and/or an aralkylcarbonyl group R 1 is hydrogenolysed to transfer a protected amino group tip in an amino group and/or cleave the aralkylcarbonyl group. This hydrogenolysis must be carried out in a manner known per se, e.g. in the presence of a noble metal catalyst such as palladium/C or platinum oxide.

Physiologically tolerable salts form compound of formula I with physiologically tolerable, organic and inorganic acids (e.g. HCl, HBr, t^SO^, methanesulfonic acid, p-toluenesulfonic acid) and bases (e.g. NaOH, KOH).

The starting compound of formula II are known compounds.

The starting compounds of formula III can e.g. is prepared by reacting L-pyroglutamic acid or an N-aralkoxycarbonyl-L-pyroglutamic acid with 2,4,5-trichlorophenol, penta-chlorophenol or N-hydroxysuccinimide in the presence of N,N<1->dicyclohexylcarbodiimide in an inert organic solvent, then as dimethylformamide. Starting from an N-aralkoxycarbonyl-L-pyroglutamic acid, the compound of formula III is hydrogenolysed, in which R is aralkoxycarbonyl in a manner known per se as indicated above. A compound of formula III is used

5 6

where R is a succinimidyl group and R is hydrogen, this is preferably formed in the reaction medium in situ.

The peptide derivatives according to the invention have anti-bacterial activity against many gram-positive and gram-negative bacteria, such as e.g. erscherichia coli, klebsiella spp., serratia marcescens, staphylococcus albus, Salmonella spp., shigella spp., streptococcus faecalis, streptococcus pyogenes, streptococcus pneumoniae

and Haemophilus influenzae. The compounds can therefore be used for the treatment and prophylaxis of bacterial infections and given parenterally.

The following table gives the results of in vitro experiments using (IR)-1-(L-pyroglutamyl-L-norvalyl-L-norvalylarnino)-ethylphosphonic acid against some representative pathogenic microorganisms.

In addition, the peptide derivatives according to the invention enhance the activity of antibiotics, preferably of 3-lactam antibiotics such as penicillins, cephalosporins or monocyclic 3-lactam antibiotics.

Examples of suitable penicillins are ampicillin, carbenicillin, penicillin G, sulbenicillin, mecillinam, pivmecillinam, pheneticillin, methicillin, propicillin, ticarcillin, amoxycillin and piperacillin.

Suitable cephalosporins are cephalexin, cefazolin, cefoxitin, cefradin, cefsulodin, cefamandole, cephaloridine, cefaloglycin, cefatrizine, cefacetril, cefuroxime, cefaclor, cefotaxirii, cefazedone, among which cephalexin is preferred. _

Examples of other antibiotics are D-cycloserine, rifampicin, phosphonomycin, gentamycin, vancomycin and kanamycin.

The antibiotics mentioned above are known compounds that can be prepared by known methods.

The present invention therefore also relates to pharmaceutical preparations which, in addition to the peptide of formula I, contain an antibiotic.

The manufacture of pharmaceutical preparations can take place in a manner known per se, e.g. by mixing the individual components with the suitable carrier materials and bringing them into a suitable galenic form. Solid or liquid substances are used as carrier materials, which are compatible with peptide derivatives and antibiotics. It can be organic or inorganic substances, which are suitable for enteral administration. Examples of such carrier materials are water, gelatin, mannitol, mineral and vegetable oils, gum arabic, propylene glycol and polyalkylene glycols. The pharmaceutical preparations can be in solid form (e.g. as lyophilisates) or in liquid form

(eg as solutions, suspensions or emulsions).

During manufacture, they can be subjected to normal pharmaceutical operations such as sterilization. Finally, they may contain additives such as preservatives, stabilisers, wetting or emulsifying agents, salts for changing the osmotic pressure or buffers. When using a buffer, the pH of the pharmaceutical preparation can of course vary within the range known from pharmaceutical practice.

The weight ratio of the peptide derivative of formula I to the antibiotic can vary within wide limits. It is expediently in the range from 1:100 to 100:1, preferably in the range from 1:64 to 64:1. Particularly preferred is a weight ratio from 1:16 to 16:1.

The daily dosage of the peptide derivatives, either alone or

in combination with an antibiotic, can vary within wide limits, and depending on factors such as the specially chosen peptide derivative, or antibiotic, the infection to be treated, etc. Thus, e.g. the daily dosage for a peptide derivative alone by parenteral application be 800-200 mg.

If the peptide derivative is given in combination with an antibiotic, the daily dosage for parenteral application can be approx. 200-2000 mg. It is clear that a daily dosage can be given as a single dose or in several partial doses and that the aforementioned dosages can both be varied upwards and downwards depending on the individual needs and adapted to the special circumstances encountered by the attending physician.

The following examples illustrate the invention.

EXAMPLES

Example' 1

8.0 g (25 mmol) of (IR)-1-(L-norvalyl-L-norvalylamino)-ethylphosphonic acid was dissolved under stirring in a mixture of 125 ml of water and 5.1 g (50 mmol) of triethylamine. 125 ml of dimethylformamide was added and the solution cooled to 0° C. After addition of 20.3 g (66 mmol) L-pyroglutamic acid-2,4,5-trichlorophenyl ester and 125 ml of dimethylformamide at 0° C, stirring was carried out under slow heating of the cooling bath to room temperature. The resulting suspension was filtered and the gelatinous precipitate washed with aqueous dimethylformamide.

The combined filtrates were evaporated under reduced pressure to dryness and the residue treated with 200 ml of diethyl ether. The diethyl ether was removed and the residue dissolved in a mixture of 500 ml of methanol and 100 ml of water. After adding 200 g of zerolit 225, SRC 13, RSO^H and stirring for 5 minutes, the mixture was filtered off. The ion exchange resin was washed with water and methanol. The washing water which was combined with the filtrate was evaporated to dryness, the residue treated with 200 ml of acetone and stirred for a few hours at room temperature. The precipitate was filtered off and, after washing with acetone and diethyl ether, gave 4.5 g of (1R)-1-(L-pyroglutamyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid,

F. 260°(s<p>altn); [a]20 = - 84.5 (c = 0.55% in trifluoroacetic acid).

Example 2

In an analogous way as in example 1, 9.27 g (30 mmol) were obtained

(L-norvalyl-L-norvalylamino)-methylphosphonic acid and 18.5 g (60 mmol) L-pyroglutamic acid-2,4,5-trichlorophenyl ester

after treatment with acetone 9.1 g of crude L-pyroglutamyl-L-norvalyl-L-norvalylamine)-methylphosphonic acid. Recrystallization from water/ethanol (1:4 v/v) gave a melting point of 230-232°C (dec); [a]p<0>= -69.6°; [a]<2>^ = -222° (c= 0.5% in water).

Example 3 • -

In an analogous manner as in example 1, but using methanol/water (2:1, v/v) in the ion exchange step, 4.2 g (12 mmol) (IR)-1-[(L-2 -amino-heptanoyl)-L<->norvalylamino]-ethylphosphonic acid and 9.3 g (30 mmol) L-pyroglutamic acid-2,4,5-trichlorophenyl ester 1.64 g crude (IR)-1-[L-pyroglutamyl- L-(2-aminoheptanoyl)-L-norvalylamino]-ethylphosphonic acid with a melting point of 225-230°C (decomposition), [a]2° _ c_ 0o0 r ,20'

v- -bo,y ^aJ 365 _

208° (c=0.48% in trifluoroacetic acid. Purification by stirring with methanol at room temperature, filtration and water with diethyl ether gave pure product; F. 235-237°C (decomposition)

[a]<20>-70.6°} [a]3g5= - 232° (c = 0.5% i.trifluoroacetic acid) .

The (IR)-1-[(L-2-aminoheptanoyl)-L-norvalylamino]-ethylphosphonic acid used as starting material was prepared as follows:

a) 3.5 g (25 mmol) L-2-aminoheptanoic acid (J.Biol.Chem. 203,

333 (1953)] was dissolved with stirring in 25 mL (25 mmol)

IN sodium hydroxide.' The resulting solution was cooled to 0°C and mixed alternately in 5 portions with a total of 10 ml

(40 mmol) 4N sodium hydroxide and 6.5 g (38 mmol) benzyl chloroformate during 30 min., the temperature being maintained at 0-10°C. The mixture was further stirred for 4 hours while slowly warming to room temperature. It was then extracted with diethyl ether, the ether extracts were washed with water, the combined aqueous extracts were acidified by the addition of 6N. HCl to pH 2.0

and extracted three times with diethyl ether. The extracts were dried over sodium sulfate, filtered and evaporated to 7.8

g N-benzyloxy-carbonyl-1-2-aminoheptanoic acid in the form of an oil. b) The oil obtained was taken up with stirring in 75 ml of di-methoxyethane, mixed with 2.9 g (25 mmol) of N-hydroxysuccinimide and, after cooling to 0°C, with 5.6 g (27.5 mmol) of dicyclohexylcarbodiimide. The mixture was stirred at 0°C and allowed to stand at this temperature overnight. The solid by-product (5.9 g of dicyclohexylurea) was filtered off, the filtrate evaporated to an oil and treated with petroleum ether. The white solid obtained was filtered off and gave 9.2 g of N-benzyloxycarbonyl-L-2-aminoheptanoic acid-N-hydroxysuccinimide, mp 65-67°C; [α]<20>- 23.6°; [a]^ = -80.9° (c = 1% in ethanol).. c) 4.48 g (20 mmol) of (IR)-1-(L-norvalylamino)-ethylphosphonic acid was dissolved with stirring in a mixture of 50 ml of water

and 4.04 g (40 mmol) triethylamine. After adding 50 ml of dimethylformamide, the solution was cooled to 0°C, mixed dropwise with 9.2 g (24 mmol) of N-benzyloxycarbonyl-L-2-aminoheptanoic acid-N-hydroxysuccinimide in 50 ml of dimethylformamide, during which the reaction mixture was kept at 0-5°C, and gradually warmed to room temperature. After filtration, the filtrate was evaporated, the residue mixed with 200 ml of water and the resulting suspension mixed with approx. 20 ml of 2N HCl. The precipitate obtained was washed with water, filtered and treated for several hours with acetone. Yield: 8.7 g (90%) (IR)-1-([N-benzyloxycarbonyl-L-(2-aminoheptanoyl)-L-norvalyl]-amino)-ethylphosphonic acid,

F. 180-190°C (split.)

d) 8.7 g (18 mmol) of (IR)-1-([N-benzyloxycarbonyl-L-2-aminoheptanoyl)-L-norvalyl]-amino)-ethylphosphonic acid was stirred

with a mixture of 20 ml of 45% hydrogen bromide in glacial acetic acid for 4 hours at room temperature. After the addition of 150 ml of diethyl ether, a gummy precipitate was obtained, which was washed with a further 150 ml of diethyl ether and taken up in 100 ml of methanol with stirring. After adding 10 ml of propylene oxide, a gelatinous precipitate was obtained. A further 100 ml of methanol was added and the mixture was allowed to stand overnight at room temperature. The precipitate was filtered off, washed first with methanol and then with diethyl ether to give 6.3 g of crude (IR)-1-[L-(2-aminoheptanoyl)-L-norvalylamino]-ethylphosphonic acid, F. 250-255 C (dec. .) After stirring with 250 ml of hot water, cooling, filtering and washing with ethanol and diethyl ether, 5.36 g of pure substance was finally obtained, F.268-272°C (dec.); [a] = -25.8°;

[0C] 365 =~89'9° (c = 0.36% in 2N HCl).

Example 4

In an analogous manner as in example 1, but using methanol/water (1:1, v/v) in the ion exchange step, 4.65 g (15 mmol) of (IR)-1-(L-alanyl-L- norvalylamino)-. ethylphosphonic acid and 11 g (35 mmol) L-pyroglutamic acid-2,4,5-trichlorophenyl ester 6.04 g of a crude product, which after purification by treatment with acetone gave 5.74 g of pure (IR)-1-(L-pyrogluta -myl-L-alanyl-L-norvalylamino)-ethylphosphonic acid, M. 262-264°C (dec.); [α]<2>° = -99.6°; [a]<2>°5= -332° (c = 0.05% in trifluoroacetic acid.

Example 5

Injection solution containing:

Example 6

Hard gelatin capsule containing the following ingredients:

Claims (8)

1. Procedure for the production of peptidylphosphonic acids with the general formula where R is hydrogen or methyl; R 2 and R 3 a lower alkyl residue which is characteristic of an α-amino acid which does not normally occur in proteins or one of the two substituents is such a lower alkyl residue and the other a lower alkyl residue which is characteristic of an α-amino acid which normally occurs in proteins; R 4 is the L-pyroglutamyl residue; the configuration of the C atom (a) is R (provided R" <*> " / H), while the configuration of the C atoms (b) and (c) is L (provided R <2> or R <3> ^ H) , and their physiologically tolerable salts, characterized in that a compound of the general formula is reacted where R^" is as indicated above; 20 30 2 3 R and R have the meanings given for R and R above, the amino groups are however protected with a hydrogenolytically cleavable protecting group and the configuration of the C atoms (a), (b) and (c) is as in the compounds of formula I, with a connection with formula where R 5 is 2,4,5-trichlorophenyl, pentachlorophenyl or succinimidyl; R is hydrogen or an aralkoxycarbonyl group and the configuration of the C atom (d) is L, hydrogenolyzes a reaction product containing a protected amino group and/or an aralkylcarbonyl group and, if desired, transfers a compound thus obtained in a physiologically tolerable salt. 2. Process according to claim 1, characterized in that starting compounds of formula II are used in which R is methyl. 3. Process according to claim 1 or 2, characterized in that starting compounds of formula II are used, in which R2(^ and R <3> ^ is a lower alkyl residue which is characteristic of α-amino acid which does not normally occur in proteins. 4. Process according to one of claims 1-3, characterized in that (IR)-1-(L-norvalyl-L-norvalyl-amino)-ethylphosphonic acid is used as starting compound with formula II. 5. Process according to one of claims 1-3, characterized in that (L-norvalyl-L-norvalylamino)-methyl-phosphonic acid is used as starting compound with formula II. 6. Process according to one of claims 1-3, characterized in that as "starting compound with formula II" (IR)-1-[L-(2-aminoheptanoyl) is used -norvalylamino]-ethylphosphonic acid. 7. Process according to one of claims 1 -• 3, characterized in that (IR)-1-(L-alanyl-L-norvalylamino) is used as starting compound with formula II -ethylphosphonic acid. 8. Process for the production of pharmaceutical preparations based on a peptidylphosphonic acid obtained according to claims 1-7, characterized by mixing a peptidylphosphonic acid with formula I or a physiologically tolerable salt thereof, if desired in the presence of an antibiotic, with a non- toxic, inert, therapeutically tolerable solid or liquid carrier and brings the resulting mixture into the desired galenic form.