GB2027433A - Aminoalkanoylaminoalkyl Phosphonic Acids - Google Patents

Abstract

This invention is concerned with compounds of the formula <IMAGE> wherein R<1> represents a hydrogen atom or the methyl or hydroxymethyl group or a mono-, di- or trihalomethyl group; R<2> represents a lower alkyl, hydroxy-(lower alkyl) or guanidino- (lower alkyl) group other than the characterising group of an alpha -amino acid of the type normally found in proteins; the configuration at the carbon atom designated as (a) is (R) when R<1> represents other than a hydrogen atom; and the configuration at the carbon atom designated as (b) is (L), and pharmaceutically acceptable salts thereof, with a process for the manufacture thereof and with pharmaceutical preparations containing same. These compounds and salts have antibacterial activity and also potentiate the activity of antibiotics.

Description

SPECIFICATION Peptide Derivatives The present invention relates to peptide derivatives. More particularly, the invention is concerned with peptide derivatives of phosphonic acids, a process for the manufacture thereof and pharmaceutical preparations containing same.

The peptide derivatives provided by the present invention are compounds of the general formula

wherein R1 represents a hydrogen atom or the methyl or hydroxymethyl group or a mono-, di- or trihalomethyl group; R2 represents a lower alkyl, hydroxy-(lower alkyl) or guanidino-(lower alkyl) group other than the characterising group of an a- amino acid of the type normally found in proteins; the configuration at the carbon atom designated as (a) is (R) when R1 represents other than a hydrogen atom; and the configuration at the carbon atom designated as (b) is (L), and pharmaceutically acceptable salts thereof.

The term "lower alkyl" is used in this Specification to mean a straight-chain or branched-chain alkyl group which preferably contains up to 8 carbon atoms. Examples of such lower alkyl groups are ethyl, propyl, butyl, tertbutyl, pentyl, hexyl etc. Examples of hydroxy (lower alkyl) groups denoted by R2 are 2hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl and the like. The term "halo" means fluoro, chloro, bromo or iodo, examples of the aforementioned halomethyl groups being chloromethyl, dichloromethyl, trifluoromethyl etc.

When R' in formula I represents other than a hydrogen atom, the configuration at the carbon atom designated as (a) is (R); that is to say, the configuration which would be obtained by replacing the carboxyl group of a naturally occurring amino acid by a phosphorus moiety.

A preferred class of peptide derivatives provided by the present invention comprises compounds of formula I in which R' represents a hydrogen atom or the methyl group and pharmaceutically acceptable salts thereof. Also preferred are compounds of formula I in which R2 represents a lower alkyl group, especially the ethyl, n-propyl or n-butyl group.

Examples of compounds of formula I hereinbefore are; ( 1 R)- 1 -[L-(a-AminobuWryl)amino]- ethylphosphonic acid, (1 R)-1 -(L-norleucylamino)-ethylphosphonic acid, (L-a-Aminobutyrylamino)-methylphosphonic acid, (L-norleucylamino)-methylphosphonic acid, (L-norvalylamino)-methylphosphonic acid, ( 1 R)- 1 L-homoa rginylamino)-ethylphosphonic acid, and (L-homoarginylamino)-methylphosphonic acid.

According to the process provided by the present invention, the peptide derivatives aforesaid (i.e. the compounds of formula I and pharmaceutically acceptable salts thereof) are manufactured by condensing a compound of the general formula

wherein R'O has any of the values accorded to R1 hereinbefore or represents a protected hydroxymethyl group:R3 and R4 each represent a hydrogen atom or a lower alkyl protecting group; and the configuration at the carbon atom designated as (a) is (R) when R' represents other than a hydrogen atom; with a protected amino acid of the general formula

wherein R5 represents a protected amino group; R20 has any of the values accorded to R2 hereinbefore or represents a protected hydroxy (lower alkyl) or guanidino-(lower alkyl) group; and the configuration at the carbon atom designated as (b) is (L), cleaving off the protecting group or groups present in the condensation product and, if desired, converting a resulting compound of formula I into a pharmaceutically acceptable salt.

The protected amino group denoted by R5 in a protected-amino acid starting material of formula Ill hereinbefore can be any protected amino group which is well-known in peptide chemistry. In a preferred embodiment of the process provided by the present invention the amino group is protected with an aralkoxycarbonyl group, particularly the benzyloxycarbonyl group, or the tert.butoxycarbonyl group. However, the amino group can also be protected with, for example, a formyl, trityl, trifluoroacetyl or 2-(biphenylyl)isopropyloxycarbonyl group or can be in the form of a phthalimido group.The protecting group present in a protected hydroxymethyl group R10 or a protected hydroxy-(lower alkyl) group R20 can be any conventional hydroxy protecting group; for example, an arnlk6xycarbonyl group (e.g. the benzyloxycarbonyl group), a lower alkanoyl group (e.g. the acetyl, propionyl and like groups), an aroyl group (e.g. the benzoyi group), a lower alkyl group (e.g. the tert.butyl group) or a lower aralkyl group (e.g. the benzyl group).

The condensation of a compound of formula II with a protected amino acid of formula Ill can be carried out according to methods known per sue in peptide chemistry; for example, according to the mixed anhyride, azide, activated ester, acid chloride, carbodiimide or EEDQ (1-ethoxycarbonyl2-ethoxy-1 ,2-dihydroquinoline) niethod.

In one method, a compound of formula II can be condensed with a protected amino acid of formula Ill in which the carboxy group is a mixed anhydride residue formed with an organic or inorganic acid. Suitably, such a protected aamino acid of formula III is treated with a tertiary base such as a tri(lower alkyl)amine (e.g.

triethylamine) or N-ethylmorpholine in an inert organic solvent (e.g. tetrahydrofuran, 1,2dimethoxyethane, dichloromethane, toluene, petroleum ether or the like) and the salt obtained is reacted with an appropriate chloroformate (e.g.

a lower alkyl chloroformate such as ethyl chloroformate orisobutyl chloroformate) at a low temperature. The mixed anhydride thus obtained is then suitably condensed in situ with a compound of formula II.

In another method, a compound of formula II can be condensed with a protected a-amino acid of formula Ill in which the carboxy group is in the form of an acid azide. This condensation is preferably carried out in an inert organic solvent such as dimethylformamide or ethyl acetate at a low temperature.

In yet another method, a compound of formula II can be condensed with a protected a-amino acid of formula Ill in which the carboxy group is in the form of an active ester group (e.g. the 4 nitrophenyl, 2,4,5-trichlorophenyl or N hydroxysuccinimide ester group). This condensation is suitably carried out in an inert solvent such as aqueous dimethylformamide or, where R3 and R4 in a compound of formula Ill both represent a lower alkoxy group, also in a lower alkanol such as aqueous ethanol.

In a further method, a compound of formula ll can be condensed with a protected a-amino acid of formula Ill in which the carboxy group is in the form of an acid chloride. This condensation is preferably carried out in the presence of a base and at a low temperature.

In yet a further method, a compound of formula II can be condensed with a protected a-amino acid of formula Ill in the presence of a carbodiimide (e.g. dicyclohexylcarbodiimide) or EEDQ. This condensation can be carried out in an inert organic solvent (e.g. methylene chloride or a lower alkanol such as methanol, ethanol etc) at room temperature or at a temperature below room temperature.

The cleavage of the protecting group or protecting groups from the condensation product is carried out in accordance with methods known per se; that is to say, methods in actual use for or described in the literature on the cleavage of protecting groups. Thus, for example, an aralkoxycarbonyl group (e.g. benzyloxycarbonyl), the tert.butoxycarbonyl group or the 2-biphenylylisopropyloxycarbonyl group can be cleaved off by hydrolysis (e.g. by treatment with hydrogen bromide in glacial acetic acid). An aralkoxycarbonyl group (e.g. benzyloxycarbonyl) can also be cleaved off by hydrogenolysis (e.g. in the presence of palladium-on-charcoal or platinum oxide). The tert.butoxycarbonyl or 2biphenylyl-isopropyloxycarbonyl group can also be cleaved off using hydrogen chloride in dioxan.

A trityl group can be cleaved off, for example, by treatment with dilute acetic acid. A phthalimido group can be converted into the amino group by hydrazinolysis. Lower alkyl protecting groups denoted by R3 and R4 can be cleaved off by treatment with hydrogen bromide in glacial acetic acid or using trimethylchlorosilane or trimethylbromosilane followed by aqueous hydrolysis. It will be appreciated that where more than one protecting group is present the cleavage of the protecting groups can be carried out in a single step or in more than one step depending on the nature of said groups. However, it is preferred to use protecting groups which can be cleaved off in a single step.

Compounds for formula I hereinbefore are amphoteric and form pharmaceutically acceptable salts with pharmaceutically acceptable strong acids (e.g. hydrochloric acid, hydrobromic acid, sulphuric acid, methanesulphonic acid, paratoluenesulphonic acid etc) and bases (e.g.

sodium hydroxide, potassium hydroxide etc).

The starting materials of formulae II and Ill are known compounds or can be prepared in analogy to the preparation of known compounds.

The peptide derivatives provided by the present invention possess an antibacterial activity against a wide range of gram-positive and gram-negative bacteria such as escherichia coli, Staphylococcus aureus, Serratia marcescens, Kiebsiella aerogenes, Streptococcus faecalis, Haemophilus influenzae, Bacillus subtilis, Salmonella typhimurium, Proteus mirabilis, Pseudomonas aeruginosa and Shigella sonnei. In addition, the present peptide derivatives potentiate the activity of antibiotics, including penicillin and cephalosporin antibiotics and D-cycloserine.

Among the antibiotics which are potentiated by the present peptide derivatives there may be mentioned amoxycillin, cephradine, cephalothin, cephalexin, carbenicillin, ampicillin, penicillin G, sulbenicillin, cephazolin, cefoxitin, rifampicin, [(R) 1 -(2-furoyloxy)-3-methylbutyl]penicillin, (6R)-6 I[(hexahydro-l H-azepin-1 -yl) methylene]amino}penicillanic acid, (pivaloyloxy)methyl (6P)-6-([hexahydro-1 H azepin-1 -yl)methylene]aminolpenicillanate, cephamandole, cephaloridin, cephaloglycin, phenethicillin, methicillin, propicillin, ticaracillin, amoxycillin arginine salt, phosphonomycin, vancomycin and kanamycin.

The present invention thus also provides a pharmaceutical preparation containing a peptide derivative aforesaid, and, if desired, an antibiotic, in association with a compatible pharmaceutical carrier material.

The carrier material present in the pharmaceutical preparations provided by this invention can be any solid or liquid carrier material which is compatible with the peptide derivatives aforesaid, and with the antibiotics when such are present, and which is suitable for therapeutic administration. The carrier material can be an organic or inorganic carrier material which is suitable for enteral (e.g. oral) or parenterai administration. Examples of such carrier materials are water, gelatin, lactose, starches, magnesium stearate, talc, vegetable oils, gum arabic, polyalkyleneglycols, petroleum jelly etc. The pharmaceutical preparations can be made up in a solid form (e.g. as tablets, dragées, suppositories or capsules) or in a liquid form (e.g.

as solutions, suspensions or emulsions). The pharmaceutical preparations may be subjected to conventional pharmaceutical operations such as sterilisation and may contain adjuvants such as preserving agents, stabilising agents, wetting agents, emulsifying agents, salts for varying the osmotic pressure or buffers. When a buffer is used, the pH of the pharmaceutical preparation will, of course, vary within the range which is well-known in pharmaceutical practice.

When the present pharmaceutical preparations contain a peptide derivative and an antibiotic, the weight ratio of peptide derivative to antibiotic can vary within wide limits. In general, the pharmaceutical preparations can contain the peptide derivative and antibiotic in a weight ratio of from 1:100 to 100:1, preferably in a weight ratio of from 1:64 to 64:1 and especially in a weight ratio of from 1:16to 16:1.

The daily dosage of peptide derivative administered alone or in combination with an antibiotic will vary within wide limits depending on factors such as the particular peptide derivative chosen, the particular antibiotic chosen, the route of administration and the infection to be treated. For example, when a peptide derivative is administered alone, a daily dosage for oral administration may amount to about 2000 mg to 4000 mg and a daily dosage for parenteral administration may amount to about 800 mg to 2000 mg. When a peptide derivative is administered in combination with an antibiotic, a daily dosage for oral administration may amount to about 750 mg to 1 500 mg of a combination of the peptide derivative and antibiotic and a daily dosage for parenteral administration may amount to about 200 mg to 2000 mg of a combination of peptide derivative and antibiotic.It will be appreciated that daily dosages can be administered in a single dosage or in divided dosages and that the dosages mentioned earlier may be varied upwards or downwards according to individual requirements and fitted to the exigencies of a particular situation as determined by the prescribing physician.

The following Examples illustrate the process provided by the present invention: Example 1 (A) The preparation of the starting material: 5.0 g (48 mmol) of L--aminobutyric acid were dissolved in 24 ml of 2-N sodium hydroxide and the solution was cooled to OOC. 12.3 g (72 mmol) of benzyl chloroformate and 1 8 ml (72 mmol) of 4-N sodium hydroxide were added alternately and portionwise while stirring over a period of 0.5 hour in such a manner that the temperature did not exceed 100C and that the pH was maintained at ca 11. The mixture was allowed to reach room temperature slowly and was then stirred overnight. The mixture was extracted with 50 ml of diethyl ether and the organic and aqueous phases were separated.The aqueous phase was acidified to pH 2 with 5-N-hydrochloric acid to give an oily mixture. This mixture was extracted with two 75 ml portions of diethyl ether. The extracts were dried over sodium sulphate and evaporated to give 9.6 g of a colourless oil. This oil was dissolved in 10 ml of ethyl acetate and 100 ml of petroleum ether (boiling point 40- 600C) were added to give an oily solution which was left to stand at OOC. There was obtained a white crystalline precipitate which was filtered off, washed with petroleum ether and dried to give 8.40 g (73%) of N-benzyloxycarbonyl-L-cr- aminobutyric acid of melting point 760--780C; [aj20=lO.90; [a]'2065- 20.30(c=1% in ethanol).

(B) The process: (i) 8.30 g (35 mmol) of N-benzyloxycarbonyl-La-amino-butyric acid and 4.02 g (35 mmol) of Nhydroxysuccinimide were dissolved in 75 ml of dimethoxyethane while stirring and the solution was cooled to OOC. 7.94 g (38.5 mmol) of dicyclohexylcarbodiimide were added and the mixture was stirred at OOC for 2 hours and then left to stand at OOC overnight. The solid byproduct was filtered off. The filtrate was evaporated to give N-benzyloxycarbonyl-L--aminobutyric acid N-hydroxy-succinimide ester in the form of a stiff gum which was used without further purification.

(ii) 4.38 g (35 mmol) of(lR)-l- aminoethylphosphonic acid were dissolved in a mixture of 40 ml of water, 7.07 g (70 mmol) of triethylamine and 40 ml of dimethylformamide while stirring. The resulting solution was cooled to OOC and a solution of 35 mmol of N benzyloxyca rbonyl-L-a-a minobutyric acid Nhydroxysuccinimide ester in 40 ml of dimethylformamide was rapidly added dropwise.

The mixture was stirred at OOC and was then allowed to come to room temperature while stirring overnight. The mixture was filtered to remove a small amount of solid. The filtrate was evaporated under an oil-pump vacuum. The residue was dissolved in a mixture of 30 ml of methanol and 10 ml of water and passed down a column of cation exchange resin (B.D.H., Zerolit 225, SRC 13, RSO3H; 150 g; freshly regenerated in the acid cycle), elution being carried out with the same solvent. The acid eluate was collected and evaporated to give a slightly gummy white solid. This solid was triturated with 100 ml of diethyl ether and the diethyl ether was decanted off. The residual solid was dissolved in a mixture of 100 ml of methanol and 50 ml of water and the resulting solution was titrated to pH 4.5 with 4-N aqueous benzylamine (titre 8.0 ml; theory 8.75 ml). The mixture solidified towards the end of the titration, and the precipitate was filtered off, washed with water and dried to give 8.84 g of the monobenzylamine salt of (1 P)-1-[(N- benzyloxycarbonyl-L-a-aminobutyryl)aminoj- ethylphosphonic acid of melting point 2280-- 231 0C (decomposition); [a]20=3 1.30; kr]2 5=105 (c=1% in glacial acetic acid).

(iii) 8.7 g (19.3 mmol) of the monobenzylamine salt of (1 R)- 1 -[(N-benzyloxycarbonyl-L-a- aminobutyryl)amino]-ethylphosphonic acid were stirred at room temperature for 6 hours with 20 ml of a mixture of 45% hydrogen bromide in glacial acetic acid and 8 ml of glacial acetic acid.

100 ml of ether were added, a gum precipitating.

This gum was washed by decantation with 100 ml of diethyl ether and dissolved in 100 ml of methanol. The solution was stirred and two 5 ml portions of propylene oxide were added to a pH of ca 5, there being thus obtained a white precipitate. The mixture was left to stand overnight, the precipitate was filtered off, washed successively with methanol and diethyl ether and dried to give 4.05 g of crude (1 R)-1-(L-a- aminobutyrylamino)-ethylphosphonic acid of melting point 2980C (decomposition).

Recrystallisation from a mixture of 750 ml of water and 1500 ml of ethanol gave a white crystalline precipitate which was washed with ethanol and then with diethyl ether and subsequentiy dried in vacuo. There were thus obtained 3.58 g (88%) of (1 P)-1 -(L-a- a mi nobutyrylamino)-ethyl-phosphonic acid of melting point 296 --298 C (decomposition); [a]020=-33.1 0; [a]s2065=-14 50 (c=1 -N sodium hydroxide, freshly prepared).

Example 2 (A) The preparation of the starting material: 5.0 g (42.5 mmol) of L-norvaline were treated with 10.2 g (60 mmoi) of benzyl chloroformate and sodium hydroxide in the manner described in Example 1(A). The crude oily product was crystallised from a mixture of 10 ml of diethyl ether and 20 ml of petroleum ether (boiling point 40 --60 C) to give 8.2 g (77%) of Nbenzyloxycarbonyl-L-norvaline of melting point 850--870C; [a]20=-9 90; [a]32065=-26.50 (c=1% in ethanol).

(B) The process: (i) In a manner analogous to that described in Example 1 (B) (i), from 8.1 g (32 mmol) of N benzyloxycarbonyl-L-norvaline, 3.68 g (32 mmol) of N-hydroxysuccinimide and 7.21 g (35 mmol) of dicyclohexylcarbodiimide there was obtained, after trituration of the oily product with 25 ml of ethanol, a white crystalline product. Addition of 25 ml of petroleum ether (boiling point 40 -- 600C) and filtration gave 9.82 g of Nbenzyloxycarbonyl-L-norvaline Nhydroxysuccinimide ester which was used directly in- the next step. A purified sample melted at 95 --97 C and showed an optical rotation [a]D20=--35.1 (c=1% in ethanol).

(ii) In a manner analogous to that described in Example 1(B) (ii), from 9.82 g (28 mmol) of Nbenzyloxycarbonyl-L-norvaline Nhydroxysuccinimide ester and 3.75 g (30 mmol) of (1 P)-1 -aminoethylphosphonic acid, but carrying out the titration with a mixture of 150 ml of methanol and 30 ml of water in place of diethyl ether, there were obtained 7.61 g of the monobenzylamine salt of (1 P)-1 -[(N benzyloxycarbonyl-L-norvalyl)amino]- ethylphosphonic acid of melting point 225 -- 2300C (decomposition); [&alpha;]D20=--29.9 ; [(22065=98.80 (c=1% in acetic acid).

Evaporation of the filtrate and trituration of the residue with 100 ml of hot water followed by filtration and washing with ethanol and then with diethyl ether gave a second crop of 1.99 g of the monobenzylamine salt of (1 P)-1-[(N- benzyloxycaronyl-L-novaly)amino]- ethylphosphonic acid of melting point 231 -- 2340C (decomposition); [aj20=-29.70; [a]22065=-99.20 (c=1 % in acetic acid). The total yield was 9.60 g (73%).

(iii) In a manner analogous to that described in Example 1(B) (iii), from 9.0 g (19 mmol) of the monobenzylamine salt of (1 P)-1-[N- benzyloxycarbonyl-L-norvalyl)amino]ethylphosphonic acid there was obtained, after recrystallisation from a mixture of 80 ml of water and 160 ml of ethanol, 3.54 g (82%) of (1 R)-1 -(Lnorvalylamino)-ethylphosphonic acid of melting point 260 --262 C (decomposition); [&alpha;]D20= 19.5 ; [a]23065=-75.30 (c=1% in water).

Example 3 (A) The preparation of the starting material: 5.0 g (38 mmol) of L-norleucine were treated with 9.7 g (57 mmol) of benzyl chloroformate and sodium hydroxide in a manner analogous to that described in Example 1(A). The product, N benzyloxycarbonyl-L-norleucine, was isolated in the form of an oil which was used in the process without crystallisation.

(B) The process: (i) In a manner analogous to that described in Example 1(B) (i), from ca 38 mmol of N benzyloxycarbonyl-L-norleucine, 4.38 g (38 mmol) of N-hydroxysuccinimide and 8.65 g (42 mmol) of dicyclohexylcarbodiimide there were obtained, after trituration of the crude oily product with 100 ml of diethyl ether, 7.47 g of Nbenzyloxycarbonyl-L-norleucine Nhydroxysuccinimide ester in the form of a crystalline solid of melting point 820--840C; [a] 020= 20.70 (c=1% in acetone).

(ii) In a manner analogous to that described in Example 1(B) (ii), from 7.40 g (20.5 mmol) of Nbenzyloxycarbonyl-L-norleucine Nhydroxysuccinimide ester and 2.56 g (20.5 mmol) of (1 R)-1-aminoethylphosphonic acid, but carrying out the ion exchange step in methanol/water (5:1) instead of methanol/water (3:1), there was obtained, after evaporation and trituration of the crude product with ether, a solid which was digested with 150 ml of hot water, cooled and filtered to give 5.64 g (74%) of (1 P)-1- [(N-benzyloxycarbonyl-L-norleucyl)amino]-ethyl- phosphonic acid of melting point 192 --194 C (decomposition); [aj020=-37.80; [a]22065=-1 26.50 (c=0.5% in acetic acid).

(iii) In a manner analogous to that described in Example 1 (B)(iii), from 5.20 g (14.0 mmol) of (1 R)-1 [(N-benzyloxycarbonyl-L-norleucyl)amino]- ethylphosphonic acid there were obtained, after recrystallisation from a mixture of 70 ml of water and 280 ml of ethanol, 3.02 g (91%) of (1 P)-1 -(Lnorleucylamino)ethylphosphonic acid of melting point 2540--2560C (decomposition); []D20=18.6 ; [a]23065=~70.1 (c=1% in water).

Example 4 (a) In a manner analogous to Example 1(B) (ii) and Example 2(B) (ii), from 17.4 g (50 mmol) of N-benzyloxycarbonyl-L-norvaline Nhydroxysuccinimide ester and 5.55 g (50 mmol) of aminomethylphosphonic acid, but carrying out the titration with benzylamine in a mixture of 250 ml of methanol and 50 ml of water, there were obtained 17.3 g of the crude monobenzylamine salt of the desired product, of melting point 195198 (decomp) (Crop 1). Evaporation of the filtrate and recrystallisation of the residue from a mixture of methanol (80 ml) and water (20 ml) gave a further 2.19 g of essentially pure product (talc), having a melting point of 1 80- 1850 (dec). The total yield was 19.5 g (86%).

Recrystallisation of a 0.5 g sample of Crop 1 from 10 ml of hot water gave a crop of 0.39 g of the pure monobenzylamine salt of (N benzyloxycarbonyl-L-norvalyl)amino- methylphosphonic acid of melting point 2032050 (dec); [(t,],20,-8.20; [a]= 21,30 (c=0.5%, in acetic acid).

(b) In a manner analogous to that described in Example 1(B) (iii), from 18.9 g (42 mmol) of the monobenzylamine salt of (N-benzyloxycarbonyl-Lnorvalyl)-aminomethyl-phosphonic acid there was obtained, after recrystallisation from a mixture of 80 ml of water and 160 ml of ethanol, 6.65 g (75%) of L-(norvalylamino)-methylphosphonic acid of melting point 273--2750 (dec); [a]0w=+61.2O, []365=+224 (c=0.54%, H2O).

Example 5 N-Benzyloxycarbonyl-L-norvaline (45.4 g 0.181 m) was stirred in methylene dichloride solution (200 mls) as dimethyl aminomethylphosphonate hydrochloride (31.76 g., 0.181 m) was added. The resulting suspension was cooled to -1 20C as dry triethylamine (25.3 ml 0.181 m) was added dropwise. After the addition was complete, the cold mixture was stirred for 1 5 minutes before E.E.D.Q. (56.8 g 0.23 m) in methylene dichloride (100 ml) was added rapidly. This mixture was stirred cold for 2 hours, and then stirred at room temperature over the weekend.

The mixture was washed with water (100 ml) then with 1 N hydrochloric acid (4 times, with 100 ml each time). The total acid wash was backextracted with methylene dichloride (2x50 ml).

The combined organic solution was washed again with water (100 ml) and finally with 1 5% KHCO2 solution (3x100 ml), then dried overanhydrous sodium sulphate. The solution was filtered and evaporated down. The residual oil was reevaporated down with benzene, to give a residue weighing 78.13 g.

The first signs of crystallisation were seen at this time. The oil was triturated with anhydrous ether (200 ml) in which it dissolved. Very soon crystallisation spread throughout the solution. The solution was refrigerated for 1 hour, the solid was filtered off, washed with ether and dried to constant weight in vacuo. There was obtained 58.2 g of a material having a melting point of 77-800C.

This was taken up in hot ethyl acetate (200 ml) and the solution filtered. To the cooled filtrate was added ether (200 ml). The mixture was seeded and refrigerated overnight. The solid was filtered off, washed with ether and dried in vacuo to give 50.64 g of a residue of m.p. 82-840C.

37.2 g of [(N-benzyloxycarbonyl-L norvalyl)amino]dimethylphosphonate was stirred in 45% hydrogen bromide in acetic acid (120 ml) for 5 hours. Initially carbon dioxide evolution was very rapid. Ether (500 ml) was added and this precipitated an oil. The mixture was stirred for 40 minutes, and then allowed to settle. The ether was decanted off and the oil was washed a further twice with ether, 500 ml each time.

The oil was taken up in methanol (300 ml) and the solution was stirred as propylene oxide (40 ml) was added. After about 5 minutes the product began to crystallise out. The mixture was adjusted to a pH of 4 and was refrigerated overnight.

The solid was filtered off and washed well with methanol, and then with ether. It was dried in vacuo, to give a residue of 20.13 g having a m.p.

of 288--9 OC (d).

This was taken up in hot water (200 ml) and filtered. To the filtrate was added 400 ml ethanol.

On standing, the material crystallised out. It was refrigerated for 2 hours. The solid was filtered off, washed with ethanol and then ether, and dried over phosphorus pentoxide in vacua. 16.66 9 were obtained of (L-norvalylamino)methylphosphonic acid of mp. 293-40C (d) [a]020=+62.70 (c=0.5% in water).

Example 6 (a) L-Nitro-homoarginine (2.33 g, 1 0 mmol, m.p. 227--2300 (dec)) was treated with 3.41 g (20 mmol) of benzyl chloroformate and sodium hydroxide in the manner described in Example 1(A). The crude oily product was extracted from the acidified solution with ethyl acetate (twice with 100 ml) instead of with diethyl ether. The crude oily product, N-benzyloxycarbonyl-o-nitro- L-homoarginine, obtained after drying over sodium sulphate and evaporation, was obtained as a gum which was used in the following step without further purification.

(b) In a manner analogous to that described in Example 1(B) (i), but using dimethyl formamide (50 ml) as solvent, from ca 4.2 g (approx. 10 mmol) of the crude N-benzyloxy-carbonyl-wnitro-L-homoarginine (obtained as above), 1.15 g (10 mmol) of N-hydroxysuccinimide and 2.27 g (11 mmol) of dicyciohexylcarbodiimide there was obtained after removal of 1.73 g of dicyclohexylene and evaporation of the filtrate, the crude product N-benzyloxycarbonyl-co-nitro- L-homoarginine N-hydroxysuccinimide ester as a yellow oil which was used in the next step without further purification.

(c) In a manner analogous to that described in Example 1(B) (ii), from ca 10 mmol of N benzyloxycarbonyl-w-nitro-l-homoarginine Nhydroxysuccinimide ester and 1.25 g (10 mmol) of (1 R)-1 -aminoethylphosphonic acid, but carrying out the treatment with ion exchange resin RS03H in 5:1 MeOH:H2O instead of 3:1. The acid eluate was evaporated and partitioned between water (300 ml) and ethyl acetate (150 ml) when some insoluble material was obtained.

This was filtered off and dried to give 1.36 g of crude product having a m.p. of 190--1930 (dec), as characterised by tic and N.M.R. (Crop 1).

The filtrate solvent layers were separated, the aqueous layer was evaporated to dryness and the residue was triturated with acetone (50 ml) to give a white precipitate. This precipitate was filtered off and washed with acetone, and dried to give a further 1.43 g (m.p. 184--8 (dec)) of the product, (1 R)-1 [(N-benzyloxycarbonyl-w-nitro-L- homoarginyl)amino]-ethylphosphonic acid (Crop 2). Crops 1 and 2 were used in the next step without further purification.

(d) In a manner analogous to that described in Example 1 (B)(iii), from 2.7 g (ca 5.7 mmol) of (1 R)-l -[(N-benzyl-oxyca rbonyl-o-n itro-l- homoarginyl)amino]-ethylphosphonic acid (Crops 1 and 2 above) there were obtained, after recrystallisation from a mixture of 15 ml of water and 150 ml ethanol, 1.23 g of (1 P)-1-(L-w-nitro- homoarginylamino)-ethylphosphonic acid of m.p.

ca 1900 (dec); [&alpha;]D20= 10.6 ; [a]22065=-43.50 (c=0.53%, H2O).

Evaporation of the filtrate and recrystallisation from water (5 ml) and ethanol (100 ml) gave a further 0.14 g of product having an m.p. ca 1900 (dec); [Cg]DO=11.2 ; [a]220s5=-43.20 (c=0.51% H20).

The total yield was 1.37 g (71%).

(e) 1.2 g (3.5 mmol) of (1 P)-1 -L-w-nitro- homoarginylamino-ethylphosphonic acid, prepared as above, was taken up in 50 ml of water, and 0.35 g of 10% Pd/charcoal was added.

The mixture was hydrogenated at ambient temperature and pressure until uptake ceased, (about 5 hours). The mixture was filtered to remove the catalyst and the filtrate was evaporated. The gummy solid residue was taken up in 15 ml of cold water and the solution was filtered and treated with 120 ml of ethanol to give a precipitate which was allowed to stand for 1 hour and then filtered off.There was obtained 0.76 g of (1 R)-1-(L-homoarginylamino) ethylphosphonic acid of melting point ca 1 950 (dec); [a]20=1 190 (c=0.5%, H2O)- Example 7 (i) In a manner analogous to that described in Example 1(B) (ii), from a solution of 45 mm,ol of N-benzyloxycarbonyl-L-&alpha;-aminobutyric acid Nhydroxysuccinimide ester in 40 ml dimethylformamide and 4.44 g (40 mmol) of aminomethylphosphonic acid, but using methanol:water of 4:1 instead of 2:1, there were obtained 14.1 g of the monobenzylamine salt of [(N-benzyloxyca rbonyl-L-x-aminobutryl)amino]- methylphosphonic acid of m.p. 185195 (dec).Evaporation of the filtrate and recrystallisation of the residue from a mixture of 50 ml of water and 50 ml of methanol gave as a white crystalline precipitate a further 1.6 g of product of m.p. 205208 (dec); [&alpha;]D20-6.1 0; [a]23065-21 .10 (c=0.51%, CH3COOH).

(ii) In a manner analogous to that described in Example 1(B) (iii) from 15.6 g (ca 36 mmol) of the monobenzylamine salt of [(N-benzyloxycarbonyl L-a-aminobutyryl)amino]-methylphosphonic acid, prepared as above, there was obtained 5.92 g of crude product of m.p. 253255 (dec).

Recrystallisation from a mixture of 60 ml of water and 120 ml of ethanol gave 4.93 g of (L-a- aminobutyryiamino)-methyiphosphonic acid of m.p. 263265 (dec); [&alpha;]D20+57.0 ; [a]22065+2120 (c=0.51%, H20).

Example 8 (i) In a manner analogous to that described in Example 3(B) (ii), from 2.52 g (7 mmol) of N benzyloxycarbonyl-L-norleucine Nhydroxysuccinimide ester and 0.78 g (7 mmol) of aminomethylphosphonic acid, but carrying out the ion exchange step in methanol/water of 2:1 instead of 5:1 and titrating the acid eluate directly with benzylamine (omitting evaporation and trituration with ether followed by dissolving in methanol/water again), there were obtained, after stirring with 1 00 ml of acetone, 0.56 g of the monobenzylamine salt of [(N-benzyloxycerbonyl- L-norleucyl)amino]-methylphosphonic acid having an m.p. of 1 85-1 890 (dec). Evaporation of the filtrate and trituration with 50 ml of diethyl ether gave a further 1.9 g of product, m.p. 170180 (dec).

(ii) In a manner analogous to that described in Example 1(B) (iii), from 2.40 g (ca 5.2 mmol) of the monobenzylamine salt of [(Nbenzyloxycarbonyl-L-norleucyl)amino]methylphosphonic acid, prepared as above, and using both crops, there was obtained 1.04 g of crude product of melting point 263266 (dec).

Recrystallisation from a mixture of 20 ml of water and 120 ml of ethanol gave 0.88 g of (Lnorleucylamino)-methylphosphonic acid of m.p.

272274 (dec); [a]20+6340; [a]32065+2310 (c=0.5%, H20).

The following Example illustrates a typical pharmaceutical preparation containing the peptide derivatives provided by the present invention: Example A A parenteral formulation containing the following ingredients can be prepared: Per 1000 ml Peptide derivative 50.0 g Chlorocresol 1.0 g Glacial acetic acid 1.2 g Sodium hydroxide solution (0.1-N) q.s. ad pH 4.5 Water for injection ad 1000 ml

Claims (30)

Claims

1. Compounds of the general formula

wherein R1 represents a hydrogen atom or the methyl or hydroxymethyl group or a mono-, di- or trihalomethyl group; R2 represents a lower alkyl, hydroxy-(lower alkyl) or guanidino-(lower alkyl) group other than the characterising group of an a- amino acid of the type normally found in proteins; the configuration at the carbon atom designated as (a) is (R) when R' represents other than a hydrogen atom; and the configuration at the carbon atom designated as (b) is (L), and pharmaceutically acceptable salts thereof.

2. Peptide derivatives as claimed in claim 1, wherein R' represents a hydrogen atom or the methyl group.

3. Peptide derivatives as claimed in claim 1 or claim 2, wherein R2 represents a lower alkyl group.

4. Peptide derivatives as claimed in claim 3, wherein R2 represents the ethyl, n-propyl or nbutyl group.

5. (1 P)-1 -[L-(a-Aminobutyryl)amino]- ethylphosphonic acid.

6. (1 R)-1-(L-Norieucylamino)-ethylphosphonic acid.

7. (1R)-l -(L-Norvalyiamino)-ethylphosphonic acid.

8. (L-a-Aminobutyrylamino)-methyl- phpsphonic acid.

9. (LNorleucylamino)-methylphosphonic acid.

1 0. (L-Norvalylamino)-methylphosphonic acid.

11. (1P)-1-(L-Homoarginylamino)- ethylphosphonic acid.

1 2. (L-Homoarginylamino)-methylphosphonic acid.

1 3. A process for the manufacture of the peptide derivatives claimed in claim 1, which process comprises condensing a compound of the general formula

wherein R'O has any of the values according to R1 hereinbefore or represents a protected hydroxy group; R3 and R4 each represent a hydrogen atom or a lower alkyl protecting group; and the configuration at the carbon atom designated as (a) is (R) when R'O represents other than a hydrogen atom, with a protected er-amino acid of the general formula

wherein R20 has any of the values accorded to R2 hereinbefore or represents a protected hydroxy-(lower alkyl) or guanidino-(lower alkyl) group;R5 represents a protected amino group; and the configuration at the carbon atom designated as (b) is (L), claving off the protecting group or groups present in the condensation product and, if desired, converting a resulting compound of formula I into a pharmaceutically acceptable salt.

14. A process as claimed in claim 13, wherein a peptide derivative in which R' represents a hydrogen atom or the methyl group is manufactured.

1 5. A process as claimed in claim 1 3 or claim 14, wherein a peptide derivative in which R2 represents a lower alkyl group,is manufactured.

1 6. A process as claimed claim 15, wherein a peptide derivative in which R2 represents the ethyl, n-propyl or n-butyl group is manufactured.

1 7. A process for the manufacture of the peptide derivatives claimed in claim 1, substantially as hereinbefore described with reference to any one of Examples 1 to 8.

18. A peptide derivative as claimed in claim 1, when manufactured by the process claimed in any one of claims 13 to 1 6 inclusive or by an obvious chemical equivalent thereof.

1 9. A pharmaceutical preparation containing a peptide derivative as claimed in any one of claims 1 to 12 inclusive in association with a compatible pharmaceutical carrier material.

20. A pharmaceutical preparation containing a peptide derivative as claimed in any of claims 1 to 12 inclusive and an antibiotic in association with a compatible pharmaceutical carrier material.

21. A pharmaceutical preparation as claimed in claim 20 wherein said antibiotic is an antibiotic as specified hereinbefore.

22. Compounds of the general formula

wherein R3, R4, R5, Rlq and R20 have the significance given in claim 13 and the configuration at the carbon atoms designated as (a) and (b) is as specified in claim 13.

23. (1 R)-1-[(N-Benzyioxywarbonyl-L-cg- aminobutyryl)aminoj-ethylphosphonic acid.

24. (1 R)- 1 -[(N-Benzyloxycarbonyl-Lnorvalyl)amino]-ethylphosphonic acid.

25. (1 R)-1-[(N-Benzyloxycarbonyl-L- norleucyl)amino]ethylphosphonic acid.

26. [(N-Benzyloxycarbonyl-L-a- aminobutyryl)amino]-methylphosphonic acid.

27. [(N-Benzyloxycarbonyl-L-norleucyl)amino]- methylphosphonic acid.

28. [(N-Benzyloxycarbonyl-L-norvalyl)amino]- methylphosphonic acid.

29. (1 R)-1 -[N-Benzyloxycarbonyl-so-nitro-L- homoarginyl)amino]-ethylphosphonic acid.

30. [(N-Benzyloxycarbonyl-w-nitro-L- homoarginyi)amino]-methylphosphonic acid.