CA1340988C - Therapeutic peptides - Google Patents

Abstract

A linear peptide which is an analog of a naturally occurring, biologically active bombesin having an active site and a binding site responsible for binding of bombesin to a receptor on a target cell, cleavage of a peptide bond in the active site of the naturally occurring peptide being unnecessary for in vivo biological activity, the analog having a non-peptide bond instead of a peptide bond between an amino acid of the active site and an adjacent amino acid, and having the same binding site as the naturally occurring peptide, so that the analog, is capable of acting as a competitive inhibitor of naturally occurring bombesin by binding to the receptor and, by virtue of the non-peptide bond, failing to exhibit the in vivo activity of naturally occurring bombesin.

Description

Therapeutic Peptides Background of the Invention This invention relates to therapeutic peptides useful, e.g., in cancer therapy.
The amphibian peptide bombesin, pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (Anastasi et al., Experientia 27:166-lEi7 (1971)), is closely related to the mammalian gastrin-releasing peptides (GRP), e.g., the porcine GRP, H2N-Ala-Pro-Val--Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arco-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-(NH2) (McDonald et al., Biochem. Biophys. Res. Commun. 90:227-233 (1979)) and human GRP, H2N-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH.,. Bombesin has been found to be an autocrine c;
or paracrine mitotic factor for a number of human cancer cell lines, including smal_1-cell lung carcinoma (SCLC) (Haveman et al., eds. Recent Results in Cancer Research - Peptide Hormones in Lun Cancer, Springer-Verlag, New York:1986). A number of these cancers a.re known to secrete peptide hormones related to GRP or bombesir.. Consequently, antagonists to bombesin have been proposed a.s agents for the treatment of these cancers.
Cuttit.ta et al. demonstrated that a specific monoclonal antibody t:o bombesin inhibited in vivo the growth of a human small-cell lung cancer cell line xenografted to nude mice (Cuttitta et al., Cancer Survey 4:707-727 (1985)). In 3T3 murine fibrobla.sts which are responsive to the mitotic effect of bombesin, Zachary and Rozengurt observed that a substance P
antagonist (Spa:ntide) acted as a bombesin antagonist (Zachary et al., Proc. Dtatl. Acad. Sci. (USA), 82:7616-7620 (1985)).

_ 13409a~

Heinz-Erian et al. replaced His at position 12 in bombesin with D-Phe and observed bombesin antagonist activity in dispersed acini from guinea pig pancreas (Heinz-Erian et al., Am. J. of Physiol.
252:G439-6442 (1987)). Rivier reported on work directed toward restricting the conformational freedom of the bioactive C-terminal decapeptide of bombesin by incorporating intramolecular disulfide bridges; however, Rivier mentioned that, so far, bombesin analogs with this modification fail to exhibit any antagonist activity (Rivier et al., "Compet:itive Antagonists of Peptide Hormones," in Abstracts of the :International Symposium on Bombesin-Like Peptides in Health and Disease, Rome (October, 1987).
Abbreviations (uncommon):
pGlu = H2C--CH-CO~- (pyroglutamate);

yr ~i Nle = H2N-CH-COON (norleucine) ( CH2 ) 3-C133 Pal - 3-pyridyl-a:lanine Nal - naphthylalanine Summary of the Invention In general, the invention features a linear (i.e., non-cyclic) peptide wlnich is an analog of a naturally occurring, biologically active bombesin having an active site and a binding site responsible for the binding of bombesin to a receptor on a target cell; the analo4 having a pseudo- or non-peptide bond instead of a peptide bond between - 2a -position 13 and position 14, the analog being capable of binding to the receptor, so that the analog is capable of acting as a competitive inhibitor of naturally occurring bombesin by binding to the receptor and, by virtue of ~ 340988 the non-peptide bond, failing to exhibit the in vivo activity of naturally occurring bombesin. (A detailed discussion of the chemistry of non-peptide bonds is given in Coy et al. (1988) Tetrahedron 44, 3:835-841).
Preferably, naturally occurring bombesin is characterized in that one or more amino acids in the amino terminal half of bornbes:in are hydrogen bonded to one or more amino acids in the ~arboxy terminal half of bombesin, and the non-peptide bond of the linear peptide decreases that hydrogen bonding, thereby destroying biological activity. It is believed that many of the li:zear peptides of the invention are analogs of bombesin whose biol~~gical activity depends at least in part on their ability t:o foam tertiary "hairpin" configurations in which amino acids in the <~mino terminal ("left") half of the molecule are hydrogen bonded to amino acids in the carboxy terminal ("right") half of the molecule, and that the pseudopeptide bond introduced according to the invention interferes with this hydrogen bonding, hindering the formation of the hairpin configuration on which activity depends. One may expect the loss of the ability to hydrogen bond to affect the biological activity of the molecule either by the loss of structural stability conferred by t:h~e transannular bonding or by the inability of the backbone to hydrogen bond to the receptor.
Additionally, t:he increased flexibility of the molecule about the reduced bond compared with the rigidity of the normal peptide amide bond =~s expected to alter the conformational integrity of the mo=_ecul.e and thus its biological activity.
It is apparent: from the above that the linear peptides for which introduction of a pseudopeptide bond is useful in creating or enhancing antagonist activity are those in which activity is associated with a site within the amino acid chain.
The pseudopepti.de bond can be introduced in a 1 3 40 98 ~

region involved in receptor binding, or in a non-binding regions it ha been shown (Nagain et al., Peptides, 81023-28 (1987)) that a psE~udopeptide bond introduced in the binding region does not prevent binding. Generally, useful classes of peptides in wh:lch this modification can be made are those in which at least one amino acid involved in the active site is located in the carbo:xy terminal half of the molecule; the non-peptide bond :ls introduced between this amino acid and one adjacent to it; .
According to one aspect of the present invention there is provided an effective bombesin antagonist peptide of formula (1)s \A1_A2_p3_A4_A5_AG_A7_A8_p9_A10_ R

All_Alt_A13..A14_N~
wherein A1 = pGlu, D or L, or is deleted;
A2 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, C1, Hr, OH or CH3), T:rp, ~3-naphthylalanine or is deleted=
A3 = Arg, D-~4rg, Lys, D-Lys or is deleted=
A4 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Meet, Val, Phe, p-X-Phe (X = F, C1, Br, OH or CH3), Trp, ~3-naphthylalanine or is deleted;

4a ~34o~ss A5 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, C1, Br, OH or C1~3), Trp, p-naphthylalanine, D-Ala or is de let ed ;s ~34~9~R., A6 - Gln, Asn, Gly, Ala, D-Ala, N-Ac-D-Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, C1, Br, OH
or CH3) , Trp, p-G:_u, ~i-naphthylalanine or is deleted;
A' - Gln, Assn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric '~ acid, Met, Val, P:he, D-Phe, p-X-Phe (X = F, C1, Br, OH or CH3), Trp, Lys, His, or (3-naphthylalanine;
A$ - Trp or Diet ;
A9 - Gln, Assn, G:Ly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, P.~e, p-X-Phe (X = F, Cl, Br, OH or CH3) , Trp or 1() (3-naphthylalanine, D or L;
Al° - Gln, As:z, G:Ly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, P:ze, p-X-Phe (X = F, C1, Br, OH or CH3), Trp, Thr, or (3-naphthy:Lalanine;
All - Gly, Ph~~, D or L;
Alz - His, Ph~~, or p-X-Phe (X = F, C1, Br, OH, CH3) , D or L;
Al3 - Gln, As:z, G:Ly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, P:ze, p-X-Phe (X = F, Cl, Br, OH or CH3) , Trp, or (3-naphthylalanine;
A14 - Gln, As:z, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, P:ze, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, or ~3-naphthylalanine;
provided that eac'.~z Rl, lZz, R3 and R4, independently, is H, Cl_lz alkyl, C~_lo phenylalkyl, COEl (where El is Cl_zo alkyl, C3_20 alkenyl , C3_zo alkinyl , phenyl , naphthyl , or C~_lo phenylalkyl ) , or COOEz (where Ez is Cl_lo <~lkyl or C~_lo phenylalkyl) , and Rl and Rz are bonded to the N-terminal amino acid of said peptide, which can be Al, A2, A3, A4, A5, A6, or A~, provided that when one of R1 or R2 is COEl or COOE2, the other must be H, and when one of R3 or R4 is COEl or COOE2, the other must be H, and further provided that when A1 -pGlu, Rl must be H and R2 must be the portion of Glu that forms t:he imine ring in pGlu; and for each of the residues A~, A8, A9, A11~ A12~ and A13, indepen~3ently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may he a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon (i.e., at least one of the subject peptide CONH bonds must be replaced by .3 non-peptide, i.e., pseudopeptide, CH2NH
bond); or a pharmaceutically acceptable salt thereof.
(Where no D- or L-isomeric designation is given herein, the naturall;~ occurring L-isomer is intended.) Pre~_'erably, an effective bombesin antagonist peptide has, for each of the residues All, A12, and A13, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue which may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom roust be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof. Most preferably, t:he bombesin antagonist peptide has A1 through A6 dE?leted each A1j and A14 is Leu and the carbon atom participating in the amide bond between A13 and A14 is a methylelre carbon, or a pharmaceutically acceptable salt thereof'.
D

~34098g Another class of peptides of the invention are bombesin-related antagonist peptides derived from litorin and of the amino acid formulas 1\
/A1_A2_A3_A4_A5_A6_A7_A8_A9 R2 ~ R4 wherein A1 is A6 of the above definition, A2 is A7 of the above definition, A3 is A8 of the above definition, A4 is A9 of the above defsnition; A5 is A10 of the above defsnition;
A6 is All of the above definition, A7 is A12 of the above definition; A8 is A1~ of the above definition and A9 is A14 of the above definition provided that the carbon atom participating in the amide bond between the A8 residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, or a pharmaceutically acceptable salt thereof.
Peptides of the invention that contain a pseudopeptide bond substitution within the active site of the naturally occurring peptide are antagonists to the biological activity of the naturally occurring bombesin peptide, with one exception which we have observed= the linear analog of bombesin BIM-26027 [Va7.lOt(CH2NH]Leul4]HN is an agonist of the biological activity of naturally occurring bombesin.
(Non-peptide bonds are symbolized herein by "fir[CH2NH]" or "t".) Therefore, a third class of peptides of the invention 1 3 40 98 g ~a are effective bomibesin agonists of the formula (1) recited above, including, for A1~, the carbon atom participating in the amide bond between the residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be a non-peptide bond, provided that the non--peptide bond may be a carbonyl carbon having been reduced to a methylene carbons further provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof. Most preferred is the bombesin agonist having the formula pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-yrAla- [CHzNH] -Val-Gly-His-Leu-Leu-NHz.
Other agonist analogues are peptides in which either the pseudopeptide bond is not located in the active site of the naturally occurring peptide, or in which two amino acid residues of the active site are replaced by statine or AHPPA.
(Statine has the chemical structure CH3~ ~CH3 C f-I
I

N HZ-C ~I-C H-C HZ-C OZ H
and statine-amide has the structure CH3~ ~CH3 CH
i and the formula A:iiPPA has the formula:
(3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid).
Therefore, a fourth class of peptides of the invention is an 2() effective bombesi:z agonist which is an analog of naturally occurring, biological_Ly active bombesin having an active site, which includes positions A9, All, Alz, A13, and A14, and a binding site responsible :Eor the binding of bombesin to a receptor on a s 8a site responsible for the binding of bombesin to a receptor on a target cell, 'the analog having either (a) a non-peptide bond outside of the active ;site of bombesin, or (b) having at least one statine or AHPPA residue in place of two naturally occurring amino acids of the active site; and further, the peptide can contain statine or AHPPA when <~11 bands between amino acid ~34~988 residues are peptide bonds and, further, when an amino acid residue is statine or AHPPA, the amino acid to the right of it in the formula is deleted, so that the analog is capable of binding to the receptor and, by virtue of the statine or AHPPA
residue, exhibiting enhanced in vivo biological activity compared to naturally occurring bombesin. Most preferred in this class is the bombe~;in agonist having the amino acid formula pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-[Stal3, Des-Metl4] -NHZ.
Therefore, a further aspect of the present invention is a peptide of the formula (2):

~A1 A2 A3 ~~a As_A,s A~-Aa As A1o R/

A11 A12 -A13 A14 N' \R

wherein A1 - pGlu, D or L or is deleted;
A2 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Va_L, Ph~=, p-X-Phe (X = F, C1, Br, OH or CH3) , Trp, (3-naphthylalanine or is deleted;
A3 - Arg, D-A:rg, Lys, D-Lys or is deleted;
A4 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, (3-naphthylalanine car is deleted;

9a 1 3 4 0 9 8 8 AS - Gln, Asn, Gly, Ala Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Plze, D-Phe, p-X-Phe (X = F, C1, Br, OH or CH3), Trp, (3-naphthylal<~nine, D-Ala or is deleted;
A6 - Gln, Asn, Gly, Ala, D-Ala, N-Ac-D-Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, C1, Br, OH, or CH3) Trp, p-Glu, (3-naphthylalanine or is deleted;
A' - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, C1, Br, OH or CH3), Trp, Lys, His, or (3-napl~thylalanine;
1 G Ag - Trp or P~tet ;
A9 - Gln, Asn, G7.y, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-:X-Phe (X = F, C1, Br, OH or CH3) , Trp or (3-naphthylalanine, D or L;
Al° - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric 1~~ acid, Met, Val, Phe, p-:X-Phe (X = F, Cl, Br, OH or CH3) , Trp or (3-naphthylalanine All - Gly, Phe~, D o:r L;
Alz - His, Phe~, or :p-X-Phe (X = F, Cl, Br, OH, CH3) , D or L;
Al3 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric 2C acid, Met, Val, Phe, p-:X-Phe (X = F, Cl, Br, OH or CH3), Trp, or ~i-naphthylalanine;~
Al4 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-:X-Phe (X = F, C1, Br, OH or CH3) , Trp, or (3-naphthylalanine;~
25 provided that each Rl, Rz, R3, and R4, independently, is H, Cl_lz alkyl, C~_lo phenylalkyl, COEl, (where El is C1_z° alkyl, C3_zo 1 3 40 98 R .
9b alkenyl , C3_zo alky:nyl , phenyl , naphthyl , or C,_lo phenylalkyl ) , (alkyl or C~_lo phe:nylalkyl) , or COOEz (where Ez is C1_lo alkyl or C~_lo phenylalkyl ) , and Rl and Rz are bonded to the N-terminal amino acid of said peptide, which can be Al, Az, A3, A4, A5, A6 or A', and further provided that when one of Rl or Rz is COEl or COOEz, the other must be' H and when one of R3 or R4 is COEl or COOEz, the other must be H, and further provided that when A1 -pGlu, R1 must be H and R:z must the portion of Glu that forms the imine ring in pGlu; and for each of the residues A', Ae, A9, All, Alz, and A13, independently; the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon (non-peptide bond), provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof; which said peptide is an analog of naturally occurring, biologically active mammalian GRP or amphibian bombesin having an active site, said active site includes the positions A9, All, Alz, A13, and A14, and a binding site responsible for the binding of said bombesin to a receptor on a target cell, said analog having either (a) said non-peptide bond at rE:sidu.e:~ other than within said active site, or (b) having at leaw;t on.e statine or AHPPA residue in place of two naturally occL:rring amino acids of said active site, and further provided that the peptide can contain statine or AHPPA
when all bonds between. amino acid residues are peptide bonds, and further provided that when an amino acid residue is statine or AHPPA, the amir.:o acid to the right of it in the formula is deleted, so that ~~aid analog is capable of binding to said receptor and, by virture of said statine or AHPPA residue, exhibiting enhanced in vivo biological activity compared to said naturally occurring bombesin.
T

~34A988 9c Yet a further aspect of the present invention is the method of synthesizing an effective bombesin antagonistic peptide containin~~ the following amino acid formula:
R~
A2 A3 Aa As As p As As Aio jRs Ass A~,i \~\

wherein, for the positions A1 through A14, Al - pGlu, D or I~ or is deleted;
AZ - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-:X-Phe (X = F, C1, Br, OH or CH3), Trp, (3-naphthylalanine o:. is deleted;
A3 - Arg, D-i~rg, Lys, D-Lys or is deleted;
l~s A4 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met Va7., Phf=_, p--X-Phe (X = F, C1, Br, OH or CH3) , Trp, (3-naphthylalanine o:_ is deleted;
AS - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, C1, Br, OH or CH3), 2G Trp, (3-naphthylalanine, D-Ala, or is deleted;
A6 - Gln, Asn, Gly, Ala, D-Ala, N-Ac-D-Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, C1, Br, OH
or CH3), Trp, pGlL~~, ~3-naphthylalanine or is deleted;

r. 1 3 40 98 g 9d A' - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, I)-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3) , Trp, Lys, His, or (3-naphthylalanine;
A8 - Trp or P~tet ;
A9 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-;K-Phe (X = F, Cl, Br, OH or CH3) , Trp or (3-naphthylalanine, D or L;
Al° - Gln, Asn, Gly, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-:K-Phe (X = F, Cl, Br, OH or CH3) , Trp, Thr, or (3-naphthy~_alanine;
All - Gly, Phe, D o:r L;
Alz - His, Phe, ox' p-X-Phe (X = F, C1, Br, OH, CH3) , D or L;
Al3 - Gln, Asn, Gl.y, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-:K-Phe (X = F, C1, Br, OH or CH3) , Trp, or 1~, (3-naphthylalanine;
A14 - Gln, Asn, G1_y, Ala, Leu, Ile, Nle, a-aminobutyric acid, Met, Val, Phe, p-:K-Phe (X = F, Cl, Br, OH or CH3), Trp, or (3-naphthylalanine~
provided that: each Rl, Rz, R3 and R4, independently, is H, Cl_lz 2C alkyl, C~_lo phenyl alkyl, COEl (where El is Cl_zo alkyl, C3_20 alkenyl , C3_zo alki:nyl , phenyl , naphthyl , or C~_1° phenylalkyl ) , or COOEz (where Ez is C1_1° alkyl or C~_lo phenyl alkyl) , and Rl and Rz are bonded to the N-terminal amino acid of said peptide, which can be Al, Az, A3, A4, A5, A6, or A', and further provided that 2~~ when one of Rl or Rz is COEl or COOEz, the other must be H, and when one of R3 or R4 is COEl or COOEz, the other must be H, and further provided that when Al - pGlu, Rl must be H and Rz must be the portion of Glu that forms the imine ring in pGlu; and a ._ 1340988 9e A' A8 A9 All A12 and A13 for each of the residu.e;~ , , , , , independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof said method comprising the steps of (a) preparing benzhydrylamine-polystyrene resin, (b) neutralizing said resin and coupling a chosen amino acid for said A14 position to said resin, (c) coupling a chosen amino acid for said A13 position to said A14-resin, (d) derivatizing the free amino group of said A13-A14-resin, and (e) coupling successively to said derivatized amino acid resin a chosen amino acid for said Alz through A1 positions by repeating steps (d) and (e) for each of said amino acid positions.
Yet a further aspect of the present invention is a method of synthesizing an effective bombesin agonist having the amino acid formula (2) recited above, wherein A1 to A14 are as defined for formula (2) above, which is an analog of naturally occurring, biologically active mammalian GRP or amphibian bombesin, wherein said active site includes the positions A9, All, Alz, A13, and A.14, and a binding site responsible for the binding of said bcmbesin to a receptor on a target cell, said method comprising either the steps of (a) preparing benzhydrylamine-pclystyrene resin, (b) neutralizing said resin and coupling a chcsen amino acid for said A14 amino acid position or alpha-t-butoxycarbonyl statine to said resin, (c) coupling a chosen amino acid for said A13 position or said statine for said A.lz pc>s:ition to said A14-resin, (d) derivatizing the free amino grcup of said A13-A1'~-resin or said statine-resin, and (e) coupling succes:~ively to said derivatized amino acid or statine resin a chosen amino acid for said Alz through A1 9f positions by repeating ;steps (d) and (e) for each of said amino acid position; provided that when said statine is coupled to said resin, said ~~tati.nc=_-resin is acetylated before derivatization to free~<~mino of groups; provided at least one statine or AHPPA residuE=_ replaces two naturally occurring amino acids of said active site.
The bomr~esin antagonists and agonists of the invention are suitable for the treatment of all forms of cancer where bombesin-related :substances act as autocrine or paracrine mitotic factors, especially pancreas and small-cell lung carcinoma.
In formula (1) , when R1, R2, R3 or R4 is an aromatic, lipophilic group, the in vivo activity can be long lasting, and delivery of the compounds of the invention to the target tissue (e. g., the lungs) can be facilitated.
Other fe~ature:~ and advantages of the invention will be apparent from the fo7_lowing description of the preferred embodiments thereof.
Description of the Preferred Embodiments We will first briefly describe the table.
Table Table I shows formulas for the pseudo-peptide analogues and results of: in vitro inhibition of [lzsl] GRP
binding to cerebral cortex and 3T3 fibroblast or murine fibroblast bombesin receptors, and bombesin-stimulated [3H]Thymidine uptake by cultured 3T3 cells.
We now describe the structure, synthesis, and use of the preferred embcdiment:s of the invention.
e+.,-."..~"z.-o a 134o98s The peptides of the invention all have a non-peptide bond in at least one of the indicated position, except for the statine or AHPPA substituted analogs, such as stal3-des Metl4 bombesin. By non-peptide bond is meant that the carbon atom 5 participating in the bond between two residues is reduced from a carbonyl carbon to ~ methylene carbon. The peptide bond reduction method which yields this non-peptide bond is described in Coy et al., European Patent Application 0 257 742 published on March 2, 1988 assigned to the same assignee as the present 10 application. Any o:ze or all of the amino acids in positions 1 through 6 of the bombesin antagonists may be deleted from the peptides, and the peptides are still active as antagonists or agonists.
The peptides of the invention can be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic, lactic, malefic, citric, malic, ascorbic, succinic, benlioc, salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxmethyl cellulose, and salts with inorganic acids such as t:he hydrohalic acids, e.g., hydrochloric acid, sulfuric acid; or phosphoric acid.
Synthesis of Bombes_Ln Antagonists The synthesis of the bombesin antagonist pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp--Ala-Val-Gly-His-Leu~[CH2-NH]Leu-NH2 follows.
Other bombesin antagonists and agonists can be prepared by making appropriate rlodifications of the following synthetic method.

The f:.rst step is the preparation of the intermediate p(~lu-Gln-Arg(tosyl)-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gli~-His(benzyloxycarbonyl)-Leu~y(CH2NH]
Leu-benzhydrylamine resin, as follows.
Benzh~~drylarnine-polystyrene resin (Vega Biochemicals, l:nc.) (0.97 g, 0.5 mmole) in the chloride ion form i.s placed in the reaction vessel of a Beckman 990B peptide s~~nthes'izer programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid (TFA) in methylene chloride (2 times for 1 and 25 min. each); (c) methylene chloride;
(d) ethanol; (e) methylene chloride; and (f) 10%
triethylamine in chloroform.
The nErutral~.zed resin is stirred with alpha-t-butoxycarbonyl(Boc)-leucine and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hour, and the resulting amino acid resin is then cycled through steps (a) to (f) in the above wash program. Boc-leucine aldehyde (1.25 mmoles), prepared by thE~ method of Fehrentz and Castro, Synthesis, p. E76 (1983), is dissolved in 5 ml of dry dimethylformamide (DMF) and added to the resin TFA salt suspension followed by the addition of 100 mg (2 mmoles) of sodium cyanaborohydride (Sasaki and Coy, Peptides 8:119-121 (1987); Coy et al., id.). After stirring for 1 hour, the resin mi~;ture is found to be negative to ninhydrin reaction (7. min.), indicating complete derivatization of the free amino group.
The fcllowing amino acids (1.5 mmole) are then coupled successively in the presence diisopropylcarbodiimide (1.5 mmole), and the resulting amino acid resin is cycled through washing/deblocking steps (a) to (f) in t:he same procedure as above:
Boc-His(benzylcxycarbonyl), Boc-Gly, Boc-Val, Boc-Ala, Boc-Trp, Boc-G.ln (coupled in the presence of equivalent of hydroxybenzotr.iazole, Boc-Asn (coupled in the presence of 1 equivalent of hydroxybenzotriazole), Boc-Gly (coupled as a 6 M
excess of the p-nitrophenylester), Boc-Leu, Boc-Arg(tosyl), Boc-Gln (coupled as a 6 M excess of the p-nitrophenylester), and pGlu. The completed resin is then washed with methanol and air dried.
The resin described above (1.6 g, 0.5 mmole) is mixed with anisole (5 ml) and anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 45 min. Excess hydrogen fluoride is evaporated rapidly under a stream of dry nitrogen, and free peptide is precipit;~ted and washed with ether. The crude peptide is dissolved in a minimum volume of 2 M acetic acid and eluted on a column (2.5 x 100 mm) of Sephadex*G-25 (Pharmacia Fine Chemicals, Inc.). Fractions containing a major component by uv absorption anti thin layer chromatography (TLC) are then pooled, evaporated i~o a small volume and applied to a column (2.5 x 50 cm) of oci=adec:ylsilane-silica (Whatman LRP-1, 15-20 ~m mesh size) .
The peptide is eluted with a linear gradient of 0-30%
acetonitrile in 0.1'-'s trifluoroacetic acid in water. Fractions are examined by TLC and analytical high performance liquid chromatography (HPLC;) and pooled to give maximum purity.
Repeated lyophi.lizat:ion o:f the solution from water gives 60 mg of the product as a white, fluffy powder.
The product is :Found to be homogeneous by HPLC and TLC. Amino acid analysis of an acid hydrolysate confirms the composition of the pepti.dc=_. The presence of the Leuyr [CH2-NH] Leu bond is demonst.ratect by fast atom bombardment mass spectrometry.
*Trade-mark pGlu-G~ln-Arg-Leu-Gly-Asn-Gln-Trp-Ala~r[CH2-NH]Val-Gly-His-Leu-Met-NH2 and pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu~[CH2NH]Met-NH2 are prepared in si~r.ilar yields in an analogous fashion by appropriately crodifyi.ng the above procedure.
A statine or AHPPA residue can be substituted in place of any two amino acids of the peptide, where the peptide contains no pseudopeptide bonds. For example, stal3-des Metl4 bombesin was prepared in an analagous fashion by first coupling statine to the resin and then proceeding with the addition of Boc-His(benzylocarbonyl). Statine or Boc-statine can be synthesized according' to the method of Rich et al., 1978, J. Organic Chem. 43; 3624; and Rich et al., 1980, J. Med. Chem. 23: 27, and AHPPA can be synthesized according to the method of Hui et al., 1987, J. Med.
Chem. _30: 1287.
Synthesis of Stal3-yes-Metl4 Bombesin Solid-phase synthesis of the peptide pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Sta-NH2 was accomplished through the use of the following procedures in which alpha-t-butoxycarbonyl statine (prepared by the procedure of Rich et al., J. Org. Chem.
1978, 43, 3624) is first coupled to methylbenzhydrylamine-polystyrene resin. After acetylation, the intermediate p-Glu-Gln-Arg(tosyl)-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His(benzyloxycarbonyl)-Sta-methylbenzhydrylamine resin is prepared. The synthetic procedure used for this preparation follows in detail:

1. Incorporation of alpha-t-butoxycarbonyl statine on methylbenzhydrylamine resin.
Methylbenzhydrylamine-polystyrene resin (Vega Biochemicals, :Lnc.) (1.0 g, 0.73 mmol) in the chloride ion form is placed in the reaction vessel of a Vega 250C Coupler peptide synthesizer. '.rhe synthesizer was programmed to perform the following reactions: (a) methylene chloride; (b) 10%
triethylformamide i:z ch:Loroform (c) methylene chloride; and (d) dimethylformamide.
The neutr;~lized resin is mixed for 18 hours with the preformed active es~~er made from alpha-t-butoxycarbonyl statine (1.46 mmol, diisopropyl carbodiimide (2 mmol), and hydroxybenzotriazolE~ hydrate (1.46 mmol in dimethylformamide at 0°C for one hour. The re;~ulting amino acid resin is washed on the synthesizer with dimethylformamide and then methylene chloride. The resin mixture at this point was found by the Kaiser ninhydrin te:~t (5 vminutes) to have an 84% level of statine incorporation on the resin.
Acetylation was performed by mixing the amino-acid resin for 15 mi_nute:~ with N-acetyl imidazole (5 mmol) in methylene chloride. Dex-i-vatization to the 94%-99% level of the free amino groups oi: the :resin was indicated by the Kaiser ninhydrin test (5 m=_nutes). The Boc-statine-resin is then washed with methylene chloride.
2. C.'oupl__ngs o:f the Remaining Amino Acids.
The peptide synthesizer is programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33%
trifluoroacetic acid (TF'A) in methylene chloride (2 items for 5 and 25 min. each) ; ~;c) methylene chloride; (d) isopropyl alcohol; (e) 10% triethylamine in chloroform; and (f) methylene chloride.
The following amino acids (2.19 mmol) are then coupled successively by dii~~opropyl carbodiimide (4 mmol) alone or ~34o9a8 diisopropyl carbodiimide (4 mmol) plus hydroxybenzotriazole hydrate (1.47 or 0.73 mmol) and the resulting peptide-resin is washed on the synthesizer' with dimethylformamide and then methylene chloride, and then cycled through the washing and 5 deblocking steps (a) to (f) in the procedure described above.
Boc-His (:benzyloxycarbonyl) (coupled in the presence of 2 equivalents hydroxybenzotriazole); Boc-Gly; Boc-Val; Boc-Ala; Boc-Trp; Boc-Gln and Boc-Asn (coupled as the preformed hydroxybenzotriazol~~ active esters made by reaction at 0°C. for 10 one hour with 1 equivalent hydroxybenzotriazole hydrate); Boc-Gly; Boc-Leu; Boc-A:rg(tosyl), Boc-Gln, and pGlu (also coupled as the preformed activ~= esters of hydroxybenzotriazole made by reaction at 0°C' for one hour with 1 equivalent hydroxybenzotriazol~~ hydrate). The completed peptide-resin is 15 then washed with mei~hanol and air dried.
The peptide-resin described above (1.60 g , 0.73 mmol) is mixed with aniso:le (2.5 mL) , dithioerythreitol (50 mg) , and anhydrous hydrogen :=luoride (30 mL) at 0°C for one hour. Excess hydrogen fluoride i:~ evaporated rapidly under a stream of dry nitrogen, and the free peptide is precipitated and washed with ether. The crude peptide is dissolved in 100 mL of 1 M acetic acid and the solution is then evaporated under reduced pressure.
The crude peptide i;~ dissolved in a minimum volume of methanol/water 1/1 <~nd t:riturated with 10 volumes of ethyl acetate.
The t:riturated peptide is applied to a column (9.4 mm I.D. x 50 cm) of oct:adec:y:lsilane-silica (Whatman Partisil 10 ODS-2 M 9). The peptide :is eluted with a linear gradient of 20-80% of 20/80 0.1% trifluo:roacetic acid/acetonitrile in 0.1%
trifluoroacetic acid in water. Fractions are examined by TLC
and analytical x' 1340g8g high performance liquid chromatography (HPLC) and pooled to give maximum purity. Lyophilization of the solution from water gives 77 mg of the product as a white fluffy powder.
Other compounds can be prepared as above and tested for effE~ctive:ness as agonists or antagonists in the following 1=est program.
Phase 1 - 3T3 l?eptid~ Stimulated [3H) Thymidine Uptaloe Assay Cell (~ulture. Stock cultures of Swiss 3T3 cells (American Type Culture Collection No. CCL 92) are grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10"s fetal calf serum in humidified atmosphere of :l0% 00,2/90% air at 37°C. For experimental use, the cells are seeded into 24-well cluster trays <ind used four days after the last change of medium. ThE~ cel.l~s are arrested in the G1/GO phase of the cell cycle by changing to serum-free DMEM 24 hours prior to the thymidine uptake assay.
Assay of DNA Synthesis. The cells are washed twice with lml aliquots of DMEM (-serum) then incubated with DMEM (-set:um), 0.5uM [methyl-3H) thymidine (20Ci/mmol.e, New England Nuclear) , bombes in ( 1nM) , and four concentrations of the test compounds (1, 10, 100, 1000nM) in a final volume of 0.5m1. After 28 hours at 37°C, [methyl-';H) thymidine incorporation into acid-insoluble pools is assayed as follows. The cells are washed twice with ice-cold 0.9% NaCl (lml aliquots), and acid solub7_e radioactivity is removed by a 30 min.
(4°C) incubation wi.tlz 5% trichloroacetic acid (TCA).
The cultures are then washed once (lml) with 95% ethanol and solubi.lized by a 30 min. incubation (lml) with O,1N
NaOH. The solubili.zed material is transferred to vials containing 15m7. Sci.n~A (Packard), and the radioactivity is determined by liquid scintillation spectrometry.
Phase _2 - Smal7_ Cel_1 Carcinoma (SCLC) - Bombesin Stimulated [3H] Thymidine U take Assay Cell C:ultur~. Cultures of the human cell carcinoma cell line (NCI-H69) (obtained from the American Type C:ulturE~ Association) are maintained in RPMI 1640 medium supplemented with 10% fetal calf serum in loo C02/90o air at: 37°C. Twenty-four hours prior to assay, the cells are washed with serum-free medium and seeded in 24-well. cluster trays.
Assay of DNR Synthesis. Bombesin (1nM), 0.5uM [methyl-3H] thymidine (20 Ci/mmole, New England Nuclear), and. four concentrations of the test compounds (1, 10, 100, 1000nM) are added to the cultures to achieve a final volume of 0.5 ml. After a 28 hr incubation at 37°C, the cells are collected onto GF/B
glass fiber filters, and the DNA is precipitated with ice-cold TCA. (3H] thymidine incorporation into acid-insoluble Fractions of DNA is determined by liquid scintillation spectrometry.
Phase 3 - Peptide-Induced Pancreatitis Male, ;~prague-Dawley rats (250g) are used for these experimenl~s. T:he test compound, or 0.9% NaCl is administered s.c. 15 min. prior to the bombesin injection. Bombesin injections are given s.c. at a dose of 10 ug/kg, and blood samples are obtained at 1 hr.30 min., 3hr. and E~hr. Plasma amylase concentration are determined by the Pantrak Amylase test.
Phase 4- In Vitro Inhibition of [125I] Gastrin Releasing F~eptide (GRP) Binding to Bombesin Rece tars _.
Membra:~es from various tissues (rat brain, rat pancreas, rat ara erior_ pituitary, SCLC, 3T3 cells) are prepared by homogenization in 50au~I TrisHCl containing 0.1% bovine serum albumin and O.lmg/ml bacitracin followed by two centrifugations (39,OOOxgxlS min., 4°C) with an intermediate resuspension in fresh buffer. For assay, aliquot:; (0.8m1) are incubated with 0.5nM
~125I~GRP ('2000 Ci/mmol, Amersham Corp.) and various concentrations of the test compounds in a final volume of 0.5m1. After a 30 minute incubation at 4°C, the binding reaction is germinated by rapid filtration through Whatmar.. GF/C filters that have been pre-soaked in 0.3% aqueous polethyleneimine to reduce the level of nonspecific binding. The filters and tubes are washed three times with 4m1 aliquots of ice-cold buffer, and the radioactivity trapped on the filters is counted by gamma-spectrometry. Specific binding is defined as the total [1251]GRP bound minus that bound in the presence of 1000nM bombesin.
Phase 5- Inhibition of Gastrin Release The stomachs, of anesthetized rats are perfused with saline collected. over 15 minute periods via pyloric cannulation while they test peptide is infused through the femoral vein for periods between 0 and 150 minutes.
Results of Tests of 'test Peptides A number of analogs of bombesin, each containing a non-peptide bond, were synthesized and tested in one or more of the above-described Phase 1 - 5 assays; the results of Phase 1, 2, and 4 tests are given in Table 1 attached hereto (analogs of bombesin are indicated by the symbol "BN"). Brain and 3T3 GRP
receptor and thymidine uptake data are expressed in IC50 (nM). Table 1 also gives results for non-peptide bond-containing ana'~ogs of three other naturally-occurring peptides, Substance P (which plays a role in the sensation of pain), Neuromedin C, whose C-terminal seven amino acids are similar to those of bombesin, and litorin, whose eight C-terminal amino acids are identical to Bombesin, with the exception of a Phe substitution for Leu at position A1~ of bombesin. ' In Table 1, the position of the nan-peptide bond is indicated by the position of the symbol, ~ (i.e., 4t is always shown preceding the amino acid which, in that peptide, is bonded to the next sequential amino acid). The nature of the non-peptide bond is specified in between brackets at the site where the peptide bond replacement is taking place in the sequence.
In Table 1, it can be seen that a preferred placement of the non-peptide bond in bombesin analogs is at the 13-14 position; two of the most active analogs (as indicated by a low GRP receptor IC50 value) are BIM-26027 and BIM-26028. However, BIM-26027 causes proliferation of cancer cells (see Table 1, under thymidine uptake), and therefore is an agonist and not an i antagonist. In general, compounds having the non-peptide bond at any position other than the active site of the peptide are agonists rather than antagonists. Table I also shows that when statine replaces the A~3 and A14 residues of bombesin, the resultant bombesin analog BIM-26096 causes proliferation of cancer cells and i:a therefore an agonist. Bombesin superagonists I
may be useful in cancer therapy, as suggested by Alexander et al., 1988, Cancer Research 48:1439-1441, and Alexander et al., 1988, Pancreas 3:2~~7-302,. Alexander et dl., show that chronic bombesin treatment inhl.bited the growth of human ductal adenocarcinoma transplanted C

into athymic mice. These results were surprising for bombesin stimulates growth of no:rm~al pancreas tissue. The demonstration of both stimulatory and inhibitory activity suggests that bombesin interacts ~3ifferently in normal and neoplastic 5 pancreatic tissues.
These observations prompted us to evaluate the affect of BIM-26096, a bom:~esin analogue which has bombesin-like agonist activity, on the in vitro growth of a pancreatic tumor cell line (AR42J, A.T.C.C. No. CRL 1492). For these 10 experiments, AR42J c~ell;~ were subcultured in to a 24-well culture plate in Du:Lbecr_o's modified Eagle's medium containing 10% fetal calf serum containing various concentrations (0.1-100nM) of BIM-26096. After a 36 hr incubation the cells were removed with a tryp:~in/EDTA solution and the number of cells 15 were determined using a Coulter Counter. The results are shown below:
Treatment Cell Count (% Control Control 100 BIM-26096 (0.1 nM) 78 20 BIM-26096 (1.0 nM) 73 BIM-26096 (10 nM) 56 BIM-26096 (100 nM) 52 These results indicate t:h<~t the bombesin agonist, BIM-26096, has in vitro antiproliferati.ve activity against the AR42J rat pancreas tumor Finally, Table :L also shows that bond placement, while important, is not only the factor influencing antagonist activity, and that ~imino acid substitutions at some positions exert influence as well; i:his is illustrated by BIM-26030, with Gly in position 11, which exhibited no antagonist activity.
Table 1 also gives negative results for analogs of Spantide ([D-Arg', D-Trp7~9, Leu"] Substance P, and Bombesin. Thus the non-peptide bond placement guidelines given herein should be used in conjunction with the routine assays described above to select useful antagonists or agonists.
In a phase 5 assay, above, the results of which are not given in Tab:Le 1, BIM-26028 was shown to be a potent inhibitor of bombesin - stimulated gastric acid secretion.
Use The peptides of the invention may be administered to a mammal, particularly a human, in one of the traditional modes (e. g., orally, parenterally, transdermally, or transmucosally), in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery (e.g., in the case of anti-cancer bombesin to the lungs) using micelles, gels and liposomes.
The bombesin antagonists and agonists of the invention are suitab7.e for the treatment of all forms of cancer where bambesin-related substances act as autocrine or pa.racrine mitotic agents, particularly small-cell lung carcinoma. The peptides can also be used for the inhibition of gastric acid secretion, the symptomatic relief and/or treatment of exocrine pancreatic aden.ocarcinoma, and the restoration of appetite to cachexic patients. The peptides can be administered to a human patient in a dosage of 0.5 ug/kg/day to 5 mg/kg/day. For some forms of cancer, e.g., small cell lung carcinoma, the preferred dosage for curative treatment is 250mg/patient/day.

Other Embodiments For example, as is mentioned above, there are a number of other peptide families from which agonists or antagonists can be made according to the invention.
Some of these families are substance P and related peptides, vasoactive inestial peptide (VIP) and related peptides, and neurotensin and related peptides. The number of peptides in each family on which antagonists or agonists can be based is large. For example, there are at least 10 currently-known peptides in the VIP
family, including sauvagine and urotensin. In addition, there have been isolated seven natural bradykinin-like ~5 peptides. Neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-~Pro-Tyr-Ile-Leu-OH) has two peptide bonds which advantageously can be replaced by non-peptide bonds:
Ile-Leu and Tyr-Ile. In addition, neurotensin antagonists can be missing any or all of the N-terminal seven amino acid residues, as it has been shown (Granier et al. (1984) Eur. J.. Hiochem. 124: 117) that they are not needed for biological activity and binding.
Screening of neurotensin antagonists can be by binding to SCLC receptors. Gastrin releasing peptides (GRP) and related peptides (e.g., Neuromedin C (GRP 18-27)) have a bond between amino acid residues 13 and 14 which can be replaced with a non-peptide bond to form a GRP
antagonist.
C

Bombesin (BN) : pGlul--Gln2-Arg3-Leu4-Glys-Asn6-Gln'-Trpe-Ala9-Vall°-Glyll_Hisl2-Leu-.3-Meth-NHZ
Substance P (SP) : Argl-I?ro2-Lys3-Pro4-Glns-Gln6-Phe'-PheB-Gly9-Leul°-Met'.1-NHz Brain GRP 3T3 GRP Thym, Receptor Receptor Uptake IC30 (nM) IC50 (nM) IC50 (nM) BIM-25025:
[yrHislz [CHzNH] Leul3, Leul4] BN (1-14) >1000 BIM-26026:
[yrAla9 [CHzNH] Vall°, Leul4] BN (1-14) >1000 1574 BIM-26027: agonist [y~Vall° [CHzNH] G1y11, Leul4] BIS (1-14 ) 0 .48 2 . 3 EC50-0.07nM
BIM-26028:
[yrLeul3 [CHzNH] Leul4] BN (1-19:) 13 BIM-26030:
[yrGlyll [CHzNH] Hislz, Leul4] BN (1-14) >1000 BIM-26034:
[ylGly' [CH2NHJ Trpa] BN (1-14) >1000 BIM-26036:
[yrD68-pGlul, Gln2, yrAla9 [CH2NH] >1000 Vall°,Phel2] (1-14) T

23a ~ ~ ~ ~ ~ 8 8 BIM-26046:
[yrGlyll [CH2NH] D-Phel'', Leul4BN (1-14 ) >1000 BIM-26048:
[yfD-Phel2 [CHzNH] Leul'3, Leul4] >1000 BN(1-14) BIM-26056:
[yrLeul° [CHzNH] Leull] Substance P >1000 (1-11) HIM-26057:
[Cys9, yrLeu'3 [CH2NH] Cysl4]BN(1-14) >1000 J
BIM-26061:

[D-pGlul, D-Alas, yrLeu' [CHzI~"H] >1000 Meta]

BN (1-14]

BIM-26062:

[~rPhel3 [CHaN'ri] Leu''4] BN (1-.1>1000 437 4) BIM-26063:

- BIM-26028 >1000 BIM-26065:

[D-Argl, D-Trp', D-Trp9, Leul >1000 [CHzNH] Nlell] SP (1-11 ) BIM-26067:

[Des-Gln', ~rLeul3 [CFi2NH] Leu''4]>1000 BN(1-14) a Brain GRP 3T3 GRP Thym, Receptor Receptor Uptake IC30 nM IC50 nM IC50 nM

BIM-26068: [~Leu'3[CH2NIH]Phe'4]BN(1-14)2.9 BIM-26070: [D-Arg', D-Try',~D-Trp9[CHZNH]Leu',>1000 Nle"]1-11SP(1-11) BIM-26071: [Tyr4, ~Leu'3[CHzNH]Met'4]BN(1-14)34 16 104 BIM-26072: = BIM-26057 >1000 BIM-26073: = BIM-28057 >1000 BIM-26074: = BIM-26067 >1000 BIM-26075: [D-Phe",~Leu'3[CH2NH]Leu'4]BN(1-14)> 1000 BIM-26076: [Phe",~Leu'3~CHZNH]Leu'4]BN(1-14)>1000 BIM-26077: [D-Ala5, ~Leu'3[CH2NH]Leu'4]BN(1-14)517 196 1001 BIM-26078: [D-Ala",~Leu~'3[CH2NH]Leu'4]BN(1-14)>1000 70 BIM-26079: [D-Arg',~Phe'[CHZNH]PheB,D-Trp9,Leu"]>1000 SP(1-11 ) BIM-26080: [D-Arg',~GIn6~:CHZNH]D-Trp',D-Trp9,Nle"]>1000 SP(1-11 ) BIM-26081: [D-Arg',~D-Trp'(CH2NH)Phe$,D-Trp9,Nle"] >1000 SP(1-11) BIM-26082: [D-Arg',D-Trp',~PheB[CHZNH]D-Trp9,Nle"]>1000 SP(1-11 ) BIM-26083: = BIM-26080 >1000 BIM-26084: = BIM-26081 >1000 BIM-26085: =BIM 26082 >1000 BIM-26086: [D-Phe'2, y~Le~a'3[CH2NH)Leu'4]BN(1-14) >1000 BIM-26088: [y~Gly9[CHZNH]Leu'°,Leu"]SP(1-11) >1000 BIM-26089: [~Glns[CHZNH]Phe',Leu"]SP(1-11) >1000 ~34098g Brain GRP 3T3 GRP Thym, Receptor Receptor Uptake C30 nM IC50(nM) IC50 nM
BIM-26090: [~Phe'[CH~]PlneB, Leu"]SP(1-11 ) BIM-26091: [~PheB(CHzNH]GIy9,Leu"]SP(1-11)1000>1000 BIM-26092: [His', ~Leu'3[c;H2NH]Leu'4]BN(5-14) (Neuromedin C) 242 466 BIM-26093: [Ac-D-Ala5, Hi:;',~Leu'3[CH2NH]Leu'4]BN(5-14)82 171 BIM-26094: [D-AIaS~", ~Leu'3[CH2NH]Leu'4]BN(1-14)1613 574 BIM-26095: [P-Glus,D-Ala",~Leu'3[CH2NH]Leu'4]BN(6-14) (Litorin) 2623 1209 BIM-26096: [Sta'3,DesMet'4]BN(1-14) 33 agonist EC50=3nM

BIM-26097: [Ac-Lys',~Leu'3[CH~NH]Met'4]BN(7-14)>1000 BIM-26098: [Lys',~Leu'3[C:HZNH]Met'4]BN(7-14) BIM-26099: [~Leu'3[CHZNI-I]Met'4]BN(1-14) BIM-26100: [P-Glus,~Phe'3[CH2NH]Leu'4]BN(8-14)(Litorin) BIM-26101: [P-Glus,Leu'3[CH2NH)L.eu'4]BN(6-14) (Litorin) IM-26102: (Leu',Thr',Phe'3[CHZNH]Met'4]BN 184 >1000 (5-14)(Neurornedin B

BIM-2610,3: [GIyZ,Thrs, y~Le~u'3[CH2NH]Met'4]BN(1-14)>1000 >1000 (A-Lytensin) BIM-26104: [His', y~Leu'3[(~H2NH]Met'4]BN(7-14)>1000 [GRP(20-27)]

Bombesin (BN): 15 0.17 p-Glu'-GIn2-Arg3-Leu4-GIyS,Asns-Gln'-Trp$-Ala9-Val'-Gly"

-His'2-Leu'3-Met'4-NHZ

Claims (20)

1. A peptide of the formula wherein A1 = pGlu, D or L or is deleted;
A2 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine or is deleted;
A3 = Arg, D-Arg, Lys, D-Lys or is deleted;
A4 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.=naphthylalanine or is deleted;
A5 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, p-naphthylalanine, D-Ala or is deleted;
A6 = Gln, Asn, Gly, Ala, D-Ala, N-Ac-D-Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X

= F, Cl, Br, OH or CH3), Trp, p-Glu, .beta.-naphthylalanine or is deleted;
A7 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, Lys, His, or .beta.-naphthylalanine;
A8 = Trp or Met;
A9 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp or .beta.-naphthylalanine, D or L;

A10 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, Thr, or .beta.-naphthylalanine;
A11 = Gly, Phe, D or L;
A12 = His, Phe, or p-X-Phe (X = F, Cl, Br, OH, CH3), D or L;
A13 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, or .beta.-naphthylalanine;

A14 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, or .beta.-naphthylalanine;
provided that each R1, R2, R3, and R4, independently, is H, C1-12 alkyl, C7-10 phenylalkyl, COE1 (where E1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkinyl, phenyl, naphthyl, or C7-10 phenylalkyl), or COOE2 (where E2 is C1-10 alkyl or C7-10 phenylalkyl), and R1 and R2 are bonded to the N-terminal amino acid of said peptide, which can be A1, A2, A3, A4, A5, A6, or A7, and further provided that when one of R1 or R2 is COE1 or COOE2, the other must be H, and when one of R3 or R4 is COE1 or COOE2, the other must be H, and further provided that when A1 = pGlu, R1 must be H and R2 must be the portion of Glu that forms the imine ring in pGlu; and for each of the residues A7, A8, A9, A11, A12, and A13, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof.

2. The peptide of claim 1 wherein A1 through A6 are deleted, each A13 and A14 is Leu, and the carbon atom participating in the amide bond between A13 and A14 is a methylene carbon; or a pharmaceutically acceptable salt thereof.

3. The peptide of claim 1 wherein, for each of said residues A11, A12, and A13 independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof.

4. A peptide of the formula wherein A1 is A6 of claim 1, A2 is A7 of claim 1, A3 is A8 of claim 1, A4 is A9 of claim 1, A5 is A10 of claim 1, A6 is A11 of claim 1, A7 is A12 of claim 1, A8 is A13 of claim 1, A9 is A14 of claim 1 and R1, R2, R3 and R4 are as defined in claim 1, provided that the carbon atom participating in the amide bond between the A8 residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue is reduced to a methylene carbons or a pharmaceutically acceptable salt thereof.

5. A peptide of the general formula of claim 1 wherein, for the residue A10 the carbon atom participating in the amide bond between the residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be a non-peptide bond, provided that said non-peptide bond is said carbonyl carbon having been reduced to a methylene carbon, further provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof.

6. A peptide having the amino acid formula:

pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-~Ala-[CH2NH]-Val-Gly-His-Leu-Leu-NH2.

7. A peptide of the formula:
wherein A1 - pGlu, D or L or is deleted;
A2 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine or is deleted;
A3 - Arg, D-Arg, Lys, D-Lys or is deleted;
A4 - Gln, Asn., Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X--Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine or is deleted;
A5 - Gln, Asn, Gly, Ala Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine, D-Ala or is deleted;
A6 - Gln, Asn, Gly, Ala, D-Ala, N-Ac-D-Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met:, Val, Phe, p-X-Phe (X = F, Cl, Br, OH, or CH3) Trp, p-Glu, .beta.-naphthylalanine or is deleted;

A7 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, Lys, His, or .beta.-naphthylalanine;
A8 - Trp or Met;
A9 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp or .beta.-naphthylalanine, D or L;
A10 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp or .beta.-naphthylalanine;
A11 - Gly, Phe, D or L;
A12 - His, Phe, or p-X-Phe (X = F, Cl, Br, OH, CH3), D or L;

A13 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, or .beta.-naphthylalanine;

A14 - Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, or .beta.-naphthylalanine;

provided that each R1, R2, R3, and R4, independently, is H, C1-12 alkyl, C7-10 phenylalkyl, COE1, (where E1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkynyl, phenyl, naphthyl, or C7-10 phenylalkyl), (alkyl or C7-10 phenylalkyl), or COOE2 (where E2 is C1-10 alkyl or C7-10 phenylalkyl), and R1 and R2 are 32a bonded to the N-terminal amino acid of said peptide, which can be A1, A2, A3, A4, A5, A6, or A7, and further provided that when one of R1 or R2 is COE1 or COOE2, the other must be H and when one of R3 or R4 is COE1 or COOE2, the other must be H, and further provided that when A1 = pGlu, R1 must be H
and R2 must be the portion of Glu that forms the imine ring in pGlu; and for each of the residues A7, A8, A9, A11, A12 and A13, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon (non-peptide bond), provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof; which said peptide is an analog of naturally occurring, biologically active mammalian GRP or amphibian bombesin having an active site, said active site includes the positions A9, A11, A12, A13, and A14, and a binding site responsible for the binding of said bombesin to a receptor on a target cell, said analog having either (a) said non-peptide bond at residues other than within said active site, or (b) having at least one statine or AHPPA
residue in place of: two naturally occurring amino acids of said active site, and further provided that the peptide can contain statine or AHPPA when all bonds between amino acid residues are peptide bonds, and further provided that when an amino acid residue is statine or AHPPA, the amino acid to the right of it in the formula is deleted, so that said analog is capable of binding to said receptor and, by virture of said 32b statine or AHPPA residue, exhibiting enhanced in vivo biological activity compared to said naturally occurring bombesin.

8. A peptide having the amino acid formula:
pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-[Sta13, Des-Met14]-NH2.

9. The method of synthesizing an effective bombesin antagonistic peptide containing the following amino acid formula:
wherein, for the positions A1 through A14, A1 = pGlu, D or L or is deleted A2 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine or is deleted;
A3 = Arg, D-Arg, Lys, D-Lys or is deleted;
A4 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine or is deleted;
A5 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine, D-Ala, or is deleted;
A6 = Gln, Asn, Gly, Ala, D-Ala, N-Ac-D-Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X
= F, Cl, Br, OH or CH3), Trp, pGlu, .beta.-naphthylalanine, or is deleted;
A7 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, D-Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, Lys, His, .beta.-naphthylalanine, A8 = Trp or Met;
A9 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine, D or L;
A10 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, Thr, or .beta.-naphthylalanine;
A11 = Gly, Phe, D or L;
A12 = His, Phe, or p-X-Phe (X = F, Cl, Br, OH or CH3), D
or L;
A13 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine;
A14 = Gln, Asn, Gly, Ala, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met, Val, Phe, p-X-Phe (X = F, Cl, Br, OH or CH3), Trp, .beta.-naphthylalanine;
provided that each R1, R2, R3 and R4, independently, is H, C1-12 alkyl, C7-10 phenylalkyl, COE1 (where E1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkinyl, phenyl, naphthyl, or C7-10 phenylalkyl), or COOE2 (where E2 is C1-10 alkyl or C7-10 phenylalkyl), and R1 and R2 are bonded to the N-terminal amino acid of said peptide, which can be A1, A2, A3, A4, A5, A6, or A7, and further provided that when one of R1 or R2 is COE1 or COOE2, the other must be H, and when one of R3 or R4 is COE1 or COOE2, the other must be H, and further provided that when A1 = pGlu, R1 must be H and R2 must be the portion of Glu that forms the imine ring in pGlu; and for each of the residues A7, A8, A9, A11, A12 and A13, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof said method comprising the steps of (a) preparing benzhydrylamine-polystyrene resin, (b) neutralizing said rein and coupling a chosen amino acid for said A14 position to said resin, (c) coupling a chosen amino acid for said A13 position to said A14-resin, (d) derivatizing the free amino group of said A13-A14-resin, and (e) coupling successively to said derivatized amino acid resin a chosen amino acid for said A12 through A1 positions by repeating steps (d) and (e) for each of said amino acid positions.

10. The method of claim 9 wherein any or all of said positions A1 through A6 are deleted.

11. A method according to claim 9 or 10 synthesizing an effective bombesin agonist of the general formula of claim 9 wherein the carbon atom participating in the amide bond between said position A10 and the nitrogen atom of the alpha amino group of said position A11 forms a non-peptide bond, and said non-peptide bond is -CH2-NH-.

12. A method of synthesizing an ezrective bombesin agonist having the amino acid formula of claim 7, wherein A1 to A14 are as defined in claim 7, which is an analog of naturally occurring, biologically active mammalian GRP or amphibian bombesin, wherein said active site includes the positions A9, A11, A12, A13, and A14, and a binding site responsible for the binding of said bombesin to a receptor on a target cell, said method comprising either the steps of (a) preparing benzhydrylamine-polystyrene resin, (b) neutralizing said resin and coupling a chosen amino acid for said A14 amino acid position or alpha-t-butoxycarbonyl statine to said resin, (c) coupling a chosen amino acid for said A13 position or said statine for said A12 position to said A14-resin, (d) derivatizing the free amino group of said A13-A14-resin or said statine-resin, and (e) coupling successively to said derivatized amino acid or statine resin a chosen amino acid for said A12 through A1 positions by repeating steps (d) and (e) for each of said amino acid positions; provided that when said statine is coupled to said resin, said statine-resin is acetylated before derivatization to free amino of groups;
provided at least one statine or AHPPA residue replaces two naturally occurring amino acids of said active site.

13. The peptide of claim 1 of the formula:
Gly-Asn-His-Trp-Ala-Val-Gly-His-~Leu[CH2NH]Leu-NH2.

14. The peptide of claim 1 of the formula:
pGLu-Gln-Trp-Ala-Val-D-Ala-His-~Phe[CH2NH]Leu-NH2.

15. The peptide of claim 1 of the formula:
pGlu-Gln-Trp-Ala-Val-Gly-His-~Phe[CH2NH]Leu-NH2.

16. The peptide of claim 1 of the formula:
pGlu-Gln-Trp-Ala-Val-Gly-His-~Leu[CH2NH]Leu-NH2.

17. The peptide of claim 1 of the formula:
D-Ala-Asn-His-Trp-Val-D-Ala-His-~Leu[CH2NH]Leu-NH2.

18. The peptide of claim 1 of the formula:
D-Ala-His-Trp-Ala-Val-D-Ala-His-~Leu[CH2NH]Leu-NH2.

19. The peptide of claim 1 of the formula:
D-Phe-His-Trp-Ala-Val-Gly-His-~Leu[CH2NH]Leu-NH2.

20. The peptide of claim 1 of the formula:
N-Ac-D-Ala-Asn-His-Trp-Ala-Val-Gly-His-~Leu[CH2NH]Leu-NH2.