AU511452B2 - Peptide derivatives - Google Patents

Description

COMMONWEALTH OF AUSTRALIA PATENU. ACT 1952-f

AT> F. HOFFMANN-LA ROCHE CO. AKTIENGESELLSCHAFT

124-184 Grenzacherstrasse, Basle, Swtzerland

hereby apply for the grant of a Patent for an invention entitled

"PEPTIDE DERIVATIVES AND PROCESS FOR THE PREPARATION THEREOF" b* p p 0 *0

0*00 p

*6U0 p 0 which is described in the accompanying complete specification. p This application is a Convention Application and is based on the p p 0 0 application numbered 200] 4/76 pp p *p 3, for a patent or similar protection made in Great Bri tain p

APPULCATION ACCETED AN At,-DV1M p

ALLOWED ~~0 p *0*

on 14th May, 1976.

0P p

Ourf address for service is: p

0 Care: SPRUSON FERGUSON P0 PATENT ATTORNEYS p. ESSO HOUSE, 127 KENT STREET SYDNEY. NEW SOUTH WALES. AUSTRALIA.

Dated this 12th day of April, 17J F. HOFFMNN-LA ROOH&

By:

TO Registered Patent, Attorney PAT. lt TIhe Commissioner of Patents

FOR1\1 4 COMMONWEALTH OF AUSTRALIA PATENTS'ACT 1952-69

DECLARATION IN SUPPORT OF A CONVENTION APPLJCATION FOR A PATENT OR PATENT OF ADDITION In support of the Convention Application made for a

patentfor an invention entitled

"PEPTIDE DERIVATIVE.S AND '?RQCESS FOR THE~ PREPARATION THEREOF"

Full name and ad- 1, Hans StUckl in, dress of Declarant.

Of 35 Im Hirshaim, Riehen, Switzerland

do solemnly and sincerely declare as follows:-. 4~.I~*ppiient~forthe.P Tc1 (or, in the case of an application by a body corporate) 1. I am authorised by F. Hoffmann.-La Roche Co. Aktiengesellschaft the applicant for the patent tomake this declaration on its behalf.

Insert country and 2. The basic application as defined by Section 141 of the Act was made in S* date of basic appli- 9 9 9 cation and name of Great Britain on the 0* foreign applicant.. 14th day of M a y, 1976 by 9 9

Roche Products Limited, Welwyn Garden City, Herts., 9998 S 9 Great Britain. *9O*

S. 9 5 9 9 I Tam 0i- nf fl-n nnt;-u info-n f-u 1-n, n~~fn- Si,. 9 I 0@ S (or where a person other than the inventor is the applicant) S 9 *9 C Full name and ad- dress of Inventor(s) 3.a) Frank Ratcliffe Atherton, b)Michael John Hall, c) Cedric Herbert Hassall, d) Robert Wilson Lambert, e) Peter Stuart Ringrose OS of S 5*5 a) -48 Parkway, Weiwy-n Garden City, Herts., Great Britain, 9, b) 15 Cannonsfield Road, Welwyn, Herts., G-reat Britain, 0 0e;- c) "Trelech", Tewin Close, Tewin Wood, Welwyn, Herts.,Great Britain, d) 6 Grange Hill, Welwyn, Herts., Great Britain, S 09 S e) 41 The Limes, Harston, Cambridgeshire, Great Britain 99@ S

are is the actual inve-itor s of the invention and tile facts upon which the applicant is/ase-entitled to make the applicaticn are as follows: Roche Products Limited, .:::*:Welw)vn*Garden City, Heits., Great Britain is the assignee of the said Frank Ratcliffe Atherton, Michael John Hall, Cedric Herbert Hassall, Robert Wilson Lambert and Peter Stuart Ringrose and F.Hoffmann-La Roche Co. Aktiengesellschaft, Basle, Switzerland, is the assignee of the said Roche Products Limited. 4. The basic application referred to in paragraph 2 of this Declaration was the first application made in a Convention country in respect of the invention the subject of the application. Declared at Ba st dao pi, 1977.

.9 MY 977

To: T 0FFIcE Siglature of Declarant The Commissioner of Patents, SPRUGON FERGUSON, SYDNEY.

(11) AU-Bl-25,008/77

(12) PATENT SPECIFICATION ABRIDGEMENT (19) AU

(21) 25,008/77 511,452 (22) 9.5.77 (23) 9.5 77 (24) 14.5.76 (31) 20014/76 (32) 14.5.76 (33) GB (43) 16.11.78 (44) 21.8.80 (51)2 C07C 103/52 A61K 37/02 (54) Peptide derivatives (71) F. Hoffmann-La Roche Co. Aktiengesellschaft (72) Atherton, Hall, Hall C.H., Lambert, R.W. and Ringrose, P.S. (74) SF (56) 10,354/76 502,919 C07F, C07C, A61K (57) As used i this specification and claims, the expression "the characterising group of an a-amino acid of the type normally found in proteins" means the residue R it a natural C-amino acid of the general formula

H CH--COOH R

which is of the type normally occurring in proteins; by "the a-amino acids of the type normally occurring in proteins" is meant thoseo-amino acids listed in Fieser and Fiester "Organic Chemistry" 3rd Edition Rheinhold N.Y. 1956 at page 419. The pepLide of formula I derivatives provided by the present invention potentiate the activity of D-cycloserine. In addition, they possess an antibacterial activity against organisms such as Escherichia coli, Klebsiella aerogenes, Streptococcus feecalis and Haemophilus influenzae. The compounds of formulae I and II are claimed per SE. Claim 1. A process for the manufacture of peptide derivatives of the general formula /2

31:2

(11) AU-B1-25,008/77 2

R3 R2 0

H2N -H-COt-NH-CH-CO-NH-- CH- P-R (I) Sn I OH

,wherein n stands for 1,2 or 3; R R and R each represent the characterising group of an <-amino acid of the type normally found in proteins with the pro- visos that R 3 represent other than a hydrogen atom and that R represent a methyl group when n stands for 1; R represents a hydroxy or methyl group; the configur- ation at the carbon atom designated as is D; the configuration at the carbon atom designated as is L when R represents other than a hydrogen atom; and the configuration at the carbon atom denoted as (c) is when R represents other than a hydrogen atom, and of pharmaceutically acceptable salts thereof, which process comprises cleaving off by methods known per se the protecting group(s) present in a compound of the general formula

R3 0 2 0 R1 0

R NH-CH--CO--NH--CH--CO H-CH-P-R (II)

R141 4

wherein n has the significance given above; R1 0 20 30 R and R 1 have any of the values accorded to R 2 3 R 2 and R hereinbefore respectively except that any amino group or amino groups present may be in protected form and any other functional group which may the present is in protected form where required; R4 0

represents a methyl group or R R 4 1 represents a hydroxy group or lower alkoxy protecting group; R represents a hydrogen atom or a protecting group; and the configurations at the carbon atoms designated as and are as defined above,

/3

3 (11) AU-B1-25,008/77

or

separating an (R,S)-diastereomeric compound corres- ponding to formula I into its diastereomers and isolating the -diastereomer, and, if desired, converting an obtained compound of formula I into a pharmaceutically acceptable salt. Claim 14. A pharmaceutical preparation containing a peptide derivative of formula I set forth in claim I in association with a compatible pharmaceutical carrier material.

COMMONWEALTH OF AUSTRALIA Form PATENTS ACT 1952-69

G"OMPLETE SPECIFICATION (OR IGIN AL) FOR OFFICE USE:

Class Int. Class Application Number Lodged

tile~ aMOfldnients, rvmIdcti ndgr Secdon 49. Complete Specification Lodged Accepted Published and is correct fp-. priuzjirui±

Priority

Related Art

*tilam~p of Applicant F. H07FMAY'N-LA ROCHE CO. AKTIENGESELLSCHAFT

Address of Applicant 124-184 Grenzacherstrasse, Basle, Switzerland

~Agttlal Inventors: FRANK RATCLIFFE ATHERTON, MICHAEL JOHN HALL, CEDRIC HER13ERT HASSALL, ROBERT WILSON LAMBERT arnd PET\2R STUART RINGROSE

Address for Service Spruson Ferguson, Patent Attorneys, Esso House, 127 Keit Street, Sydney, New South Wales, 2000 Austral ia.

etomlete Specification for thie invention entitled

"PEPTIDE DERIVATIVES AND PROCESS POR THE PREPARAT ION THERE OF"

The following statement is a full description of this invention, including Ake best method of performing it known to -meAs

"rv

JI

1t IRAN 4105/24

The present invention relates to peptide derivatives.

More particularly, the invention is concerned with peptide

derivatives of phosphonic and phosphinic acids, a process for

the manufacture thereof and pharmaceutical preparations

containing sa.e

The peptide derivatives provided by the present invention

are compounds of the general formula S

R3 R 2 R 1 O :I I 7INH-CH--P-R II 4 H2N-CH--CO NH-CH-CO b (I) I n OH

wherein n stards for 1, 2 or 3;

R R and R each represent the

characterising group of an a-amino

acid of the type normally found in R3 proteins with the provisos that R represents other than a hydrogen atom and

that R 3 represents a methyl group when n

stands for 1; R' represents a hydroxy or

methyl group; the configuration at the

carbon atom designated as is D; the

configuration at the carbon atom designated 2 as is L when R 2 represents other than a

hydrogen atom; and the configuration at

the carbon atom denoted as is when

r ,17 7 2

R represents other than a hydrogen atom, and pharm-

aceutically acceptable salts thereof.

As used in this specification and claims, the

expression "the characterising group of an a-amino acid of the

type normally found in proteins" means the residue R in a

natural a-amino acid of the general formula

H2N-- 2 CH COOH

R

which is of the type normally occurring in proteins; by "the

a-amino acids of the type normally occurring in proteins" is

meant those a-amino acids listed in Fieser and Fiester "Organic

Chemistry" 3rd Edition Rheinhold N.Y. 1956 at page 419. Thus,

for example, if the amino acid is glycine then R represents a

hydrogen atom and if the amino acid is alanine then R

S: represents the methyl group. In valine R represents the

isopropyl group, in leucine R represents the isobutyl group

and in glutamic acid R represents the 2-carboxyethyl group S

and in phenylalanine R represents the benzyl group. R can also o

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.

When R in formula I represents other than a

hydrogen atom, the configuration at the carbon atom denoted

as is that is to say, the configuration which would

:be obtained by replacing the carboxyl group of a naturally

occurring L a-amino acid by a phosphorus moiety.

It will he appreciated that when n in formila I 2 hereinbefore stands for 2 or 3, the value of P can be the

1? same or different.

L_

Preferred compounds of formula I hereinbefore are those

in which R 4 represents a hydroxy group. Also preferred are

those compounds of formula I in which R represents a methyl

group as well as those compounds of formula I in which R 2

represents a methyl group and R 3 represents a methyl, isopropyl

or isobutyl group.

Examnples of compounds of formula I are the following:

(1R)-1-(D-alanyl-L-alanylamino)-ethylphosphonic acid,

(1R)-1-(D-alanyl-L-alanyl-L-alanylamino)-ethylphosphonic

acid,

S (lR)-1-(D-valyl-L-alanyl-L-alanylamino)-ethylphosphonic

S: acid,

(1R)-1-(D-leucyl-L-alanyl-L-alanylamino)-ethylphosphonic

acid and 9

15 (1R)-1-(D-alanyl-L-alanyl-L-alanyl-L-alanylamino)-

-ethylphosphonic acid.

According to the process provided by the present

invention, the peptide derivatives aforesaid the

compounds of formula I and their pharmaceutically acceptable

salts) are manufactured by

cleaving off by methods known per se the protecting

group(s) present in a compound of the general formula

R3 0 R2 0 R10 O

s I R -NH-CH-C00 1 NH-CH-CO 1 NH-CH-P-R 40 (II) c n 41 R

wherein n has the significance given 10 and R have any of any of 5 earlier; earlier; R R and R have

1-I r

the values accorded to R 2 and R 3 hereinbefore

respectively except that any amino group or

amino groups present may be in protected form

and any other functional group which may be

present is in protected form where required;

R 4 0 represents a methyl group or R41; R

represents a hydroxy group or lower alkoxy

protecting group; R represents a hydrogen atom or a protecting group; the configuration at the carbon atom designated as is D; the configuration at the carbon atom designated 20 as is L when R represents other than a

hydrogen atom; and the configuration at the carbon atom designated as is when R 1 0

I5 represents other than a hydrogen atom,

or

separating an (R,S)-diastereomeric compound cor-

responding to formula I into its diastereomers and isolating

the (R)-diastereomer,

and, if desired, converting an obtained compound of formula I

into a pharmaceutically acceptable salt.

An amino group or amino groups which may be present in R 0

R and R 3 0 in formula II 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 benzyloxycarbonyl 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 I 0 R 2 0 and R 3 0 in formula II can be protected by i conventional carboxy-protecting or hydroxy-protecting group respectively. For example, a carboxy group may be protected by conversion into an alkyl ester a tertbutyl ester) or an aralkyl ester a benzyl ester). Again, for example, a hydroxy group may be protected, for example, by means of an aralkoxycarbonyl group benzyloxycarbonyl), an alkanoyl group acetyl, propionyl etc), an aroyl group (e.g.

benzoyl), an alkyl group tertbutyl) or an aralkyl group

benzyl). The protection of other functional groups

present in R R 2 0 and R 3 0 may be carried out in a known manner.

The protecting group denoted by R 5 in formula II can be any

of the amino-protecting groups mentioned earlier in connection wh R10 20 with R, R and R *g

The cleavage of the protecting group or protecting groups

present in a compound of formula II is carried out in accordance

with methods known per se; that is to say, methods in actual

*0 use for or described in the literature on the cleavage of

protecting groups. Thus, for example, an aralkoxycarbonyl

*20 group benzyloxycarbonyl) or a tertbutoxycarbonyl group

may be cleaved off by hydrolysis treatment with a mixture

of hydrogen bromide and glacial acetic acid). An aralkoxy-

carbonyl group benzyloxycarbonyl) can also be cleaved

off by hydrogenolysis in the presence of palladium-on-

-charcoal or palladium oxide). The tertbutoxycarbonyl group

may also be cleaved off by means of hydrogen chloride in

dioxan. A lower alkoxy group denoted by R 4 0 and/or R 4 1 can

be a straight-chain or branched-chain alkoxy group preferably

containing from 1 to 6 carbon atoms methoxy, ethoxy,

6

_3

-r

propoxy, isopropoxy, butoxy etc) and may be converted into a

hydroxy group by treatment with a mixture of hydrogen bromide

in glacial acetic acid or by means of trimethylchlorosilane

followed by aqueous hydrolysis. It will be appreciated that

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 the protecting groups present.

The separation of an diastereomeric compound cor-

responding to formula I into its diastereomers and isolation

of the (R)-diast-reomer can be carried out according to known methods; for example, by fractional crystallisation or by high

pressure liquid chromatography. S oa

•Compounds a of formula I are amphoteric in nature and form

pharmaceutically acceptable salts with strong acids (e.g.

methanesulphonic acid, paratoluenesulphonic acid, hydrochloric

acid, hydrobromic acid, sulphuric acid etc) and with bases sodium hydroxide etc). a.*

.S The starting materials of formula II hereinbefore may be

prepared, for example, by condensing a compound of the general 0

0 formula

R2 0 R10 0 I I 11 40 H NH-CH -CO- NH-CH-P-R (III) I41 m R

,wherein m stands for zero, 1, 2 or 3;

R R2 0 R 4 0 and R 4 1 have the significance

given earlier; the configuration at the carbon atom designated as is L when

R 2 0 represents other than a hydrogen atom;

and the configuration at the carbon atom

designated as is when R represents

other than a hydrogen atom,

with an appropriately protected a-amino acid, ai appropriately

protected dipeptide, an appropriately protected tripeptide, an

appropriately protected tetrapeptide or a reactive derivative

thereof as the case may require.

Thus, a compound of formula III in which m stands for

zero can be condensed with an appropriately protected dipeptide

or a reactive derivative thereof to give a compound of formula

*0 II in which n stands for 1, or with an appropriately protected Se S if 09 tripeptide or a reactive derivative thereof to give a compound

e 3.5 of formula II in which n stands for 2 or with an appropriately S 0

protected tetrapeptide or a reactive derivative thereof to give S a compound of formula II in which n stands for 3.

9 Again, a compound of formula III in which m stands for 1

can be condensed with an appropriately protected a-amino acid

20 or a reactive derivative thereof to give a compound of formula

II in which n stands for 1, or with an appropriately protected o 9

dipeptide or a reactive derivative thereof to give a compound

of formula II in which n stands for 2 or with an appropriately 9 0 protected tripeptide or a reactive derivative thereof to give

a compound of formula II in which n stands for 3.

Yet again-, a compound of formul. III in which m stands

for 2 can be condensed with an appropriately protected a-amino

-ru I

acid or a reactive derivative thereof to give a compound of

formula II in which n stands for 2 or with an appropriately

protected dipeptid-7 or a reactive derivative thereof to give a

compound of formula II in which n stands for 3.

Finally, a compound of formula III in which m stands for

3 can be condensed with an appropriately protected a-amino acid

or a reactive derivative thereof to give a compound of

formula II in which n stands for 3.

Alternatively, the compounds of formula II can be

prepared by carrying out the foregoing condensation using an

compound corresponding to formula III and separating the

comnound from the resulting product in a manner S S known per se; for example, by crystallisation, chromatography Ur

or fractional crystallisation using a suitable base such as

a-methylbenzylamine. SS S

S

The aforementioned condensation can be carried out in

accordance with methods which are known per se in peptide *r S

0P chemistry; for example, by the mixed anhydride, azide, S activated ester or acid chloride method. 5

In one method, an appropriate compound of formula III

can be condensed with an appropriately protected a-amino acid,

.:so 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

9

CsF--I~~ alkyl)-amine triethylamine, or N-ethylmorpholine in an inert organic solvent tetrahydrofuran, 1,2-dimethoxy- ethane, dichloromethane, toluene, petroleum ether or mixtures thereof) and the resulting salt is reacted with a chloroformic acid ester the ethyl or isobutyl ester) at a low temperature. The mixed anhydride obtained is then suitably condensed in situ with the compound of formula III.

In another method, an appropriate compound of formula III

can be condensed with an appropriately protected a-amino acid,

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.

15 In yet another method, an appropriate compound of formula

S* III can be condensed with an appropriately protected a-amino

acid, di-, tri- or tetrapeptide as the case may require in

which the terminal carboxy function is in the form of an active

ester group the p-nitrophenyl, 2,4,5-trichlorophenyl or

S 20 N-hydroxysuccinimide ester group). This condensation is

suitably carried out either in an inert organic solvent such 40 41 as dimethylformamide 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 formula

III can be condensed with an appropriately protected a-amino

acid, di-, tri- or tetrapeptide as the case may require in which the terminal carboxy functio: is in the form of an acid chloride. This condensation is preferably carried out in the presence of a base and at a low temperature.

The peptide/derivatives provided by the present invention

potentiate the activity of D-cycloserine. In addition, they

possess an antibacterial activity against organisms such as

Escherichia coli, Klebsiella aerogenes, Streptococcus faecalis

and Haemophiius influenzae.

The present peptide derivatives may accordingly be used

as medicaments; for example, in the form of pharmaceutical

preparations which contain them in association with a

compatible pharmaceutical carrier material. This carrier

material can be an organic or inorganic carrier material S" suitable for enteral oral) or parenteral administration.

15 Examples of such carrier materials are water, lactose, starch, S* di magnesium stearate, gum arabic, gelatin, polyalkyleneglycols petroleum jelly and the like. The pharmaceutical preparations

can be made up in a solid form as tablets, dragees,

suppositories or capsules) or in a liquid form as

S 20 solutions, suspensions or emulsions). The pharmaceutical

Spreparations may be subjected to conventional pharmaceutical operations such as sterilisation and may contain adjuvants such

as preservatives, stabilisers, wetting agents or salts for

altering the osmotic pressure.

The peptide derivatives provided by the present invention

can be administered in combination with D-cycloserine.

Alternatively, the peptide derivative anr D-cycloserine can be

4- 11

'-JW IU-- -~Blg

administered separately, if necessary by different routes. The amount of the peptide derivatives to be administe'ed as

well as the ratio in which the peptide der.vative and D-cclo-

serine can be administered can vary wirhin wide limits

depending on such factors as the particular derivative chosen,

the route of administration and the organism to be combatted.

For example, the peptide derivative and D-cycloserine may be

administered in a ratio of from ca 100:1 to 1:100.

.0e4

0

8

o 0

o" "*a

Qef

t o* oo e

-u

The following Examples illustrate the process provided

by the present invention

Example 1

The process:

4.3 g (7.4 mmol) of the benzylamine salt of

-benzyloxycarbonyl-D-alanyl-L-alanyl-L-alanyl)-amino]-ethyl-

phosphonic acid were added to 12 ml of a stirred 45% sclution

of hydrogen bromide in glacial acetic acid and washed in with

4 ml of glacial acetic acid. The mixture was stirred at room

temperature for 6 hours and then 100 ml of ether were added

while stirring. The supernatant was decanted and the residue

was again treated with 100 ml of ether. The resulting gum was

taken up in 30 ml of methanol and the solution treated with a

solution of 3 ml of propylene oxide in 5 ml of methanol to give

15 a white precipitate. After standing overnight at room

temperature, the precipitate was filtered off, washed succen- .9 sively sith methanol and ether and then dried. Crystallisation

fron water/ethanol gave 1.96 g of (1R)-l-(D-alanyl-L-alanyl-L- 0 -32 0 C -alanylamino)-ettylphosphonic acid of melting point 31

20 (decomposition); [20 -107° (c 0.5% in 1 N sodium Dhydroxide) hydroxide)

The preparation of the starting ma-erial:

a A 2.7 g (10 mmol) of (1R)-l-(L-alanyl-L-alanylamino)-

-ethylphosphonic acid were stirred in 50 ml of water at 50 C

while 2,0 g (20 mmol) of triethylaminr followed by 50 ml of

ethanol were added. The resulting clear solution was cooled

to 0°C and there were added thereto 3.8 g (12 mmol) of solid

N-hydroxysuccinimide ester of N-benzyloxycarbonyl-D-alanine in a single portion. This N-hydroxysuccinimlde ester was washed in with 25 ml of ethanol. The mixture was stirred for 2 hours at 0°C and then for 16 hours at room temperature when a clear solution was obtained. The solvents were removed by evaporation and the residue was partitioned between 150 ml of water and 100 ml of chloroform. The aqueous layer was Washed with a further 100 ml of chloroform and the solvent extracts were back-washed with 50 ml of water. The aqueous extracts were combined and evaporated. The residue was taken up in a mixture of 40 ml of water and 40 ml of methanol and passed down a column of cation exchange resin (sulfonated polystyrene, freshly regenerated in the acid cycle).

Elution was carried out with the same solvent system. The *4 a 4* S 4 acid eluate obtained was evaporated, the residue taken up in *4

15 250 ml of water and extracted twice with 100 ml of ether each 4*aB a time. The ether extracts were back-washed with 50 ml of water.

4 4 ~4 4 The combined aqueous extracts were concentrated to ca 100 ml. 4 4

St An equal amount of methanol was added c.nd the resulting solution a 4 4* 4 4 was titrated to pH 4.5 with 1-M aqueous benzylamine. The solution

was evaporated to dryness and the solid obtained was recrystal- *4 S pUt lised from a mixture of 100 ml of hot water to which was added *0 4 *4~ 400 ml of ethanol followed by 500 ml of ether. There were 4

a. .4 obtained 4.38 g of the benzylamine salt of (1R)-l-[(N-benz/loxy- 4*4 4

carbonyl-D-alanyl-L-alanyl-L-alanyl)-amino] -ethylphosphonic acid -81 25 of melting point 240°-243°C (decomposition); [a]D "38.1 S .t

(c 0.53% in acetic acid).

PA 16

I Example 2

The process:

In a manner analogous to that described in Example 1,

from the benzylamine salt of (IR)-1-[(N-benzyloxycarbonyl-D-

valyl-L-alanyl-L-alanyl)-amino]-ethylphosphonic acid there was

obtained (1R)-l-(D-valyl-L-alanyl-L-alanylamino)-ethylphosphonic

acid of melting point 30io-304 0 C (decomposition); [a] 220 0 D -1050 (c 0.45 in 1 N sodium hydroxide)

The preparation of the starting material:

3.8 g (15 mmol) of N-benzyloxycarbonyl-D-valine were

stirred in 200 ml of petroleum ether (boiling point 600°-830), 9 9L 4 1.5 g (15 mmol) of triethylamine were added and the mixture 4 9

*444 4C was cooled to -5 C. 2.1 g (15 mmol) of isobutyl chloroformate 4*4

were added and the mixture was maintained at -5 0 C for 9 4J 4 9' 15 minutes. While stirring this mixture at -5 0 C there was added 4 3

.4 4 4 dropwise a solution of g (10 mmol) of (lR)-1-(L-alanyl-L- 44

alanylamino)-ethylphosphonic acid in a mixture of 2.0 g

mmol) of triethylamine and 15 ml of water. This solution was

washed in with 5 ml of water. The stirring was continued at 0*

a 20 -5 C to 0 C for a further 2 hours and then overnight at room r pU

temperature. 150 ml of water were added and the aqueous and 44

94 44 organic phases were separated. The aqueous phase was evaporated, 4 4

the residue taken up in a mixture of 40 ml of water and 40 ml

of methanol and the residue passed down a column of cation

exchange resin freshly regeneraced in the acid cycle. The

column was eluted with the same solvent mixture and the acid

fraction was evaporated and re-evaporated with five 50 ml

portions of water. The residue was triturated with 100 ml of

ether and the solid filtered off. The-solid was taken up in a mixture of 400 ml. of methanol and 400 ml of water and the solution was titrated to ph 4.5 with 4 M aqueous benzylamine.

The solution was evaporated to dryness and the resulting solid

was crystallised from a mixture of 100 ml of methanol and

150 ml of to give 3.24 of the benzylanine salt of

(lR)-l.-[(N-bezyloxvcarbonyl-D-valyl-L-alanyl-L-alanyl'-amino]-

-ethylphosphonic acid of meltin- point 238'-244' (decomposi-

tion).

Example 3

The process:

In a manner analogous to that described in Example 1,

p S from the benzqlamine salt of (IP)-l-i(N-benzyoxycarbonyl-

b -o D-alanyl-L-alanyl-L-alanyl-L-alanyl)-amino]-ethylphosphonic pp* acid there was obtained (lR)-l-(D-aanyl-L-alanyl--L-alanyl-

15 L-alanylamino)-ethllphosphonic acid of melting point 323o-

325CC (Gecomposition); r -0.48 in i N :D D D sodium hydrcxih)

The preparation of the starting material:

In a manner analogous to that described in part of

20 Example 1, from the N-hydroxysuccinimide ester of N-benzyloxy-

carbonyl-L-alanine and (1P-l-(L-alanyl-L-alanylarino)-ethyl- a phosphonic acid there was obtained (lR)-l-(N-benzyloxv-

carbonyl-L-alanyl-L-alanyl-L-alanyl)-amino] -ethylphosphonic

acid of melting point 255'-257'C (decomposition); l0 62.00 D (c 0.4% in glacial acetic acid).

In a manner analogous to that described in part of

Example 1, from (N-benzyloxycarbonyl-L-alanyl-L-alanyl-

16

IF

-L-alanyl)-aminol-ethylphosphonic acid there was obtained

(lR)-l-(L-alanyl-L-alanyl-L-alanylamino)-ethylphosphonic

acid of melting point 312'-313'C (decomposition); [a] 20 D (c l 0. 53 in 1 N sodium hydroxide).

Sn a manner analogoub to that described in part (B)

o 1:iyamnple i, but with acidification to pH 2 and further

solvent extraction with ether replacing passage through the

r: in usecquent solvent extraction with ether, u'd from the

T-hydroxysuccinimide ester of N-benzyloxycarbonyl-D-alanine

i; anac 1.I)-1-(L-alanyl-L-alanyl-L-alanylamino)-ethylphosphonic

acid there w.s obtained the benzylamine salt of abonzyloxycarhonyl-D-alanyl-L-alanyl-L-alanyl-L-alanyl)-amino]

,tthylph ;sphonic acid of melting point 272u-277 0 C (decomposition);

i !l 47." (c 0,5% in acetic cid).

'1xample 4

.d ecanted off. This procedure was repeated twice using 80 ml

l0,8: q of dimethyl (N-benzyloxycarbonyl-D-

1 eucy -L-alanyl-L-alanyl -amino -ethylphosphonate were

.f dissolved in 30 ml of a 35r solution of hydrogen bromide

2 in qacial acetic acid anl the mixture was stirred at room 4.

rop temperature for 4 hours. 130 ml of ether were ihen added

Swhile stirring, the stirring was discontinued and the ether decanted off. This procedure was repeated twice using 80 ml of ether each time. The residue was dissolved in 70 ml of

methanol and to the resulting solution were added 10 ml of

propylene oxide. After refrigerating overnight, the resulting

white precipitate was filtered off and washed with ethanol and

ether. The product was dried in vacuo to a constant weight of

7.99 g; melting point 2930-295 0 C (decomposition). Recrystal- lisation from 500 ml of cold water and from 700 ml of ethanol

gave 6.67 g of (1R)-1-(D-leucyl-L-alanyl-L-alanylamino)-

-ethylphosphonic acid of melting point 300°-3020C (decomposi- tion) =20-129,3" (c 1% in water) D

The preparation of the starting material:

12.9 q (0.03 mol) of dimethyl (1R)-l-[(N-benzyloxy- carbony]-L-alanyl-L-alanyl) -aminol-ethylphosphonate were

dissolved in 150 ml of methanol containing 0.032 mol of

1. hydrogen chloride. The solution was hydrogenated at room

temperature and pressure in the presence of 1 g of

palladium-on-charcoal until the hydrogen uptake ceased.

The catalyst was filtered off, the filtrate evaporated in vacuo and the oily hydrochloride evaporated twice with

15 ethyl acetate.

Ca.

SThe product obtained according to the preceding paragraph and 10.9 g (0.03 mol) of the N-hydroxysuccinimide ester of

N-benzyloxycarbonyl-D-leucine were stirred in the presence

of 100 ml of dry dimethylformamide. While stirring and 20 maintaining the temperature below 15'C, 4.2 ml of dry a triethylamine were added dropwise. The mixture was then see stirred overnight at room temperature. The triethylamine

hydrochlorid- was filtered off and washed with a small amount of dimethylformamide. The filtrate was evaporated

2L under an oil-pump vacuum and at a bath temperature of below

The residual oil was treated with 50 ml of water and

the resulting mixture extracted with four 50 ml portions of

chloroform. The combined organic phases were washed with

a small volume of 20% potassium carbonate solution and then

18

rr~l r~ i

W

dried over sodium sulphate. The sodium sulphate was filtered

off and the filtrate evaporated under a water-pump vacuum.

The residue was re-evaporated twice with ethyl acetate.

Trituration with dry ether. filtration and vacuum drying

S yielded 15.5 g of dimethyl (lR)-l-[(N-benzyloxycarbonyl-D-

leucyl-L-alanyl-L-alanyl)-aminc. -thylphosphonate of melting

point 163 0 -167 0 C. After recrystallisation from acetonitrile, 0 0 the meltinc point was 173 -176 C; [a]20 -36.6 (c 1% in

methanol).

Example

The process:

In a manner analogous to that described in Example C. 9.92 g of dimethyl (1R)-l-[(N-benzyloxycarbonyl-D-

alanyl-L-alanyl)-amino]-ethylphosphonate were treated with

*CC 15 45 ml of a 35% solution of hydrogen bromide in acetic acid. o Working-up in the same manner as described in Example 4(A) CC C 4 gave 6.16 g of crude product of melting point 282 0 -285°C C (decomposition) which, after recrystallisation from water/ethanol,

yielded 5.47 g of pure (1R)-l-(D-alanyl-L-alanylamino)-

eC 20 ethylphosphonic acid of melting point 292 0 -293 0C (decomposition); CC

4 a]20 -101.20 (c 1% in water). C D C CCC

The preparation of the starting material:

In a manner analogous to that described in Example 4(B)

from 7.82 g (0,03 mol) of dimethyl (1R)-l-(L-alanylamino)-

-ethylphosphonate hydrochloride and 9.6 g (0,03 mol) of the

N-hydroxysuccinimide ester of N-benzyloxycarbonyl-D-alanine

there were obtained 11.1 g of dimethyl (1R)-1-[(N-benzyloxy-

carbonyl-D-alanyl-L-alanyl)-amino]-ethylphosphonate of melting point 115 0 -117C; [a]20 -28.40 (c 0.5% in methanol).

The following Example illustrates a typical pharmaceutical

preparation containing a peptide derivative provided by the

present invention:

r Example 6

A 1000 ml injection solution containing the following

ingredients was prepared:

Per 1000 ml

(IP)-1-(D-Alanyl-L-alanylamino)- -ethylphosphonic acid 100.0 g

Chlorocresol 1.0 g

Acetic acid (glacial) 1.2 g

o Sodium hydroxide solution (0.1 N) o q.s. pH o 0 15 Water for injection ad 1000 ml o *S

The (1R)-1-(D-alanyl-L-alanylamino)-ethylphosphonic

acid was suspended in 500 ml of water for injection. The

chlorocresol was dissolved in 200 ml of water for injection

and added to the first solution. The acetic acid was then

added while stirring. A 0.1 N solution of sodium hydroxide in water for injection was added while stirring until a pH

value of 4.5 was obtained. The solution was then made up to

1000 ml with water for injection, filtered through a sterile

0.22 micron membrane filter and filled into ampoules. The

ampoules were sealed and then sterilised by autoclaving at

121°C for 20 minutes.

1) A process for the manufacture of peptide

derivatives of the general formula

R3 R2 R O

H -CH-CO--NH-CH-CO--NH-CH-P-R (I) 2 J I n OH

wherein n stands for 1, 2 or 3; R R2 and R each represent the characterising

group of an a-amino acid of the type o 9. a* normally found in proteins with the aa aI provisos that R 3 represent other than a a hydrogen atom and that R 3 represent a

*0 methyl group when n stands for 1; R4 *9 a. represents a hydroxy or methyl group; the configuration at the carbon atom designated a4 as is D; the configuration at the carbon *O a

atom designated as is L when R 2 represents 4* other then a hydrogen atom; and the configuration 00 990 0 at the carbon atom denoted as is when R 1

represents other than a hydrogen atom,

and of pharmaceutically acceptable salts thereof, which

process comprises

cleaving off by methods known per se the

protecting group(s) present in a compound of the general

formula

R3 0 R2 0 R10O 5 R--NH--CH-CO CH-CO 11-R -NH-CH-P (II) n (c) 41 R

wherein n has the significance given above 10 20 30 R R and R have any of the values accorded 2 3 to R 1 R and R hereinbefore respectively except

that any amino group or amino groups preaent

may be in protected form and any other functional

group which may the present is in protected form *0 0 where required; R 4 0 represents a methyl group or 41 41 @0@0 R R represents a hydroxy group or lower

:u alkoxy protecting group; R represents a

hydrogen atom or a protecting group; and the

*i 0* S configurations at the carbon atoms designated

.000 as and are as defined abcve, CC

e

*S or

*C

e separating an (R,S)-diastereomeric compound S S Ce corresponding to formula I into its diastereomers and

isolating the (R)-diastereomer,

and, if desired, converting an obtained compound of formula

I into a pharmaceutically acceptable salt.

22

i: i- i.

2) A process according to claim 1, wherein there is

manafactured a peptide derivative in which R 4 represents a

hydroxy group.

3) A process according to claim 1 or claim 2, wherein

there is manufactured a peptide derivative in which R

represents a methyl group.

4) A process according to claim 1, claim 2 or claim 3,

wherein there is manufactured a peptide derivative in which R 2

represents a methyl group and R 3 represents a methyl, isopropyl

or isobutvl group.

A process according to claim 1, wherein (1R)-l-(D-alanyl-

-L-alanylamino)-ethylphosphonic acid is manufactured.

6) A process according to claim 1, wherein

-alanyl-L-alanyl-L-alanylamino)-ethylphosphonic acid is

manufactured.

7) A process according to claim 1, wherein

-valyl-L-alanyl-L-alanylamino)-ethylphosphonic acid is

Smanufactured.

S' 8) A process according to claim 1, wherein .0

-leucyl-L-alanyl-L-alanylamino)-ethylphosphonic acid is

manufactured-

9) A process according to claim 1, wherein

-alanyl-L-alanyl-L-alanyl-L-alanylamino)-ethylphosphonic is

manufactured.

23

I

M I n Mp I0_

A process according to claim 1, wherein a compound of

formula II is obtained by condensing a compound of the general

formula

R2 0 1 0 R R 4

H- NH-CH--N N-CH-P-R (II) b) (c) R4

wherein m stands for zero, 1, 2 or 3;

R 10 20 I R 0 R 40 and R 41 have the significance

given in claim 1; the configuration at

the carbon atom designated as is L when

R 2 0 represents other than a hydrogen atom;

0* 10 and the configuration at the carbon atom *0

designated as is when R represents

other than a hydrogen atom, 0* o With an appropriately protected a-amino acid, an appropriately 0 1

protected dipeptide, an appropriately protected tripeptide, an

appropriately protected tetr peptide or a reactive derivative

thereof as the case may require.

11) A process for the manufacture of the peptide derivatives

0 of formula I set forth in claim 1, substantially as hereinbefore 0

described with reference to any one of Examples 1 to

0000 0 0. 00 0

24

i

12) A process for the manufacture of pharmaceutical

preparations which process comprises mixing a compound of

formula I set forth in claim 1, or a pharmaceutically

acceptable salt thereof, as active ingredient, Niith

non-toxic, inert, therapeutically compatible solid or

liquid carriers commonly used in such preparations and

bringing the resulting mixture into a suitable pharmaceutical

dosage form.

13) A process for the manufacture of pharmaceutical

preparations which process comprises mixing a ccmpound of

formula i set forth in claim 1, or a pharmaceutically

acceptable salt thereof, and D-cycloserine as active

ingredients, wiith non-toxic, inert, therapeutically

compatible solid or liquid carriers commonly used in such

15 preparations and bringing the resulting mixture into a eu** suitable pharmaceutical dosage form. 00

S 0

00

b g

e *e

o 0 0 0. 0

ee*0

0000 @0* 00 0

25

~1

I

14) A pharmaceutical preparation containing a peptide

derivative of formula I set forth in claim 1 in association

with a compatible pharmaceutical carrier material.

A pharmaceutical preparation containing a peptide

derivative of formula I set forth in claim 1, D-cycloserine

and a compatible pharmaceutical carrier material.

16) A peptide derivative of formula I set forth in claim 1,

when manufactured by the process claimed in any one of

claims 1 to 11 ar by an obeious chemical equivalent theref.

o

Sa

0.0as

kOa

26

I

17) A compound of the general formula

R 30 R 20 R 10 0

R 5 NH--CH-CO--CO 11 NH--CH--P--R L 14 R

wherein n has the significance given in

claim 1; R 1 0 R 2 0 and R3 0 have any of the values accorded to R1, R and R 3 hereinbefore

respectively except that any amino group or

amino groups present may be in protected

form and any other functional group which may

be present is in protected form where required; 40 41 41 10 R represents a methyl group or R R

S• represents a hydroxy group or lower alkoxy

protecting group; R 5 represents a hydrogen

atom or a protecting group; the S

configuration at the carbon atom designated S

as is D; the configuration at the carbon

atom designated as is L when R 2 0 represents S. other than a hydrogen atom; and the configuration at the carbon atom desigi ated as

I is when R represents other than a

hydrogen atom.

Claims (1)

1. 8) Peptide derivatives of the general formula 3 2 R R2 R11 0 I "i 1 11 4 HN-CH-CO NH-CH-CO--NH--CH--P-R (I I n OH

   1 2 ,wherein n stands for 1, 2 or 3; R R

   and R 3 each represent the characterising

   group of an a-amino acid of the type

   normally found in proteins with the

   provisos that R 3 represents other than a

   hydrogen atom and that R 3 represents a

   methyl group when n stands for 1; R

   *o r represents a hydroxy or methyl group; the

   configuration at the carbon atom designated C we..r as is D; the configuration at the

   4 as a carbon atom designated as is L when R a oer C represents other than a hydrogen atom; S e a 15 and the configuration at the carbon atom

   denoted as is when R represents

   ao other than a hydrogen om, Ow

   we and pharmaceutically acceptable salts thereof.

   Os wOO 19) Peptide derivatives according to claim 18. wherein R w 20 represents a hydroxy group. BLOC a

   Peptide derivatives according to claim 18 or claim 19,

   wherein R represents a methyl group.

   21) Peptide derivatives according to claim 18, claim 19 or

   cla 20, wherein R2 represents a methyl group and R 3 represents

   a methyl isopropyl or isobutyl group.

   22) (D-Alanyl-I-alanylami.no)-ethylphosphonic acid.

   23) Anyl-L-alanyl-L-alanylamino)-ethylphospho-

   flic atrid.

   24) (D-Valyl-I, -alanyl -L-alanylamino)-ethylphosphonic

   a cid.

   (1IP)-1-(lm.Luc yl-L-alanyl.-L-alanylamino)-ethylphosphonic

   acid. ftn ft

   S rn

   26) (i (D-Alan yX-L-a Iany1-L-alany l-L-alanylam ino) 0 ft... -ot.hylphosp1ioP1ic acid.

   ft S ft..

   DATED this TWELFTH day of APRIL, 1977 0S

   F. HOFFMANN-LA ROCHE CO. AKTIENGESELLSCHAFT

   0O

   Patent Attorneys for the Applicant f. ft. SPRUSON FERGUSON

   ft *0 ft f

   29

   i 1- I