MC1348A1 - PEPTIDE DERIVATIVES - Google Patents

Description

1

The present invention relates to peptide derivatives. More particularly, it relates to peptide derivatives of phosphonic acids, a process for their preparation, and pharmaceutical preparations containing them. The invention also relates to the use of these derivatives.

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

3 2 ' 1

R R R ' 0

L 1 1 1 11 OH

10 R — NH — CH — NH — CH — CO — NH — CH — P _

(c) (b) (a) OH

1

in which R represents a hydrogen atom or the

2 3

methyl group, R and R each represent a lower alkyl group other than the characteristic group 15 of an alpha-amino acid of the type normally found in proteins or one of the substituents R and R^ represents such a lower alkyl group and the other represents the characteristic group of an alpha-amino acid of the type normally found in proteins, R^" represents the L-pyroglutamyl group, the configuration at the carbon atom designated by (a) is (R)

HAS

where R represents the methyl group and the configuration at the carbon atoms designated by (b) and (c) is L 2 3

when R or R, as the case may be, represents something other than a hydrogen atom, and their pharmaceutically acceptable salts.

The term "lower alkyl" is used in this description to refer to a straight-chain or branched-chain alkyl group containing up to 8 carbon atoms. Examples of lower alkyl groups that are other than the characteristic groups of alpha-amino acids of the type normally found in proteins are ethyl, propyl, nutyl, pentyl, hexyl, etc. The term "characteristic group of an alpha-amino acid of the type normally found in proteins" is used in this description.

2

denotes residue R in a natural alpha-amino acid of the general formula

R

i

H2N-CH-C00H

5, which is of the type normally found in proteins. Thus, for example, if the alpha-amino acid is glycine, then R represents a hydrogen atom, and if the alpha-amino acid is alanine, then R represents the methyl group. Furthermore, for example, in me-10-thionine, R represents the 2-methylthioethyl group, in serine, R represents the hydroxymethyl group, and in tyrosine, R represents the p-hydroxybenzyl group. R can also represent a residue that is bonded to the nitrogen atom of the amino group (with the loss of one of the bonded hydrogen atoms) to form a nitrogen-containing ring, as in proline.

When R in formula I represents the methyl group, the configuration at the carbon atom designated by (a) is (R); that is, the configuration which would be obtained by replacing the carboxyl group of an alpha-amino acid existing in nature with a phosphorus portion.

-1

Compounds of formula I in which R represents the methyl group are preferred.

2 3

25 also compounds of formula I in which R and R

each represent a lower alkyl group other than the characteristic group of an alpha-amino acid of the type normally found in proteins.

Examples of compounds with formula I above 30 are as follows:

(1R)-l-(L-pyroglutamyl-L-norvalylamino-ethylphosphonic acid,

(L-pyroglutamyl-L-norvalylamino)-methylphosphonic acid,

35 acid (lR)-l--/L-pyroglutamyl-L-(2-aminoheptanoyl)-

L-norvalylamine β7-ethylphosphonic acid and ^

b

3

(iR)-l-(L-pyroglutamyl-L-alanyl-L-norvalyl-amino)-ethylphosphonic acid.

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

R30 R20 R1 0

OH

H2N-CH-C0-NH-CH-C0-NH-CH-P II

10 (c) (b) (a) OH

1

in which R has the meaning indicated above,

R2*"* and R3<^ have any of the meanings

2 3

indicated for R and R above, except that any amino group present is protected by a protecting group 15 susceptible to cleavage by hydrogenolysis and the configuration at the carbon atoms designated by (a), (b) and (c) is as specified above, with a compound of the general formula

CH0— CH0

He

20 C (d) CH — CT III

0 N ^ OR

'r6

5

in which R represents a 2,4,5-trichloro- group

Fi phenyl, pentachlorophenyl or succinimidyl, R represents a hydrogen atom or an aralkoxy-car- group

bonyl and the configuration at the carbon atom designated by (d) is L, by subjecting a condensation product obtained that contains a protected amino group and/or an aralcoxycarbonyl group R^ to hydrogenolysis and, if desired, by transforming a compound of formula

I obtained it in a pharmaceutically acceptable salt.

Examples of protecting groups susceptible to cleavage by hydrogenolysis that can be u-

4

20

used to protect an amino group present in R

30

or R in formula II are aralkoxycarbonyl groups (e.g., benzyloxycarbonyl). An amino group can also be protected by a nitro group, such as 5 in the case of the nitroarginyl group.

The condensation of a compound of formula II with a compound of formula III can be carried out under conditions well known in peptide chemistry for condensations involving activated esters. For example, the condensation can be performed in an organic solvent such as aqueous dimethylformamide at approximately 0°C. In a preferred embodiment of the process, a compound of formula II is condensed with a compound of

C

15 formula III in which R represents the 2,4,5-trichlorophenyl group.

A condensation product containing a protected amino group and/or an aralkoxycarbonyl group R^ is subjected to hydrogenolysis to transform the protected amino group into an amino group and to eliminate the aralkoxycarbonyl group represented by R^, as appropriate. This hydrogenolysis can be carried out using techniques that are themselves known; for example, in the presence of a noble metal catalyst such as palladium/carbon or platinum oxide.

Pharmaceutically acceptable salts of formula I compounds are formed with pharmaceutically acceptable strong acids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, 30 methanesulfonic acid, p-toluenesulfonic acid, etc.) and bases (e.g. sodium hydroxide, potassium hydroxide, etc.).

The starting materials for formula II above are known.

35 The starting materials of formula III above

5

can be prepared, for example, by reacting L-pyroglutamic acid or N-aralcoxycarbonyl-L-pyroglutamic acid with 2,4,5-trichlorophenol, pentachlorophenol, or N-hydroxysuccinimide in the presence of N,N'-dicyclohexylcarbodiimide and an inert organic solvent such as dimethylformamide. When N-aralcoxycarbonyl-L-pyroglutamic acid is used in this reaction, the resulting compound of

formula III in which R represents an aralkoxy-10 carbonyl group can be hydrogenolyzed in the manner described above to give a corresponding compound of formu-

g the III in which R represents a hydrogen atom.

When using a compound of formula III in which

5 6

R represents a succinimidyl group and R represents

15 a hydrogen atom, this compound is preferably for-

îû 5 in the condensation mixture.

The peptide derivatives provided by the present invention possess antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria, including Escherichia coli, Klebsiella spp., Serratia marcescens, Staphylococcus albus, Salmonella spp., Shigella spp., Streptococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, and Haemophilus influenzae. They can therefore be used in the prevention and treatment of bacterial infections. These peptide derivatives can be administered parenterally.

The following table gives the results obtained in an in vitro test using (1R)-1-(L-py-30 roglutamyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid against representative organisms.

6

Painting

5

10

Organization

M.I.C. ( µg/ml)

E. coli

0.25

Klebsiella spp.

16

S. marcescens

30

S. albus

• 16

Salmonella spp.

20

Shigella spp.

1.1

S. faecalis

0.05

S. pyogenes

16

S. pneumoniae

2.4

H. influenzae

0.2

15 The peptide derivatives provided by the present invention also potentiate the activity of antibiotics.

Among the antibiotics that are potentiated by the present peptide derivatives, the 20 beta-lactam antibiotics such as penicillins are preferred.

cephalosporins or monocyclic beta-lactam antibiotics.

Examples of usable penicillins include: ampicillin, carbenicillin, penicillin G, sulbeni-25-cillin, mecillinam, pivmecillinam, phenethicillin,

methicillin, propicillin, ticarcillin, amoxycillin, piperacillin, etc.

Examples of usable cephalosporins include: cephalexin, cephalozin, cefoxitin, cephradine, cefsulodine, cephamandole, cephaloridine, cephaloglycine,

Cefatrizine, cefacetrile, cefuroxime, cefaclor, cefotaxime, cefazedone, etc. Cephalexin is preferred.

Examples of other antibiotics include: D-cycloserine, rifampicin, phosphonomycin, gentamicin, vancomycin, kanamycin, etc.

7

The antibiotics mentioned above are known substances and can be obtained through processes that are themselves known.

The present invention therefore also provides a pharmaceutical preparation containing a pep-tide derivative described above and, if desired, an antibiotic, in association with a compatible pharmaceutical vehicle.

The vehicle in the pharmaceutical preparations provided by the present invention may be any solid or liquid material usable as a vehicle that is compatible with the peptide derivatives described above, and with antibiotics when present, and that is suitable for therapeutic administration. The vehicle may be an organic or inorganic material usable for parenteral administration. Examples of such vehicles are water, gelatin, mannitol, mineral oils, vegetable oils, acacia gum, propylene glycol, and polyalkylene glycols. The pharmaceutical preparations may be presented in a solid form (e.g., as lyophilized powders) or in a liquid form (e.g., as solutions, suspensions, or emulsions). Pharmaceutical preparations may undergo standard pharmaceutical processes such as sterilization and may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents, salts to adjust osmotic pressure, or buffers. When a buffer is used, the pH of the pharmaceutical preparation will obviously vary within a range that is well-established in pharmaceutical practice.

When these pharmaceutical preparations contain a peptide derivative and an antibiotic, the weight ratio of the peptide derivative to the antibiotic

8

may vary within wide limits. In general, pharmaceutical preparations may contain the peptide derivative and the antibiotic in a weight ratio of between 1:100 and 100:1, preferably in a weight ratio of between 1:64 and 64:1 and especially in a weight ratio of between 1:16 and 16:1.

The daily dose of a peptide derivative, administered alone or in combination with an antibiotic, will vary within a wide range depending on factors such as the specific peptide derivative chosen, the specific antibiotic chosen, and the infection being treated. For example, when a peptide derivative is administered alone, a daily parenteral dose may range from approximately 800 to 2000 mg. When a peptide derivative is administered in combination with an antibiotic, a daily parenteral dose may range from approximately 200 to 2000 mg of a combination of peptide derivative and antibiotic. It should be understood that daily doses may be administered all at once or in divided doses, and that the doses mentioned above may vary up or down depending on individual needs and the requirements of a particular situation, as determined by the treating physician.

The following examples illustrate how the peptide derivatives provided by the invention can be prepared.

Example 1

8.0 g (25 mmol) of (1R)-1-(L-norvalyl-L-norvalylamino)-ethylphosphonic acid is dissolved in a me-

"z layer of 125 cm of water and 5.1 g (50 mmol) of sorted-

3

thylamine, while stirring. 125 cm³ of dimethylformamide is added and the solution is cooled to 0°C. 20.3 g (66 mmol) of 2,4,5-trichlorophenyl ester of L-pyroglutamic acid is added and washed with 125 cm³ of dimethylformamide at 0°C. The

9

The mixture is stirred while the surrounding cooling bath slowly warms to room temperature, and stirring is continued overnight at room temperature. The resulting suspension is filtered, and the gelatinous precipitate is washed with aqueous dimethylformamide. The filtrate and combined washing liquids are evaporated to dryness under reduced pressure, and the residue is triturated with 200 mL of ethyl oxide. The ethyl oxide and solid matter are removed by decantation.

'3 10 residual is dissolved in a mixture of 500 cm of

3

methanol and 100 cm³ of water are added. 200 g of Zerolit 225, SRC 13, RSO₄H ion exchange resin are added and the mixture is stirred for approximately 5 minutes, then filtered. The resin is washed with aqueous methanol and evaporated.

15 dry the filtrate and the combined washing liquids.

3

The residue is triturated with 250 ml of acetone and then stirred at room temperature for several hours. The precipitate is separated by filtration and washed with acetone and then with ethyl oxide, yielding 4.5 g of (iR)-l-(L-pyroglutamyl-L-

norvalylamino)-ethylphosphonic acid with a melting point of 260°C (decomposition); = -84.5° (c = 0.55%

in trifluoroacetic acid).

Example 2

25. In a manner analogous to that described in Example 1, from 9.27 g (30 mmols) of (-L-norvalyl-L-norvalyl-amino)-methylphosphonic acid and 18.5 g (60 mmols) of the 2,4,5-trichlorophenyl ester of L-pyroglutamic acid, 9.1 g of crude product with a melting point of 205-210°C (decomposition) is obtained after trituration with acetone; = -58.9°

= -178° (c = 0.5% in water). Recrystallization from a water/ethanol mixture (1:4 v/v) gives (L-pyroglutamyl-L-norvalyl-norvalylamino)-35-methylphosphonic acid with a melting point of 230-232°C (de

composition); = -69.6°; 35^ = -222° (c =

10

0.5% in water).

Example 3

In a manner analogous to that described in Example 1, starting from 4.2 g (12 mmols) of (1R)-1-r~(L-2-aminoheptanoyl)-L-norvalylamin£7-ethylphosphonic acid and 9.3 g (30 mmols) of 2,4,5-trichlorophenyl ester of L-pyroglutamic acid, but using a methanol/water mixture (2:1 v/v) in the ion exchange step, 1.64 g of crude product is obtained.

PO

melting point of 225-230°C (decomposition); =

-63.9°; 355 = -208° (c = 0.48% in trifluoroacid

(roacetic). Purification by stirring with methanol at room temperature overnight, filtration, washing the filtration residue with methanol and then with ethyl oxide gives (1R)-1-/L-pyroglutamyl-L-(2-aminoheptanol)-L-norvalyl-mino7-ethylphosphonic acid with a melting point of 235-237°C (decomposition); /ô^_7D2° = -70.6°; /~c<_7 365 ~ "232° (c = 0.5% in trifluoroacetic acid).

The (1R)-1-(L-2-aminoheptanoyl)-L-norvalyl-amino7-ethylphosphonic acid used as a starting material can be prepared as follows:

(a) 3.6 g (25 mmol) of L-2- acid are dissolved

aminoheptanoic acid (Biol. Chem. 203, 333 (1953)) in

___

25 cm³ (25 mmol) of sodium hydroxide N₂O is stirred. The resulting solution is cooled to 0°C while 10 cm³ (40 mmol) of sodium hydroxide 4N and 6.5 g (38 mmol) of benzyl chloroformate are alternately added in 5 portions over a period of 0.5 hours, with the temperature maintained at 0–10°C. The mixture is stirred for a further 4 hours while the temperature is allowed to rise to room temperature. The mixture is then treated by ethyl oxide extraction, and the ethyl oxide extracts are backwashed with water. The combined aqueous extracts are acidified to approximately pH 2.0 with

11

10

15

20

25

30

of 6N hydrochloric acid. The oily mixture is treated by three-time ethyl oxide extraction, the combined ethyl oxide extracts are dried over sodium sulfate, filtered and evaporated to give approximately 7.8 g of N-benzyloxycarbonyl-L-2-aminoheptanoic acid in the form of an oil.

(b) The oil obtained according to the preceding paragraph is reconstituted in 75 cm³ of dimethoxyethane while being stirred, and 2.9 g (25 mmol) of N-hydroxysuccinimide is added. The mixture is cooled to 0°C, and 5.6 g (27.5 mmol) of dicyclohexylcarbodiimide is added. The resulting mixture is stirred at 0°C overnight. The solid by-product (dicyclohexylurea; 5.9 g) is separated by filtration, and the filtrate is evaporated to obtain an oil, which is triturated with petroleum ether to obtain a sticky white solid. This is left at 0°C overnight and then filtered to obtain 9.2 g of N- ester

N-benzyloxycarbonyl-L-2-ami- acid hydroxysuccinimide

PO

noheptanoic with a melting point of 65-67°C •• r«Jv = -23.6°; /~c£7 = -80.9° (c = 1% in ethanol).

(c) 4.48 g (20 mmol) of (lR)-l-(L-norvalylamino)-ethylphosphonic acid is dissolved in a mixture of 50 cm³ of water and 4.04 g (40 mmol) of sorted

•3

thylamine is stirred. 50 cm³ of dimethylformamide is added and the solution is cooled to 0°C. 9.2 g (24 mmol) of N-benzyloxycarbonyl-L-2-aminoheptanoic acid N-hydroxysuccinimide ester in 50 cm³ of dimethylformamide is rapidly added while maintaining the internal temperature at 0-5°C. The mixture is stirred while the cooling bath slowly warms to room temperature, and stirring is continued at room temperature overnight. The mixture is then filtered and the filtrate is evaporated.

~Z

to obtain a white, gelatinous residue. 200 ml of water is added to this residue to obtain a suspension which is

12

3

treated with approximately 20 cm of 2N hydrochloric acid, a thick precipitate is obtained. This precipitate is stirred at room temperature for several hours, separated by filtration, and washed with water, yielding 11.9 g of a thick solid. This solid is stirred with acetone for several hours, yielding 8.7 g (90%) of (1R)-1-N-benzyloxy-carbonyl-L-(2-aminoheptanoyl)-L-norvalyl-7-amino-7-ethylphosphonic acid with a melting point of 180-190°C (decomposition), which is used in the next step without further purification.

(d) 8.7 g (18 mmol) of (1R)-1-α-N-benzyloxycarbonyl-L-(2-aminoheptanoyl)-L-norvalyl-amino7-ethylphosphonic acid is stirred with a mixture of 20 cm³ of 45% α-15 hydrobromic acid in glacial acetic acid

at room temperature for 4 hours. 150 cm³ of ethyl oxide is added to precipitate a gum which is washed by decantation with an additional amount of 3

a 150 cm³ ethyl oxide residue. The residual gum

3

20 is dissolved in 100 cm of methanol while being stirred

3

and we add 10 cm of propylene oxide, a precipitate

3

a gelatinous substance is obtained. Another 100 mL of methanol is added, and the mixture is left at room temperature overnight. The precipitate is separated by filtration and washed first with methanol and then with

ethyl oxide is used to obtain 6.3 g of crude product with a melting point of 250-255°C (decomposition). The mixture is then shaken.

3

This raw product is placed in 250 cm of hot water, cooled, filtered, washed with ethanol and then with ethyl oxide, and finally dried to obtain

5.36 g of (1R)-1-/L-2-aminoheptanoyl)-L-norvalylami-no7-ethylphosphonic acid with a melting point of 268-272°C (decomposition); μc<.7 3^ = -89.0° (c = 0.36% in 2N hydrochloric acid). Further purification of a sample gives a substance with a melting point of 170-272°C (decomposition); μc<.7 3^ = -25.6°; μc<.7 = -90.7° (c = 0.34% in 2N hydrochloric acid).

13

10

15

20

25

Example 4

Similar to that described in Example 1, starting with 4.65 g (15 mmol) of (1R)-l-(L-alanyl-L-norvalylamino)-ethylphosphonic acid and 11 g (35 mmol) of the 2,4,5-trichlorophenyl ester of L-pyroglutamic acid, but using a methanol/water mixture (l/l v/v) for the ion exchange step, and carrying out the elution with the same solvent system, an acidic eluate is obtained, yielding a solid collection in the collection vessel. By filtration, 2.76 g of crude product with a melting point of 272–274°C (decomposition) is obtained. Evaporation of the filtrate and trituration of the residue with acetone yields an additional 3.28 g of crude product with a melting point of 268–270°C (decomposition). The above crude products are combined and stirred at room temperature with 75 cm³ of acetone for 3 hours. After filtration, 5.74 g of (1R)-1-(L(pyroglutamyl-L-alanyl-L-norvanylamino)-ethylphosphonic acid with a melting point of 262–264°C (decomposition) are obtained.

position); £~cAJ q° = -99.6”; /o<7 §°5 = "332°

(c = 0.5% in trifluoroacetic acid).

The following examples illustrate typical pharmaceutical preparations containing the peptide derivatives provided by the present invention.

Example A

A parenteral composition containing the following ingredients can be prepared:

Example B

Hard gelatin capsules can be prepared

For 1000 cm'

Peptide derivative

Sodium hydroxide solution (0.2N); q.s. for

Water for injection, supplement to

pH 6.5, nN A 3

1000 cm'

50.0 g

14

containing the following ingredients:

Peptide derivative

Cephalexin

Corn starch

Microcrystalline cellulose

Sodium dioctyl sulfosuccinate

Stearic acid

Per capsule 125.0 mg 125.0 mg 20.0 mg 34.0 mg 0.5 mg 5.5 mg

Total contents of one capsule: 310.0 mg

15

Claims (1)

DEMANDS 1) A process for the preparation of peptide derivatives of the general formula R3 R2 R1 0 _ OH L t i I II R — NH—CH—CO—NH — CH — CO — NH —CH—P (c) (b) (c) OH •i in which R represents a hydrogen atom or the 2 3 methyl group, R and R each represent a lower alkyl group other than the characteristic group 10 of an alpha-amino acid of the type normally found 2 3 in proteins or one of the substituents R and R represents such a lower alkyl group and the other represents the characteristic group of an alpha-amino acid of the type normally found in proteins, R^" represents the L-pyroglutamyl group, the configuration at the carbon atom designated by (a) is (R) -i when R represents the methyl group and the configuration 2 to the carbon atom designated by (b) is L when R or R as the case may be represents something other than a hydrogen atom, and of their pharmaceutically acceptable salts, according to which a compound of the generalized formula is condensed R30 R20 R1 0 I l I ll/°H HoN-CH-C0-NH-CH-C0-NH-CH-P,11 2 ^ 25 OH -j in which R has the meaning indicated above, R20 and R30 have any of the following meanings 2 3 indicated for R and R above except that any amino group present is protected by a protecting group 30 susceptible to cleavage by hydrogenolysis, and the configuration at the carbon atoms designated by (a), (b) and (c) is as specified above, with a compound of the general formula Îl 16 CH0 — CH0 I 2 I 2 C (d) CH — C / \ / \ OR5 0 N I 5 R6 5 in which R represents a 2,4,5-trichlorophenyl, pentachlorophenyl, or succinimidyl group, R^ represents a hydrogen atom or an arlkoxycarbonyl group, and the configuration at the carbon atom designated by 10(d) is L, a condensation product obtained containing a protected amino group and/or an arlkoxycarbonyl group R^ is subjected to hydrogenolysis and, if desired, a compound of formula I obtained is transformed into a pharmaceutically acceptable salt. 15 2) A method according to claim 1, characterized -i in that R in a compound of formula II is a methyl group. / 3) A method according to one of claims 1 or 2, characterized in that R2^ and R3^ in a compound of 20 formula I each represent a lower alkyl group other than the characteristic group of an alpha-amino-a-acid of the type normally found in proteins. 4) A process according to any one of claims 1 to 3, characterized in that it uses the acid (iR)-l-(L-nor- 25 valyl-amino)-ethylphosphonic as starting compound of formula II. 5) A method according to any one of claims 1 to 3, characterized in that the acid (1R)-1- is used r(L-2-amino-heptanoyl)-L-norvalylamin£7~ethylpho sphoni-30 as a starting compound of formula II. 6) A process according to any one of claims 1 to 3, characterized in that (iR)-l-(ala-nyl-L-norvalylamino)-ethylphosphonic acid is used as the starting compound of formula II. 17 7) A process according to claim 1, characterized in that (L-norvalyl-L-norvalylamino)-methylphosphonic acid is used as the starting compound of formula II. 8) A process for preparing pharmaceutical compositions containing a peptide derivative of formula I given in claim 1, wherein the peptide derivative or its salt is mixed with a pharmaceutically acceptable vehicle and, if desired, with an antibiotic. 9) A pharmaceutical composition containing a peptide derivative of formula I given in claim 1 in association with a compatible vehicle and, if desired, an antibiotic. 10) The compounds of formula I given in claim 1 prepared by a process according to any one of claims 1 to 3. 11) The (iR)-l-(L-pyroglutamyl-L-norvalyl-L-norvalyl-amino)-ethylphosphonic acid prepared by the process according to claim 4 or by an obvious chemical equivalent. 12) The (L-pyroglutamyl-L-norvalyl-L-norvalylami-no)-methylphosphonic acid prepared by the process according to claim 7 or by an obvious chemical equivalent. 13) The (1R)-1-f~L-pyroglutamyl-L-(2-aminohep-tanoyl)-L-norvalylamino7-ethylphosphonic acid prepared by the process according to claim 5 or by an obvious chemical equivalent. 14) The (iR)-l-(L-pyroglutamyl-L-alanyl-L-nor-vanyl-amino)-ethylphosphonic acid prepared by the process according to claim 6 or by an obvious chemical equivalent.