MC1242A1 - PEPTIDIC DERIVATIVES, PROCESS FOR THEIR PREPARATION AND THEIR APPLICATION IN PHARMACEUTICAL PREPARATIONS - Google Patents

Description

1

The present invention relates to peptide derivatives. More particularly, the invention relates to peptide derivatives of phosphonic acids, a process for their preparation, and pharmaceutical preparations containing them.⁵ The peptide derivatives provided by the invention are compounds of the general formula

R2 R1 0 OH

1 1 1/

H9N-CH CO NH CH P (I)

10 OH

where R"5* represents a hydrogen atom, a methyl group or hydro-

2

xymethyl or a mono-, di- or trihalomethyl group, R represents a lower alkyl, a hydroxy-(lower alkyl) or guanidino-(lower alkyl) group other than the group characterizing 15 of an alpha-amino acid of the type normally encountered in proteins; the configuration at the carbon atom designated as (a) is (R) when R^" represents something other than a hydrogen atom; and the configuration at the carbon atom designated as (b) is (L),

20 and their salts usable in pharmacies.

In this description, the term "lower alkyl"

is used to describe a straight-chain or branched alkyl group preferably having up to 8 carbon atoms. Examples of such lower alkyl groups include ethyl, propy-

25. For example, butyl, tert-butyl, pentyl, hexyl, etc.

/■ 2

Examples of hydroxy-(lower alkyl) groups denoted by R include 2-hydroxyechyl, 3-hydroxypropyl, 4-hydroxybutyl, etc. The term "halo" represents a fluoro, chloro, bromo, or iodo group, and examples of the halomethyl groups mentioned above include chloromethyl, dichloromethyl, trifluoromethyl, etc.

When R"*" in formula I represents something other than a hydrogen atom, the configuration at the carbon atom designated by . (a) is (R); that is to say, it is the configuration 35 that would be obtained by replacing the carboxyl group of an alpha-amino acid found in nature with a phosphorus fraction.

As a preferred class of peptide derivatives supplied by

2

The invention requires mentioning the compounds of formula I where R"1"

represents a hydrogen atom or the methyl group, and their salts usable in pharmacy. Compounds are also preferred

2

formula I or R represents a lower alkyl group, in particular the ethyl, n-propyl or n-butyl group.

Examples of compounds with formula I include: Xi' acid (1R) -l-^/IL-fct-aminobutyryl ) amino/-ethylphospho~

fuck,

(1R)-l-(L-norleucylamino)-ethylphosphonic acid, 10 (IR)-l-(L-norvalylamino)-ethylphosphonic acid,

L ' acid' (1R) -l-/L-(o( -aminobutyryl) amino/-methylphospho-

fuck,

(1R)-l-(L-norleucylamino)-methylphosphonic acid, (1R)-l-(L-norvalylamino)-methylphosphonic acid, (1R)-l-(L-homoarginylamino)-ethylphosphonic acid,

And

(1R)-l-(L-homoarginylamino)-methylphosphonic acid

According to the invention, the above-mentioned peptide derivatives are prepared (i.e., compounds of formula I and

2o their salts usable in pharmacy) by a process in which a compound of general formula is condensed.

R10 0 ^

OR3.

H0N— fcH P^ (II)

25 ^ OR

where R^° has one of the values assigned to R"*" above, where R1 represents a protected hydroxymethyl group; R3 and R^ each represent a hydrogen atom or a lower alkyl protecting group; and the configuration at carbon atom (a) is (R) when-

30 that R"*"° represents something other than a hydrogen atom;

with a protected alpha-amino acid of general formula

R20

5 I X

R Cil C ^ (III)

35

(b) \

OH

0.20

C, "2Q

where R represents a protected amino group; R has one of the values

3

7

granted to R" above or represents a corresponding hydroxy-(lower alkyl) or guanidino-(lower alkyl) group in protected form; and the configuration at the carbon atom called (b) is (L),

5 we separate the protecting group or groups in the condensation product and, if desired, we transform a compound of formula I obtained into a salt usable in pharmacy.

The protected amino group denoted by R in a protected amino acid starting product of formula III above can be any well-known protected amino group in peptide chemistry. In a preferred illustration of the process provided by the invention, the amino group is protected by an arCl-oxycarbonyl group, in particular the benzyloxycarbonyl group, or the tert-butoxycarbonyl group. However, the amino group can also be protected with, for example, a formyl, trityl, or tri-group.

fluoroacetyl or 2-(biphenyl)-isopropyloxycarbon, or it may be in the form of a phthali group. The protecting group present in a protected hydroxymethyl group R or a hydroxy- group

' 20

(lower alkyl) protected R can be any group

20 classic hydroxy protectant; for example, an aralkoxycarbonyl group (e.g. the benzyloxycarbonyl group), a lower alkanoyl group (e.g. acetyl, propionyl, etc. groups), an aroyl group (e.g. the benzoyl group), a lower alkyl group (e.g. the tert-butyl group or a 25 lower aralkyl group (e.g. the benzyl group).

The condensation of a compound of formula II with a protected alpha-amino acid of formula III can be carried out using processes well known in peptide chemistry; for example, according to the mixed anhydride process, the azohydride process, the activated ester process, the acid chloride process, the carbodiimide process, or the EEDQ (1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) process.

In one process, a compound of formula II can be condensed with a protected alpha-amino acid of formula III, where the carboxy group is a mixed anhydride residue formed with an organic or inorganic acid. It is convenient to treat such a protected alpha-amino acid with a tertiary base such as a tri(lower alkyl)amine (e.g., triethylamine) or N-ethyl raorpholine.

4

in an inert organic solvent (e.g. tetrahydrofuran, 1,2-dimethoxyethane, dichloromethane, toluene,

petroleum ether, etc.), and the resulting salt is reacted with a suitable chloroformate (for example, a lower 5-alcohol chloroformate such as ethyl chloroformate or isobutyl chloroformate) at low temperature. The mixed anhydride thus obtained is then suitably condensed in situ with a compound of formula II.

In another process, a compound of formula II can be condensed with a protected alpha-amino acid of formula III where the t»

The carboxy group is in the form of an acid azothydride. This condensation is preferably carried out in an inert organic solvent such as DMF or ethyl acetate at low temperature.

There is yet another method where one can condense a compound of formula II with an alpha-amino acid of formula III where the carboxy group is in the form of an active ester group (for example the 4-nitrophenyl, 2,4,5-trichlorophenyl or N-hydroxysuccinimidester group). This condensation is conveniently carried out in an inert solvent such as aqueous DMF or, where 20 R3 and in a compound of formula II both represent a lower alkoxy group, also in a lower alkanol such as aqueous ethanol.

In another process, a compound of formula II can be condensed with a protected alpha-amino acid of formula III where the

25. The carboxy group is in the form of an acid chloride. This con-\.

densification is preferably carried out in the presence of a base and at low temperature.

In yet another process, a compound of formula II can be condensed with a protected alpha-amino acid of formula III in the presence of a carbodiimide (e.g., dicyclohexylcarbodiimide) or EEDQo. This condensation can be carried out in an inert organic solvent (e.g., methyl chloride or a lower alkanool such as methanol, ethanol, etc.) at room temperature or below. The cleavage of the protecting group(s) of the condensation product is carried out according to conventional methods; that is, the methods actually used or described in the literature.

5

Methods for cleaving protecting groups. Thus, for example, an aralcoxycarbonyl group (e.g., benzyloxycarbonyl), a tert-butoxycarbonyl group, or a 2-biphenyl-isopropyloxycarbonyl group can be separated by hydrolysis (e.g., by treatment with hydrobromic acid in glacial acetic acid). An aralcoxycarbonyl group (e.g., benzyloxycarbonyl) can also be separated by hydrogenolysis (e.g., in the presence of palladium on carbon or platinum oxide). A tert-butoxycarbonyl or 2-biphenyl-isopropyloxycarbonyl group can also be separated using hydrochloric acid in dioxane. A trityl group can be separated, for example, by treatment with dilute acetic acid. A phthalimido group can be converted to an amino group by hydrazinolysis. The lower alkyl protecting groups, denoted by R 15, can be separated by treatment with hydrobromic acid in glacial acetic acid, or with trimethylhydrorosilane or trimethylbromosilane followed by aqueous hydrolysis. It should be noted that when 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 preferable to use protecting groups that can be separated in a single step.

The compounds of formula I above are amphoteric and form usable pharmaceutical salts with the strong acids usable in pharmacy (for example hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, para-toluenesulfonic acid, etc.) and the bases usable in pharmacy (for example sodium hydroxide, potassium hydroxide, etc.).

30 The starting products of formulas II and III are known compounds or can be prepared by analogy with the preparation of known compounds.

•The peptide derivatives provided by the invention possess antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria such as Escherichia coli, Staphylococcus aureus, Serratia marcescens, Klebsiella aeroquenes, Streptococcus faecalis, Haemophilus influenzae, Bacillus subtilis,

6

Salmonella tvphimuriumr Proteus mirabilis. Pseudomonas aeruginosa and Shiqella Sonnei.

Furthermore, the present peptide derivatives enhance the activity of antibiotics including penicillin, cephalosporin antibiotics, and D-cycloserine. Among the antibiotics whose action is enhanced by the present peptide derivatives, we can mention amoxicillin, cephradine, cephalothin, cephalexin, carbenicillin, ampicillin, penicillin G, sulbenicillin, cephazoline, cefoxytinoin, rifampicin, (R)-1-(2-furoyloxy)-3-methylbutylpenicillin, and (6R)-1-hexahydro-1H-azepin(1-yl)-methylene/ami-

penicillanic acid, (pivaloyloxy)methyl (6R)-6-^/*(hexahydro-.lH~ azepin-l-yl)methylene/iamino/penicillanate, cephamandole, cephaloridine, cephaloglycine, phenethicillin, methicillin-15 line, propicillin, ticaracillin, amoxicillin arginine salt, phosphonomycin, vancomycin and cannamycin*

The invention therefore also provides a pharmaceutical preparation containing a peptide derivative mentioned above and, if desired, an antibiotic, in association with a compatible pharmaceutical carrier.

The carrier present in the pharmaceutical preparations provided by the invention may be any solid or liquid carrier that is compatible with the aforementioned peptide derivatives, and with antibiotics when present, and suitable for therapeutic administration. The carrier may

A pharmaceutical preparation can be an organic or inorganic carrier suitable for enteral (e.g., oral) or partial enteral administration. Examples of such carriers include water, gelatin, lactose, starches, magnesium stearate, talc, vegetable oils/acacia gum, polyalkylene glycols, petrolatum, etc. Pharmaceutical preparations can be in solid form (e.g., tablets, coated tablets, suppositories, or capsules) or in liquid form (e.g., solutions, suspensions, or emulsions). Pharmaceutical preparations may be subjected to standard pharmaceutical operations such as sterilization and may contain excipients such as preservatives, antacids, and other active ingredients.

7

Stabilizers, wetting agents, emulsifying agents, salts to vary osrootic pressure, or buffers. When using a buffer, the pH of the pharmaceutical preparation can naturally vary within a range known in pharmaceutical practice.

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

The daily dose of peptide derivative administered alone 15 or in combination with an antibiotic varies 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 treated0 For example, when administering a peptide derivative alone, the daily dose for oral administration may be about 2000 mg to 4000 mg and the daily dose for parenteral administration may be about 800 mg to 2000 mg. When a peptide derivative is administered in combination with an antibiotic, the daily dose for oral administration may be approximately 750 mg to 1500 mg of the combination of the peptide derivative and the antibiotic, and the daily dose for parenteral administration may be approximately 200 mg to 2000 mg of the combination of the peptide derivative and the antibiotic. It should be noted that the daily doses may be administered once or several times, and that the figures mentioned above may be adjusted up or down according to individual needs and may be adapted to the requirements of a particular situation at the discretion of the treating physician.

The following examples illustrate the process provided by

35 the invention -{L

8

EXAMPLE 1

^ Preparation of the starting product:

5.0 g (48 mM) of L-alpha-aminobutyric acid is dissolved in 24 mL of 2 N sodium hydroxide, and the solution is cooled to 0°C. Alternately and in several stages, 12.3 g (72 mM) of benzyl chloroformate and 18 mL (72 mM) of 4 N sodium hydroxide are added while stirring for 30 minutes to ensure that the temperature does not exceed 10°C and the pH remains around 11. The mixture is allowed to slowly reach room temperature and then stirred overnight. The mixture is extracted with 50 mL of diethyl ether, and the aqueous and organic phases are separated. The aqueous phase is acidified to pH 2 with 5 N hydrochloric acid to give an oily mixture. This mixture is extracted with two 75 mL fractions of diethyl ether. The 15 extracts are dried on sodium sulfate and evaporated to give

9.6 g of a colorless oil. This oil is dissolved in 10 ml of ethyl acetate, and 100 ml of petroleum ether (boiling point 40-60°C) is added to give an oily solution, which is left to stand at 0°C. A white crystalline precipitate is obtained, which is separated by filtration, washed with petroleum ether, and dried to give 8.40 g (73%) of N-benzyloxycarbonyl L-aminobutyric acid (boiling point 76-78°C); 10.9%; 20.3%.

(c = 1% in ethanol).

(B) Procedure:

25 (i) 8.30 g (35 mM) of N-benzoyloxycarbonyl-L-alpha-aminobutyric acid and 4.02 g (35 mM) of N-hydroxysuccinimide are dissolved in 75 mL of dimethoxyethane while stirring, and the solution is chilled to 0°C. 7.94 g (38.5 mM) of dicyclohexylcarbodiimide is added, and the mixture is stirred at 0°C for 2 h, then left to stand at 0°C overnight. The sub-

solid product. The filtrate is evaporated to give the N-hydro-xysuccinimidester of N-benzyloxycarbonyl-L-alpha-aminobutyric acid in the form of a thick gum which is used without further purification.

35 (ii) 4.38 g (35 mM) of (1R)-1-aminoethylphosphonic acid is dissolved in a mixture of 40 mL of water, 7.07 g (70 mM) of triethylamine, and 40 mL of DMF while stirring. The solution is cooled.

9

The solution is obtained at 0°C, and a 35 mM solution of N-hydroxysuccinimide ester of N-benzyloxycarbonyl-L-aminobutyric acid is rapidly added dropwise to 40 mL of DMF. The mixture is stirred at 0°C and then allowed to return to room temperature while stirring overnight. The mixture is filtered to remove a small amount of solid. The filtrate is evaporated under vacuum using an oil pump. The residue is dissolved in a mixture of 30 mL of methanol and 10 mL of water and passed through a cation exchange resin column (BDH, 10 Zerolit 225, SRC 13, RSO^H; 150 g; freshly regenerated in the acid cycle), with elution carried out using the same solvent. The eluate is collected and evaporated to give a slightly gummy white solid. This solid is triturated with 100 mL of diethyl ether and the diethyl ether is separated by decantation. The residual solid is dissolved in a mixture of 100 mL of methanol and 50 mL of water and the resulting solution, at pH 4.5, is titrated with 4 N aqueous benzylamine (8.0 mL titration; 8.75 mL theoretical quantity). The mixture solidifies towards the end of the titration, and the precipitate is separated by filtration, washed with water, and dried to give 8.84 g of the monobenzylamine salt of (1R)-1-α-benzyloxycarbonyl-L-alpha-aminobutyryl)amine-β-ethylphosphonic acid. (Pf 228° -231°C (dec.); -31.3°;) &2° = ~i05° (c =

1% in glacial acetic acid)

(iii) 8.7 g (19.3 mM) of the monobenzylamine salt of (1R)-1/(N-benzyloxycarbonyl-1/2-aminobutyryl)aminoethylphosphonic acid is stirred at room temperature for 6 hours with 20 mL of a mixture of 45% hydrobromic acid in glacial acetic acid and 8 mL of glacial acetic acid. 100 mL of ether is added, and a gum precipitates. This gum is washed by decantation with 100 mL of diethyl ether and dissolved in 100 mL of methanol. The solution is stirred, and two 5 mL fractions of propylene oxide at a pH close to 5 are added, resulting in a white precipitate. The mixture is allowed to stand overnight, the precipitate is separated by filtration, and then washed successively with

♦5 r methanol and diethyl ether and dried to give 4.05 g of crude (1R)-1-(L-alpha-aminobutyryl-amino)-ethylphosphonic acid

298° (dec.). Recrystallization from a mixture of 750 m.

10

Mixing 1500 ml of water with ethanol produces a white crystalline precipitate which is washed with ethanol, then with diethyl ether, and finally dried under vacuum. This yields 3.58 g (88%).

of (1R)-1- (L-^-aminobutyrylamino)-ethylphosphonic acid of P^ 29 6-5 298° (dec.) ; -33.1° ; = = sodium hydroxide 1 N, freshly prepared).

EXAMPLE 2

(A) Preparation of the starting product:

5.0 g (42.5 mM) of L-norvaline is treated with 10.2 g 10⁻¹⁰ (60 mM) of benzyl chloroformate and sodium hydroxide as described in Example 1 (A). The crude oily product is crystallized from a mixture of 10 mL of diethyl ether and 20 mL of petroleum ether (P₂, 40–60°C) to give 8.2 g (77%).

^ o of N-benzyloxycarbonyl-L-norvaline at P„ 85°-87°C ; £&/y. = -9.9° ;

9 n

15 ^*^365 = "26.5° (c = 1% in ethanol).

• (b) Process

(i) In a manner analogous to what was described in example 1(B)

(i) , starting from 8.1 g (32 mMol) of N-benzyloxycarbonyl-L-norva-line, 3.68 g (<32 mMol) of N-hydroxysuccinimide and 7.21 g (35

20 mMol) of dicyclohexylcarbodiimide yields, after trituration of the oily product with 25 ml of ethanol, a white crystalline product. After the addition of 25 ml of petroleum ether (Rg^ 40-60°C) and filtration, 9.82 g of N-hydroxysuccimidester of N-benzyloxycarbonyl-L-norvaline is obtained, which is used directly in the next step. A purified sample melts at 95-97°C and exhibits a rotation of -35.1° (c = 1% in ethanol).

(ii) In a manner analogous to that described in Example 1(B)(ii), starting from 9.82 g (28 mMol) of N-hydroxysuccinimidester of N-benzyloxycarbonyl-L-norvaline and 3.75 g (30 mMol) of (1R) acid

30 1-aminoethylphosphonic acid, but by performing the trituration with a mixture of 150 ml of methanol and 30 ml of water instead of diethyl ether, 7.61 g of the monobenzylamine salt of (1R)-1-(N-benzyloxycarbonyl-L-norvalyl)amino-ethylphosphonic acid is obtained. Pf 225°-230°C (dec.) /^°= -29.9° ; = -98.8° (c = 1%

35 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 then with diethyl ether gives a second collection.

11

1.99 g of monobenzylamine salt of (1R)-l~/N-benzyloxycarbonyl-L-norvalyl)amino7-ethylphosphonic acid at 231°-234°C (decomposition); - "■ 29.7°; = -99.2° (c = 1% in acetic acid). Total yield: 9.60 g (73%).

5 (iii) In a manner analogous to what has been described in the example

1(B) (iii), starting from 9.0 g (19 mMol) of the monobenzylamine salt of (1R)-l-/N-benzyloxycarbonyl-L-norvalyl)amino7~ethyl-phosphonic acid, after recrystallization from a mixture of 80 ml of water and 160 ml of ethanol, 3.54 g (82%) of (1R)-1-(L-norvalylamino)-ethylphosphonic acid is obtained at P^ 260°-262°C (decomposition); -19.5°; ~ -75.3° (c = 1% in water).

EXAMPLE 3

(A) Preparation of the starting product 15 In a manner similar to what was described in the example

1 (A) 5.0 g (38 mM) of L-norleucine is treated with 9.7 g (57 mM) of benzyl chloroformate and sodium hydroxide. The product, N-benzyloxycarbonyl-L-norleucine, is isolated as an oil which is used in the process without crystallization. 20 (B) Process

(i) Analogously to what was stated in Example 1(B)(i), starting from approximately 38 mM of N-benzyloxycarbonyl-L-norleucine, 4.38 g (38 mM) of N-hydroxysuccinmide and 8.65 g (42 mM) of dicyclohexylcarbodiimide, after trituration of the pro-

25 crude oily milk with 100 ml of diethyl ether, 7.47 g of N-hydro-

N-benzyloxycarbonyl-L-norleucine xysuccinimidester in the form

20

of a crystalline solid of P^ 82°-840C ; = -20.7° (c = 1% in acetone).

(ii) Analogously to what was described in Example 1(B) 30 (ii), starting from 7.40 g (20.5 mMol) of N-hydroxysuccinimidester of N-benzyloxycarbonyl-L-norleucine and 2.56 g (20.5 mMol) of (1R)-1-aminoethylphosphonic acid, but carrying out the ion exchange step in methanol/water (5:1) instead of methanol/water (3:1), after evaporation and trituration of the crude product 35 with ether, a solid is obtained which is digested with 150 mL of hot water, cooled and filtered to give 5.64 g (74%) of (1R)-1-/N-benzyloxycarbonyl-L-norleucyl)amino/-ethylphosphonic acid of P^ 192-

THERE

194°C (decomposition); /%720= -37.8°; /V355 = -126.5° (c = 0.5% in acetic acid).

(iii) Analogously to what was described in Example 1 (B) (iii), starting from 5.20 g (14.0 mMol) of (1R)-1-(N-5 benzyloxycarbonyl-L-norleucyl)aminoethylphosphonic acid, after recrystallization from a mixture of 70 mL of water and 280 mL of ethanol, yields 3.02 g (91%) of (1R)-1-(L-norleucyl-amino)ethylphosphonic acid with a decomposition temperature of 254°–256°C; α₇ = -18.6°C; α₃ = -70.1°C (c = 1% in water). 10 EXAMPLE 4

(a) In a manner analogous to what was stated in Example 1(B)(ii) and Example 2(B)(ii), starting with 17.4 g (50 mMol) of N-hydroxysuccinimidester of N-benzyloxycarbonyl-L-norvaline and 5.55 g (50 mMol) of aminomethylphosphonic acid, but carrying out the

15. Titration of benzylamine in a mixture of 250 ml of methanol and 50 ml of water yields 17.3 g of the crude monobenzylamine salt of the desired product, with a pH of 195-198° (dec.) (sample 1). After evaporation of the filtrate and recrystallization of the residue from a mixture of methanol (80 ml) and water (20 ml), another 20.19 g of a very pure product (thin-layer chromatography) with a pH of 180-185° (dec.) is obtained. Total yield: 19.5 g (86%)

Recrystallization of 0.5 g of the harvest sample from 10 ml of hot water gives a harvest of 0.39 g of the pure monobenzylamine salt of the acid (N-benzyloxycarbonyl-L-norvalyl)

2o

25 amino-methylphosphonic of P^ 203-205° (dec.) ; ^Ç7n = -8.2° ;

OH

/*ê?365 = "21/3° (c = 0.5% in acetic acid).

(b) In a manner analogous to what was described in Example 1 (B) (iii), starting from 18.9 g (42 mMol) of the monobenzylamine salt of (N-benzyloxycarbonyl-L-norvalyl)-aminomethylphosphonic acid

30 obtains, after recrystallization from a mixture of 80 ml of water and 160 ml of ethanol, 6.65 g (75%) of L-(norvalylamino)methylphosphonic acid of P^ '273-275° (dec) ; = + 61.2° , /^y^gg =

+ 224° (c = 0.54%, H2O).

EXAMPLE 5

35 N-benzyloxycarbonyl-L-norvaline (45.4 g,

0.181 mol) in a methylene dichloride solution (200 ml)

while adding dimethyl-aminomethylphosphonate hydrochloride

13

(31.76 g, 0.181 mol). The resulting suspension is cooled to -12°C by adding dry triethylamine dropwise (25.3 ml, 0.181 mol). After the addition is complete, the cold mixture is stirred for 15 minutes before rapidly adding E.E.D.Q. (56.8 g, 0.23 mol) to methylene dichloride (100 ml). This mixture is stirred at room temperature for 2 hours and then at room temperature over the weekend.

The mixture is washed with water (100 ml) and then with hydrochloric acid (4 times, 100 ml each time). The total acidic wash water is re-extracted with methylene dichloride (2 x 50 ml). The organic solution is washed again with water (100 ml) and finally with a 15% KI-ICO^ solution (3 x 100 ml); then dried over anhydrous sodium sulfate. The solution is filtered and concentrated. The residual oil is reconcentrated with benzene to give a residue weighing 78.13 g.

The first signs of crystallization are then observed. The oil is triturated with anhydrous ether (200 ml) in which it dissolves. Very quickly, the crystallization spreads throughout the solution. The solution is cooled for 1 hour, the solid is separated by filtration, washed with ether, and dried to constant weight under vacuum. 58.2 g of a product at 77-80°C is obtained.

This product is absorbed into very hot ethyl acetate (200 ml) and the solution is filtered. Ether (200 ml) is added to the cooled filtrate. The mixture is inoculated and cooled overnight. The solid is separated by filtration, washed with ether, and dried under vacuum to give 50.64 g of a residue at 82-84°C.

• 37.2 g of (N-benzyloxycarbonyl-L-norvalyl)aminodimethylphosphonate is stirred in 45% hydrobromic acid in 120 ml of acetic acid for 5 h. Initially, the release of carbon dioxide is very rapid. Ether (500 ml) is added, causing an oil to precipitate. The mixture is stirred for 40 min, then allowed to settle. The ether is separated by decantation, and the oil is washed twice more with ether (500 ml each time).

The oil is absorbed into methanol (300 ml) and the solution is shaken while adding propylene oxide (40 ml). After

14

After approximately 5 minutes, the product begins to separate by crystallization. The mixture is adjusted to pH 4 and cooled overnight.

The solid is separated by filtration and carefully washed with methanol, then with ether. It is dried under vacuum, to give a residue of 20.13 g, having a P^ of 288-289°C (dec.).

This is absorbed into very hot water (200 ml) and filtered. 400 ml of ethanol is added to the filtrate. After letting

After 10 minutes of rest, the product separates by crystallization. It is cooled for 2 hours. The solid is separated by filtration, washed with ethanol and then ether, and dried under phosphorus pentoxide in a vacuum. 16.66 g of (L-norvalylamino)-methylphosphonic acid with a boiling point of 293-4°C (d) and a concentration of +62.7°C (c = 0.5% in water) is obtained.

15 EXAMPLE 6

(a) L-nitro-homoarginine (2.33 g, 10 mM, P^227-230°, (dec)) is treated with 3.41 g (20 mM) of benzyl chloroform and sodium hydroxide in the manner described in Example 1 (A). The crude oily product is extracted from the acidified solution with 20% ethyl acetate (twice with 100 mL) instead of diethyl ether. The crude oily product, N-benzyloxycarbonyl-W-nitro-L-homoarginine, obtained after drying over sodium sulfate and evaporation, is in the form of a gum which is used in the next step without further purification.

25 (b) In a manner analogous to what was described in example 1 (B)

(i), but using dimethylformamide (50 ml) as solvent, from about 4.2 g (approx. 10 mMol) of crude N-benzyloxycarbonyl-W-nitro-L-homoarginine (obtained as above), 1.15 g (10 mMol) of N-hydroxysuccinimide and 2.27 g (11 mMol) are obtained

30 of dicyclohexylcarbodiimide, and after removal of 1.73 g of dicyclohexyl xylene and evaporation of the filtrate, the crude N-hydroxysuccinimidester of N-benzyloxycarbonyl-W-nitro-L-homoarginine in the form of a yellow oil which is used in the next step without further purification.

35 (c) In a manner analogous to what was described in example 1(B)

(ii) , approximately 10 mMol of N-hydroxysuccinimidester of N-benzyloxycarbonyl-W-nitro-L-homoarginine is reacted with 1.25 g of (1R) acid

15

-1-aminoethylphosphonic acid, but the treatment is carried out with an RSO3H ion-exchange resin in aqueous methanol (5:1 instead of 3:1). The acidic eluate is evaporated and divided between water (300 mL) and ethyl acetate (150 mL), yielding an insoluble material. This is separated by filtration and dried to give 1.36 g of crude product with a P^ of 190-193° (dec.), characterized by thin-layer chromatography and NMR (sample 1).

The layers of the filtered solvent are separated, the aqueous layer is evaporated to dryness, and the residue is triturated with acetone (50 mL) to give a white precipitate. This precipitate is separated by filtration, washed with acetone, and dried to yield another 1.43 g (P^ 184-188° dec) of the product, (1R)-1/^N-benzyloxycarbonyl-1-nitro-1-homo-arginyl)amino-15-ethylphosphonic acid (harvest 2). Harvests 1 and 2 are used in the next step without further purification.

(d) Analogously to what was described in Example 1(B)(iii), starting from 2.7 g (approximately 5.7 mMol) of (1R)-1-/N-benzylpxycarbonyl-U-nitro-L-hcmoarginyl)amine-7-ethylphosphonic acid 20 (harvests 1 and 2' above) after recrystallization

from a mixture of 15 ml of water and 150 ml of ethanol, 1.23 g of (1R)-1-(L-CJ-nitro-homoarginylamino)-ethylphosphonic acid of P^ approximately 190° (dec); -10.6°; /ÔÇ/355 = -43.5° (c = 0.53%

h2°). .

25 Evaporation of the filtrate and recrystallization from water (5 ml) and ethanol (100 ml) yields an additional temperature of approximately 190° (dec); /°C/q

0.14 g of product having a P^ of approximately 190° (dec); -11.2'

^365 = ~43'2° (c = °'51% H20) *

Total yield: 1.37 g (71%).

30 (e) 1.2 g (3.5 mM) of (îr)-i-l-u/-nitro-homoarginylamino-ethylphosphonic acid prepared as above is absorbed into 50 mL of water, and 0.35 g of 10% Pd on charcoal is added. The mixture is hydrogenated at ambient temperature and pressure until the absorption is complete (approximately 5 h). The mixture is filtered to remove the catalyst, and the filtrate is evaporated. The gummy solid residue is absorbed into 15 mL of cold water, then the solution is filtered and treated with 120 mL of ethanol to give a precipitate

16

which is left to rest for 1 hour and then separated by filtration.

We obtain 0.76 g of (1R)-1-(L-homoarginylamino)-ethylphos-

20

phonic temperature of Pf approximately 195° (dec); ~ -11.9° (c = 0.5% H2O)

EXAMPLE 7

5 (i) Analogously to what was described in Example 1(B)(ii), starting from a 45 mM solution of N-hydroxysuccinamide ester of N-benzyloxycarbonyl-L-α-aminobutyric acid in 40 mL of dimethylformamide and 4.44 g (40 mM) of aminomethylphosphonic acid, but using methanol:water in a 4:1 ratio instead of 2:1, 14.1 g of the monobenzylamine salt of (N-benzyloxycarbonyl-L-α-aminobutyryl)aminomethylphosphonic acid of P₁₈₅₁₀ ... supplement of 1.6.g 15 of product in the form of a white crystalline precipitate of P^ 205-208° (dec); -6.1°; -21.1° (c = 0.51% CH3CO0H) .

(ii) In a manner analogous to what was described in example 1(B)

(iii), starting from 15.6 g (approx. 36 mMol) of the monobenzylamine salt of the acid ^(N-benzyloxycarbonyl-L-sC-aminobutyryl) amino/-

20 methylphosphonic acid prepared as above gives 5.92 g of crude product, P^ 253-255° (dec). Recrystallization from a mixture of 60 ml of water and 120 ml of ethanol gives 4.93 g of L~«C-aminobutyrylamino)-methylphosphonic acid of Pf 263-265° (dec); /&l°+ 57.0° ; ^355 + 212° (c = 0.51% H2O).

25 EXAMPLE 8

(i) In a manner analogous to what was described in example 3(B)

(ii) , starting from 2.52 g (7 mMol) of N-hydroxysuccinimidester of N-benzyloxycarbonyl-L-norleucine and 0.78 g (7 mMol) of aminomethylphosphonic acid, but proceeding with the ion exchange step

30 in methanol/water at a ratio of 2:1 instead of 5:1 and by titrating the acid eluate directly with benzylamine (omitting evaporation and trituration with ether followed again by dissolution in methanol/water), 0.56 g of the monobenzylamine salt of α-35(N-benzyloxycarbonyl-L-norleucyl)amino-methylphosphonic acid (Pf185-189° dec) is obtained after stirring with 100 ml of acetone. After evaporation of the filtrate and trituration with 50 ml of diethyl ether, an additional 1.9 g of

17

of product, P^ 170-180° (dec.).

(ii) Analogously to what was described in Example 1(B)(iii), starting from 2.40 g (approximately 5.2 mMol) of the monobenzylamine salt of L-(H-benzyloxycarbonyl-L-norleucyl) 5-aminomethylphosphonic acid, prepared as above, and using both harvests, 1.04 g of crude product with a Pf of 263-266° (dec.) is obtained. After recrystallization from a mixture of 20 ml of water and 120 ml of ethanol, 0.88 g of L-norleucyl-aminomethylphosphonic acid with a Pf of 272-274° (dec.); 10 + 63.4°; 355 + 231° (c = 0.5%, H2O) is obtained.

The following example illustrates a characteristic pharmaceutical preparation containing the peptide derivatives provided by the invention.

EXAMPLE A

15. A parenteral formulation can be prepared containing the following ingredients:

Per 1000 ml

Peptide derivative of formula I 50.0 g

Chlorocresol 1.0 g pQ

Glacial acetic acid 1.2 g

Sodium hydroxide solution (0.1 N)

q.s. ad pH 4.5

Injectable water ad 1000 ml

25

18

Claims (1)

CLAIMS 1, General formula compounds .2 "1 0 f f I! . CH CO NH CH P OH 5 H0N—CH CO NH CH P (I) 2 <a> \0H where R*1" represents a hydrogen atom, a methyl or hydroxymethyl group, or a mono-, di-, or trihalomethyl group, R represents a lower alkyl, a hydroxy-(lower alkyl) group, or a guanidino-(lower alkyl) group other than the characterizing group of an alpha-amino acid of the type normally encountered in proteins; the configuration at the carbon atom designated as (a) is (R) when R"*" represents something other than a hydrogen atom, and the configuration at the carbon atom designated as (b) is 15 (L). and their salts that can be used in pharmacies. 2„ Peptide derivatives as claimed in the x 2 claim 1, or R represents a lower alkyl or lower hydroxyalkyl group other than the characterizing group of an alpha-amino acid of the type normally found in proteins. 3. Peptide derivatives according to claim 1 or 2, where R"*" represents a hydrogen atom or the methyl group. 4. Peptide derivatives according to any one of the claims 2 ✓ 1-3, where R represents a lower alkyl group. 2 25 5. Peptide derivatives according to claim 4, where R represents the ethyl, n-propyl or n-butyl group. 6. (1R)-l-/t-Ccx-Aminobutyryl) amino/-ethylphosphonic acid and its salts usable in pharmacy. 7. (1R)-l-(L-Norleucylamino)-ethylphosphonic acid 30 and its salts usable in pharmacy. 8. (1R)-l-(L-Norvalylamino)-ethylphosphonic acid and its salts usable in pharmacy. 9. L-(cc-Aminobutyrylamino)-methylphosphonic acid and its salts usable in pharmacy. 35 10° L-Norleucylaitiino-methylphosphonic acid and its salts usable in pharmacy "11. (L-Norvalylamino)-methylphosphonic acid and its salts usable in pharmacy. 19 12. (1R)-l-(L-HoiTioarginylBiino)-ethylphosphonic acid and its salts usable in pharmacy, 13. (L-Homoarginylamino)-methylphosphonic acid and its salts usable in pharmacy 14» General formula compounds R20 R10 O OR3 5 L 1 'I ^ r CH CO NH CH P R" CH CO NH CH P IV (b) (a) \ ^ OR where R3 and R^ each represent a hydrogen atom or a group 5 lower alkyl protector, R represents a protected amino group, R"*"0 has one of the values assigned to R"1" above or represents 20 a protected hydroxymethyl group, and R has one of the values assigned to R above or represents a corresponding lower hydroxyalkoyl or guanidino(lower alkyl) group in protected form; the configuration at carbon atom (a) is (R) when R10 represents something other than hydrogen and the configuration at the carbon atom (b) is (L). 15. (1R)-l-/*(N-Benzyloxycarbonyl-L-norvalyl)amino 20 ethylphosphonic acid. 16. The (1R)-l~/"(N-Benzyloxycarbonyl-W-nitro~L-homo-arginyl)aminq7-ethylphosphonic acid. 17. Peptide derivatives according to any one of claims 1 to 13 as pharmaceutical agents, 25 18. Peptide derivatives according to claim 17 as antibacterial agents or as agents enhancing the activity of antibiotics. 19. A process for preparing the peptide derivatives claimed in claim 1, a process in which a compound of general formula 30 is condensed R10 O GOLD 35 HON— CH—P ^ (II) (al \ 4 OR . ^ where R"*"0 is one of the values assigned to R"*" above or represents a protected hydroxymethyl group; R^ and R^ each represent a 20 15 hydrogen atom or a lower alkyl protecting group; and the configuration at the carbon atom (a) is (r) when R"'"0 represents something other than a hydrogen atom; with a protected alpha-amino acid of general formula 0.20 0 X, , R^— CH C (III) (b) \ OH 0.5 5 ^ 20 where R represents a protected amino group; r to one of the values 10 2 assigned to R above or represents a corresponding hydroxy-(lower alkyl) or guanidino-(lower alkyl) group in protected form; and the configuration at the carbon atom (b) is (L), we separate the protective group(s) present s) in the condensation product and, if desired, a compound of formula I obtained is transformed into a salt usable in pharmacy. 20. Pharmaceutical preparation containing a peptide derivative as claimed in any one of claims 1 to 13, or a salt usable in pharmacies from this body, ... in association with a compatible pharmaceutical support. 20 21. Pharmaceutical preparation containing a peptide derivative as claimed in any one of claims 1 to 13, or a body salt usable in pharmacy, and an antibiotic, in association with a compatible pharmaceutical carrier. 22. Peptide derivative as claimed in claim 1, or salt of this substance usable in pharmacy, when prepared by the process claimed in claim 19 or by an obvious chemical equivalent of this process. Original in step®8 30' _. containing References __ added word crossed out word harm Ai Industrial Property Consultant 26bl1, Boul. Princess Charlotte MONTE-CARLO By proxy <£© 'T. ScxsT'èVé CWxor^V^. sr K ex.e