PT98498A - METHOD FOR PREPARING POLYEPEPTIDES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM - Google Patents

Description

I I

Description relating to the patent of invention of THE WELLCOME FOUNDATION LIMITED, British, industrial and commercial, headquartered at Unicom House, 160 Euston Road, London NW1 2 BP, England, (inventors: Frederick Charles Kull and Johann Jakob Leban, residing in the United States of America) for " PROCESS FOR THE PREPARATION OF POLYPEPTIDES AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM ".

description

The present invention relates to polypeptide compounds which possess antagonistic properties against bombesin, or peptides of the bombesin type, and are useful in the treatment of diseases, particularly human small cell lung cancer, in Zollinger-Ellison syndrome or cancer pancreatic. The invention therefore discloses the polypeptides, the processes for their preparation, pharmaceutical compositions containing them and their use in medicine. Bombesin is a tetradecapeptide originally isolated from the skin of a frog. It has the formula

Glp-Gln-Arg-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 The gastrin releasing peptide is a peptide with 27 amino acids isolated from pork casings. The last 10 amino acids at the C-terminal end of the gastrin releasing peptide correspond, with a modification of amino acid (3)

namely: to the last 10 amino acids of bombesin, H-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH 2

It has been reported (JH Walsh and JR Reeve, Petides 6, (3), 63-68 (1985)) that bombesin and bombesin-like peptides, such as the gastrin releasing peptide, are secreted by human lung cancer cells of small cells (SCLC). It has been found (PJ Woll and E. Rozengurt, PNAS 85, 1859-1863, (1988)) that gastrin releasing factor antagonists would bind competitively to bombesin receptors in animals and therefore would be useful in the treatment of SCLC and / or in the control of clinical symptoms associated with this disease and due to hypersecretion of this peptide hormone.

Bombesin analogs have been shown to inhibit gastrin-releasing peptide binding to a SCLC cell line and to inhibit SCLC cell growth in vitro and in vivo (S. Mahmoud et al., Cancer Research, 5, 1798-1802 (1991) .

A number of bombesi-13-14 antagonists have already been disclosed, for example [Leu -HP (CH 2 -NH) -Leu] bombesin and [Ala-Cl ^-N--Val-Leu] bombesin (Coy et al. 4-Pyridyl-CO-His-Trp-Ala-Val-D-Ala-His-Leu-Me, D-Ala-His-Leu-NHe, 3-Pyridyl-CO-His-Trp-Ala-Val-D-Ala-His-Leu-NHe, 4-Pyridyl- Ala-His-MeLeu-OMe, 4-Pyridyl-C0-His-Trp-Ala-Val-D-Ala-Lys (Z) -MeLeu-Me, 3-Indolyl-C0-His-Trp-Ala-Val-D 4-Pyridyl-CO-His-Trp-Ala-Val-D-Ala-His-Me-Leu-NHMe, 4-Pyridyl-CO-His-Trp-Ala-Val-D-Ala -Lys (Z) -Leu-NHMe, 4-Pyridyl-CO-His-Trp-Ala-Val-D-Ala-Lys (COCH2 Ph) -Leu-NHMe and 4-Pyridyl-CO-His-Trp-Ala-Val -D-Ala-Lys (COCH2CH2Ph) -Leu-NHMe. (European Patent Application No. 345990A).

In formula (I) below and throughout this specification 2 1 i

Descriptively, amino acid residues are designated by their standard abbreviations (Pure and applied Chemistry, 1974, 40, 317-331; European Journal of Bio-Chemistry, 1984, 138, 9-37).

For the avoidance of doubt, it should be clarified that: amino acid symbols refer to the L configuration, except where indicated otherwise by the letters D or DL before the symbol, and separated by a hyphen, (R) and (S) are the standard designations for the molecular configuration.

The following abbreviations are used: = psi (CH2 NH)

Ada = 1-adamantanecarboxylic acid CPenc = aminocyclopentanecarboxylic acid

Mox = methoxynin

Des NHgPro = 1-cyclopentanecarboxylic acid

D-tBuGly = D-t-butyl-glycine (t-D-leucine) D-tBuGly = D-t-butyl-glycine

When the Ada, CPenc, Mox, des-NHg Pro, des-NI-Tyr groups are in a polypeptide chain, they are present in the carbonyl form.

The compounds of the present invention inhibit the production of the gastrin releasing peptide in mammalian cells and are therefore useful for controlling the clinical symptoms of the disease causing the secretion or hypersecretion of the gastrin releasing peptide (e.g., SCLC).

It has now been discovered that another group of polypeptides has potent bombesin antagonist activity.

Accordingly, the present invention discloses a polypeptide of the formula (I): (I) 1 2 3 4 5 6 7 XX TrpX X 3 X X X NH 2 8 η

in which X represents a group X Arg (or D-Arg) XyX

O and Xo represents des-NH2 Pro, TyrPro, des-NH2 TyrPro, Ada, Pro,

D-Pro or is missing; g X is Gly, Ala, D-Ala or is absent; 10 X represents Asn, Phe, D-Phe, or Phe or D-Phe substituted by one or more halogen atoms; or X represents an A- (CH 2) n -CO- group in which A represents a group containing 1 to 3 rings of which at least one ring is aromatic, each of which ring systems is optionally substituted; and the alkylene group is optionally substituted by 1 to 4 groups selected from amino, hydroxy, alkoxy having 1 to 4 carbon atoms and alkyl having 1 to 4 carbon atoms, optionally substituted by halogen and n is 0 to 0 or X is wherein A represents an optionally substituted aromatic residue containing 1 to 3 rings and the alkylene group is optionally substituted by 1 to 4 groups selected from amino, alkoxy having 1 to 4 carbon atoms and with 1 to 4 carbon atoms optionally substituted by halogen and n is 1 to 4, or X represents cyclopentylcarbonyl substituted by a group (as defined above); 1 φ X represents His, ThiAla or is missing; X represents Ala, D-Ala, CPenc, D-tBuGly or Pro;

O

Jr is Vai or Vai substituted by one or more halogen atoms; X represents Gly, Ala, B-Ala, sarcosine, pro, D-Pro or D-Phe; X represents His or ThiAla; represents D-proy, Pro-, 2-pyrrolidinyl-3-hydroxypropionyl or D-pro; X represents Nle, leu, Phe, Val, Mox, D-Phe, or Phe, or D-Phe substituted by 1 or more halogen or naphthyl-Ala or naphthyl-D-Ala atoms or a water-repellent substituted aromatic amino acid , or aralkylamine, or is absent. A is preferably phenyl, naphthyl, phenothiazinyl or indolyl. A is preferably phenyl or naphthyl. - 4 -

tun

Suitable substituents for the aromatic ring Ar include hydroxy, phenyl, halogen, alkyl of 1 to 4 carbon atoms or alkoxy of 1 to 4 carbon atoms optionally substituted by halogen.

Preferably η I 2.

Preferably X terminates by a radical of -NH2 + g

Preferably X represents des-NHâ, "Tyr or deso-NHâ," Pro. X preferably represents Gly or D-Ala. X is preferably D-Phe. preferably X represents des-NÎ ± Phe, des-NHgTyr, des-NE-TyrPro (or D-Pro) Arg (or D-Arg). 1

Preferably X represents His or ThiAla; Preferably X is ala, Pro;

Preferably X is Val or Hexafluorvaline; Preferably X represents D-Ala, D-Phe; Preferably X represents His, ThiAla; g preferably X represents D-Pro2, PrcA1, Pro, D-Pro; Preferably X represents Nle or Phe, Leu, methoxynin, 2-naphthyl-2-alanine.

Preferred polypeptides of the present invention include: N - ((R) -2- (6-methoxy-2-naphthyl) propionyl) -HysTrpAlaValD-AlaHisD-ProVNle -NH2 N - ((S) -2- (6-Methoxy- 2-Naphthyl) propionyl) -HisTrpAlaValD-AlaHisD-ProVNle-NH2 N - ((S) -B-Phenylbutyryl-HisTrpAlaValD-AlaHisD-proWle-NH2 N - ((R) -3-Phenylbutyryl) -HisTrpAlaValD-AlaHisD-ProVNle-NH2 N - ((S) -3,3,3-Trifluoro-2-thienyl) - N - ((3-Phenyl) propionyl) -HysTrpAlaValD- (R) -3,3,3-Trifluoro-2-methoxy-2-Phenyl-Propionyl) -HysTrpAla-Val-D-prenyl] -N- (2-methoxy-2-phenyl-propionyl) NH2 N-3 - (((4'-Hydroxy) Phenyl) propionyl) -ProD-ArgGlyD-PheHisTrpAla-ValGlyHisD-proH / Nle-NH2 N - (((4'-Hydroxy) -3-phenyl) propionyl) -ProD -ArgHisTrpAlaValD-AlaHisD-ProyLeu-NH2 5 *

Ν - ((3-Phenyl) propionyl) -HisTrpAlaValD-AlaHisD-ProYmox-NH2 N - ((3-Phenyl (Propionyl) -HisTrpAlaValD-proYPhe -NH2 N - ((3-Phenyl) Propionyl) TrpAlaValD-AlaHisD-Pr oYLeu -NH 2 N - ((3'-Trifluoromethyl) Phenyl) Propionyl) -HysTrpAlaValD-AlaHis-D-ProYLeu- NH2 N - ((desamino-Pro) -D-ArgD-AlaD-PheHisTrpAlaValGlyHisD-proYNle-NH2 N- ((3-Phenyl) propionyl) - (3- (2-Thi) -Ala) TrpAlaValD-AlaHisD-ProfLeu- -NH2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValGlyHisD-ProVNle-NH2 N - ((desamino-Pro) -D-ArgD-AlaD-PheHisTrpAlaValD-AlaHisD-ProYNle- ) -HisTrpAlaValD-AlaHisD-ProYNle-NHg

TyrProD-ArgGlyD-PheHisTrpAlaValGlyHisD-ProYNle-NH2 D-ArgGlyD-PheHisTrpAlaValGlyHisD-Prc * PNle -NH2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisD-ProPhe-NH2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValD -NH 2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValD-PheHisD-ProYPhe-NH2 D-PheHisTrpAlaValD-AlaHisD-proYPhe -NH2 N- ((2-Naphthyl) (3-Phenyl) Propionyl) -D-ProArgGlyD-PheHisTrpAlaValD-AlaHisD-ProYPhe-NH2 N - ((3-Phenyl) Propionyl) - (3- (2-Thi) -Ala) -TrpAlaValD-AlaHisD-ProtyPhe- NH2 N-3 - (((4-Hydroxy) Phenyl) Propionyl) -HysTrpAlaValD-AlaHisD-ProV-Phe-NH 2 NH 2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisD-ProV-Phe-NH2 N - (((2 ', 6'-Dichloro) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-ProYNle-NH2 N - ((((3,4-Dichloro) -2-phenyl) Acetyl) - HysTrpAlaValD -AlaHisD-ProHN-NH2 N- (1-Naphthoyl) -HisTrpAlaValD-AlaHisD-proVNle-NH2 N- (1-Naphthoyl) -HisTrpAlaValD-AlaHisD-AlaHisD- N - ((3,7-Dihydroxy) -2-naphthoyl) -HysTrpAlaValD-AlaHisD-ProtyNle-NH2 6 (

Ν - (((3,4-Dihydroxy) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-ProVNle-NH2 N- (2- (3-Pyridyl) Acetyl) -HisTrpAlaValD-AlaHisD-proVNle- N - (((3-Fluoro) -3-Phenyl) Propionyl) -HysTrpAlaValD-AlaHisD-ProtyNle-NH2 N - (((4-Fluoro- 3-methoxy) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-PrctyNle-NH2 N - (((R) - (-) - 2-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisD-ProVPhe-nh2 N - (((S) - (+) - 2-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisD-ProH2 Phe-Nh2 N - (((Trans) -2-Phenyl) Cyclopropanoyl) -HisTrpAlaValD-AlaHisD-_proV ) / phe-NH 2 N- (3- (10-Phenethiazinyl) Propionyl) -HysTrpAlaValD-AlaHisD-Pro-Phe-2 N - ((3-Methyl-3-Phenyl) butyryl) -HisTrpAlaValD-AlaHisD-ProH / Phe -NH2 N - (((2'T-Trifluoromethyl) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-ProyPhe-NH2 N - (((2'-Trifluoromethyl) -2- -ProtyPhe-NH2 N - (((4-trifluoromethyl) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD - γγοθθ-ΝΗ2 N - (((2 ', 3'-Difluoro) -2- -HisTrpAlaValD-AlaHisD-Pro-Phe-NH2 N - (((2 ', 4-Difluoro) -2-Phenyl) Acetyl) -HysTrpAlaValD-AlaHisD-Pro-Phe-NH2 N - (((2, 6-Difluoro) -2- Phe-NH 2 N- ((2-Amino) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-Pro'VPhe -NH2 N- (1-naphthoyl) -HisTrpAlaValD-AlaHisD-ProtyPhe-NHg N - ((( 3 ', 41,5'-Trimethoxy) -3-Phenyl) Propionyl) -HysTrpAlaValD-AlaHisD-Pro2 Phe-NH2 N - ((6'-Methoxy) -2- (2-Naphthoyl) Propionyl) HysTrpAlaValD- AlaHisD-Pro-Phe-NÎ ± - (((3-Trifluoromethyl) -3-Phenyl) Propionyl) -HisTrpAlaValD-AlaHiβ-Pro-Phe-NH2-7 - Î ± - (((S) -3 "(Phenyl) butyryl) -HisTrpAlaValD-AlaHisD-ProlyPhe-NH2 - (((4'-Hetoxy) -3-phenyl) propionyl) Hydroxy) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-PrcPhe-NH2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisProPhe-Nα - ((2-Methyl-2-Phenyl ) Propionyl) -HisTrpAlaValD-AlaHisD-ProVPhe-nh2 N- (3- (1-Naphthyl) Propionyl) -HisTrpAlaValD-AlaHisD-PrcWPhe-NH2 N - (((R) -3-Phenyl) butyryl) -HisTrpAlaValD-AlaHisD -ProyPhe-NH 2 N - ((9-Fluoroenoyl) 1-Carbonyl) -HysTrpAlaValD-Ala HisD-ProH4? He-NH2 N - (((2'-Methoxy) -3-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisD-ProVPhe -NH2 N - (((2 ', 5'-Dimethoxy) -3-Phenyl ) Propionyl) -HysTrpAlaValD-AlaHisD-ProAvhe-NH2 N - ((3-Phenyl) Propionyl) -HisTrpAlaValD-AlaHisD-Pro-Tyr-NH2 N - (((2 ', 3'Dimethoxy) -S-Phenylpropionyl- (3- (2-Pyrrolidinyl-3-hydroxy) propionyl) -Phe-NH2 ((isoquinolylcarbonyl) -HisTrpAlaValD -AlaHisD-ProyPhe-NH2 N- ((3-Phenyl) Propionyl) -HisTrpAla't / ValD-AlaHisD-ProH'Phe-NH 2

Pharmaceutically acceptable salts or precursors of the polypeptides of formula (I) are also included within the scope of the present invention. Suitable pharmaceutically acceptable salts are acid addition salts when the polypeptide is sufficiently basic, for example, to contain one or more basic residues such as histidine. A suitable pharmaceutically acceptable acid addition salt according to the invention may be formed with an inorganic acid, for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid, or with an organic acid, for example acetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid or trifluoroacetic acid. The invention also discloses, as an additional feature, one or more of the compounds preferred together with the

their pharmaceutically acceptable acid addition salts.

The polypeptides of the present invention may be prepared by any process well known in the art of peptide chemistry as being applicable to the synthesis of analogous compounds. Thus, for example, a polypeptide according to the invention can be obtained by processes analogous to those described in " Solid Phase Peptide Synthesis " by Stewart and Young (published by Pierce Chemical Company, Illinois, 1984), " Principles of Peptide Synthesis " (published by Springer-Verlag, Berlin, 1984), " Practice of Peptide Synthesis " Berlin, 1984), and " The Synthesis of a Tetrapeptide (J.Am.Chem.Soc., 83, 2149 (1963)).

Preferred processes for the production of a polypeptide according to the invention include, for example: (a) the removal of one or more conventional peptide blocking groups from a blocked polypeptide, so as to produce a polypeptide according to the invention of formula I;

(b) forming an amide bond through the coupling of two peptidic units containing one carboxylic acid group, or a reactive derivative thereof, and the other containing an amino group, such that a polypeptide is formed, blocked or unblocked, having the sequence indicated in formula I, whereafter, if necessary, the blocking groups are removed using process (a) above.

In process (a) there may be as many blocking groups in the starting compound as the radicals requiring protection, for example some or all of the groups which exist in the product in the form of free hydroxy groups or basic amino groups (both primary amino groups as secondary). The blocking group or blocking groups may be chosen from those described in the usual manuals on the peptide chemistry described above. In the same works there are also various methods for removing the blocking group (s).

In process (a) a blocking group suitable for - 9 -

a basic amino group (either at the N-terminal or an amino acid side chain) is for example an arylmethoxycarbonyl group, which may be removed by hydrogenation in the presence of a catalyst, for example palladium on activated carbon, or may be removed by treatment with an inorganic acid, for example anhydrous hydrogen fluoride or hydrogen bromide.

In process (a) a particularly suitable blocking group for a basic amino group is for example an alkoxycarbonyl group, for example a Boc group, which may be removed by treatment with an organic acid, for example trifluoroacetic acid, or may be removed by treatment with an inorganic acid, for example anhydrous hydrogen chloride or hydrogen bromide; or for example a 9-fluorenylmethoxycarbonyl group, which may be removed by treatment with an organic base, for example piperidine.

In process (a) a blocking group particularly suitable for the basic amino group on the histidine side chain is for example an arylsulfonyl group, for example a tosyl group, which may be removed by treatment with hydroxylamine, for example an N-hydroxytriazole, particularly 1-hydroxybenzotriazole, benzyloxymethyl or t-butyloxymethyl.

In process (a) a suitable blocking group for a hydroxy group is for example an arylmethyl group, for example a benzyl group, which may be removed by treatment with an inorganic acid, for example anhydrous hydrogen fluoride, or may be removed by hydrogenation in the presence of a catalyst, for example palladium on activated charcoal; or may be for example an esterifying group, for example an acetyl or benzoyl group, which may be removed by hydrolysis with a base, for example sodium hydroxide.

In process (a) a blocking group suitable for a carboxy group is for example an esterifying group, for example an arylmethyl group, for example a benzyl group, which may be removed by treatment with an inorganic acid, for example hydrogen fluoride anhydride, or may be removed by hydrogenation in the presence of a catalyst, for example

activated charcoal; or an alkyl, alkyl of 1 to 6 carbon atoms, for example a t-butyl group, which may be removed by treatment with an organic acid, for example trifluoroacetic acid.

In process (a) particularly convenient protection for a carboxy group at the C-terminus is provided by the formation of for example an ester, for example the ester formed by the coupling of the C-terminal amino acid and a resin, for example a styrene resin rectylated hydroxymethylated di-vinylbenzene; or by the formation of, for example, an amide, for example the amide formed by the coupling of the C-terminal amino acid and a resin, for example a recylated methylbenzhydrylamino-styrene-divinylbenzene resin.

In process (b) any conventional peptide coupling reaction can be employed, for example those described in the conventional manuals on peptide chemistry already indicated above.

In process (b) it is to be understood that a polypeptide moiety may contain only one amino acid, either blocked or unblocked.

In process (b) a suitable coupling reaction is for example a coupling reaction in solution, for example an active ester coupling, an azide coupling or a coupling involving Î ±, Î'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and BOP benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate.

In process (b) an appropriate reactive derivative of the peptidic moiety containing a carboxylic acid group is for example an acyl halide, for example an acyl chloride formed by the reaction of the acid and an inorganic acid chloride, for example thionyl chloride ; a mixed anhydride, for example an anhydride formed by the reaction of the acid and a halo-genoformate, for example isobutyl chloroformate; or an acyl azide, for example an azide formed by the reaction of the acid and an azide, such as diphenylphosphoryl azide. 11

In process (b) a particularly suitable reactive derivative of the peptidic moiety containing a carboxylic acid group is for example the reaction product of the acid and a carbodiimide, for example Ν, Ν-dicyclohexylcarbodiimide or N, N- [ diisopropylcarbodiimide, or is the reaction product of the acid, an N-hydroxytriazole, for example 1-hydroxybenzotriazole, and a carbodiimide, for example N-dicyclohexylcarbodiimide or N, Î'-diisopropylcarbodiimide.

In process (b) a preferred strategy is for example to use solid phase synthesis in which the amino acid which becomes the C-terminal amino acid of an α-polypeptide of the invention is protected in the α-amino group and, if necessary, the side chain and I coupled to a solid support, for example a resin, for example a hydroxy methylated styrene resin or a methylbenzhydrylamino-styrene-divinylbenzine crosslinked through an ester or amide bond respectively, after the The amino acid to be bound to the C-terminal amino acid is blocked in the test group and, if necessary, in the side chain and coupled to the C-terminal amino acid that remains This gradual process of unlocking the α-amino group and coupling to the next amino acid is repeated in order to obtain a blocked or unblocked polypeptide attached to the solid support. or non-blocked, can be released from the solid support of the hydroxymethylated resin for example by hydrolysis, for example an acidic hydrolysis, for example with an organic acid, for example trifluoroacetic acid, or for example with an inorganic acid, for example hydrogen fluoride or anhydrous hydrogen bromide; or the polypeptide is released for example by alcoholysis, for example methanolysis, in the presence of a base, for example an organic base, for example diisopropylethylamine, whereafter the blocking groups are removed using the above process (a).

When a methyl benzhydrylamine resin is used, the blocked or unblocked polypeptide can be released from the 12

solid support, for example by treatment with an inorganic acid, for example hydrogen fluoride, whereafter, if necessary, the blocking groups are removed using the above process (a).

Process (b) Another preferred strategy is for example to use a solid phase synthesis in which an amino acid, which becomes a linker within the chain of amino acids forming a polypeptide according to the invention, is blocked in the Î ± -amino group and, if necessary, the side chain and is coupled to a solid support, for example a resin as described above, whereupon the Î ± -amino group blocking group is removed. The amino acid which binds to the amino acid which has been coupled to the solid support is blocked in the < / RTI > group and if necessary in the side chain and coupled to the amino acid which remains coupled to the solid support. This gradual process of unlocking the αξ-amino group and coupling to the next amino acid is repeated in order to obtain a blocked or unblocked polypeptide attached to the solid support. The blocked or unblocked polypeptide can be released from the solid support, for example using one of the methods described above, after which another peptide moiety can be coupled using a coupling reaction in solution as described for process (b) above , and after which if necessary the blocking groups are removed using process (a) above.

The polypeptides of the present invention have a bombesin antagonist effect, which can be demonstrated by their ability to inhibit bombesin C stimulated mitogenesis of the Swiss 3T3 mouse fibroblast cells, as determined by the absorption of [H] - thymidine.

According to a further feature of the invention there is shown a pharmaceutical composition comprising a polypeptide of formula I, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable salt or carrier. . The composition may be in an appropriate form -

for oral use, for example a tablet, capsule, or aqueous or oily solution, suspension or emulsion; for nasal use eg an inhaler, a nasal spray or nasal drops; for vaginal or rectal use, for example a suppository; for administration by inhalation, for example as a finely divided powder or a liquid aerosol; for sublingual or buccal use, for example, a tablet or capsule; or for parenteral (including intravenous, subcutaneous, intramuscular, intravascular or infusion) use eg a sterile aqueous or oily solution or suspension.

In general the compositions described above can be prepared in a conventional manner using conventional excipients. However, in the case of a composition for oral administration, it may be desirable for the composition to include a coating to protect the polypeptide active ingredient from the action of the enzymes in the stomach. The composition of the invention may also contain, in addition to the polypeptide according to the invention, one or more known antitumor substances chosen from, for example, mitotic inhibitors, for example vinblastine; alkylating agents, for example cis-platin, carboplatin and cyclophosphamide; antimetabolites, for example 5-fluorouracil, cytosine arabinole and hydroxyurea; intercalary antibiotics, for example adriamycin and bleomycin; enzymes, for example asparaginase, topoisomerase inhibitors, for example etoposide, and biological response modifiers, for example interferon.

A preferred composition according to the invention is for example a composition suitable for oral administration in the form of a dosage unit, for example in the form of a tablet or capsule containing from 2.5 to 500 mg and preferably 10 to 100 mg of polypeptide in each dosage unit, or a composition suitable for parenteral administration containing 0.5 to 100 mg of the polypeptide per ml, and preferably 1 to 10 mg of the polypeptide per ml of the solution.

A parenteral composition is preferably a solution in isotonic saline or isotonic dextrose,

It is preferred that the parenteral composition may be a composition intended for slow release, in which case the amount of the polypeptide per dosage unit is generally greater than that required when a conventional injectable formulation is used. A preferred slow release formulation is for example a sustained release formulation, for example a formulation of the type disclosed in U.S. Patents 4,767,628 and 5,004,602. A preferred slow release parenteral formulation contains from 10 to 100mg of the polypeptide per unit of dosage. Another preferred slow release formulation is a microencapsulated polypeptide using a biodegradable biocompatible copolymer.

These compositions are preferably administered intravenously, although administration may also be effected by subcutaneous, intramuscular or in-transdermal injection.

Polypeptides are therapeutic agents that require specialized pharmaceutical formulations for efficient, safe and convenient use by patients. Since even small oligopeptides, such as thyrotropin releasing hormone (TRH), have very low oral activity, and large molecules are inactivated by endopeptidases in the gastrointestinal tract, long-term treatment requires injection or daily administration from of small portable infusion pumps. On the other hand, there are a number of potential attempts for self-administration methods (for example, nasal application, suppositories) or for sustained release formulations, such as injectable microcapsule suspensions and implants.

Suitable medicaments for transdermal administration may take the form of an aqueous solution of a compound of formula (I), optionally buffered, and may be provided by passive diffusion or electrically assisted transport, for example iontophoresis (see for example Pharmaceutical Research (3), 318 (1986)). 15

The composition according to the invention will normally be administered so that a daily oral dose contains from 0.1 mg / kg to 50 mg / kg, most preferably 0.1 mg / kg to 25 mg / kg, and a dose parenterally contains 20 microgram / kg to 10 mg / kg, more preferably 100 micrograms to 10 mg / kg.

According to another feature of the invention there is disclosed a method for producing a bombesin antagonist effect in a warm-blooded animal, such as man, in need of such treatment, which comprises administering to said animal an effective amount of a polypeptide of formula I, or a pharmaceutically acceptable salt thereof. The invention also discloses the use of this polypeptide of formula 1, or a pharmaceutically acceptable salt thereof, in the production of a novel medicament for use in the treatment of a disease or clinical condition mediated by bombesin or by a bombesin-type peptide.

The compounds of the present invention are useful for inhibiting the binding of the gastrin releasing peptide to cells. The compounds of the present invention also inhibit the growth of cancer cells.

A polypeptide according to the invention is useful in the treatment of malignancies, for example malignant diseases of the lung, such as human small cell lung cancer, e.g. malignant diseases of the pituitary gland, adrenal glands, pancreas or the skin. In particular a polypeptide according to the invention is useful for the symptomatic relief and / or for the treatment of exocrine pancreatic adenocarcinoma. A polypeptide according to the invention is useful in the treatment of conditions associated with an overproduction of bombesin or bombesin-like peptides (such as the gastrin releasing factor), for example the overproduction of gastrin in the intestine. in animals has been linked to the suppression of growth hormone and pro-lactin release. The polypeptides according to the invention can therefore be used to promote the availability of

growth hormone in humans or animals requiring this treatment. The polypeptides according to the invention may also be used in the treatment of conditions associated with the deficiency of the normal physiological control of the regulation of gastric acid secretion.

According to other aspects of the invention, there are disclosed: (a) polypeptides according to the invention for use in therapy; (b) pharmaceutical formulations containing polypeptides according to the invention, at least one pharmaceutical carrier or excipient and optionally one or more other therapeutic ingredients; (c) the use of the polypeptides according to the invention in the manufacture of a medicament for the treatment of cancer; (d) the use of a polypeptide according to the invention in the manufacture of a medicament for the treatment of: (I) entercutaneous fistula

(II) type II diabetes, type I (III) diabetes Zollinger-Ellison syndrome (IV) pancreatic (V) acromegaly (VI) psoriasis carcinoma, eg chronic plaque psoriasis (VII) small postoperative intestinal fistulas VIII) syndrome of depression (IX) malignant insulinoma (X) adenoma of cells of the growth hormone of the pituitary (XI). (e) a method of inhibiting the growth of cells which are sensitive to the growth promoting activity of the gastrin releasing peptide in a mammal (such as man) which requires this treatment, which comprises administering to said mammal a growth inhibitory amount of a polypeptide according to the invention,

to said mammal.

The following examples serve to illustrate the preparation of the polypeptides of the present invention and their biological properties:

EXPERIMENTAL SECTION

Materials; The following abbreviations are generally used BOC = t-butyloxycarbonyl TLC = thin layer chromatography NMR = nuclear magnetic resonance MS = mass spectrometry.

The articles below were obtained from Advanced Chemtech, Inc., P. 0. Box 1403, Louisville, KY 40201 U.S.A .: p-methylbenzhydrylamine resin. HCl (substitution ranges 0.56-0.94 meq / g), trifluoroacetic acid, N-Boc-D-Alanine, N-Boc-L-Alanine, N-Boc-N-Tosyl-Arginine, N N-Boc-L-Leucinol, N-Boc-L-Norleucine, N-Boc-D-Phenylalanine, N-Boc-L-Phenylalanine, N-Boc-D N-Boc-L-Proline, N-Boc-D-Prolinol, N-Boc-D-Sarcosine, N-Boc-L-Tryptophan and N-Boc-L-valine. N-Boc-L-im-CBZ-L-histidine, N-Boc-p-thienyl-L-alanine, and 3- (2-naphthyl) -D-alanine were purchased from Bachem, Inc., 3132 Kashiwa Street, Torrance, CA 90505, USA. L-Methoxynin (O-methyl-L-homoserine) was obtained from Biohellas S.A., 10. Parnithos Street, 154 52 P. Phsychiko, Athens, Greece. Leucine methyl ester hydrochloride, (+) - 6-methoxy-o-methyl-2-naphthaleneacetic acid and (S) -6-methoxy-t-methyl-2-naphthaleneacetic acid were supplied by Sigma Chemical Company, P. 0. Box 14508, St. Louis, MO 63178, USA Di-t-butyl dicarbonate, 4-fluorophenylalanine, N-hydroxysuccinimide ester with 3- (4-hydroxyphenyl) - 18 -

and both (R) and (S) -3-phenylbutyric acid were obtained from Fluka Chemical Corp., 980 South Street, Ronkonkoma, NY 11779, USA.

The following products were obtained from Aldrich Chemical Co., Inc., 1001 West Saint Paul Avenue, Milwaukee, W1 53233, USA: 3-phenylpropionic acid (hydrocinnamic acid), (R) and (S) 3-trifluor-2-methoxy-2-phenylpropionic acid, dicyclohexylamine, 1-cyclopentanecarboxylic acid, 4-methylmorpholine, 1-methylimidazole, sodium cyanoborohydride and diisoprostylethylamine.

Dichloromethane (CH2 Cl2), N, N-dimethylformamide (DMF) and N-methylpyrrolidone were used as solvents for the synthesis of the solid phase peptides, which were obtained from Baxter Healthcare Corp., Burdick & Jackson Division, Muskegon, MI 49442, USA. The BOP coupling reagent, benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate, was obtained from Richelieu Biotechnologies, Inc., 5726 Laurier Blvd., St-Hyacinthe, QC, J2S 3V8 Canada. Hydrofluoric acid was purchased from Matheson Gas Products, P. 0. Box 85, 932 Paterson Plank Road, East Rutherford, NJ 07073, USA.

PREPARATION OF THE PEPTIDES OF THE PRESENT INVENTION

Step I. Preparation of Boc-amino acids:

Amino acids which were obtained without the t-butyloxycarbonyl blocking moiety at the amino group (L-methoxynin, 4-fluorophenylalanine) were protected as described below. One equivalent of the amino acid was dissolved in 5% aqueous sodium carbonate / 1,4-dioxane and cooled to 0 ° C. 2 equivalents of di-t-butyl dicarbonate in 1,4-dioxane was added dropwise to the cold amino acid mixture. After the addition was complete the mixture was allowed to warm to 25 ° C and stirred for 12 hours. After the reaction was complete, which was followed by TLC, the dioxane was evaporated. The aqueous mixture was acidified with 1N HCl to pH = 2, whereupon crystals formed, in most cases, 60% yield. The solid was isolated by filtration and characterized by TLC, NMR and MS, generally being sufficiently pure to proceed without further purification. In extreme cases, such as in the case of L-methoxynin, where the product removed from the acidic aqueous solution was a liquid, the product was removed from the aqueous solution with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate and the ethyl acetate was evaporated. The liquid residue was dissolved in a small portion of ethyl acetate. 1 equivalent of dicyclohexylamine was added to the mixture, then diluted with a large excess of diethyl ether. After cooling the dicyclohexylamine salt of the Boc-amino acid crystallized and separated. The solid was isolated by filtration (yield 20-30%) characterized (TLC, NMR, MS) and was used without further purification.

Step II. Preparation of Boc-D-proline The procedure below is a variant of the reaction carried out by Mancuso et al in J. Org. Chem. 43, 2481 (1978).

In a 500 ml 3 neck flask, 25 ml of a 2.0 M solution of oxalyl chloride in methylene chloride (50 mmol, solution obtained from Aldrich) were dissolved in 60 ml of anhydrous dichloromethane at -60 ° C , under nitrogen atmosphere. Dimethyl sulfoxide (10 ml, 140 mmol), Aldrich) was dissolved in 25 ml of anhydrous dichloromethane and added dropwise to the solution at -60 ° C by means of a separatory funnel. This mixture was then stirred for 15 minutes after complete addition. Boc-D-prolinol (10 g, 50 mmol, Advanced Chemtech) was dissolved in 60 mL of anhydrous dichloromethane and cooled to -60 ° C in a 500 mL round bottom flask under a nitrogen atmosphere. To facilitate the combination of the contents of both cold balloons the nitrogen line was arranged in series to feed both balloons. The oxalyl chloride / DMSO mixture was added gradually to the alcohol solution by transfer aided with α-

under pressure through a double-ended needle. By manually controlling the nitrogen flow the contents were transferred from one flask to another, very slowly but not drop by drop. After the addition was complete this mixture was stirred for 20 minutes. Finally, 20 ml of triethylamine (140 mmol, Kodak) was slowly added, keeping the temperature still at -60 ° C. The mixture was then naturally warmed to room temperature. The mixture was washed with 50 ml of water. This aqueous phase was then extracted 3 times with 70 ml of dichloromethane. The organic extracts were successively washed with 50 ml each of sodium hydrogen carbonate media in 5N hydrochloric acid and water. The aqueous washing solutions were extracted with 50 ml of dichloromethane, which was then combined with the remaining organic phase. After drying the organic phase with anhydrous sodium sulfate, the solvent was removed by evaporation on a rotary evaporator. The residue was dried on a vacuum pump for 3 hours.

According to the NMR spectra, the characteristic peak -OH of Boc-D-prolinol (£ 4.5 ppm) was no longer visible. A clear syllable at 9 9.4 ppm evidences the presence of the Boc-D-proline aldehyde proton. TLC indicated a mixed product containing practically no side products and was therefore used without further purification. >

Step III. Preparation of Resin Boc-D-ProH * (CH3 NH) X-MBHA I). The p-methylbenzhydrylamine resin in the chlorohydrate form of Advanced Chemtect ™ s (p-MBHA) was used. The substitution of the oscillated resin between 0.90-0.97 meq / g. 10 g of p-MBHA resin (9.0-9.7 mmol) were placed on a manual agitator of peptides (Milligen). The resin was washed 2 times (3 and 5 minutes) with 10% Î ±, β-diisopropyl-ethylamine (DIEA, aldrich) in dichloromethane with stirring. The resin was then rinsed 2 times with dichloromethane. The neutralization of the. hydrochloric acid from the resin was indicated by a Kaiser nitrate test, when a very dark blue color was observed. The resin was then treated with 2 equivalents of each of the Boc-norleucine reagents (Bachem, Torrance, CA), benzotriazol-1-yloxy-tris (dimethylamino) -phosphonium hexafluorophosphate (BOP, Richelieu Biotechnologies of Canada) and 4-methylmorpholine (Aldrich); all dissolved in 50 ml of N, N-dimethylformamide (DMF). This mixture was stirred 1 hour and then washed twice with dichloromethane. The coupling was checked by a Kaiser test (colorless) compared to the result of the previous Kaiser test (blue). Release of the Boc-norleucine group was accomplished by shaking the resin in the presence of 50% trifluoroacetic acid (TFA, Advanced Chemtech) in dichloromethane twice over 5 and 20 minutes. After washing the resin 1 time with dichloromethane the excess TFA was neutralized with 10% DIEA / dichloromethane solution for 3 and 5 minutes while stirring simultaneously. After two more washes with dichloromethane the resin was retested qualitatively again with ninhydrin (very dark blue). The resin was then stirred for 2 hours with 2 equivalents of Boc-D-proline in 50 ml of 2% glacial acetic acid in dimethylformamide. Over the two hour period, 3 equivalents of sodium cyanoborohydride (Aldrich) were slowly and gradually added. After washing twice with dichloromethane the coupling was checked with the ninhydrin (colorless) assay. The resin was then ready for conventional solid phase peptide synthesis.

The following processes can be used to prepare any Boc-D-Pro-CHO-N-OX-MBHA resin in which X represents any amino acid with a primary amino group. A. Boc-D-Pro-V / (CI) NH2-OH may be synthesized by the process of

Martinez, et al., J. Med. Chem., 28, 1874 (1985), or D.

Tourwe, et al., In Peptides 1988: Proceedings of the 20th 22

European Peptide Symposium, Ed. Jung, Bayer; Walter de Gruyter, p.562-4. B. This resin may be prepared by coupling

(Or 1-methylimidazole) in N-methyl-pyrrolidone for 2 hours.

Preparation of Intermediate Compounds

Preparation of Ethyl 3- (1-butoxycarbonyl) -2-pyrrolidinyl) -3-hydroxypropionate:

Freshly activated zinc powder (0.79 g, 12 mmol, Aldrich) and benzene (50 ml) were placed in a 250 ml round-bottom flask, provided with two necks, under a nitrogen atmosphere. This flask was connected to a Dean-Stark apparatus and 25 ml of benzene was distilled into the trap. A solution of Boc-D-proline (1.90 g, 9.5 mmol) and ethyl bromoacetate (2.0 g, 12 mmol, Aldrich) was added dropwise under reflux. An iodine crystal was added to start the reaction, after half of the solution had already been added. After the addition was complete the mixture was heated to reflux for 3 hours, cooled and washed thoroughly with 0.5N hydrochloric acid. The aqueous solution was extracted with ether (25 ml) and the combined organic solution was washed successively with water (30 ml) and saturated sodium hydrogen carbonate solution (30 ml), then dried over anhydrous sodium sulfate and concentrated in vacuo. A yellow oil obtained was chromatographed on silica gel (with hexane / ethyl acetate) by isolating a colorless oil (1.12 g, 41%). Mass spectral analysis gave results at 288 (m + 1, 20%), 232 (m-tBu + 1.40%), and 188 (m-CO2 Bu +1, 100%). 1 H-NMR (CDCl3: Î'4.15 (br m, 3H), 3.92 (m, 1H), 3.48 (m, 1H), 3.26 (m, 1H), 2.40 (m, 2H) 2.00 (br m, 4H), 1.45 (s, 9H), 1.25 (t, 3H)

Preparation of 3- (1- (t-butoxycarbonyl) -2-pyrrolidinyl) -3-hydroxypropionioic acid:

A mixture of ethyl 3- (1- (t-butoxycarbonyl) -2-pyrrolidinyl) -3-hydroxypropionate (3.06 g, 10.6 mmol) and caustic soda (0.80 g, 20 mmol) in water: tetrahydrofuran: methanol (3: 3: 1, 35 ml) was stirred at 25 ° C for 2 hours. The mixture was concentrated sparingly (30 ° C) under vacuum. The aqueous solution was acidified with 1.0 N hydrochloric acid to pH 5 and was then extracted with ethyl acetate. The organic washing solutions were dried over magnesium sulfate, filtered and concentrated in vacuo. This residue was recrystallized from hexane / ethyl acetate to give 1.19 g (47%) of a colorless solid (A, melting point 122-124 ° C). The mother liquors were concentrated to a pale yellow viscous oil (B). Mass spectral analysis gave the results: 260 (m + 1, 60%); 204 (m-tBu + 1, 100%), I60 (m-100 + 1, 10%). 1 H-NMR (DMSO): δ 4.09 (m, 1H); 3.57 (br s, 1H); 3.31 (m, 1H); 3.15 (m, 1H) 2.10-2.40 (m, 2H); 1.60-2.00 (m, 4H); 1.38 (s, 9H). Elemental Analysis: Theory, 55.58% C, 8.16% H, 5.40% N; HPLC: a major NOK PAK peak with 50% methanol / 10% TFA / 0.1% triethylamine (A = ratio 93: 7, B = 1: 2 ratio, observed in expanded HPLC). The 2 products were determined to be different stereoisomers in the hydroxy position. Both stereoisomers were used in individual peptides; the most active of the 2 is N - ((3-Phenyl) Propionyl) -HysTrpAlaValD-AlaHis (3- (2-Pyrrolidinyl-3-Hydroxy) Propionyl) -Phe-NH2.

Step IV. Peptide synthesis and purification

The peptides were synthesized using an improved version of the solid phase method described by R.B. Merrifield, " Solid Phase Peptide Synthesis I. The Synthesis of a Tetrapeptide ", J. Am. Chem. Soc., 83, 2149 (1963), using a peptide synthesizer of Applied Biosystems model 430A.

For the synthesis of " pseudopeptides ", the resin approximates -

For example, Boc-D-Pro (IC50Phe-MBH) was loaded onto the synthesizer and a standard unblocking program (TFA / C Cl ClCn-neutralization (diisopropylethylamine / CH₂Cl₂) supplied by Applied Biosystems ( 850 Lincoln Center Drive, Foster City, CA 94404 USA). ≫

Boc-blocked amino acids were coupled to the resin using a modified program to adapt to the BOP coupling process, as described by Dung Le-Nguyen, Annie Heik, and Bertrand Castro, J. Chem. Soc., Perkins Trans. 1, 1915 (1987). The coupling protocol involved the dissolution of one mmol of the Boc-blocked amino acid, 1 mmol BOP and 1 ml 1M 1-methylimidazole in 7 ml dimethylformamide. The mixture was added to 0.5 mmol of the resin, mixed for one hour and filtered. Subsequently, a series of washes were performed with dimethylformamide and dichloromethane.

After the peptide was fixed on the resin, it was deblocked and separated from the resin with liquid hydrofluoric acid containing 10% anisole, a variation of the method described by S. Sakakibara et al., Bull. Chem. Soc. Jap., 40, 2164 (1967). The peptide and resin were then washed with ethyl acetate and then the peptide was extracted from the resin with a% aqueous acetic acid solution. The peptide solution was then lyophilized to give the peptide as an anhydrous solid. i

The papytides were then purified by reverse phase liquid chromatography using a Vydac 218TP1022 column in a Waters Delta Prep 3000 system equipped with a Gilson model 116 ultraviolet detector. Purification was performed by equilibrating the column with 0.1% TFA in water and developing with a linear acetonitrile gradient of 1-40% in 20 minutes at a flow rate of 20 ml / min. Samples were collected manually which were assayed for purity on a Spectra-Physics analytical HPLC system (including SP87OO, SP8440, SP8780, and SP4200) using a Vydac 218TP54 column. A flow rate of 1.5 ml / min was employed. using a 10% TFA gradient / acetonitrile from 10-60% ACN in 10 min. 25

Synthesis of N - ((3-Phenyl) Propionyl) HisTrpAlaValDAlaHisDPro-psi (CH 2 NH) Phe-NH 2

Methyl benzhydrylamine (MBHA) resin (5.0 g, 4.7 mmol Advanced Chemtech) was washed twice (30 minutes) with diisopropylethylamine at 1055 (DIEA, Aldrich) in dichloromethane (DCM) on a stirrer of Milligen peptides. The resin was then washed with DCM and N, N-dimethylformamide (DMF). A solution containing 2.5 g of Boc-Phe (9.4 mmol, Chemtech) 4.3 g of BOP reagent (9.4 mmol,

Richilieu Biotechnologies) and 0.95 ml of 4-methylmorpholine (8.6% mmole, Aldrich) were shaken together with the resin for 1 h. The resin was then washed successively with methanol and DCM and the coupling was checked by means of a qualitative Kaiser assay with ninhydrin.

A part of Boc-Phe-MBHA (3.06 g, 2.8 mmol) was deblocked by treatment with 5055 trifluoroacetic acid (TFA, Chemtech) in DCM for periods of 5 and 20 min. The resin was washed 2 times with DCM. The acid was then neutralized by stirring for 3 and 5 min. with 1 055 solution of DIEA and washed with DCM and DMF. The HgN-Phe-MBHA was stirred in a solution of 155 / N-methylpyrrolidone acetic acid containing 1.2 g of Boc-D-proline (6.0 mmol, synthesis described above) for 2 h. Sodium cyanoborohydride (0.54 g, 8.62 mmol, Aldrich) was added in 4 portions over 2 h (approximately every 20 min). The resin was washed with methanol and DCM and assayed with ninhydrin. Alternatively the same resin may be prepared as described in the above alternate process.

Subsequent amino acids (Boc-His (Z), Boc-P-Ala, Boc-Val, Boc-Ala, Boc-Trp, Boc-His (Z) and 3-phenylpropionic acid) were added in sequence by means of an automatic peptide synthesizer Applied Biosystems (model 430A) was prepared by the same procedure described for coupling, unlocking and neutralization involving Boc-Phe (using 0.5 mmol of the resin and 1 mmol of each of the compounds, amino acid, BOP and 4 methylmorpholine). All amino acid couplings performed with the 430A apparatus were checked by qualitative

of Kaiser in resin samples supplied by the instrument after completion of each of the couplings. The peptide (approximately 1.0 g) was separated from the MBHA resin by treatment of the peptide-resin with hydrogen fluoride (approximately 10 ml) at 0 ° C for 1 hr. The peptide was precipitated and filtered with the MBHA resin with ethyl acetate. The peptide was then extracted from the resin using acetic acid in 1% aqueous solution and isolated by lyophilization of this extract.

A part of the peptide (100 mg) was purified on a Vydac C-18 preparative column (Chemtech) using a 0.1% TFA to 0.1% TFA / acetonitrile gradient. The collected fractions were checked by means of a Vydac C-18 analytical column (Chemtech) and the samples evidencing pure papyde were combined and lyophilized. Approximately 15 mg of the peptide was isolated. After characterization by FAB-mass spectrum (MH + = 1081.7) and amino acid analysis [Ala (2.19),

His (1.68), Val (1.12)], there remained 12.2 mg (12% yield based on HPLC). Analytical Methods Purity was checked by analytical HPLC using a Spectra-Physics analytical HPLC system, including SP8700, SP8440 SP878O and SP4200. A Vydac 218TP54 column with a flow rate of 1.5 ml / min was used. of a gradient of 0.1% TFA / acetonitrile. The correct composition of each peptide was confirmed by amino acid analysis and mass spectroscopy (FAB). Amino acid analysis was performed according to the following procedure. Approximately 1-10 nanomoles of the peptide were placed in an acid washed pyrex test tube. The peptide was hydrolysed under a nitrogen atmosphere under reduced pressure with 6N hydrochloric acid containing 1% phenol for 1 h at 150 ° C. The peptide was then dried with a 2: 2: 1 mixture of ethanol: water: triethylamine, and subjected to the formation of derivatives with a 7: 1: 1: 1 ethanol: water: triethylamine: isothiocyanate solution

phenyl. The amino acids in the derivative form were analyzed by reverse phase chromatography.

Mass spectra were obtained by fast atom bombardment (FAB) on a VG 70SQ mass spectrometer of EBQQ geometry using a VG 11-150J Data System for data acquisition. The mass spectrometer was operated at 7 kvolts of acceleration potential and with a resolution of 1000 (definition of 10 / vale). The FAB cannon used in the experiments was the Ion Tech FAB11N working at a potential of 7 kvolt and a current of 1 milliampere. Xenon was used as the bombardment gas at a pressure of 1 x 10 millibar as pressure at the source. The sample of interest was dissolved in glycerin prior to analysis by FAB-MS.

Biological results

Peptides were evaluated for their activity of inhibiting GRP binding to Swiss 3T3 cells. The antagonistic activity was measured by inhibition of mitogenic stimulation of immobile R0Z 3T3 cells. 3T3 R0Z cells were obtained from Enrique Rozengurt, Imperial Cancer Research Fund, P. 0. Box 123, Lincoln's Inn Fields, London WC2A 3PX, England. Cells were incubated for 18 h with 10 ng / ml BN, either alone or in the presence of an antagonist. The cells were then pulse-labeled for 2 h with H-thymidine and washed. The incorporated H was then counted. Bombesin exposure resulted in a higher cpm value relative to the control means up to a maximal response. The potency of the antagonists was measured by inhibition of this maximum response in the reverse direction to the base level. IC50 values were determined from titration curves. Analogous and available IC50 values are listed in Table I. -

Table I

(1.85), Propionyl) -HisTrpAlaValD-Ala His (1.79), HisD-Ala His (1.79), Ala (1.85) (1.97), Propionyl) -HysTrpAlaValD-Ala-His (1.81), N - ((S) -2- (6-Methoxy-2-Naphthyl) - 1.77x108) N-NH 2 Val (1.00) N - ((S) -3-Phenylbutyryl) -HysTrp-4.71x10-8 1061.6 Ala (1.92), AlaValD-AlaHisD-Prdl / Nle-NH2 Hisd.71), Val (1.00) N - ((3-Phenylbutyryl) -HysTrp-7.54x10-8 1061.6 Ala (1.77), AlaValD-AlaHisD-ProfNle- (S) -3,3,3-Trifluoro-D-Proza-3-yl) - N - ((S) -3,3,3-Trifluoro- -2-Methoxy-1132 Ala (2.40), -2-Phenyl-Propionyl) -HisTrpAla-His (1.15), Val-AlaHisD-Pro-Nle-NH2 Assay Val (1.00) N - ((R) -3 , 3,3-Trifluoro-2-Methoxy-1131.3 Ala (1.90), -2-Phenyl-Propionyl) -HisTrpAla-His (1.19), ValD-AlaHisD- Assay Val (1.00) 29 >

Ν - (((4'-Hydroxy) -3-Phenyl) - 1.99x10 Propionyl) -ProD-ArgGlyD-PheHis-TrpAlaValGlyHisD-PrcH / Nle-NH2-8 N - (((4'-Hydroxy) -3-Phenyl ) - 7-96x10 1508.2 Propionyl) -ProD-ArgGlyD-PheHis- TrpAlaValGlyHisProWle-NH2 -8 N - ((3-Phenyl) Propionyl) -HisTrp-3 · 43 · 10 -1049.4 AlaValD-AlaHisD-Pro2Methoxynin- (3-Phenyl) Propionyl) -HisTrp 8.30x10 4 1081.7 AlaValD-AlaHisD-PrcA |> Phe-NH2 N - ((3-Phenyl) Propionyl) -HysTrp- 2.72x10-8 922.0 AlaValD-AlaHisD-Prc-Leu -NH 2 N - ((3-Phenyl) Propionyl) -HysTrp-6.92x10 ProValD-ProHisD-Pro1j / Leu-NH2

Ala (1.00), Arg (1.05), Gly (2.15), His (1.73), Phe (1.02), Pro (1.10), Val (0.95)

Al (0.96), Arg (1.07), Gly (2.08), His (1.78), Phe (1.03), Pro (1.17), Val (0.91) Ala (2.09), His (1.84), Val

Ala (2.19), His (1.68), Val (1.12)

Al (2.02), His (0.75), Val (1.00) 1099.9 Hls (1.73), Pro (2.06), Val (1.00) N - (((3'-Trifluoromethyl) -3-Phenyl) 2.70x10-9 1116.0 Propionyl ) -HisTrpAlaValD-AlaHisD-ProVLeu-NH2

Ala (1.92), Hls (1.57), Val (1.00)

4.70x10 -9 1064.0

Ala (I.96), His (0.75), Val (1.00) N - ((3-Phenyl) Propionyl) - (3- (2-Thienyl) -Ala-TrpAlaValD-AlaHisD- N - ((1-Cyclopentyl) carboxyl) -D-ArgD-AlaD-PheHisTrpAlaValGly-HisD-ProWle-NH2 1.75x10-8 1371.6 Ala (1.93),

Arg (1.04), Gly (1.03), His (1.91), Phe (1.04), Val (1.04) N - ((3-Phenyl) Propionyl) - HysTrp-AlaValGlyHisD- Cyclopentyl) carboxyl) -D-

ArgD-AlaD-PheHisTrpAlaValD-

Pr02Nle-NH2 2.00x10- ^ 1034.1 Ala (0.99),

Glyd.11), His (1.94), Val (0.96) 1.73x10-8 1386.7 Ala (2.90),

Arg (1.07), Hls (1.93), Phe (1.05), Val (0.05) N - ((3-Phenyl) Propionyl) - HysTrp-AlaValD-AlaHisD-ProHlNle-NH2

TyrProD-ArgGlyD-PheHisTrpAla

Val-GlyHisD-PrcH, Nle-NH2 3.10x10 -9 1048.2 Ala (2.02),

His (1.90), Val (1.08) 8.54x10-8 1522.2 Ala (1.03),

Arg (0.97), Gly (2.07), His (2.03), Phe (1.00), Pro (0.93), Tyr (0.87), Val (1.11) - 31

2.00x10-7 1262.5

D-ArgGlyD-PheHisTrpAlaValGly HisD-ProyNle-NHL

(4-fluoroPhe) -NHL Q N - ((3-Phenyl) Propionyl) -HysTrp-3.65x10 -109 AlaValD-AlaHisD- ProPhe-NH "

Ala (0.97), Arg (1.07), Gly (1.96), His (1.94), Phed .04), Val (1.04) Ala (1.87), His (0.86), Val d- (2.31), Phed (11), Pro (1.24), Val (1.00) N - ((3-Phenyl) Propionyl) HysTrp-AlaValD-AlaHisD-ProV (3- (2-Naphthyl) -D-Ala) -NH 2 N - ((3-Phenyl) Propionyl) -HysTrp-AlaValD-PheHisD-ProV | / Phe-NH2 2.65x10 '4.32x10 -8 132.4 1157.4 D-PheHisTrpAlaValD-AlaHisD-PrdPhe-NHg <5.47x10 &quot; 10 1093.2

(1.96), Val (1.00), Al (1.98), Phe (0.92), Val (1.00), Val -

ArgGlyD-PheHisTrpAlaValD-Ala HisD-PrctyPhe-NH2

Arg (1.02), Gly (0.94), His (1.54), Phe (0.85), Pro (0.94), Val (1.00) N - ((3-Phenyl) propionyl) -3- (2- 3.65x10- -Ala) -TrpAlaValD-Ala HisD-PrcH? Phe-NH 2

Ala (1.92), His (0.59) Val (1.00) N - ((3-Phenyl) Propionyl) -HisTrp- &lt; 5.55x10 AlaVal-Sarcosine) -HisD-Pr d2 Phe-nh2

Ala (1.02), His (2.06), Val (1.00) N - (((4'-Hydroxy) -3-Phenyl) -propionyl) -HisTrpAlaValI) - AlaHisD-PrcWPhe-NH2 &lt; 5.47x10-10 1098

Ala (2.04), His (1.96), Val (1.00) N - (((2 ', 61-Dichloro) -2-phenyl) -

Acetyl) -HisTrpAlaValD-AlaHisD-AlaHisD-ΡΓ0ψΝ1β-ΝΗ2 N - (((4'-Acetyl) -HisTrpAlaValD- Hydroxy) -2-Phenyl) Acetyl) -HisTrpAlaValD-Ala-HisD-prc * and Nle-NH2 N- (1-naphthoyl) -HisTrpAlaValD-AlaHisD-Pro-Nle-NHg 9.07x10- 1103 Ala (1.85),

His (I.83), Val (1.00) <3.44x10-10 1103.5 Ala (2.06),

His (1.79), Val d.00)

1.91x10 "9 1050.1 N / A

9.35x10 "10 1069.9 N / A 33 -

Î ± - ((3,7-Dihydroxy) -2-Naphthoyl) -HisTrpAlaValD-AlaHisD-ProWle-nh2 2.36x10 &quot; 8 1101.6 Ala (1.34), His (2.05), Val (1.00) N - 2,3-Dihydroxy) -2-Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-θθψΝ1β-ΝΗ2 1.22x10-7 1065.5 Ala (2.23), His (1.90), Val (1.00) N- (2- (3- (2.26), His (2.00), Val (1.00) N- (2- (2-Thienyl) Azetil) -His (R) -pyrrolidin-1-yl] (2.63), Val (1.00) N - (((3-Fluoro) -3-Phenyl) Propi-onyl) -HysTrpAlaValD-AlaHisD-AlaHisD-Prdf Nle-NH2 2.98x10-8 1040 Ala (2.15) (1.00) N - ((3,4-Dihydroxy) -2-Phenyl) Acetyl) -HysTrpAlaValD-AlaHisD--PrdψNle- NH2 4.63x108 1079 Ala (2.13), HisO.43), Val (1.00) N - (((R) - (-) - 2-Phenyl) Propionyl) HisTrpAlaValD-AlaHisD-PrdyPhe- 0 9.25x10 ιυ 1081.2 Ala (2.07), His (1.51), Val (1.00) N - (((S) - (+) - 2-Phenyl) Propionyl) HisTrpAlaValD-AlaHisD-ProtyPhe-nh2 2.77x1o- 9 1081.1 Ala (2.05), His (1.44), ValO .00) N - (((Trans) -2-Phenyl) Cyclopropane (1.99), His (1.44), Val (1.00) â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ34 -

N - (3- (10-Phenothiazinyl) propion-2.66x10 1202.1 nyl) -HisTrpAlaValD-AlaHisD-Pqqq / Phe-NH2 N - ((3-Methyl-3-Phenyl) butyryl) - 1.44x10-8 1109.5 -HisTrpAlaValD-AlaHisD-ProyPhe-nh2 N - (((2, -Trifluoromethyl) -2-3-50 x 10-7 1135.4

Phenyl) Acetyl) -HisTrpAlaValD-AlaHisD-ProyPhe-NH2 N - (((3, -Trifluoromethyl) -2- 8.81x10 8 1135.4

Phenyl) Acetyl) -HisTrpAlaValD- -AlaHisD-Pr (> yPhe-NH2 N - (((4-Trifluoromethyl) -2- 1.41x107) 1135.5-Phenyl) Aoethyl) -HisTrpAlaValD- AlaHisD-pro-Phe- NH2 N - (((2 ', 3'-Difluoro) -2-phenyl) -3-6x10 &quot; 7 1103.4

Acetyl) -HisTrpAlaValD-AlaHisD-ProPhe-NH2 N - ((2 ', 4'-Difluoro) -2-Phenyl) - 1.80x10-7 1103-2

Acetyl) -HisTrpAlaValD-AlaHisD--Prc-Phe-NHg N - (((2 ', 6'-Difluoro) -2-phenyl) -5,7,7,10,10-

Acetyl) -HisTrpAlaValD-AlaHisD--Prc-Phe-NH2 N - (((2-Amino) -2-Phenyl) Acetyl) -7 · 30 × 10- → 1082.2 -HisTrpAlaValD-AlaHisD-PrcWPhe-nh2

Al (2.10), His (1.58), His (1.58), Val (1.00) Ala (2.07), His (1.63), Val (1.00) Ala (2.10), His (1.63), ValO. ), Val (1.00) Ala (2.24), His (1.68), Val (1.00) Ala (2.16), His (1.63), Val d-00) Ala (2.16), His (1.62) -Val (1.00 ) Ala (2.11), His (1.69), Val (1.00) N / A-35 Ν- (1-Naphthoyl) -HisTrpAlaValD- 1.81x10 &quot; 12 AlaHisD-Prd * VPhe-NH2 1103.9 Ala (2.06) ), Val (1.00) N - (((3 ', 4', 5'-Trimethoxy) -3-1.11 x 10-Phenyl) Propionyl) -HistrpAla-Val-AlaHisD-Pro-Phe-NH2 1171.8 Ala (1.86) , His (1.71), Val (1.00) K - ((6'-Methoxy) -2- (2-naphthoyl) -3,4x10-11 Propionyl) -HisTrpAlaValD-AlaHisD-PrdPhe-NH2 1161.8 Ala (1.87) , His (1.87), Val (1.00) N - (((3'-Trifluoromethyl) -3- [3- (4-oxo-1H-imidazolyl) propionyl) -HysTrpAlaValD - ΑΗΐ Ηΐ3ΐ) -ΡηοψΡ1ΐΘ-ΝΗ2 1149.8 Ala (2.17) , His (1.50), Val (1.00) N - (((S) -3-Phenyl) butyryl) -His- 5.47x10-11 TrpAlaValD-AlaHisD-PrcfyPhe-NH2 1095.9 Ala (1.83), His (1.82), Val (1.00) N - (((4'-Methoxy) -3-phenyl) -5.40 x 10- 12 Propionyl) -HysTrpAlaValD-Ala-HisD-PrO2 Phe-NH2 1111.9 Ala (1.84), Hisd .76), Val (1.00) N - (((S) -2-Hydroxy) -2-Phenyl) - 5.53x10- (Acetyl) -HisTrpAlaValD-AlaHisD-PrcPhe-NH2 1083.8 Ala (1.92), His (1.64), Val (d) N - ((3-Phenyl) Propionyl) -HysTrp-9.25x10- , Val (1.00), His (1.55), Val (1.00), His (1.55), Val (1.00) N - ((2-Methyl-2-Phenyl)

Ala (2.22), His (1.44), Val (1.00)

Ala (2.68), His (1.38), Val (1.00)

Ala (2.71), His (1.08), Val (1.00) N- (3- (1-Naphthyl) Propionyl) -His- 1.77x10-8 1131

TrpAlaValD-AlaHisD-Prc-Phe-NH2 N - (((R) -3-Phenyl) butyryl) -Hi3Trp-9.12x10 &quot; 9 1095

AlaValD-AlaHisD-Prc2 Phe-NH2 N - ((9-Fluoroenyl) -1-Carbonyl) -8.66x109 9 1158

HisTrpAlaValD-AlaHisD-Pro-Phe-Nh2 N - (((2-Methoxy) -3-Phenyl) -propionyl) -HisTrpAlaValD-Ala HisD-ProVPhe-NH2 9.00x10-9 1111 Ala (2.07), His ( 1.35), Val (1.00)

Ala (2.23), Hisd. 67), Val (1.00)

AT

Ala (2.25), His (1.66), Yal (1.00) N - (((21,5'-Dimethoxy) -3-phenyl) - 8.76x10-9 1141.7

Propionyl) -HisTrpAlaValD-AlaHisD-

ProvVPhe -NH 2 N - ((3-Phenyl) Propionyl) -HysTrp- 1.82x107 ® 1097.7

AlaValD-AlaHisD-ProS / Tyr-MH2 N - (((2 ', 3'-Dimethoxy) -3-phenyl) -3' 50χ1θ-9 1141.7

Propionyl) -HisTrpAlaValD-AlaHisD-ProVPhe-NH2

N - ((3-Phenyl) Propionyl-HisTrpAla- 1.76x10 "® 1139.6 N / A

Vald-AlaHis (3- (2-Pyrrolidinyl) -Hydroxy) Propionyl) -Phe-NH2 (Isoquinolyl-Carbonyl) -HisTrp AlaValD-AlaHisD-ProVPhe-NH2 3.44x109 9 1104.5 Ala (1.82),

His (1.32), Val (1.00) N - ((3-Phenyl) Propionyl) -HysTrp-Ala3 Val-AlaHis D-Pro-Phe-NH2 7.50x10 1067.1

Ala (1.00), His (3.17) 37-8

Claims (2)

(I) In which B 1 is X is a group of formula X Arg (or D-Arg) X and X wherein X is des-NH 4 Pro, wherein R 1, R 2, R 3, TyrPro, des-NH2TyrPro, Ada, Pro, D-Pro or is non-existent, X is Gly, Ala, D-Ala or non-existent and X is Asn, Phe, D-Phe or Phe or D-Phe substituted by one or more halogen atoms; or X is a group of formula A- (CH2) n -CO- wherein A is a group containing 1 to 3 rings of which at least one is aromatic, each ring system being optionally substituted and the alkylene group being optionally substituted by one to four groups selected from amino, hydroxy, C1 -C4 alkoxy and C1 -C4 alkyl optionally substituted by halogen and n is 0 to 4; or X is cyclopentylcarbonyl substituted by a group of the formula X Arg (or D-Arg) X and X as defined above; 1-X is His, ThiAla or is non-existent; X2 is Ala, D-Ala, CPenc, D-tBuGly or Pro; X1 is Val or is substituted by one or more halogen atoms; 4, X and Gly, Ala, D-Ala, sarcosine, Pro, D-Pro or D-Phe; X3 is His or ThiAla; XD is D-Proy, Proy, 2-pyrrolidinyl-3-hydroxypropionyl or D-Pro; and X is Nle, Leu Phe, Val, Mox, D-Phe, Phe or D-Phe substituted by one or more halogen atoms or naphthylAla or naphthyl-D-Ala or a hydrophobic substituted aromatic amino acid or aralkyl-amino or is non-existent , or the pharmaceutically acceptable salts thereof, characterized in that the amino acids and / or amino acid derivatives are condensed in the desired sequence and / or peptide fragments containing these amino acids or their derivatives in the desired sequence to give peptide, activating either an end carboxylic acid group or an amino group at the end to make the peptide bond and protecting the remaining groups and optionally deprotecting the resulting peptide and / or transforming the resulting peptide into a desired peptide. pharmaceutically acceptable salt. A process according to claim 1, characterized in that, as a final product, a polypeptide of formula (I) wherein X represents a phenyl, naphthyl, phenothiazinyl or indolyl group is optionally substituted by substituted- hydroxy, phenyl, halogen atoms, C1-4 alkoxy or C1-4 alkoxy optionally substituted by halogen; n is 2; and X, X, X, X, X, X, X are as defined in claim 1, or a pharmaceutically acceptable salt thereof. 3. A process according to claim 1, characterized in that, as a final product, a polypeptide of formula (I) wherein X is des-NH 2 Phe, des-NH 2 Tyr, des-NE 3 Tyr Pro (or D- Pro) Arg (or D-Arg) and XI is His or ThiAla; 2, X and Ala, Pro; X is Val or hexafluorvaline; X4 is D-Ala, D-Phe; X1 is His, ThiAla; X ^ is D-Prdy, Prod /, Pro, D-Pro; and X is Nle or Phe, Leu Methoxynin, 2-naphthyl-2-alanine, or a pharmaceutically acceptable salt thereof. A process according to any one of claims 1 to 3, characterized in that one of the following polypeptides is obtained as the final polypeptide: a) N - [(3-phenyl) propionyl] -1H-pyrrola-Val-AlaHisD-ProHdPhe- (B) N - [(4'-hydroxy) -3-phenyl] -propionyl] -HysTrpAlaValD-AlaHisD-ProyPhe-NH2, c) N- (1-naphthoyl) -HisTrpAlaValD-AlaHisD-ProV | 4 &gt; e) -N - [(4-methoxy) -3-phenyl] -propionyl] -Hydro-AlaHisD-ProWhe-NH2, e) N - [(2-methyl-2-phenyl) -propionyl] -HisTrpAlaValD-AlaHisD-ΡΓθΨΡ1ιβ-ΝΗ2 or one of its pharmaceutically acceptable salts. A process for the preparation of pharmaceutical compositions, preferably in the form of a tablet or capsule, for inhibiting the development of cells which are sensitive to the growth promoting activity of the gastrin releasing peptide (GRP), to inhibit the binding of peptide in cells capable of carrying out said binding or for the prophylaxis or treatment of cancer in mammals, especially humans, characterized in that at least one polypeptide of formula (I) is incorporated when prepared according to any one of claims of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier and optionally one or more other pharmaceutically active agents. The applicant claims the priority of the British patent application filed on 31 July 1990, under No. 2. 9016810.5. - 40