IE42467B1 - Peptide derivatives of phosponic and phospinic acids - Google Patents

Description

The present invention relates to peptide derivatives of phosphonic and phosphinic acids, for example for the preparation of peptide derivatives as claimed in Patent Specification No. fro» which the present Application is divided.

According to the present invention there are provided compounds of formula wherein R10 represents a hydrogen atom or a lower alkyl, lower cycloalkyl, (lower cycloalkyl - (lower alkyl), aryl or aryl - (lower alkyl) group (said groups being . optionally substituted as the case may require by one or more amino, hydroxy, mercapto, methylthio, carboxy or guanidino groups so as to form the chara cterising group of an a - amino acid of the type found in proteins); R20 and R30 (where appropriate in combination with R’ ) each represent the charac terising group of an a - acid of the type found in 4246? — 3 — ' proteins; and R represents a hydrogen atom or Is in combination with R30 to form a characterising group of an a - amino acid with the proviso that R30 cannot represent a hydrogen atom when n is zero and R3-0 is a hydrogen atom or the phenyl group, and that any amino group or amino groups present in R10, R20, R30 and R3® may be in protected form and any other functional group which may be present is optionally 40 in protected form; R represents a methyl group or 41 41 R ; R represents a hydroxy group or lower alkoxy protecting group; R3 represents a hydrogen atom or a protecting group, with the proviso that at least one of R10, R20, R30, R5, R40 and R41 is a protecting group or is in protected form; n stands for zero, 1, or 3; single asterisks denote that when R20 or R30 represents other than a hydrogen atom, the configuration at the carbon atom so-marked is L; and the double asterisk denotes that, when represents other than a hydrogen atom, the configuration at the carbon atom so-marked is that which would be obtained by replacing the carboxyl group of a naturally occurring L-α amino acid by a phosphorus moiety /Hereinafter referred to as in (R) - configuration/, and their salts.

As used in this Specification, the termlower alkyl means a straight-chain or branched-chain alkyl group which contains from 1 to 6 carbon atoms (e.g. methyl, ethyl, propyl or isobutyl). The term lower cycloalkyl comprises cyclic alkyl groups which contain from 3 to 6 carbon atoms. The term aryl preferably comprises mono-nuclear groups such as phenyl, which may be substituted in one or more positions with hydroxy, halogen, nitro, lower alkyl or lower alkoxy substitutents. The term halogen means fluorine, chlorine, bromine and iodine and the term lower alkoxy means groups of the structure -0-(lower alkyl) wherein the - 4 lower alkyl group is as defined earlier. The expression the characterising group of an u - amino - acid of the type found in proteins is used to mean the residue R in a natural a - amino acid of the general formula H ’J—-CH COOII I R which is of the type occurring in proteins. Thus, for example, if the amino - acid is alanine, then the residue R represents the methyl group, in leucine the residue R represents the isobutyl group and in glutamic acid the residue R represents the 2 - carboxyethyl group. R can also represent a residue which is linked with the amino nitrogen (With the loss of one of the hydrogen atoms attached thereto), thus forming a nitrogen-containing ring such as in proline and pyroglutamic acid.

It will be appreciated that when n in formula I stands for 2 or 3 the values of R can be the same or different.

The amino group or amino groups which may be present in R3·0, R20 and R30 In formula I can be protected with any amino-protecting group which is well-known in peptide chemistry. Especially suitable amino-protecting groups for the purpose of the present invention are aralkoxycarbonyl groups, particularly the benzylcarbonyl group, and the tertbutoxycarbonyl group. The amino-protecting group may also be a formyl, trityl or trifluoroacetyl group. Any carboxy or hydroxy group which may be present in R^, R2® and R30 in formula I can be protected by a conventional carboxy-protecting or hydroxy-protecting group respectively. For example, a carboxy group may be protected by conversion into an alkyl ester (e.g. a tertbutyl ester) or an aralkyl ester (e.g. a benzyl ester). Again, for example, a hydroxy group'may be protected, for 43467 - 5 example, by means of an aralkoxy-carbonyl group (e.g. benzyloxycarbonyl), an alkanoyl group (e.g. acetyl or propionyl), an aroyl group (e.g. benzoyl), an alkyl group (e.g. tertbutyl) or an aralkyl group (e.g. benzyl). The protection of other functional groups present in p3°, R(II) * * * * * * * * 20 and R30 may be carried out in a known manner.

The protecting group denoted by R in formula I can be any of the amino-protecting groups mentioned earlier in connection with R10, R20 and R30.

The compounds of formula X hereinbefore may be prepared, for example, by condensing a compound of the general formula •NH,20 CH* CO .10 -NH-CH* * II P-R' (II) wherein 40 41 R ,R , R ,R , n and the single and double asterisk have the significance given earlier, with an appropriately protected a - amino acid, an appropriately protected dipeptide, an appropriately protected tripeptide, an appropriately protected tetrapeptide or a reactive derivative thereof as the case may require.

Thus, when a compound of formula II in which n stands for zero is used, such a compound can be condensed with an appropriately protected a - amino acid or a reactive derivative thereof to give a compound of formula I in which n stands for zero, or with an appropriately protected dipeptide or a reactive derivative thereof to give a compound of formula I in which n stands for 1, or with an appropriately protected tripeptide or a reactive derivative thereof to give a compound of formula I in , 43467 *“ I - 6 which n stands for 2 or with an appropriately protected tetrapeptide or a reactive derivative thereof to give a compound of formula I in which n stands for 3.

Again, a compound of formula II in which n stands 5 for 1 can be condensed with an appropriately protected a - amino acid or a reactive derivative thereof to give a compound of formula I in which n stands for 1, or with an appropriately protected dipeptide or a reactive derivative thereof to give a compound of formula I in which n stands for 2 or with an appropriately protected tripeptide or a reactive derivative thereof to give a compound of formula I in which n stands for 3.

Yet again,a compound of formula II in which n stands for 2 can be condensed with an appropriately protected a - amino acid or a reactive derivative thereof to give a compound of formula I in which n stands for 2 or with an appropriately protected dipeptide or a reactive derivative thereof to give a compound of formula I in which n stands for 3.

Finally, a compound of formula II in which n stands for 3 can be condensed with an appropriately protected a amino acid or a reactive derivative thereof to give a compound of formula I in which n stands for 3.

Alternatively, the compounds of formula I can be prepared by carrying out the foregoing condensation using an R,S)', compound corresponding to formula II and separating the (R) compound from the resulting (R,S) product in a manner known per se; for example, by crystallisation, chromatography or fractional crystallisation using a suitable base such as a - methylbenzylamine.

The aforementioned condensation can be carried out in accordance with methods which are known per se in peptide chemistry; for example, by the mixed anhydride, azide, activated ester or acid chloride method. 43467 - 7 In one method, an appropriate compound of formula II can be condensed with an appropriately protected amino acid, di -, tri - or tetrapeptide as the case may require in which the terminal carboxy function is a mixed anhydride residue formed with an organic or inorganic acid. Suitably, such an amino acid, di -, tri or tetrapeptide carrying a free carboxy function 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), 1,2 - dimethoxyethane, dichloromethane, toluene, petroleum ether or mixtures thereof and the resulting salt is reacted with a chloroformic acid ester (e.g. the ethyl or isobutyl ester) at a low temperature. The mixed anhydride obtained is then suitably condensed in situ with the compound of formula II.

In another method, an appropriate compound of formula II can be condensed with an appropriately protected amino acid, di -, tri - or tetrapeptide as the case may require in which the terminal 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, an appropriate compound of formula II can be condensed with an appropriately protected amino acid, di -, tri - or tetrapeptide as the case may require in whieh the terminal carboxy function is in the form of an active ester group (e.g. the p - nitrophenyl, 2,4,5 - trichlorophenyl or N - hydroxysuccinimide ester group). This condensation is suitably carried out either in an inert organic solvent such as dimethylform40 41 amide or, in the case where R and/or R represents a lower alkoxy group, in an aqueous alkanol (e.g. aqueous ethanol).

In a further method, an appropriate compound of - 8 formula II can be condensed with an appropriately protected amino acid, di -, tri - or tetrapeptide as the case may require in which the terminal carboxy function is in the form of an acid chloride. This condensation is pre5 ferably carried out in the presence of a base and at a low temperature.

The following Examples illustrate the present invention.

Example 1. 33.3 g (0.30 mol) of aminomethylphosphonic acid were dissolved in a mixture of 1.5 litres of water and 750 ml of ethanol. The solution was cooled to 10°C, treated portion wise with 75.6 g (0.90 mol) of solid sodium bicarbonate while stirring and then cooled to 0° C. A reagent solution of 96 g (0.30 mol) of the Nhydroxysuccinimide ester of N - benzyloxycarbonyl - L alanine in 1 litre of hot ethanol was added dropwise rapidly during ca 10 minutes while maintaining the internal temperature below 5°c. The reagent was washed in with two 200 ml portions of ethanol. The heterogeneous mixture was stirred for a further 2 hours at 0°C and then for 24 hours at room temperature. An almost clear solution was obtained. Evaporation at room temperature followed by re-evaporation with 400 ml of water at room temperature gave a gummy solid. This solid was dissolved in 1.5 litres of water, extracted with 1.5 litres of chloroform, then With two 500 ml portions of chloroform, acidified with 2-N hydrochloric acid to give a pH value of 2 and again extracted with chloroform. Thin layer chromatography showed that the desired starting material was in the aqueous fraction. This fraction was concentrated to ca 300 ml and passed down a soluffln of cation exchange resin (B.D.H., Zerolit 225 (registered Trade Mark), SCR 13, RSO^H; 1.5 kg; freshly - 9 regenerated in the acid cycle). Elution with 1 litre of water followed by three 500 ml portions of water gave four acid fractions, only the first two of which contained substantial amounts /according to thin layer chromatograph^ of desired starting material. These two fractions were combined, evaporated and re-evaporated with water until substantially free of hydrogen chloride. There was obtained a final residue of /IN - benzyloxycarbonyl - Xi - alanyl)amino/ - methylphosphonic acid, which was converted to the benzylamine salt as follows: The latter residue was dissolved in 700 ml of water and titrated with 1-N benzylamine to give a pH value of 4.5; titre 240 ml; theory 300 ml. The precipitate was filtered off and crystallised from 800 ml of water. The crystals were filtered off, washed with water until the filtrate was chloride-free, then washed successively with ethanol and ether and dried. There were obtained 52 g (41% yield) of the benzylamine salt of /Jn benzyloxycarbonyl - L - alanyl)amino7 - methylphosphonic acid of melting point 2OO°-2O1°C (decomposition); /α7ρ2Ο=-6.7° (c=l% in acetic acid). Concentration of the mother liquors gave a further crop of 4.2 g; melting point 199°-2O1°C (decomposition); /α7θ2θ=-7.4° (c=l% in acetic acid).

Example 2. 24.2 g (0.24 mol) of triethylamine were added to 53.5 g (0.24 mol) of N - benzloxycarbonyl - L - alanine in 2 litres of dry toluene and the mixture was cooled to -5°C. 32.8 g (0.24 mol) of isobutvl chloroformate were added dropwise while stirring and the mixture was maintained at -5°C for a further 25 minutes. While stirring this mixture at -5°C, there was added dropwise a solution of 6.66 g (0.060 mol) of aminomethylphosphonic acid in 60 ml of 2 - N sodium hydroxide and the stirring continued 43467 - 10 for a further 3 hours at -5°C. The mixture was then allowed to warm to room temperature and stirred overnight. The aqueous layer was separated,back-extracted with toluene and adjusted to pH 9.5 with 45 ml of 2 - N sodium hydroxide The solution was evaporated at room temperature to remove triethylamine. The residue was dissolved in 200 ml of water three times and re-evaporated each time. The final residue was dissolved In 500 ml of water and the resulting solution extracted three times with 350 ml portions of chloroform. The aqueous layer was adjusted to pH 2.5 with mi of 2 - N hydrochloric acid and then extracted successively with three 350 mi portions of ether and three 350 ml portions of chloroform. The aqueous layer was evaporated at room temperature and the resulting white solid dissolved in 50 ml of water and 20 ml of 2 - N ammonium hydroxide and then passed down a column of cation exchange resin (B.D.H., Zerolit 225, SRC 13, RSO^H; 250 g; freshly regenerated in the acid cycle). The column was eluted with water and the acid eluate evaporated. The residue was evaporated at room temperature three times with 100 ml of water each time to remove hydrogen chloride. There was obtained a final residue of - benzyloxycarbonyl - L alanyl)amino/ - methylphosphonic acid, which was converted to its benzylamine salt as follows: The latter residue was titrated with 36 ml of 1 M benzylamine to pH 4. Evaporation gave a White solid which was purified by crystallisation from water. There were obtained two 0.9 g crops of the benzylamine salt of /(N - benzyloxycarbonyl - L - alanyl)aminq7 - methyl30 phosphonic acid with respective melting points of 193°195°C (decomposition) and 196°-199°C (decomposition),/α7ρ2θ=-6.0° (c=l% in acetic acid) 43467 - 11 Example 3 .

In a manner analogous to Example 2, starting from N - benzyloxycarbonyl - L - valine there was obtained the benzylamine salt of /(N - benzyloxycarbonyl - L valyl)amino/ - methylphosphonic acid of melting point 235°-237°C (decomposition); /q7D2O=-5.7° (c=0.1% acetic acid).

Example 4.

In a manner analogous to Example 1 starting from the N -r hydroxysuccinimide ester of N - benzylcarbonyl L - leucine there was obtained the benzylamine salt of /“(N - benzyloxycarbonyl - L - leucyl)amino7 - methylphosphonic acid of melting point 175°-178°C (decomposition) ; /q7D20=-10.1° (c=O.77% in acetic acid).

Example 5.

In a manner analogous to Example 1, starting from 2 6 the N - hydroxysuccinimide ester of N ,N - bis(benzyloxy— 2 6 carbonyl) -- L - lysine there was obtained /N ,N bis(benzyloxycarbonyl) - L - lysyl)aminq7 - methylphosphonic acid of melting point 16O°-162°C (decomposition): Zq7D2Oas”9,55°(c=O.5% in ethanol).

Example 6.

In a manner analogous to Example 1, but with the ion exchange carried out in methanol/water instead of water, starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - phenylalanine there was obtained /[N - benzyloxycarbonyl - L - phenylalanyl)amino/ methylphosphonic acid of melting point 181°-182°C (decomposition); /ά7θ20=-11.9ο (c=1.0% in methanol).

Example 7. 0.91 g CO.005 mol) of (L - alanylamino) methylphosphonic acid were dissolved in 25 ml of water - 12 and 12.5 ml of ethanol and treated with 1.26 g (0.015 mol) of solid sodium bicarbonate to give a clear solution.

The solution was stirred at 0°C while a warm solution of 1.6 g (0.005 mol) of the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine in 16 ml of ethanol was added and then washed in with two 5 ml portions of ethanol. The initially heterogeneous mixture became homogeneous within 5 minutes. The mixture was stirred at 0°C for 2 hours and then at room temperature for 16 hours. The ethanol was evaporated and the residue re-evaporated with water. The residue was dissolved in 100 ml of water and extracted with 100 ml of chloroform followed by two 50 ml portions of chloroform. The aqueous layer was acidified with ca 15 ml of 2 - N hydrochloric acid to pH 2 and extracted with 100 ml of chloroform and then two 50 ml portions of chloroform. Thin layer chromatography showed that the product was in the aqueous phase. The aqueous phase was concentrated until a solid began to precipitate.

The minimum amount of 2 Ή ammonium hydroxide to give a clear solution was added. The solution was passed down a column of cation exchange resin (B.D.H., Zerolit 225, SCR 13, RSO^H; 150 g; freshly regenerated in the acid cycle) and eluted with water. The acid eluates which contained only the desired starting material (according to thin layer chromatography; three 100 ml portions) were combined,evaporated and re-evaporated with water to remove hydrogen chloride. There was obtained a crude residue of/(N - benzyloxycarbonyl L - alanyl - L - alanyl)aminq7 - methylphosphonic acid, which was converted to the benzylamine salt as follows: The latter residue was taken up in water and titrated to pH 4.5 with 4-N benzylamine; titre 1.6 ml; theory 1.25 ml. The product crystallised on standing and was digested with warm water, cooled and stood overnight.

The resulting precipitate was filtered off and washed with 25 ml of water until free from chloride ions /benzylamine hydrochloride/. The solid was washed successively - 13 with ethanol and ether and dried. There was obtained 1.085 g of the benzylamine salt of /IN - benzyloxycarbonyl - L alanyl - L - alanyl)amino/ - methylphosphonic acid of melting point 232°-234°C (decomposition); /07D2O=-22.1° (c=0,5% in acetic acid). Concentration of the mother liquors gave a further 0.3 g of the benzylamine salt with a melting point of 232°r234°C (decomposition). Recrystallisation of the first crop from 60 ml of water gave 0.71 g of pure benzylamine salt of melting point 232°-234°C (decomposition); /07D2O=-2O.3° (c=0.5% in acetic acid).

Example 8.

In a manner analogous to Example 7, starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and (L - alanyl - L - alanylamino) - methylphosphonic acid there was obtained the benzylamine salt of /”(N - benzyloxycarbonyl - L - alanyl - L - alanyl - L alanyl)amino/ - methylphosphonic acid of melting point 249°-251°C (decomposition); /a7D2O=-32.2° (e=0.5% in acetic acid).

Example 9. 2.8 g (0.036 mol) of solid sodium carbonate were added at 5°C to a solution of 1.96 g (0.018 mol) of (aminomethyl) - methylphosphinic acid in 72 ml of water and 36 ml of ethanol, a clear solution resulting. The solution was stirred at 0°C while a warm solution of 5.76 g (0.018 mol) of the N - hydroxysuccinimide ester of N benzyloxycarbonyl - L - alanine in 36 ml of ethanol was added and washed in with two 10 ml portions of warm ethanol. The heterogeneous mixture was stirred at O°C for 2 hours and then overnight at room temperature. The mixture was evaporated and then re-evaporated with 50 ml of water to remove ethanol. The residue was dissolved in 150 ml of water and extracted once with 150 ml of chloroform and twice with 30 ml portions of chloroform. The aqueous layer - 14 was acidified with 18 ml of 2 - N hydrochloric acid and again extracted once with 150 ml of chloroform and twice with 30 ml portions of chloroform. The aqueous layer was separated, evaporated and then taken up in 10 ml of water and 10 ml of 2 · B ammonium hydroxide. The solution was passed down a column of cation exchange resin (B.D.H., Zerolit 225, SCR 13, RSO^H; 150 g; freshly regenerated in the acid cycle) and eluted with water. There were collected four 100 ml acid fractions. The first two fractions were combined and evaporated to give a gum. This was re-evaporated with water to remove hydrogen chloride and there remained a sticky solid which was triturated with ethyl/ dioxane (1:1); There were obtained 1.8 g of a white Solid of melting point 118°-121°C (decomposition). Concentration of the mother liquors gave a further 2.02 g of solid of melting point 126°-13O°C (decomposition). Recrystallisation of the second crop from dioxane/ether gave 1.82 g of{/(N benzyloxycarbonyl - L - alanyl)amino/methyl} - methylphosphinic acid of melting point 129O-131°C (decomposit20 ion); /m7d =-26.0 g (c=l% in water).

Example 10 14.1 g (0.168 mol) of solid sodium bicarbonate were added to a solution of 7 g (0.056 mol) of (IR,S) - 1 aminoethylphosphonic acid in 280 ml of water and 140 ml of ethanol while stirring at 0°C. While stirring this mixture at 0°C, a solution of 17.9 g (0.056 mol) of the N hydroxysuccinimide ester of N - benzyloxycarbonyl - L alanine in 140 ml of warm ethanol was added dropwise over ca 15 minutes. The latter solution was washed in with 70 ml of ethanol. The heterogeneous mixture was stirred for 1 hour at 0°C and then for a further 16 hours at room temperature, the mixture becoming homogeneous. The mixture was evaporated and re-evaporated with 200 ml of water to give a gum which was dissolved in 500 ml of water. The 43467 - 15 solution was extracted firstly with 500 ml of chloroform and then with two 250 ml portions of chloroform, acidified to pH 2 with ca 80 mi of 2 - N hydrochloric acid and again extracted with 500 ml of chloroform followed by two 250 ml portions of chloroform. The aqueous layer was concentrated and passed down a column of cation exchange resin (B.D.H., Zerolit 225, SCR 13, RSO3H; 750 g; freshly regenerated in the acid cycle). The column was eluted with water and there were collected six 250 ml fractions. The first four fractions were combined, evaporated and reevaporated with water to remove hydrogen chloride. There was obtained a final residue of (IR,S) - 1 - /IN benzyloxycarbonyl - L - alanyl) - aminq7 - ethylphosphonic acid which was separated as follows; The latter residue was dissolved in 400 ml of water and titrated with 1 - M benzylamine to pH 4.5; titre 75 ml; theory 56 ml. The resulting solution was concentrated and crystallised from water to give 5.3 g of the benzylamine salt of (IS) - 1 - /TN - benzyloxycarbonyl - L alanyl)amino/ - ethylphosphonic acid of melting point 210°215°c. Concentration of the mother liquors followed by further reerystallisation from water gave the benzylamine salt of (IR) - 1 - /(N - benzyloxycarbonyl - L - alanyl)aminq7 - ethylphosphonic acid in a first crop of 0.59 g /melting point 226°-228°C' (decomposition); /ct/D2O=-32.3° (c=l% in acetic acid)_7 and a second crop of 0.825 g /melting point 225°-227°C (decomposition); /q/D2O=-33.0° (c= 1% in acetic acid)/. Reerystallisation of the first crop from water gave 0.333 g of pure benzylamine salt of the R - stereoisomer; melting point 226°-228°C (decomposition); /q7D2O=-33.1° (c=l% in acetic acid).

Example 11.

A solution of 30 g (0.24 mol) of (IR,S) - 1 aminoethyIphosphonic acid in 120 ml (0.48 mol) of 4 - 16 484 67 Ν - sodium hydroxide was stirred at 14°C while 180 ml (0.72 mol) of a solution of 4 - N - sodium hydroxide and 102 g (0.60 mol) of benzyl chloroformate were added alternately in four portions. The stirring was continued b and after a further 2 hours the temperature has risen to 20°C. The mixture was stirred for a further 16 hours at room temperature. 600 ml of ether were then added and the mixture was stirred vigorously for 2 hours to extract the excess benzyl chloroformate. The layers were separated and the aqueous layer was acidified to pH 2 with ca llO ml of 5 - N hydrochloric acid while maintaining the temperature below 10°C, The resulting slurry was concentrated to a low bulk to remove carbon dioxide. The residue was disso Ived in 100 ml of 2 - N sodium hydroxide and 50 mi of water, passed down, a column of cation exchange resin (B.D. H., Zerolit 225, SCR 13, RSO^H; 750 g; freshly regenerated in the acid cycle) and eluted with water. There were obtai ned ca 3.2 litres of acid eluate which were evaporated at room temperature and re-evaporated with three 500 ml portions of water. The residue was dissolved in Water and allowed to crystallise. The crystals were filtered off, washed with ice-cold water and dried; yield 39.2 g; melting point li0°-113° (decomposition). Evaporation of the combined filtrates followed by crystallisation from 75 ml of water and 10 ml of methanol and refrigeration, gave a further yield of 6,51 g; melting point 110°-li2°C (decomposition) . There was obtained a total of 45.71 g of (IR,S) - 1 - (benzyloxycarhonylamino) - ethylphosphonic acid, which was characterised as the monobenzylamine salt of melting point 196O-197°C (decomposition). 42.2 g (163 mmol) of (IR,S) - 1 - (benzyloxycarbonyl - amino) - ethylphosphonic acid were dissolved in 100 ml of methanol. The solution was treated with a solution of 30.8 g (81.5 mmol) of quinine trihydrate in 100 ml of methanol and the mixture was stored for 3 hours at room temperature and then overnight at 0°C, The quinine <»467 - 17 salt of CIS) - 1 - (benzyloxycarbonylamino) - ethylphosphonic acid was filtered off and washed with methanol.

The combined filtrates were evaporated and the residue was dissolved in 300 ml of 2 - N ammonium hydroxide. The solution was extracted with three 300 ml portions of chloroform. Each chloroform extract was back-washed with 150 ml of water. The aqueous extracts were combined, concentrated and then passed down a column of cation exchange resin (B.D.H., Zerolit 225, SRC 13, RS03H; 750 g; freshly regenerated in the acid cycle). Elution with water gave ca 2.3 litres of acid eluate, which was evaporated. The residue was re-evaporated firstly with three 200 ml portions of water and then with three 300 ml portions of methanol to give ca 24 g of a residual gum. This gum was dissolved in 100 ml of dry methanol and treated with a solution of dehydroabietylamine /82 mmol? freshly regenerated from 28.4 g (82 mmol) of dehydroabietylamine acetate with ammonium hydroxide/petroleum ether/. The mixture was stood at 0°C, filtered and the filtrate was washed with methanol and ether. There were obtained 47.4 g of crude dehydroabietylamine salt of (IR) - 1 - (benzyloxycarbonyl - amino) - ethylphosphonic acid of melting point 189°-194°c (decomposition); /0.7^^=+16·8° ( c-0.5% in methanol). Further recrystallisation from methanol and water gave 33.0 g of the pure dehydroabietylamine salt of (IR) - 1 - (benzyloxycarbonyl - amino) - ethylphosphonic acid of melting point 2O2°-2O5°C (decomposition); /o7D2<3= +18.1° (c=0.5% in methanol). 8.0 g (14 mmol) of the dehydroabietylamine salt of (IR) - 1 - (benzyloxycarbonyl - amino) - ethylphosphonic acid were partitioned between 100 ml of 2 - N ammonium hydroxide and 100 ml of petroleum ether (boiling point range 60°-80°C). The mixture was shaken vigorously and then the layers were separated. The aqueous layer was extracted with two 50 ml portions of petroleum ether. Each petroleum ether extract was then back-extracted with two - 18 50 ml portions of water. The aqueous extracts were combined and evaporated at room temperature to give an oil. This oil was dissolved in water, passed down a column of cation exchange resin (B.D.H., Zerolit 225, SRC 13, RSO^H; 250 g; freshly regenerated in the aoid cycle) and eluted with water. There were obtained 800 ml of an acid fraction which was then concentrated to 400 ml. To this concentrate Were added successively 2.0 g of 10% palladium-on-charcoal catalyst, 400 ml of methanol and 0.2 ml of glacial acetic aoid. The mixture was then hydrogenated. The catalyst was filtered off and the solvent evaporated. The residue was re-evaporated with three 100 ml portions of n - propanol and triturated with ether to give a solid of melting point ca 285°-288°C (decomposition). Recrystallisation from water and ethanol gave 1.0 g of (IR) - 1 - aminoethylphosphonic acid of melting point 294°-295°C (decomposition); = -16.9° (c=2% In 1 - N sodium hydroxide). 0.4 g (3.2 mmol) of (IR) - 1 - aminoethylphosphonic acid in 14 ml of water and 7 ml of ethanol were stirred at 10°C while 0.806 g (9.6 mmol) of sodium bicarbonate were added portionwise. The mixture was then stirred at 0°C while a hot solution of 1.024 g (3.2 mmol) of the Nhydroxysuccinimide ester of N- benzyloxycarbonyl - L alanine in 8 mi of ethanol was added rapidly dropwise.

The mixture was stirred for 3 hours at 0°C and then for 16 hours at room temperature. The mixture was worked up in a manner analogous to Example 10 a) by passing down a column of cation exchange resin and converting to the benzylamine salt. There were obtained 0.26 g of the benzyla30 mine salt of (IR) - 1 - (N - benzyloxycarbonyl - L alanyl)amino - ethylphosphonic acid of melting point 229°-231°C (decomposition); = -34.2° (c=l% in glacial acetic acid). 43467 Τ 19 Example 12.

In a manner analogous to Example 7, starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and (IR) - 1 - (L - alanylamino) ethylphosphonic acid there was obtained the benzylamine salt of (IR) - 1 - /(Ν - benzyloxycarbonyl - L - alanyl L alanyl) amin<27 - ethylphosphonic acid of melting point 247°- 25O°C (decomposition); /q7D2O=-45.1° (c=0.5% in acetic acid).

Example 13. 9.6 g (0.03 mol) of the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L «· alanine and 5.26 g (0.03 mol) of dimethyl aminomethylphosphonate hydrochloride were stirred in 65 ml of dry dimethylformamide.

While stirring and maintaining the temperature below 20° C, there were added dropwise 4.2 ml of dry triethylamine. The mixture was then stirred overnight at room temperature. The triethylamine hydrochloride was filtered off and washed with a little dimethylformamide. The filtrate was evaporated under an oil-pump vacuum and at a bath temperature below 40°C. The residual oil was treated with 40 ml of water and the resulting mixture extracted with four 40 ml portions of chloroform. The combined organic phases were washed with a small volume of a strong potassium carbonate solution and then dried over sodium sulphate. The sodium sulphate was filtered and the filtrate evaporated first under a water-pump vacuum and then under an oil-pump vacuum. There were obtained 11.0 g of dimethyl /In - benzyloxycarbonyl - L - alanyl)aminq7 - methylphosphonate as an oil with the expected N.M.R. spectrum.

Example 14.

In a manner analogous to Example 13, starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl . 42467 - 20 - L - alanine and diethyl aminomethylphosphonate hydrochloride there was obtained diethyl /'(N - benzyloxycarbonyl - L - alanyl)amino/ - methylphosphonate as a solid of melting point 72O-74OC.

. Example 15.

In a manner analogous to Example 13, starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - phenylalanine and dimethyl aminomethylphosphonate hydrochloride there was Obtained dimethyl /(N -benzyloxycarbonyl - L - phenylalanyl)amino/ - methylphosphonate as an oil with the expected N.M.R. spectrum.

Example 16.

In a manner analogous to Example 13, starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - leucine and dimethyl aminomethylphosphonate hydrochloride there was obtained dimethyl /“(N - benzyloxycarbonyl - L - leucyl)amino/ -methylphosphonate as a crystalline solid of melting point 9O°-91°C; /α/02θ=-24. 3° (c=l% in methanol), Example 17.

In a manner analogous to Example 13 starting from 64.0 g of the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and 35.1 g of dimethyl amino25 methylphosphonate hydrochloride, there were obtained 73.3 g of dimethyl /(N - benzyloxycarbonyl - L - alanyl)amino7 - methylphosphonate as an oil. This oil was refluxed in a mixture of 200 ml of trimethylchlorosilane and 100 ml of acetonitrile under exclusion of moisture for 100 hours.

The mixture was then cooled, filtered and evaporated in vacuo on a rotary evaporator. The residue was re-evapora43487 - 21 ted several times with toluene. The final residue was dissolved in 250 ml of dioxane and treated with 25 ml of water. Crystallisation began after several minutes and this was completed by storing overnight. The separated solid was filtered off, washed with ethyl acetate and dried in vacuo. There were obtained 29.0 g of solid of melting point 147°-148°C (decomposition). A further 28.0 g of solid of the same melting point were obtained by concentration of the mother liquors and treatment with ethyl acetate. The combined solids were recrystallised by dissolving in 1.5 parts by volume of tepid methanol, filtering and then adding 15 parts by volume of ethyl acetate to the filtrate. There were obtained 39.5 g of pure /XN - benzyloxycarbonyl - L - alanyl) - amino7 - methylphosphonic acid of melting point 153°-155°C (decomposition); /α7η2Ο=-28.9° (c=l% in water) .

Example 18.

In a manner analogous to Example 17, starting from the N - hydroxysuecinimide ester of N - benzyloxycarbonyl - L - phenylalanine and dimethyl aminomethylphosphonate hydrochloride there was obtained /(N benzyloxycarbonyl - L - phenylalanyl)aminq7 - methylphosphonic acid of melting point 183°-184°C (decomposition) /q7D20=-10.9° (c=l% in methanol).

Example 19.

In a manner analogous to Example 17 starting from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - leucine and dimethyl aminomethylphosphonate hydrochloride there was obtained /Xn - benzyloxycarbonyl - I· - leucyl)amino7 - methylphosphonic acid of melting point 129°-13O°C; /q7D?'°=-29.2° (c=l% in water). - 22 Example 20.

In a manner analogous to Example 13 starting from 64.0 g of the, N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and 35.1 g of dimethyl amino5 methylphosphonate.hydrochloride, there were obtained 73.3 g of dimethyl /[N - benzyloxycarbonyl - L - alanyl)amino/ - methylphosphonate as an oil. This oil was dissolved in 350 ml of methanol and treated with 40 ml of 5 N methanolic hydrogen chloride. The mixture was then hydrogenated for several hours afc room temperature under atmospheric pressure and a soda-lime trap and in the presence of 6 g of 10% palladium-on-charcoal catalyst until hydrogen uptake ceased. The catalyst was filtered off and the filtrate evaporated in vacuo to 100 ml. After the addition of 300 ml of ethyl acetate, crystallisation began and was completed by storing overnight at 0°C. The solid was filtered off, washed successively with ethyl acetate/methanol and ethyl acetate and then dried in vacuo There were obtained 40.9 g of solid which was recry20 stallised from methanol/ethyl acetate to yield 40.5 g of dimethyl (L - alanylamino) - methylphosphonate hydrochloride of melting point 168°-170°C (decomposition); /57^^=-5.05^ (c=l% in water, . 24.65 g of dimethyl (L - alanylamino) - methyl25 phosphonate hydrochloride and 32.0 g of the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine were stirred in 200 ml of dry dimethylformamide while 14 ml of dry triethylamine were added dropwise at 20°C.

The mixture vzas stirred overnight and then the triethyl30 amine hydrochloride filtered off ind washed with a small amount of dimethylformamide. The filtrate was evaporated under an oil-pump vacuum, the residue treated with 150 ml of water and then extracted with four 125 ml portions of chloroform. The combined chloroform layers were washed with a 20% potassium carbonate solution, separated and dried over sodium sulphate. The sodium sulphate was - 23 filtered off, the filtrate evaporated firstly under a water-pump vacuum and then under an oil pump vacuum and the residue taken up in 100 ml of ethyl acetate.

About 100 ml of ether were added to a faint turbidity when crystallisation began. After refrigeration overnight, the solid was filtered off, washed successively with ethyl acetate/ether (1:1) and ether and then dried in vacuo. Recrystallisation from 200 ml of boiling ethyl acetate by the addition of 200 ml of ether gave 27.8 g of dimethyl Z/N - benzyloxycarbonyl ,-1- alanyl - L alanyl)amino7 “ methylphosphonate of melting point 106°108°C? /&702θ=-37.4° (c=l% in glacial acetic acid).

Example 21.

In a manner analogous to Example 20 from dimethyl (L - alanylamino) - methylphosphonate hydrochloride and the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - leucine there was obtained dimethyl /(Ν benzyloxycarbonyl - L - leucyl - L - alanyl)aminq/ methylphosphonate of melting point 117°-119°C,· /ajq2°=“ 42.55° (c=l% in methanol).

Example 22 In a manner analogous to Example 20 from dimethyl (L - leucylamino) - methylphosphonate hydrochloride and the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine there was obtained dimethyl /“(N ~ benzyloxycarbonyl - L - alanyl - L - leucyl)amino7 methylphosphonate of melting point 163°~165°Cj /a7D2O=51.6° (c=l% in methanol).

Example 23.

In a manner analogous to Example 20 but using the 2,4,5 - trichlorophenyl ester of N - benzyloxycarbonyl - L - alanine instead of the N - hydroxysuccinimide ester 43467 -24 there was obtained dimethyl //N - benzyloxycarbonyl L - alanyl - L - alanyl)amino/ - methylphosphonate of melting point 106°-108°C; /jx7D2°=-36.7° (c=l% in glacial acetic acid).

Example 24 In a manner analogous to the first part of Example 20, from the N - hydroxysuccinimide ester of N benzyloxycarbonyl - L - alanine and dimethyl aminomethylphosphonate hydrochloride there was obtained dimethyl (L — alanylamino) - methylphosphonate hydrochloride.

Example 25. 139.7 g (0.5 moi) of dimethyl 1 - benzylami.no ethylphosphonate hydrochloride wore dissolved in 1000 ml of methanol. The solution was hydrogenated at room temperature and atmospheric pressure in the presence of g of 10% palladium-on-charcoal for several hours until the hydrogen uptake ceased. The catalyst was filtered off and the filtrate evaporated in vacuo. The residue of dimethyl 1 - aminoethylphosphonate hydrochloride was dissolved in 500 ml of dry dimethyl formamide and then treated with 160 g (0.5 mol) of the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine. While stirring and maintaining the temperature below 0°C, there were added dropwise 70 ml of dry triethylamine. The mixture was then stirred overnight at room temperature.

Further processing analogous to that given in Example 13 yielded a residue which, on treatment with 600 ml of dry ether, gave 72,5 g of dimethyl (IS) - 1 //N — benzyloxycarbonyl - L - alanyl) amino7 - ethyl30 phosphonate of melting point 134c-i35°C? /5/^^^=+14.9° (c=l% in methanol). Evaporation of the mother liquors gave ca 100 g of a gum consisting substantially of the corresponding R - isomer. 43467 - 25 Example 26.

In a manner analogous to that given in Example 13 from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl — L - alanine and dimethyl a - aminobenzylphosphonate hydrochloride, there was obtained an isomeric mixture. After chromatography on silica gel with an isopropanol/ethyl acetate eluant, followed by recrystallisation from ethyl acetate/ether, there were isolated dimethyl (IS) - a - /IN - benzyloxycarbonyl - L - alanyl)aminq7 τ· benzylphosphonate of melting point 103°-105°C Γ/ό)7ο2θ=-46.6ο fc=l% in methanol)/7 and dimethyl (lR) a - ZIN - benzyloxycarbonyl - L - alanyl)aminq7 benzylphosphonate of melting point 12Ο°-122°Ο//όί/ρ2Ο=+12.3° (c=l% in methanol)/7 Example 27. 100 g of the gum obtained according to Example 25 were dissolved in 500 ml of methanol containing 0.3 mol of hydrogen chloride. The solution was hydrogenated at room temperature and atmospheric pressure in the presence of 8 g of 10% palladium-on-charcoal until hydrogen uptake ceased. The catalyst was filtered off, the filtrate evaporated in-vacuo and the residue triturated with acetone. The solid was filtered off, washed with acetone and dried in vacuo. After reerystallisation from methanol/ether, there were obtained 42 g of dimethyl (IR) - 1 - (L - alanylamino) - ethylphosphonate hydrochloride of melting point 195°-198°C (decomposition); /&7D2O=-51.1° (c=l% in water).

In a manner analogous to that given in Example 13 from 13 g of dimethyl (lR) - 1 - (L - alanylamino) ethylphosphonate hydrochloride and 16 g of the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - I» - alanine there were obtained 16 g of dimethyl (IR) - 1 - /(N benzyloxycarbonyl - L - alanyl - L - alanyl)amino/ “ - 26 ethylphosphonate of melting point 149°-151°C; n20=65.5° (c=l% in methanol).

Example 28 In a manner analogous to that given in Example 27 from dimethyl (IR) - 1 - (L - alanylamino) - ethylphosphonate hydrochloride and the N - hydroxysuccinimide ester of N - benzyloxycarbonylglycine there was obtained dimethyl (IR) - 1 - //N - benzyloxycarbonyl - glycyl L - alanyl)aminq7 - ethylphosphonate as an oil with the expected N.M.R. spectrum.

Example 29.

In a manner analogous to that given in Example 27 from dimethyl (IR) - 1 - (L - alanylamino) - ethylphosphonate hydrochloride and the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - G - proline there was obtained dimethyl (IR) - 1 - /(N - benzyloxycarbonyl L - prolyl - L - alanyl)amino/ - ethylphosphonate as an oil with the expected N.M.R. spectrum.

Example 30.

In a manner analogous to that given in Example 27 from dimethyl (IR) - 1 - /IN - benzyloxycarbonyl - L alanyl - L - alanyl)amino/ - ethylphosphonate there was obtained as a gum dimethyl (IR) -1- (L - alanyl -L alanylamino) - ethylphosphonate hydrochloride. The latter compound was processed with the N - hydroxysuccinimide ester of N - benzyloxycarbonylglycine in a manner analogous to Example 13 to yield dimethyl (IR) - 1 - / (N benzyloxycarbonylglycyl - L - alanyl - L - alanyl)amino/ethylphosphonate of melting point 162°-164°Cr· /a7n2O=30 55.0° (c=l% in methanol). 2467 - 27 Example 31.

In a manner analogous to that given in Example 30 from dimethyl (IR) * 1 - (L - alanyl - L - alanylamino) ethylphosphonate hydrochloride and the N - hydroxysuccini5 mide ester of N - benzyloxycarbonyl - L - proline there was obtained dimethyl (IR) - 1 - /(N - benzyloxycarbonyl L prolyl - L - alanyl - L ·<· alanyl) amino/ - ethylphosphonate of melting point 181°-183°C; /g7D20=-100.3° (c=l% in methanol).

Example 32.

In a manner analogous to that given in Example 27 from dimethyl (IR) - 1 - /JN τ benzyloxycarbonyl glycyl - L - alanyl)amino/ - ethylphosphonate there was obtained dimethyl (IR) - 1 - (glycyl - L - alanylamino) 15 ethylphosphonate hydrochloride. The latter compound was processed with the N - hydroxysuccinimide ester of N benzyloxycarbonylglycine in a manner analogous to Example 13 to yield dimethyl (IR) - 1 - /IN - benzyloxycarbonylglycyl glycyl - L - alanyl)aminq7 - ethylphos20 phonate of melting point 124°-127°C; /α7ρ20=-36.6° (c=l% in methanol).

Example 33.

In a manner analogous to that given in Example 2 by the reaction of (1R,S) - 1 - aminoethylphosphonic acid with the mixed anhydride obtained from N - benzyloxycarbonylglycine and Isobutyl chloroformate there was obtained the benzylamine salt of (1R,S) - 1 - /'(M - benzyloxycarbonyl - glycyl)amino7 - ethylphosphonic acid of melting point 2O4°-2O6°C (decomposition). 2.1 g of the latter compound were converted to the free acid by ion exchange. The resulting acid was titrated with {+) -a - methylbenzylamine to pH 4.0, evaporated - 28 and then re-evaporated with methanol. The residue was crystallised from a mixture of 10 ml of methanol and 0.5 ml of water at 0°C to give 0.85 g of the crude (+) - a methylbenzylamine salt of melting point 202°-203°C (decom· position). Recrystallisation from butanol/water gave the (+) — a - methylbenzylamine salt of (IR) - 1 -/(N benzyloxycarbonyl - glycyl)aminq7 - ethylphosphonic acid of melting point 2O3°-2O4°C (decomposition) ; 14.8° (c=0.9% in water).

Example 34. 2.5 g (20 mmol) of (IR) - 1 - aminoethylphosphonic acid were stirred in 5 ml of water at 0°C while 5.6 ml (40 mmol) of triethylamine and 10 ml of dimethylformamide were added. 7.65 g (25 mmol) of solid N - hydroxysuccini15 mide ester of li - benzyloxycarbonylglycine were added in a single portion. The mixture was stirred for 3 hours at 0°C and then for 16 hours at room temperature. The mixture was worked up in a manner analogous to that given in Example 11. There were obtained 4.3 g of the benzylamine salt of (IR) - 1 - Z~(N - benzyloxycarbonyl - glycyl)aming7 - ethylphosphonic acid of melting point 198°-200°C (decomposition) ,- /q7D2O=-16.6° (c=l% in water).

Example 35. 0.88 g (7.0 mmol) of (IR) - 1 - aminoethylphos25 phonic acid were stirred in 100 ml of water at 5°C while 1.41 g (14 mmol) of triethylamine and 100 ml of ethanol were added. 2.42 g (7.0 mmol) of solid N - hydroxysuccinimide ester of N - benzyloxycarbonyl - Xj - proline were added and washed in with 50 ml of ethanol. The mixture was stirred at 0°C for 2 hours and then at room temperature for 72 hours. The mixture was woj’ked up in essentially the same manner as given in Example 11. After recrystal]1sation from methanol/ether, there were obtained 2.3 g of 43467 - 29 -τ the benzylamine salt of (IR) - 1 - /IN - benzyloxycarbonyl - L - prolyl)amino/ - ethylphosphonic acid of melting point 2O6°-2O9°C (decomposition); /q7D2O=-53.1° (c=0. 6% in glacial acetic acid).

Example 36 In a manner analogous to that given in Example 34, β from the N - hydroxysuccinimide ester of Ν , N - bis(benzyloxycarbonyl) - L - lysine there was obtained (lR) - 1 2 6 /IN ,N - bis - (benzyloxycarbonyl) - L - lysyl)aminq7 ~ ethylphosphonic acid of melting point 195°-197°C (decomposition) ; /0(7^20=-17.50 (c=0.5% in ethanol).

Example 37.

In a manner analogous to that given in Example 34 from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - leucine there was obtained the benzylamine salt of (lR) - 1 - /IN - benzyloxycarbonyl - L - leucyl)amine/ - ethylphosphonic acid of melting point 228°-23O°c (decomposition); .0° (c=0.5% in glacial acetic acid).

Example 38.

From N * benzyloxycarbonyl - L - valine, N - ethylmorpholine and (IR, - 1 - aminoethylphosphonic acid there was obtained in a manner analogous to that given in Example 2 the benzylamine salt of (IR) - 1 - ZIN - benzyloxycarbonyl - L - valyl)amino/ - ethylphosphonic acid of melting point 251°-252°C (decomposition); /57^20,-25.4° (c=0.5% in glacial acetic acid).

Example 39.

In a manner analogous to that given in Example 34 from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - phenylalanine there was obtained (IR) 1 ~ /IN - benzyloxycarbonyl - 1 - phenylalanyl)amino7 ~ 467 - 30 ethylphosphonic acid of melting point 212°-215°C (decomposition) ; /VD20_16 3o (c=0>5% ,n ethanol).

Example 40 In a manner analogous to that given in Example 7 but with ion-exchange in methanol/water, from the N hydroxysuccinimide ester of N - benzyloxycarbonyl - L phenylalanine there was obtained the benzylamine salt of /(N - benzyloxycarbonyl - L - phenylalanyl - L - alanyl)aming7 - methylphosphonic acid of melting point 233°234°C (decomposition); /&702θ=-2.7° (c=0.6% in glacial acetic acid).

Example 41 In a manner analogous to that given in Example 7 but with ion-exchange in methanol, from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - phenylalanine and (L - phenylalanylamino) - methylphosphonic acid there was obtained /IN - benzyloxycarbonyl - L phenylalanyl - L - phenylalanyl)amino/ - methylphosphonic acid of melting point 2OO°C-21O°C (decomposition).

Example 42 In a manner analogous to that given in Example 7 but with ion-exchange in methanol/water, from the N hydroxysuccinimide ester of N - benzyloxycarbonyl - L alanine and (L - phenylalanylamino) - methylphosphonic acid there was obtained the benzylamine salt of /(N benzyloxycarbonyl - L - alanyl - L - phenyl - alanyl)amino/ - methylphosphonic acid of melting point 232°234°C (decomposition); /ά/ο2Ο=+3.Οθ (c=0.6% in glacial acetic acid).

Example 43 In a manner analogous to that given in Example 34 but with ion «.-exchange in ethanol/water, from the N 43467 - 31 hydroxysuccinimide ester of N - benzyloxycarbonyl - L phenylalanine and (IR) - 1 - (L - alanylamino) - ethylphosphonic acid there was obtained (IR) - 1 - /IN benzyloxycarbonyl - L - phenylalanyl - L - alanyl)amin67 ethylphosphonic acid of melting point 22Οθ·*221°Ο (decomposition) ; /57^^=^27.1° (c=l.l% in glacial acetic acid).

Example 44 In a manner analogous to that given in Example 2 but using petroleum ether instead of toluene and methanol/ water for the ion-exchange, from N - benzyloxycarbonyl L - valine and (IR) - 1 - (L - alanylamino) - ethylphosphonic acid there was obtained the benzylamine salt of (IR) - 1 - /XN - benzyloxycarbonyl - L - valyl - L alanyl)amino/ - ethylphosphonic acid of melting point 25O°-251°C (decomposition); /tj7D2O=-47.2° (c=l% in glacial acetic acid).

Example 45 In a manner analogous to that given in Example 35 from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and (IR) - 1 τ (L - alanyl - L alanylamino) - ethylphosphonic acid there was obtained (IR) r· 1 - /IN - benzyloxycarbonyl - L - alanyl - L alanyl - L - alanyl)amino7 - ethylphosphonic acid of melting point 255°-257°C (decomposition); /q7D2(2=-62.0° (c=0.4% in glacial acetic acid).

Example 46 In a manner analogous to that given in Example 45 from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and (IR) - 1 - (L - alanyl - L alanyl - L - alanyl - amino) - ethylphosphonic acid there was obtained, on acidification and without recourse to ion-exchange, the free acid. The latter was filtered off, washed with water and acetone and dried to give the - 32 pure free acid, namely (IR) - 1 - /(N - benzyloxycarbonyl - X, - alanyl,- L - alanyl - L - alanyl)aminp7 - ethylphosphonic acid of melting point 27O°-275°C (decomposition) ; /q7D2O=-71.7° (c=O.54% in 1 - N sodium hydroxide).

Example 47 In a manner analogous to that given in Example 11 from the N - hydroxysuccinimide ester of N - benzyloxycarbonyl - L - alanine and (1R,S) - 1 amino - 2 - phenyl - ethylphosphonic acid there was 10 obtained a diasteromeric mixture of (1R,S) - 1 - £(N benzyloxycarbonyl - L - alanyl)amino7 - 2 - phenyl ethylphosphonic acid. The mixture was prepared by conversion to the benzylamine salts and crystallisation from water. There was obtained the benzylamine salt of (IR) - 1 - /(N - benzyloxycarbonyl - I. - alanyl)aminoT” 2 - phenyl — ethylphosphonic acid of melting point 223°226°C (decomposition); Za7D2O=-46.5° (c=O.53% in glacial acetic acid),

Claims (13)

1. CLAIMS:1. Compounds of formula wherein R 10 represents a hydrogen atom or a lower alkyl, lower cyeloalkyl, (lower cyeloalkyl - (lower alkyl), aryl or aryl - (lower alkyl) group (said groups being optionally substituted as the case may require by one or more amino, hydroxy, mercapto, methylthio, carboxy or guanidino groups so as to form the characterising group of an a - amino acid of the type found in proteins); R 20 and R 30 (where appropriate 30 1 in combination with R ) each represent the characterising group of an a - amino acid of the type 30' found in proteins; and R represents a hydrogen atom or is in combination with R 30 to form a characterising group of an a - amino acid with the proviso that R cannot represent a hydrogen atom when n is zero and R 10 is a hydrogen atom or the phenyl group, and that any amino group or amino groups present in R 10 , R 20 , R 30 and R 30 may be in protected form and any other functional group which may be present is optionally in protected form; R represents a methyl 41 41 group or R ; R represents a hydroxy group or lower alkoxy protecting group; R 3 represents a hydrogen atom or a protecting group,with the proviso that at least one of R 10 , R 20 , R 3 °, R 5 , R 40 and R 41 is a protecting group or is in protected form; tt stands for zero, 1, 2 or 3; single asterisks denote that 43467 - 34 10 when R 20 or R 30 represents other than a hydrogen atom, the configuration at the carbon atom somarked is L; and the double asterisk denotes that, when R 3-0 represents other than a hydrogen atom, the configuration at the carbon atom so-marked is that which would be obtained by replacing the carboxyl group of a naturally occurring L - a - amino acid by a phosphorus moiety and their salts.

2. Compounds according to claim 1, wherein R 3 represents a formyl group.

3. Compounds according to claim 1, wherein R 5 represents a benzyloxycarbonyl group.

4. Compounds of the general formula R 5 -NH „30 R 11 0 -CH-CO* -NH:-CH-CO * -NH--CH-P—-R . * ι '41 (la) 15 wherein

5. 7. A process for the manufacture of compounds of formula I as claimed in Claim 1, which process comprises condensing a compound of the general formula R 20 _ R 10 0 H· -NH-CH* -CO -NH-CH* * ,40 (II) wherein 5 20 30 40 41 R , R' , R , R and R have the significance given in Claim 1; R 3-3- represents a hydrogen atom or a lower alkyl aryl or aryl - (lower alkyl) group; and m stands for zero, 1 or 2; the single asterisks denote 20 that, when R 20 or R 30 represents other than a hydrogen atom, the configuration at the carbon atom somarked is L; and the double asterisk denotes that, when R 3-3 · represents other than a hydrogen atom, the configuration at the carbon atom so-marked is that 25 which would be obtained by replacing the carboxyl group of a naturally occurring L - a - amino acid by a phosphorus moiety, and their salts. 43467 - 35 5. Compounds according to Claim 4, wherein R 3 represents a formyl group.

6. Compounds according to Claim 4, wherein R 3 represents a benzyloxycarbonyl group.

7. Wherein the condensation is carried out using an (R,S) compound corresponding to formula II and the (R) compound 8. Wherein the condensation is carried out using an (R,S) compound corresponding to formula Ila and the (R) compound is separated from the resulting (R,S) product in a manner known per se.

8. A process for the manufacture of compounds of formula Ia as claimed in Claim 4, which comprises condensing a compound of general formula ,20 R 11 0 -NH-CH-C0* -NH-CH— P-R * * (Ila) wherein 43467 R 11 , R 20 , R 40 , R 43 ·, and the single and double asterisk have the significance given in Claim 4, with an appropriately protected a- amino acid, an appropriately protected dipeptide, an appropriately protected ,. tripeptide or a reactive derivative thereof as the case may require.

9. A modification of the process according to Claim

10. A modification of the process according to Claim 10 is separated from the resulting (R,S) product in a manner known per se. 10 R 10 , R 20 , R 40 , R 41 , n and the single and double asterisks have the significance given in Claim 1, with an appropriately protected a - amino acid, an appropriately protected dipeptide, an appropriately protected tripeptide or a reactive derivative thereof as the ca3e 15 may require.

11. A process for the manufacture of the compounds as set forth in Claim 1 substantially as hereinbefore described with reference to any of the Examples. 2q

12. Compounds as set forth in Claim 1 when manufactured by a process claimed in any one of Claims 7, 9 and 11.

13. Compounds as set forth in Claim 4 when manufactured by a process claimed in either of Claims 8 and 10.