NO771237L - PROCEDURES FOR PREPARING POLYPEPTIDES - Google Patents

Description

"Method for the Preparation of Polypeptides"

This invention relates to the production of polypeptide derivatives which possess analgesic properties.

An article by Kosterlitz et al in Nature, 1975, 258, 577-579 describes the identification of two related penta-peptides called "enkephalins" extracted from pig brain.

These two peptides have the structures respectively

H-Tyr-Gly-Gly-Phe-Met-OH and H-Tyr-Gly-Gly-Phé-Leu-OH and have been found to have potent opiate-like properties in isolated organ preparations. Although these two peptides produce transient analgesic effects when injected into the cerebral ventricle of cats, they are inactive when administered by more conventional routes, e.g. intravenously, on laboratory animals in standard analgesic trials.

It has now been found that when certain amino acid modifications are made in the enkephalin molecule, compounds are obtained which have analgesic activity when dosed intravenously in a standard test for analgesic activity.

According to the invention, a polypeptide derivative with the formula is produced:

where

R 2 "'" means hydrogen or an alkyl radical of 1 to 6 carbon atoms;

R means hydrogen or a radical selected from an alkanoyl radical of 1 to 3 carbon atoms, an alkoxycarbonyl radical of 2 to 6 carbon atoms or an amino acid residue which is Arg, Gly,

Glu, Asp, Phe, 3~Ala, or Lys or D-Lys, both of which are optionally substituted on the e-amino group with an alkoxycarbonyl radical with 2 to. 6 carbon atoms, or R 2 means 2 to 6 α-amino acid residues which are linked together via normal peptide bonds, these amino acid residues being independently selected from Gly, Lys, Pro, Phe, Gin, Glu, Leu, Arg and Asp, being Lys and The Arg residues are possibly alternatively linked together via their ε-amino and -guanidino radicals respectively, and the Asp and Glu residues are optionally alternatively

bound together via their 3~ and ycarboxy radicals, respectively; B is D-Ser or D-Thr, both of which are optionally substituted

3~0H with an alkyl radical of 1 to 6 carbon atoms, or B

is D-Ala, D-Leu, D-Asp, D-Lys, D-Try, D-Met, 3~Ala or Azala; E is Gly or Azgly, both of which are optionally substituted

the ot-amino radical, or 3~Ala which is optionally substituted on the 3-amino radical, with an alkyl radical of 1 to 6 carbon atoms;

F is Phe, hexahydroPhe, Azphe or hexahydroAzphe, all of which may optionally be substituted on the α-amino radical with a

alkyl radical of 1 to 6 carbon atoms;

G is Leu, D-Leu, Met, D-Met, Nie, D-Nle, Ile or D-Ile,

all of which may optionally be substituted on the α-amino radical by an alkyl radical with 1 to 6 carbon atoms, or G is

Pro, Azleu or Azpro;

K means a hydroxy or amino radical, a radical of the formula OR 3 , where R 3 is an alkyl-y alkenyl or hydroxy-alkyl radical of up to 6 carbon atoms, an aminoalkyl radical of up to 6 carbon atoms or an alkylaminoalkyl radical of up to 10 carbon atoms, where the nitrogen atom in both optionally substituted with a benzyloxycarbonyl radical,

3-

or R means a phenyl radical or a radical of the formula:

where R 4 is an alkanoyl radical with 1 to 20 carbon atoms, 5 5 or K means a radical with the formula NHR where R is an alkyl or cycloalkyl radical with 1 to 6 carbon atoms, a radical with the formula NR 6 R 7 , where R 6 and R 7 are alkyl radicals with 1 to 6 carbon atoms, or R and R 7 are bonded together to form a 5- or 6-membered ring optionally containing 4 a heteroatom, a radical of the formula -NH-CH£~CH&0OR4, where R has the meaning given above, a hydroxyalkylamino radical of up to 6 carbon atoms, or an (alkylamino)alkylamino or (d8alkylamino)alkylamino radical of up to 10 carbon atoms, or K means a radical of the formula Gly-OR<8>, D-Thr -OR<8>, Thr-OR<8>, D-Ala-OR<8> or 8 8 Ala-OR , where R is a hydrogen atom, an alkyl radical of 1 to 6 carbon atoms or a radical of the above

indicated formula II, III or IV, where R 4 has the meanings indicated above, or K is an alkyl radical with 1 to 6

carbon atoms; or

G and K together mean the D or L forms of a radical with the formula:

where n is an integer from 1 to 4, or a radical with

the formula:

and where the polypeptide of formula I contains a basic function, the pharmaceutically acceptable acid addition salts thereof, and where the polypeptide of formula I contains an acid function, the pharmaceutically acceptable base addition salts thereof.

In the above formula I and throughout this description and claims, the amino acid residues are denoted by their standard abbreviations (Pure and Applied Chemistry, 1974, 40, 317-331). An a-aza amino acid residue is one where the a-CH of an amino acid is replaced by nitrogen. The abbreviation for an a-aza amino acid is derived from the abbreviation for the corresponding amino acid by introducing "Az" as a prefix. Thus Azala means aza-alanine,

Azgly means aza-glycine, Azphe means aza-phenylalanine, Azleu

means aza-leucine, and Azpro means aza-proline. The abbreviations hexahydroPhe and Phe(6H) mean a phenylalanine residue where the benzene ring has been replaced by a cyclohexane ring. The abbreviations hexahydroAzphe and Azphe(6H) mean the corresponding a-aza-amino acid. When the configuration of a particular amino acid is not specified, the amino acid (except for glycine and the a-aza amino acids which contain no asymmetric center adjacent to the carboxyl group) has the natural L configuration.

A particular meaning for R"<*>" is hydrogen or a methyl radical. A particular meaning for R 2 is hydrogen or an acetyl radical or an Arg-, Gly-, Glu-, Asp-, Phe-, 3~Ala-, D-Lys-, Lys- (which is optionally substituted on e -amino group with a t-butoxycarbonyl radical) , H-Glu-Gly-OH H-Lys-, H-Phe-I H-Lys-, H-Gly-Gly-Gly—I H-Lys-, H-Asp-1 H-Lys-, H-Lys-l H-Lys-, H-Gly-Gly-, H-Ley-Ley-Leu-, H-Arg-Pro-Lys-, H-Pro-Gln-Gln-, H -Gly-Gly-Gly-, H-Lys-Gly-Gly-Gly-, H-Asp-Gly-Gly-Gly-, H-Arg-Pro-Lys-Pro-Gln-Gln-or H-Gly-Gly -Gly-Gly-Gly-Gly residue. ;A special meaning for B is D-Ser which is optionally substituted on its 3-OH with a t-butyl radical, D-Ala, D-Thr, D-Leu, D-Asp, D-Lys, D-Try, D -Met, 3~Ala or Azala. ;A special meaning for E is Gly, 3_Ala or Azgly.;A special meaning for F is Phe, hexahydroPhe or Azphe. ;A particular meaning for G is Leu, D-Leu, Met, NLe,;Pro, Azleu or Azpro.;A particular meaning for K is a hydroxy-, amino-, methoxy-, t-butoxy-, isopentyloxy-, allyloxy -, 2-hydroxy-ethoxy-, 2-aminoethoxy-, 2-(methylamino)ethoxy-, 2-(N-phenoxy-carbonylamino)ethoxy-, 2-(N-methyl-N-phenoxycarbonylamino)-ethoxy-, phenoxy -, 1,3-diacetoxy-prop-2-yloxy-, 2,3-diacetoxy-proposky-, 1,3-dihexanoyloxyprop-2-yloxy-, 2,3-dipalmitoyloxy-propoxy-, 2-acetoxyethoxy-, 2 -palmitoyloxyethoxy-, ethylamino-, cyclohexylamino-, diethylamino-, pyrrolidino-, morpholino-, 2-hydroxyethylamino-, 2-(methylamino).ethylamino-, 2-(dimethylamino)-ethylamino- or methyl radical, or a Gly- OCH^, Thr-OH, D-Thr-OH, Ala-OCH3 or D-Ala-OCH3 residue. ;A special meaning for G and K together is the D and L forms for a radical with the formula: or a radical with the formula: ; The following 7 groups of compounds fall under the above definitions of the new compounds produced according to the invention: ;Group 1. Those where B is D-Ala.;Group 2. Those where B is D-Ser.;Group 3. Those where B is D-Ser substituted on (3-OH with an alkyl radical of 1 to 6 carbon atoms. ;Group 4. Those where B is Azala.;Group 5. Those where B is D-Thr which is optionally substituted on ;3- OH with an alkyl radical of 1 to 6 carbon atoms.;Group 6. Those where B is D-Leu or 3-Ala.;Group 7. Those where B is D-Asp, D-Lys, D-Try or D-Met. ;Under each of the above 7 groups of compounds fall the following 4 subgroups: ;Subgroup 1. Those where R is hydrogen and R means 2 to 6,;and especially 3, α-amino acid residues which are bound together via normal peptide bonds, as these amino acid residues independently of each other are selected from Gly, Lys, Pro, Phe, Gin, Glu, Leu, Arg and Asp, with the Lys and Arg residues optionally alternatively linked together via their e-amino and -guanidino radicals respectively, o g The Asp and Gin residues are optionally alternatively bound together via sine and ycarboxy radicals, respectively. ;2 I " 3 ;A particular meaning for R is a H-Glu-Gly-OH H-Lys, ; H-Gly-Gly, H-Leu-Leu-Leu, H-Arg-Pro-Lys, H-Pro-Gln-Gln, H-Gly-Gly-Gly, H-Lys-Gly-Gly-Gly, H- Asp-Gly-Gly-Gly, H-Arg-Pro-Lys-Pro-Gln-Gln or H-Gly-Gly-Gly-Gly-Gly-Gly residue, and a preferred meaning for R 2 is a H-Leu-Leu -Leu or H-Gly-Gly-Gly residue. ;1 2 ;Subgroup 2 . Those where R is hydrogen and R means an Arg,;Gly, Glu, Asp, Phe, p-Ala or a Lys or D-Lys which are both optionally substituted on the ε-amino group with a H-dlu-Gly-OH, H -Phe-, H-Gly-Gly-Gly-, H-Asp- or H-Lys- residue. ;1 2 ;Subgroup 3. Those where R and R are both hydrogen.;Subgroup 4. Those where R"<*>" means an alkyl radical with 1 to 6

2

carbon atoms and R is hydrogen.

Under each of the above described 7 groups and

4 subgroups fall the following 9 classes of compounds:

Class 1. Those where G is Pro.

Class 2. Those where G is Azpro.

Below classes 1 and 2 is a preferred value for K

3 3

an amino radical, a radical of the formula OR where R is an alkyl, alkenyl, hydroxyalkyl 1 or aminoalkyl radical of up to 6 carbon atoms or an alkylaminoalkyl radical of up to 10 carbon atoms, a phenyl radical, a radical of the formula

5 5

NHR where R is an alkyl radical with. 1 to 6 carbon atoms or

6 7 6 7

a radical with the formula NR R where R and R are alkyl radicals with 1 to 6 carbon atoms or R 6 . and R 7 are bonded together to form a ring optionally containing a heteroatom.

Class 3. Those in which G and K together mean the D or L forms of a radical of the above formulas V or VI where n has the above meaning, or G and K together mean a radical of the above formula VII.

Class 4. Those where G is Met, Leu or NLe and K is an (alkylamino)alkylamino or (dialkylamino)alkylamino radical of up to 10 carbon atoms.

Class 5. Those where G is Met, Leu or NLe and K is an alkyl radical of 1 to 6 carbon atoms.

Class 6. Those where G is Met, Leu or NLe and K is a hydroxy or amino radical or a radical of the formula OR 3 where R 3 is an alkyl, alkenyl, hydroxyalkyl or aminoalkyl radical of up to 6 carbon atoms, an alkylaminoalkyl radical of up to 10 carbon atoms or a phenyl radical.

Class 7. Those where G is Met, Leu or NLe and K is a radical of the formula NHR^ where R^ is an alkyl radical of 1 to 6 carbon-6 7 6 V

atoms or a radical with the formula NR R where R and R are

6 7 alkyl radicals with 1 to 6 carbon atoms or R and R are bound together to form a 5- or 6-membered ring which optionally contains a further heteroatom.

Class 8. Those where G is Met, Leu or Ile and K is Gly-OR g,

g o n R 8

D-Thr-OR , Thr-OR , D-Ala-OR or Ala-OR where R is hydrogen or an alkyl radical of 1 to 6 carbon atoms.

Class 9. Those where G is Met, Leu or Ile and K is a radical with the above formula II, III or IV where R 4 has the above meaning..

Particular compounds according to the invention are indicated in examples 1-9 3, and of these the preferred compounds are as follows:

A particularly pharmaceutically acceptable acid addition salt of the compounds prepared according to the invention is e.g. a hydrochloride, phosphate, citrate, acetate or trifluoroacetate.

A particularly pharmaceutically acceptable base addition salt of the compounds prepared according to the invention is e.g. an ammonium or meglumine salt.

The polypeptide derivatives of formula I are produced by methods known per se for the production of chemically analogous compounds. The following methods, where R 1 , R 2, B, E,

3 4 5 6 V

F, G, K, R, R, R, R, R and n have the above meanings are used to prepare the compounds: (a) Removal of one or more common peptide protecting groups from a protected polypeptide to form a compound of formula I. (b) For the preparation of the compounds where K is different from a hydroxy radical, or when K means a radical

g 8 R 8

with the formula Gly-OR , D-Thr-OR , Thr-OR , D-Ala-OR or Ala-OR 8 , where R 8 is different from a hydrogen atom, and for those compounds where G and K together mean radicals of formula V , VI and VII, reaction of a carboxylic acid with the formula:

or

or a. activated derivative thereof, with a suitable alcohol or an amine.

In method (a), there may be as many protective groups in the starting material as there are radicals that may require protection, e.g. the radicals that exist in the product as free OH radicals or basic NH radicals.

In method (a), the protecting group or groups may be those described in a standard textbook on peptide chemistry, for example: M. Bodansky and M.A. Ondetti, "Peptide Synthesis", Interscience, New York, 1966, Chapter IV; F. M. Finn and K. Kaufmann, "The Proteins", Volume II, edited by H. Neurath and R. L. Hill, Academic Press Inc.,

New York, 19 7.6, p. 106; "Amino-acids, Peptides and Proteins"

(Specialist Periodical Reports), The Chemical Society, London, Volumes 1 to 8. Various methods for removing the protecting groups are also described in these books.

In method (a), a particularly suitable NH-protecting group is a benzyloxycarbonyl radical, and a particularly suitable OH-protecting group is a benzyl radical. Both of these protective groups can be easily removed by hydrogenolysis, e.g. by hydrogenolysis in the presence of a palladium-on-carbon catalyst, e.g. 5% w/w palladium-on-carbon, in a diluent or solvent such as water, methanol, ethanol, butanol, dimethylformamide, acetic acid, chloroform or a mixture of any of these. The reaction can optionally be carried out in the presence of an acid such as hydrogen chloride or toluene-p-sulphonic acid, and is most appropriately carried out at atmospheric pressure.

In method (a), another particularly suitable NH-protecting group is a t-butoxycarbonyl radical and a further particularly suitable OH-protecting group is a t-butyl radical. Both of these protecting groups can be easily removed by treatment with an acid such as hydrogen chloride or trifluoroacetic acid. The hydrogen chloride can be used in the form of an aqueous solution, e.g. at a concentration between IN and a saturated solution, or the hydrogen chloride may alternatively be used as a solution in a diluent or solvent such as ethyl acetate, methanol, acetic acid, ether, dioxane or a mixture of any of these. The concentration used can be any concentration up to the saturation point of the diluent or solvent, and is preferably in the range 2 to 6N. The reaction is preferably carried out at a temperature between 0°C and ambient temperature, and can take place in the presence of a flushing agent such as 2-mercaptoethanol. The trifluoroacetic acid can be used directly without diluent or solvent or it can be diluted with 5-10% water. The reaction is preferably carried out at ambient temperature, possibly in the presence of a flushing agent such as 2-mercaptoethanol.

In method (a), a further particularly suitable NH protecting group is a benzyloxycarbonyl or t-butoxycarbonyl radical, and a particularly suitable OH protecting group is a t-butyl radical. These protective groups can be easily removed by treatment with HBr in acetic acid. A preferred concentration is 1 to 3N, and a particularly preferred concentration is 2N.

In method (a), a particularly suitable protecting group for a carboxy group is an ester, e.g. a methyl or ethyl ester. The ester group is conveniently removed by base hydrogenolysis, for example, with sodium or potassium hydroxide in a diluent or solvent such as aqueous methanol, aqueous cellosolve, aqueous dioxane or aqueous dimethylformamide. The reaction is suitably carried out at ambient temperature.

In method (b), a suitable activated derivative of the starting material is e.g. an ester or an anhydride. When using an activated derivative, the reaction can be carried out by bringing the activated derivative into contact with a suitable alcohol or the suitable amine in the presence of a diluent or solvent. In those cases where the starting material is the free acid of formula X or XI, the reaction with the appropriate alcohol or the appropriate amine is conveniently effected by means of a standard peptide coupling reagent such as N,N'-dicyclohexylcarbodiimide. The method is particularly suitable for the preparation of compounds of formula I where K is an amino radical or a radical of formula NHR5 or NR<5>R<6.> In this case an ester is mixed, e.g. a methyl ester, of the compound of formula X with an excess of ammonia or the suitably substituted amine in a solvent such as methanol or dimethylformamide at a temperature between 0°C and ambient temperature for up to 3 days.

The starting materials used in the method

according to the invention, can be prepared from known compounds by standard peptide-coupling reactions, standard peptide-protection reactions and standard peptide-deprotection reactions which are well known to those skilled in the art, e.g. as stated in example 94. Particular reference should be made to the production of intermediate no. 175 where the methyl ketone was produced from

the corresponding acid by a Dakin-West reaction with acetic anhydride in pyridine.

As stated above, they have produced new compounds

according to the invention analgesic effect in warm-blooded animals. This can be demonstrated by a standard test for detecting an analgesic effect, such as the hot plate test on mice

(Eddy and Leimbach, J. Pharmac. Exp. Therap., 1953, 107, 385-393). This experiment is carried out as follows: Groups of three female mice weighing 22-25 g are used to test each compound. Each mouse is placed on a heated, thermostatically controlled copper surface with a temperature of 56°C, and the time it takes for the mouse to react to the heat (e.g. by licking its hind paws) is recorded. Normal reaction times are in the range of 3 to 5 seconds.

Each of the three mice is then given an intravenous dose of 100 mg/kg of a solution of the test compound. At the times 5, 10 and 30 minutes after the administration, each mouse is placed back on the hot plate, and the reaction time is determined. If the mouse does not react after 20 seconds, the mouse is removed from the heating plate. In this case, the compound is considered to have maximum activity

at this dose.

A compound that produces an average increase in reaction time of at least 3 seconds is considered active.

An active compound is then tested again at lower doses.

All the compounds described in the examples,

are active in this test at a dose equal to or less

than 100 mg/kg of the free acid or base. The intravenous LD^o. in mice for the compounds prepared according to the invention is also considerably higher than the lowest dose that produces the maximum analgesic effect, as shown in the following table:

The new polypeptide derivatives produced according to the invention can, together with a non-toxic pharmaceutically acceptable diluent or carrier, be prepared in the form of a pharmaceutical preparation.

The pharmaceutical preparation can e.g. be in a form suitable for parenteral administration, for which purpose it may be prepared by known methods in the form of sterile, injectable, aqueous or oily solutions or suspensions."

In addition to the polypeptide derivative, the pharmaceutical preparation may also contain one or more known agents selected from other painkillers, e.g. aspirin, paracetamol, phenacetin, codeine, pethidine and morphine, anti-inflammatory agents, e.g. naproxen, indomethacin and ibuprofen, neuroleptics such as chlorpromazine, prochlorperazine, trifluoperazine and haloperidol and other anesthetics and sedatives such as chlordiazepoxide, phenobarbitone and amylobarbitone.

A preferred pharmaceutical preparation is one suitable for intravenous, intramuscular or subcutaneous injection, e.g. in a sterile, aqueous solution containing between 1 and 50 mg/ml of active ingredient.

The pharmaceutical preparation will normally be administered to humans for the treatment or prevention of pain in such a dose that each patient receives an intramuscular or subcutaneous dose of between 2 and 150 mg of active ingredient or an intravenous dose of between 1 and 100 mg of active ingredient.

The preparation can be administered according to a schedule determined by the biological half-life of the polypeptide derivative, e.g. with intervals of from 0.5 to 4 times the biological half-life, e.g. 2 to 6 times a day. The preparation is particularly suitable for relieving the pain that occurs during and immediately after a surgical operation and in this situation will normally be administered during the operation itself and in the first post-operative period.

The injectable preparation may be administered in a single dose, either directly at the site of injection or in a prearranged arrangement for intravenous infusion, or it may

administered more slowly in dilute solution as a component of the intravenous infusion fluid.

The invention is illustrated by means of the following examples 1 to 94.

In the following examples, R,, refers to rising thin-layer chromatography on silica gel plates ("Kieselgel G"). The solvent systems used in this chromatography were butan-1-ol/acetic acid/water (4:1:5 v/v) (R^A), butan-1-ol/acetic acid/water/pyridine (15:3 :12:10 v/v (R^B), butan-2-ol/3% w/v aqueous ammonium hydroxide (3:1 v/v)

(R^C), acetonitrile/water (3:1 v/v) (R^D),

acetone/chloroform (1:1 v/v) (R^E), chloroform/ethanol (1:4 v/v) (R^F) , cyclohexane/ethyl acetate (1:1 v/v)

(R^G), cyclohexane/ethyl acetate/methanol (1:1:1 v/v) (R^H), chloroform/methanol/water (11:8:2 v/v) (R^K) , chloroform/ methanol (19:1 vol/vol) (R^P) and chloroform/methanol (9:1 vol/vol (R^Q). (Some of these solvents were also used for column chromatography on silica and are referred to in the footnotes as solvents G, K, Q and P). In all cases the plates were examined under ultraviolet light and treated with fluorescamine, ninhydrin and chlorine-starch-iodine reagents. Unless otherwise indicated, the indication of an R^ means that a single spot was found by these methods.

Acid hydrolysates for all products described in Examples 1 to 93 were prepared by heating the peptide or protected peptide with 6N hydrochloric acid containing 1% w/v phenol in a closed, evacuated tube for 16 hours at 110°C. The amino acid composition of each hydrolyzate was determined with a so-called LoCarte Amino-Acid Analyzer and was in each case in accordance with the expected composition.

In the examples, the following symbols are used:

Z - benzyloxycarbonyl

Bzl - benzyl

OCp - 2, 4,5-trichlorophenoxy

Boe - t-butoxycarbonyl

ONp - p-nitrophenoxy

ONSu- succinimido-oxy

TosOH-toluene-p-sulfonic acid

DMF - dimethylformamide

TFA - trifluoroacetic acid

Examples 1-42

The protected polypeptide was subjected to reductive cleavage using one of the methods H1 to H1 described below. The polypeptide derivatives indicated in Table I were thus obtained.

St. The starting material (indicated by number in Table I) was dissolved in ethanol containing up to 25% water and, under an atmosphere of nitrogen, 5% w/w palladium-on-carbon catalyst (150 mg/g) was added, and a mild stream of hydrogen was bubbled through the stirred reaction mixture at 20-25°C. A time of 5 to 6 hours was sufficient to ensure that the reaction was complete. The hydrogen atmosphere was replaced with nitrogen before removal of the catalyst by filtration through a layer of diatomaceous earth. The product was isolated by evaporation of the filtrate in vacuo.

H2. As in H1, but using aqueous methanol instead of aqueous ethanol.

' H3. As in H1, but using methanol as reaction medium.

H4. As in H1, but using aqueous ethanol with the addition of an equivalent of hydrogen chloride.

H5. As in H1, but using aqueous methanol with the addition of an equivalent of hydrogen chloride.

H6. As in H1, but using dimethylformamide with the addition of one equivalent of toluene-p-sulfonic acid.

H7. As in H1, but using methanol with the addition of an equivalent of hydrogen chloride.

H8. As in H1, but using a dimethylformamide/butanol mixture as reaction medium.

H9. As in H1, but using aqueous 90 or 95% volume/volume acetic acid as reaction medium.

HlO. As in H9, but with the addition of 1 to 2 equivalents of hydrogen chloride.

Hello. As in H1, but using aqueous dimethylformamide as reaction medium.

H12. As in H1, but using a mixture of aqueous ethanol and chloroform as reaction medium.

H13. As in H1, but using methanol/dimethylformamide as reaction medium.

Examples 43-69

The protected polypeptide was subjected to cleavage with hydrogen chloride using one of the methods E1 to E10 described below. The polypeptide derivatives listed in Table II were thus obtained.

El. The starting material (indicated by number in Table II) was dissolved in ethyl acetate, and a solution of hydrogen chloride in ethyl acetate in an amount sufficient to give a total strength of 2N to 6N acid was added. The reaction was allowed to take place at 20-25°C for a period of 1 to 2 hours, with too long a reaction time being avoided to minimize unwanted side reactions. The hydrochloride of the product often precipitated out of solution and was collected by filtration. Otherwise, the isolation was carried out by evaporation in a vacuum.

E2. As in El, but using methanol as reaction- . medium instead of ethyl acetate.

E3. As in El, but using hydrogen chloride in methanol/ethyl acetate.

E4. As in E1, but using acetic acid to dissolve the starting material and then adding hydrogen chloride in ethyl acetate solution.

E5. As in E2, but under nitrogen and in the presence of a flushing agent, e.g. 2-mercaptoethanol.

E6. As in El, but using diethyl ether as reaction medium instead of ethyl acetate.

E7. As in El, but using dioxane as reaction medium instead of ethyl acetate.

E8. As in El, but using acetic acid as reaction medium instead of ethyl acetate.

E9. As in El, but under nitrogen and in the presence of a flushing agent (2-mercaptoethanol).

ElO. The protected material was stirred with concentrated hydrochloric acid (about 10 ml/g) at 0°C for 10 minutes. Excess acid was then distilled off in vacuum at as low a temperature as possible, and the product was obtained by freeze-drying.

Examples 70-88

The protected polypeptide was subjected to cleavage with trifluoroacetic acid using one of the methods described below. The polypeptide derivatives listed in Table III were thus prepared.

Fl The starting material (indicated by number in Table III) was dissolved in trifluoroacetic acid (10 ml/g), and the reaction was allowed to take place at 20-25°C for a period of 1-2 hours. The product was isolated as the trifluoroacetate by evaporation of the solution in vacuo. Optionally, freeze drying in the presence of a small excess of hydrochloric acid was used to form the hydrochloride.

F2. As in Fl, but using aqueous 90 or 95% v/v trifluoroacetic acid.

F3. As in Fl, but using aqueous 90 or 95% v/v trifluoroacetic acid under nitrogen in the presence of a flushing agent, e.g. 2-mercaptoethanol.

Example 89

Aqueous N sodium hydroxide (1.0 mL, 3 equivalents)

was added to a solution of H-Tyr-D-Ser-Gly-Phe(6H)-Leu-OMe (starting material 110) (235 mg, 330 µmol) in methanol (5 mL), and the mixture was stirred at ambient temperature for 3 hours. The solution was evaporated in vacuo, diluted with water and acidified with dilute hydrochloric acid. After clarification by filtration, the solution was freeze-dried, and the product was desalted by being passed down a "Sephadex" G15 column in aqueous 5% v/v acetic acid and a final freeze-drying to give H-Tyr-D-Ser-Gly -Phe (6H)-Leu-OH, RfC 0.38.

Example 90

When treating. H-MeTyr-D-Ser-Gly-Phe(6H)-Leu-OMe (starting material 102) in aqueous solution with sodium hydroxide (2 equivalents) and working up as in Example 89 gave H-MeTyr-D-Ser-Gly- Phe(6H)-Leu-OH, RfCO,44.

Example 91 Ac-Tyr-D-Ala-Gly-Phe-Leu-OMe (starting material 123)

(140 mg, 224 ymol) was dissolved in hot dioxane (10 mL) and allowed to cool to ambient temperature before adding water (2 mL)

and aqueous N sodium hydroxide (0.75 mL, 3.5 equivalents). The mixture was vigorously stirred at ambient temperature for 2.5 hours,

and most of the dioxane was distilled off in vacuo. The remaining solution was acidified by adding N hydrochloric acid; and the resulting suspension was concentrated in vacuo. A clear solution was obtained by adding aqueous ammonia, along with aqueous 0.05M ammonium acetate. Excess ammonia was removed by evaporation, and purification was carried out on a column of "Sephadex" G25 in aqueous 0.05M ammonium acetate. Freeze-drying of material from the appropriate fractions gave Ac-Tyr-D-Ala-Gly-Phe-Leu-OH, RfD 0.56, RfD 0.47.

Example 9 2

A solution of H-MeTyr-D-Ala-Gly-Phe-Leu-OMe.HCl (Example 1) (100 mg, 157 µmol) in methanol (15 mL) was cooled to 4°C and saturated with dry ammonia. After storage at 4°C for 2 days, the solution was evaporated in vacuo and the residue was purified chromatographically on a column of silicic acid using system Q, followed by system K, as eluents. Evaporation of the appropriate fractions and freeze drying of the residue gave H-MeTyr-D-Ala-Gly-Phe-Leu-NH2, RfH 0.22, RfK 0.70.

Example 9 3

Ethylamine (50 equivalents) was added to a solution of H-Tyr-D-Ala-Gly-Phe-Leu-OMé (Example 47) (100 mg) in dimethylformamide (1 mL), and the mixture was stirred at 4°C in 3 days. The solvent was distilled off in vacuo, and the residue was freed from traces of impurities by chromatography on a column of silicic acid using system K as eluent. Evaporation of the appropriate fractions and freeze drying of the residue gave H-Tyr-D-Ala-Gly-Phe-Leu-NHEt, RfD 0.62, RfH 0.50, RfK 0.90.

Example 9 4

This example describes the preparation, from known compounds, of all the numbered starting materials in examples 1 to 93. The starting materials are arranged in the tables according to the number of amino acid residues that each contains. In each table, each compound is identified by a specific number which is used whether it is used in the preparation of another starting material as described in this example, or in the preparation of a compound described in one of examples 1 to 93.

In the tables, the procedures are indicated with symbols

Hl to H13, El to E10 and Fl to F3, the same as described in

respectively examples 1-42, 43-69 and 70-88. The other process symbols have the meanings given below.

Active- Ester- Reactions

Eel. A solution of the appropriate amino component (1 mmol) and the desired active ester (1.1 mmol) in dimethylformamide (minimum volume) was kept at 20-25°C until a positive reaction was no longer obtained with ninhydrin (15-50 hours). The reaction mixture was evaporated in vacuo to an extent sufficient to allow isolation of the crude product, either by precipitation by addition of a suitable organic solvent or by treatment with water or a mixture of water and a suitable organic solvent, e.g. ether. If necessary, further purification was carried out as indicated in the tables.

A2. As in Al except that ethyl acetate at 4°C was used as reaction medium.

A3. As in Al, but with the addition of 1-hydroxybenzotriazole (approx. 0.2 mmol). . A4. As in Al, but the reaction mixture was kept at 4°C. A5. As in A1, but with the addition of 1-hydroxybenzotriazole (ca. 0.2 mmol), and the whole was kept at 4°C.

Mixed- Anhydride- Reactions

Bl. A solution of the appropriate carboxy component (1 mmol) in dimethylformamide (ca. 5 mL) was cooled to -20° to -40°C and treated with N-methylmorpholine (1.05 mmol), followed by isobutyl chloroformate (1, 05 mmol). The mixture was stirred at -20 to -40°C for 5 minutes before adding a solution of the amino component

(1 mmol) in dimethylformamide. When a salt of the amino component was used, N-methylmorpholine (1 mmol) was also added. The mixture was stirred at 20-25°C for up to 24 hours to complete the reaction and was then evaporated in vacuo. The residue was partitioned between dilute aqueous citric acid and a suitable immiscible organic solvent, usually ethyl acetate. The product in the organic phase was washed in turn with aqueous citric acid and with aqueous sodium bicarbonate and was finally isolated by evaporation

of the dried solution in vacuo. If a suitable solvent could not be found, the washing was carried out on the solid which was then isolated by filtration after a final washing with water.

B2. As in B1, but using triethylamine as tertiary base.

B3. As in B1, but using ethyl chloroformate

to form the mixed anhydride.

B4. As in B1, but under formation of the.mixed.anhydride with ethyl chloroformate and N-methylmorpholine in tetrahydrofuran.

B5. As in B1, but with formation of the mixed anhydride with ethyl chloroformate and use of triethylamine as tertiary base.

Azide Reactions

Cl. The hydrazide (1 mmol) of the carboxy component was dissolved in dimethylformamide (ca. 5 ml), cooled to -20°C and converted to its azide by treatment with a 6M solution of hydrogen chloride (5-6 mmol) in dioxane, followed by t- butyl nitrite (1.2 mmol). Disappearance of the hydrazide from the solution was checked by

to apply some spots on filter paper and examine with iron(III) chloride/potassium iron(III) cyanide shower. During 10 minutes, the mixture, which was stirred at -10°C, was neutralized by adding an equivalent amount of triethylamine (ie 5-6 mmol), and the desired amino component (1 mmol), dissolved in dimethylformamide (3 ml) , was added. When a salt of the amino component was used, the required amount of triethylamine (1 mmol) was also added at this step. The reaction mixture was then stirred at 0-4°C for 18-24 hours. After filtration, the filtrate was evaporated in vacuo, and the residue was dissolved, if possible, in a non-water-miscible solvent, was washed in turn with aqueous citric acid, aqueous sodium bicarbonate and water, and was recovered by evaporation or filtration.

N,N'-dicyclohexylcarbodiimide reactions

Dr. The appropriate carboxy component (1 mmol) was dissolved in dimethylformamide (5 ml) and stirred at 4°C, while N,N'-dicyclohexylcarbodiimide (1.1 mmol) and the desired amino component

(]>mmol) was added. The mixture was then stirred at 4°C (or in some cases at 20-25°C) until a positive ninhydrin reaction was no longer obtained (up to 18 hours). If the amino component was in the form of its salt, triethylamine (1 mmol) was also added at the beginning of the reaction. After filtration, the product was isolated by evaporating the filtrate in vacuo, redissolving the residue in a suitable organic solvent, e.g. ethyl acetate, and washing in turn with aqueous citric acid, aqueous sodium bicarbonate and water, followed by evaporation of the dried extract, or by precipitation by addition of a suitable agent, e.g. petroleum ether (b.p. 60-80°C).

D2. As in D1, but with the addition of 1-hydroxybenzotriazole (2 mmol) to the reaction mixture.

D3. The appropriate carboxy compound (1 mmol) was dissolved

in dry tetrahydrofuran (2 mL), and N-hydroxysuccinimide (1 mmol) was added, followed by N,N'-dicyclohexylcarbodiimide (1 mmol) in tetrahydrofuran (1 mL). After 15 minutes, the amino component (1 mmol) was added, and the mixture was stirred at 20-25°C for 15-25 hours. After filtration, the product was isolated by evaporation in a vacuum, dissolution again in a suitable solvent, e.g. ethyl acetate, and washing in turn with aqueous citric acid, aqueous sodium bicarbonate and water, followed by evaporation of the dried extract in vacuo.

Reductions

Gl. For the preparation of the hexahydrophenylalanine derivatives, the appropriate phenylalanine-containing intermediate (as indicated in the tables) (1 mmol) was dissolved in aqueous 80% acetic acid, possibly containing some hydrochloric acid, Adam's platinum oxide catalyst (50-150 mg) was added with the usual precautions, and the reduction was carried out by passing a stream of hydrogen through the stirred mixture at 20-25°C until the reaction was complete (15-50 hours). The catalyst was removed by filtration through a layer of diatomaceous earth, and the crude product was isolated by evaporation of the filtrate in vacuo.

Ester hydrolyses

Jl. The ester (1 mmol) was dissolved in aqueous 75% v/v acetone (10-15 mL) containing sodium hydroxide (1.1 to 1.2 mmol), and the solution was stirred at 20-25°C until no more some reaction as judged by thin layer chromatography (2-3 hours). Most of the acetone was distilled off in vacuo, and the remaining aqueous solution was. washed with ethyl acetate."It was then acidified, e.g. by addition of citric acid, and the product was either extracted into a suitable solvent, e.g. ethyl acetate, or isolated by evaporation if this was not possible. The extracts were washed with saturated saline, dried and evaporated in vacuo to give the desired carboxy compound.

J2. As in Jl, but using a water/methanol/acetone mixture.

J3. As in Jl, but using aqueous methanol as reaction medium.

J4. Hydrolysis was carried out with an equivalent of sodium hydroxide

in water, and the product was isolated as the sodium salt by evaporation in vacuo.

J5. As in Jl, but using aqueous dioxane as reaction medium.

Acylations

At. To an ice-cold solution of the appropriate glyceryl mpnoester (1 mmol) in chloroform (1 ml) containing pyridine

(3 mmol) was added acetyl chloride (3 mmol), and the mixture was stirred at 20-25°C for 18 hours. The solvent was distilled off in vacuo, and the residue was re-dissolved in ethyl acetate and was washed in turn with water, saturated aqueous sodium bicarbonate and water. The dried extract was evaporated in vacuo to give the crude product

which was triturated with a small amount of methanol and collected by filtration.

The glyceryl monoester used for this acylation was obtained as follows. The corresponding isopropylidene-protected ester (50 mmol) in 2-methoxyethanol (300 mL) was heated on a steam bath for 6 h with boric acid (500 mmol). The solvent was evaporated in vacuo and replaced with ethyl acetate. After washing with water, the solution was dried and evaporated in vacuo to give the ester as an oil which was further purified by chromatography on a silica column.

K2. As in Kl, but using palmitoyl chloride instead of acetyl chloride.

K3. The benzylidene-protected glyceryl monoester (1 mmol) and boric acid (4 mmol) in trimethylborate (5 mL) were heated on a steam bath for 20 minutes. The trimethyl borate was distilled off, and the residue was heated for a further 20 minutes on a steam bath.

It was then redissolved in ethyl acetate (30 ml), washed with water and recovered as free glyceryl monoester by evaporation. Acetylation was then carried out using acetyl chloride which

in Kl.

K4. As in K3, but using hexanoyl chloride for the final acylation.

F forest strings

Lee. The anhydrous protected amino acid (10 mmol) was dissolved in dry pyridine (10 mL) at 0°C, and benzenesulfonyl chloride (10 mmol) was added. The mixture was stirred at 0°C for 15 minutes before adding the appropriate alcohol (as indicated in the table) (10 mmol). The whole was then stirred at 4°C for 18 hours before distilling off the pyridine in vacuo. The residue, dissolved in ethyl acetate (150 ml), was washed in turn with water, N hydrochloric acid, water,

2N aqueous potassium bicarbonate and finally with water. Evaporation of the ethyl acetate after drying gave the crude product.

L2. To a stirred solution of the appropriate protected amino acid (5 mmol) in pyridine (5 mL) at 0°C was added phenol (10 mmol) and N,N<1->dicyclohexylcarbodiimide (10 mmol) and the whole was stirred at 4°C for 18 hours. After filtering and evaporating the pyridine in vacuo, the residue was dissolved in ethyl acetate (100 ml) and washed in turn with water, aqueous 2N potassium bicarbonate and water. The dried extract was evaporated to give the crude ester.

L3. The appropriate protected amino acid (10 mmol) was dissolved in acetonitrile at 0°C, and the desired alcohol (10 mL) was added along with pyridine (20 mmol) and N,N'-dicyclohexylcarbodiimide (11 mmol). The mixture was stirred at 4°C for 18 hours and then filtered and evaporated in vacuo. The residue was taken up in ethyl acetate and washed in turn with aqueous citric acid, aqueous potassium bicarbonate and water. Evaporation of the dried ethyl

acetate extract gave the crude ester.

L4. As in L3, but using acetone as reaction medium instead of acetonitrile.

Turnover with ammonia

Ms. • The indicated peptide methyl ester was dissolved in the minimum amount of dimethylformamide and kept in contact with an excess of concentrated ethanolic ammonia for 3 days at 20-25°C. The product was isolated by eluting with water.

Reaction with hydrazine

Nine. The peptide methyl ester (10 mmol) was dissolved in dimethylformamide (25 mL) and aqueous 60% hydrazine hydrate (50 mmol) was added. The mixture was stirred at 20-25°C for 18 hours, optionally concentrated to approx. half volume in vacuo, and the product was precipitated by addition of water.

Other reactions

Pl. Boc-Tyr(But)-D-Ala-Gly-Phe-Leu-OH (starting material no. 161) (729 mg, 1 mmol), neat dry pyridine (0.55 mL, 7 mmol) and freshly distilled acetic anhydride (0 .9 ml, 6.7 mmol) were stirred together at 20-22°C for 10 minutes. The resulting solution was heated at 90-92°C for 6 hours and then cooled and treated with water (15 mL). The gum that separated was freed from the supernatant by decantation, washed with water (5 x 15 mL) by decantation, and dissolved in ethyl acetate (50 mL). The solution was washed successively with 10% w/v aqueous citric acid (4 x 10 ml), water (1 x 5 ml), 10% w/v aqueous potassium bicarbonate (3 x 10 ml) and water (3 x 10 ml), then dried ( anhydrous magnesium sulfate) and evaporated. The nuclear magnetic resonance spectrum of the resulting solid residue (530 mg) showed that it was a mixture of the L, D, L, L and L, D, L, D forms of

Ql. Z-Tyr(Bufc)-OH (isolated from 55.2 g, 100 mmol, of its dicyclohexylamine salt) was dissolved in dry tetrahydrofuran (300 mL), and methyl iodide (50 mL, 800 mmol) was added. Sodium hydride (8.6 g, 80% w/w dispersion in oil, i.e. 300 mmol) was then added and the mixture was heated under reflux for 18 hours in a bath at 75°C. Excess sodium hydride was then destroyed by addition of ethyl acetate to the cooled suspension, followed by water to give an almost clear solution. This was concentrated in vacuo to give an aqueous solution which was further diluted with water (150 ml) and

washed twice with ether to remove any methyl ester present. The aqueous solution was brought to pH 3 by addition of citric acid and extracted with ethyl acetate (400 ml,

200 ml x 2). The extracts were washed in turn with water (100 ml), aqueous 10% w/v sodium thiosulphate (100 ml) and then with water until the wash waters were neutral. evaporation of the extract after drying over magnesium sulfate gave an oil which was suspended in hot petrol (200 ml, b.p. 60-80°C) and treated with ethyl acetate to give a clear solution. On cooling to 4°C,

Z-MeTyrfB.uS-0H separated as a solid, m.p. 96-99°C.

This preparation was based on the methylation process described by J.R. McDermott and N.L. Benoiton, Can. J. Chem., 1973, 51, 1915.

R1..Z-NHNH-C0-C1 (3.87 g., 17 mmol) was added to a solution of Phe-Azleu-NH2.HC1 (5.33 g, 17 mmol) and triethylamine (2.45 mL, 17 mmol) in chloroform (50 mL). The mixture was kept at ambient temperature for 16 h, and ethyl acetate (400 mL) was then added. The solution was washed alternately with water and aqueous 20% w/v citric acid, dried over sodium sulfate and evaporated in vacuo. The residue was purified by silica gel column chromatography using chloroform, 2% v/v methanol in chloroform and systems P and Q as eluents. Z-Azgly-Phe-Azleu-NH2 was obtained as a solid, m.p. 116-120°C (split.)

Sl. A solution of Z-Tyr(Bzl)-OCp (17.5 g, 30 mmol) and Boc-NMeNH 2 (4.38 g, 30 mmol) in dimethylformamide (50 mL) was kept for 18 h at ambient temperature. After dilution with ethyl acetate (500 ml), the solution was washed in turn with water, aqueous 20% w/v citric acid and water. Evaporation of the dried extract in vacuo gave a solid which was treated directly with a solution of hydrogen chloride (100 mmol) in ethyl acetate for 2 hours at ambient temperature. Evaporation of the solvent in vacuo gave Z-Tyr(Bzl)-NHNHMe.HCl; sm.p. 223-224°C. RfD 0.82, RF 0.64, R£Q0.63.

The preceding hydrochloride (9.4 g, 20 mmol) was dissolved in chloroform (200 mL) and triethylamine (2.8 mL, 20 mmol) was added, followed by methyl isocyanatoacetate (2.3 g, 20 mmol). The mixture was stirred for 18 hours at ambient temperature before removal of the solvent by evaporation in vacuo. The residue was dissolved in ethyl acetate and washed in turn with water, aqueous 20% w/v citric acid, saturated aqueous sodium bicarbonate and water. Evaporation of the dried extract in vacuum gave Z-Tyr(Bzl)-Azala-Gly-OMe which after crystallization from methanol/ether had m.p. 107-108°C.

In Tables IV to XIX, the special number for each starting material is given in the first column and is followed in the next or the next two columns by its structure. The "Method" column describes the particular method used and then indicates the number or structure of the starting material on which the method was performed. The next columns give information on R^ values, and the last column refers to special points indicated immediately after Table XIX. Thus, e.g.,

in Table IV, starting material No. i3, the structure of which is given in the second and third columns, prepared by carrying out procedure H1 on starting material No. 24. The product was recrystallized from isopropanol/ether and had m.p. 181-183°C.

Claims (1)

Method for producing a physiologically active polypeptide with the formula: where R" <*> " means hydrogen or an alkyl radical of 1 to 6 carbon atoms; R means hydrogen or a radical selected from an alkanoyl radical with 1 to 3 carbon atoms, an alkoxycarbonyl radical with 2 to 6 carbon atoms or an amino acid residue which is Arg, Gly, Glu, Asp, Phe, 3~ Ala or a Lys or D-Lys both of which are optionally substituted on the e-amino group with a Alkoxycarbonyl radical with 2 to 6 carbon atoms, or R 2 means 2 to 6 α-amino acid residues which are linked together via normal peptide bonds, these amino acid residues being independently selected from Gly, Lys, Pro, Phe, Gin, Glu, Leu, Arg and Asp, The Lys and Arg residues are possibly alternatively linked together via their ε-amino- and -guanidino radicals respectively, and the Asp and Glu residues are optionally alternatively linked together via their 3- and y-carboxy radicals respectively; B is D-Ser or D-Thr, both of which are optionally substituted 3~ 0H with an alkyl radical of 1 to 6 carbon atoms, or B is D-Ala, D-Leu, D-Asp, D-Lys, D-Trp, D-Met, 3-Ala or Azalea; E is Gly or Azgly, both of which are optionally substituted on α-amino- the radical, or 3-Ala optionally substituted on the 3-amino radical, with an alkyl radical of 1 to 6 carbon atoms; F is Phe, hexahydroPhe, Azphe or hexahydroAzphe, which all can optionally be substituted on the α-amino radical with et alkyl radical of 1 to 6 carbon atoms; G is Leu, D-Leu, Met, D-Met, Nie, D-Nle, Ile or D-Ile> all of which may optionally be substituted on the α-amino radical with an alkyl radical of 1 to 6 carbon atoms, or G is Pro, Azleu or Azpro; K means a hydroxy or amino radical, a radical with the formula OR where R is an alkyl, alkenyl or hydroxy alkyl radical with up to 6 carbon atoms, an aminoalkyl radical with up to 6 carbon atoms or an alkylaminoalkyl radical with ■ up to 10 carbon atoms, where in both the nitrogen atom is optionally substituted with a benzyloxycarbonyl radical, or R <3> means a phenyl radical or a radical with the formula: where R 4 is an alkanoyl radical with 1-20 carbon atoms, or K means, a radical with the formula NHR 5 where R 5 is an alkyl or cycloalkyl radical with 1 to 6 carbon atoms, a radical with the formula NR 6 R 7 where R 6 and R 7 are alkyl radicals with .1 to 6 carbon atoms, or R and R are bonded together to form a 5- or 6-membered ring optionally containing a further heteroatom, a radical with the formula -NH-CHo CHo 0R <4> 4 12 . where R has the above meaning, a hydroxyalkylamino radical with up to 6 carbon atoms or an (alkylamino)alkylamino or (dialkylamino)alkylamino radical with up to 10 carbon atoms, or K means a radical with the formula Gly-OR8, D-Thr -OR8, Thr-OR <8> , D-Ala-OR <8> or Ala-OR <8> where R 8 is a hydrogen atom, an alkyl radical with 1 to 6 carbon atoms or a radical of formula II, III or IV indicated above where R 4 has the above meaning, or K is an alkyl radical with 1 to 6 carbon atoms; or G and K together mean the D or L forms of a radical with the formula: where n is an integer from 1 to 4, or a radical with the formula: ~ and when the polypeptide of formula I contains a basic function, the pharmaceutically acceptable acid addition salts thereof, and when the polypeptide of formula I contains an acidic function, the pharmaceutically acceptable base addition salts thereof, characterized by (a) removing one or more conventional peptide protecting groups from a protected polypeptide to form the compound of formula I; or b) for the preparation of the compounds where K is different from a hydroxy radical or, when K means a radical with the formula Gly-OR8, D-Thr-OR8, Thr-OR <8> , D-Ala-OR <8> or Ala -OR <8> , R g is different from a hydrogen atom, and for the preparation of the compounds where G and K together mean radicals with the above-mentioned formulas V, VI or VII, reaction of a carboxylic acid with the formula: or an activated derivative thereof, with a suitable alcohol or amine, and optionally converting the compound produced into a pharmaceutically acceptable salt thereof.