AU2006225304B2 - Novel exendin agonist compounds - Google Patents

Description

ID

1 0 NOVEL EXENDIN AGONIST COMPOUNDS Field of the Invention SThe present invention relates to novel compounds which have activity as exendin agonists. These compounds are SN 5 useful in treatment of Type I and II diabetes, in treatment I of disorders which would be benefited by agents which lower q plasma glucose levels and in treatment of disorders which would be benefited with agents useful in delaying and/or slowing gastric emptying.

BACKGROUND

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.

Exendin The exendins are peptides that are found in venom of the 0o Gila-monster, a lizard endogenous to Arizona and Northern Mexico. Exendin-3 [SEQ. ID. NO. 11 is present in the venom of Heloderma horridum, and exendin-4 [SEQ. ID. NO. 2] is present in the venom of Heloderma suspectum (Eng, et al., 2 c J. Biol. Chem., 265:20259-62, 1990; Eng., et al., J.

O Biol. Chem., 267:7402-05, 1992). The amino acid sequence of exendin-3 is shown in Figure 1. The amino acid sequence of exendin-4 is shown in Figure 2. The exendins have some S sequence similarity to several members of the glucagon-like Speptide family, with the highest homology, 53%, being to V GLP-1[7-36]NH, [SEQ. ID. NO. 3] (Goke, et al., J. Biol. Chem., N 268:19650-55, 1993). GLP-1[7-36]NH,, also known as Sproglucagon[78-107] or simply "GLP-1" as used most often to herein, has an insulinotropic effect, stimulating insulin secretion from pancreatic P-cells; GLP-1 also inhibits glucagon secretion from pancreatic a-cells (Orsov, et al., Diabetes, 42:658-61, 1993; D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). The amino acid \S sequence of GLP-1 is shown in Figure 3. GLP-1 is reported to inhibit gastric emptying (Willms B, et al., J Clin Endocrinol Metab 81 327-32, 1996; Wettergren A, et al., Dig Dis Sci 38 665-73, 1993), and gastric acid secretion.

Schjoldager BT, et al., Dig Dis Sci 34 703-8, 1989; 0 O'Halloran DJ, et al., J Endocrinol 126 169-73, 1990; Wettergren A, et al., Dig Dis Sci 38 665-73, 1993).

GLP-1[7-37], which has an additional glycine residue at its carboxy terminus, also stimulates insulin secretion in humans (Orsov, et al., Diabetes, 42:658-61, 1993).

A transmembrane G-protein adenylate-cyclase-coupled receptor believed to be responsible for the insulinotropic effect of GLP-1 has been cloned from a p-cell line (Thorens, Proc. Natl. Acad. Sci. USA 89:8641-45, 1992), herein after referred to as the "cloned GLP-1 receptor." Exendin-4 is o reportedly acts at GLP-1 receptors on insulin-secreting PTC1 cells, at dispersed acinar cells from guinea pig pancreas, and at parietal cells from stomach; the peptide is also 3 reported to stimulate somatostatin release and inhibit o gastrin release in isolated stomachs (Goke, et al., J. Biol.

C Chem. 268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol., 69:183-91, 1994; Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3 and exendin-4 were reportedly found to 0 stimulate cAMP production in, and amylase release from, I pancreatic acinar cells (Malhotra, et al., Regulatory N- Peptides,41:149-56, 1992; Raufman, et al., J. Biol. Chem.

0 267:21432-37, 1992; Singh, et al., Regul. Pept. 53:47-59, S0 1994). Based on their insulinotropic activities, the use of exendin-3 and exendin-4 for the treatment of diabetes mellitus and the prevention of hyperglycemia has been proposed (Eng, U.S. Patent No. 5,424,286).

Agents which serve to delay gastric emptying have found a place in medicine as diagnostic aids in gastro-intestinal radiologic examinations. For example, glucagon is a polypeptide hormone which is produced by the a cells of the pancreatic islets of Langerhans. It is a hyperglycemic agent which mobilizes glucose by activating hepatic glycogenolysis.

o It can to a lesser extent stimulate the secretion of pancreatic insulin. Glucagon is used in the treatment of insulin-induced hypoglycemia, for example, when administration of glucose intravenously is not possible.

However, as glucagon reduces the motility of the gastro- 2s intestinal tract it is also used as a diagnostic aid in gastro-intestinal radiological examinations. Glucagon has also been used in several studies to treat various painful gastro-intestinal disorders associated with spasm. Daniel, et al. (Br. Med. 3:720, 1974) reported quicker symptomatic relief of acute diverticulitis in patients treated with glucagon compared with those who had been treated with analgesics or antispasmodics. A review by Glauser, et al. Am. Coll. Emergency Physns, 8:228, 1979)

ID

84 described relief of acute esophageal food obstruction o following glucagon therapy. In another study glucagon C significantly relieved pain and tenderness in 21 patients with biliary tract disease compared with 22 patients treated with placebo Stower, et al., Br. J. Surg., 69:591-2, 1982).

Methods for regulating gastrointestinal motility using amylin agonists are described in International Application 0No. PCT/US94/10225, published March 16, 1995.

o 1 Methods for regulating gastrointestinal motility using exendin agonists are described in U.S. Patent Application Serial No. 08/908,867, filed August 8, 1997 entitled "Methods for Regulating Gastrointestinal Motility," which application is a continuation-in-part of U.S. Patent Application Serial No. 08/694,954 filed August 8, 1996.

Methods for reducing food intake using exendin agonists are described in U.S. Patent Application Serial No.

09/003,869, filed January 7, 1998, entitled "Use of Exendin and Agonists Thereof for the Reduction of Food Intake," which claims the benefit of U.S. Provisional Application Nos.

60/034,905, filed January 7, 1997, 60/055,404, filed August 7, 1997, 60/065,442, filed November 14, 1997 and 60/066,029, filed November 14, 1997.

Novel exendin agonist compounds are described in PCT Application Serial No. PCT/US98/16387 filed August 6, 1998, entitled "Novel Exendin Agonist Compounds," claiming the benefit of U.S. Provisional Application Serial No.

60/055,404, filed August 8, 1997. Other novel exendin agonist compounds are described in PCT Application Serial No.

filed November 13, 1998, entitled "Novel Exendin Agonist Compounds," which claims the benefit of U.S.

Provisional Application No. 60/066,029 filed November 14, 1997.

IND Summary of the Invention Q According to a first embodiment of the invention, there is provided apeptide having the amino acid sequence: Xaaj Gly Xaa 3 Gly Xaa 5 Xaa 6 Xaa 7 Xaa 8 Xaa 9 Xaalo Xaaj 1 Xaa 12 Xaa 13 Xaa 14 Xaa 15 Xaa 16 Xaa 1 Ala Xaal 9 Xaa 2 0 Xaa 2 l Xaa 22 Xaa 23 Xaa 24 Xaa 25 Xaa 26 X I-Z 1 ri wherein IND Xaaj is His or 4-imidazopropionyl; Xaa 3 is Asp or Giu; Xaa 5 is Ala or Thr; Xaa6 is Phe or naphthylaianine; Xaa 7 is Thr or Ser; Xaa 8 is Ala, Ser or Thr; Xaa 9 is Asp or Giu; XaajO is Ala, Leu, Ilie, Val, pentyiglycine or Met; Xaaj I is Ala or Ser; Xaa 1 2 is Ala or Lys; Xaa 13 is Ala or Gin; Xaa 14 is Leu, pentyiglycine, or Met; Xaa 15 is Ala or Giu; Xaa 16 is Ala or Glu; Xaa 17 is Ala or Giu; Xaal 9 is Ala or Val; Xaa 2 0 is Ala or Arg; Xaa 2 l is Ala, Leu or Lys- NW'-R where R is Lys, Arg, C I-C I straight chain or branched aikanoyl or cycloalkyikanoyl; Xaa 22 is Phe or naphthylaianine; Xaa 23 is Ilie or Val; Xaa 24 is Ala, Glu or Asp; Xaa 25 is Trp or Phe; Xaa 26 is Ala or Leu; R:\PA L Specifications\5066721D2spec.doc:gcc

O

X

1 is Lys Asn, Lys- NHE-R Asn, Lys- NH'-R Ala, where R is Lys, Arg, SC i-Clo straight chain or branched alkanoyl O Zi is -OH,

S-NH

2 Gly-Z 2 Gly Gly-Z 2 SGly Gly Xaa 31

-Z

2 Gly Gly Xaa 31 Ser-Z 2 ND Gly Gly Xaa 31 Ser Ser-Z 2 to Gly Gly Xaa 31 Ser Ser Gly-Z 2 Gly Gly Xaa 31 Ser Ser Gly Ala-Z 2 Gly Gly Xaa 31 Ser Ser Gly Ala Xaa 36

-Z

2 Gly Gly Xaa 31 Ser Ser Gly Ala Xaa 36 Xaa 37

-Z

2 or Gly Gly Xaa 31 Ser Ser Gly Ala Xaa 36 Xaa 37 Xaa 3

-Z

2 wherein Xaa3i, Xaa 36 Xaa 37 and Xaa 38 are independently selected from the group consisting of Pro, homoproline, thioproline, and N-alkylalanine; and

Z

2 is -OH or -NH2; provided that no more than three of Xaas, Xaa 8 Xaalo, Xaa Xaa 12 Xaa 13 Xaals, Xaa 16 Xaa 17 Xaal9, Xaa20, Xaa 21 Xaa 24 and Xaa26 are Ala; and pharmaceutically acceptable salts thereof.

According to a second embodiment of the invention, there is provided acomposition comprising a compound in accordance with the first embodiment of the present invention in a pharmaceutically acceptable carrier.

According to a third embodiment of the invention, there is provided amethod for the treatment of diabetes mellitus comprising administering a therapeutically effective amount of a compound in accordance with the first embodiment of the present invention.

According to a fourth embodiment of the invention, there is provided the use of any one of the compounds in accordance with the first embodiment of the present invention in the manufacture of a medicament for the treatment of diabetes mellitus.

[R:\PAL Specificaions\06672]D2spec.do c:gcc According to a fifth embodiment of the invention, there is provided amethod for treating hyperglycemia comprising administering a therapeutically effective amount of a compound in accordance with the first embodiment of the present invention.

According to a sixth embodiment of the invention, there is provided the use of the compound in accordance with the first embodiment of the present invention for the manufacture of a medicament for the treatment of hyperglycemia.

In addition, there is herein disclosed novel exendin agonist compounds which exhibit advantageous properties which include effects in slowing gastric emptying and lowering plasma glucose levels.

In addition, there is herein disclosed compounds of the formula [SEQ. ID. NO. 4]: Xaa, Xaa 2 Xaa 3 Gly Xaa s Xaa 6 Xaa, Xaa, Xaa, Xaa, 1 Xaan, Xaa 1 2 Xaa,, Xaa 2 Xaa 5 Xaaje Xaa,, Ala Xaa,q Xaa 20 Xaa 2 1 Xaa 22 Xaa 2 Xaa 24 Xaa 2 Xaa 26 Xaa,, Xaa 2 wherein Xaa, Xaa 2 Xaa 3 Xaa 5 Xaa 6 Xaa, Xaa 8 Xaa, Xaao Xaan 1 Xaa 1 2 Xaa 13 Xaa, 4 30 Xaa 15 Xaa,6 Xaa 17 Xaa, 9 Xaa 20 Xaa 21 His, Arg or Tyr; Ser, Gly, Ala or Thr; Asp or Glu; Ala or Thr; Ala, Phe, Tyr or naphthylalanine; Thr or Ser; Ala, Ser or Thr; Asp or Glu; Ala, Leu, Ile, Val, pentylglycine Ala or Ser; Ala or Lys; Ala or Gin; Ala, Leu, lie, pentylglycine, Val Ala or Glu; Ala or Glu; Ala or Glu; Ala or Val; Ala or Arg; Ala or Leu; or Met; or Met; [RAPAL Spec i fication s\506672 D2spec.doc:gcc IND 6 C1 Xaa,2 2 is Phe, Tyr or naphthylalanine; U Xaa, 3 is Ile, Val, Leu, pentyiglycine, tert-butylglycine 0or Met; Xaa 24 is Ala, Glu or Asp; 2 is Ala, Trp, Phe, Tyr or naphthylalanine; Xaa 26 is Ala or Leu; Xaa 27 is Ala or Lys; Xaa 28 is Ala or Asn; IND Z, s -OH, '0 -NH, Gly-Z 2 Gly Gly-Z 2 Gly Gly Xaa 3 l-Z 2 Gly Gly Xaa 3 1 Ser-Z 2 iSGly Gly Xaa 3 l Ser Ser-Z 2 Gly Gly Xaa 3 1 Ser Ser Gly-Z 2 Gly Gly Xaa 3 1 Ser Ser Gly Ala-Z 2 Gly Gly Xaa 3 1 Ser Ser Gly Ala Xaa 36

-Z

2 Gly Gly Xaa 3 1 Ser Ser Gly Ala Xaa 2 6 Xaa 3 2 or Gly Gly Xaa 3 1 Ser Ser Cly Ala Xaa 36 Xaa 3 7 Xaa 38

Z

2 Xaa 3 l, Xaa 36 Xaa 37 and Xaa 3 8 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and

Z

2 is -OH or -NH 2 provided that no more than three of Xaa Xaa., Xaa 6 Xaa 8 Xaal., Xaa, 1 Xaa 12 Xaa 13 Xaa 1 4 Xaal,, Xaa, 1 Xaa 1 Xa aj 9 Xaa 20 Xaa 2 l Xaa 24 Xaa 2 Xaa, 6 Xaa 2 and Xaa 2 are Ala.

In addition, there is herein disclosed o ~pharmaceutical ly acceptable salts of the compounds of formu.La and pharmaceutical compositions including said compounds and salts thereof.

In addition, there is herein disclosed narrower genera of compounds having peptides of various 0 lengths, for example genera of compounds which do not include peptides having a length of 28, 29 or 30 amino acid residues, respectively. In addition, there is herein disclosed narrower genera of compounds having particular amino acid sequences, for example, compounds of the formula

[SEQ.

ID. NO. 4]: Caa Xaa X 3 GyXa Xaa, Xaa, Xaa 8 Xaa, Xaa 10 Xaal 1 Xaa 1 2 Xaa,, Xaa 14 Xaa 15 Xaa,, Xaa 1 Ala Xaa,, Xaa,, Xaa 2 Xaa,l Xaa 2 Xaa.

3 Xaa 2 4 Xaa.

5 Xaa 2 Xaa 2 7 Xaa,,-Z 1 wherein Xaa, is His or Arg; Xaa 2 is Gly or Ala; Xaa 3 is Asp or Glu; Xaa, is Ala or Thr; Xaa, is Ala, Phe or naphthylalanine; Xaa, is Thr or Ser; Xaa, is Ala, Ser or Thr; IC9 Xaa, is Asp or Glu; Xaa 10 is Ala, Leu or pentyiglycine; Xaaj, is Ala or Ser; Xaa,, is Ala or Lys; Xaa 13 is Ala or Gln; Xaaj, is Ala, Leu or pentylglycine; Xaa,, is Ala or Glu; Xaa 1 6 is Ala or Glu; Xaa 1 is Ala or Glu; Xaa 1 9 is Ala or Val; Xaa 20 is Ala or Arg; 00 Xaa 2 l is Ala or Leu; Xaa 22 is Phe or naphthylalanine; z Xaa 23 is Ile, Val or tert-butyiglycine; Xaa 24 is Ala, Glu or Asp; Xaa 25 is Ala, Trp, or Phe; Xaa 26 is Ala or Leu; C) Xaa 27 is Ala or Lys; tn Xaa 2 g is Ala or Asn; Z, is -CR, C) 10-NH 2 CI GlY-Z 2 Gly Gly -Z 2 Gly Gly Xaa 31

-Z

2 Gly Gly Xaa 31 Ser-Z 2 is Gly Gly Xaa 3 1 Ser Ser-Z 2 Gly Gly Xaa 3 1 Ser Ser GIY-Z 2 Gly Gly Xaa 3 1 Ser Ser Gly Ala-Z 2 Gly Gly Xaa 31 Ser Scr Gly Ala Xaa 36 -Z2, Gly Gly Xaa 3 1 Ser Scr Gly Ala Xaa 3 6 Xaa 3 7 -Z2 or Gly Gly Xaa 3 l Ser Ser Gly Ala Xaa 36 Xaa 37 Xaa 3 8-Z2; Xaa 3 l Xaa 36 Xaa 37 and Xaa 3 8 are independently selected from the group consisting of Pro, homoproline, thioproline and N-methylylalanine; and

Z

2 is -OH or -NH 2 excluding SEQ ID NO: 42; and provided that no more than three of Xaa 3 Xaa 5 Xaa 6 Xaa8, Xaai1o, Xaa 11 Xaa 1 2 Xaa 13 Xaa 14 Xaa 1 5 Xaa 16 Xaa 17 Xaa 19 Xaa2o, Xaa 2 1 Xaa 24 Xaa 2 5, Xaa 26 Xaa 27 and Xaa 2 8 are Ala; and pharmaceutically acceptable salts thereof.

Also disclosed are compounds of the fomula (11) [SEQ. ID. NO. 66]: Xaa 1 Xaa 2 Xaa 3 Gly Xaa 5 Xaa 6 Xaa 7 Xaa8 Xaa 9 Xaajo 1837776 U:NB 9 Xaa, 1 Xaa 1 Xaa,, Xaa 4 Xaa 15 Xaa 16 Xaa 1 Ala Xaajq Xaa 20 0Xaa-j Xaa 9 Xaa, Xaa 2 Xaa,,, Xaa 2 x~ wherein Xaaj is His, Arg, Tyr or 4-imidazopropionyl; Xaa 2 i S Ser, Gly, Ala or Thr; Xaa 3 is Asp or Glu; Xaa~, is Ala or Thr; Xaa, is Ala, Phe, Tyr or naphthylalanine; IND Xaa, is Thr or Ser; Xaa 8 is Ala, Ser or Thr; 0Xaa, is Asp or Glu; Xaa 10 is Ala, Leu, Ile, Val, pentyiglycine or Met; Xaal 1 is Ala or Ser; Xaa 12 is Ala or Lys; Xaa, 3 is Ala or Gin; Xaa 14 is Ala, Leu, Ile, pentylglycine, Val or Met; Xaa 15 is Ala or Glu; Xaaj, is Ala or Glu; Xaaj, is Ala or Glu; Xaaj, is Ala or Val; 0 Xaa 2 0 is Ala or Arg; Xaa 2 1 is Ala, Leu or Lys-NHc-R where R is Lys, Arg, straight chain or branched alkanoyl or cycloalkylalkanoyl; Xaa 22 is Phe, Tyr or naphthylalanine; Xaa 23 is Ile, Val, Leu, pentyiglycine, tert-butylglycine or Met; Xaa 2 4 is Ala, Glu or Asp; Xaa 25 is Ala, Trp, Phe, Tyr or naphthylalanine; Xaa,, is Ala or Leu; X, 'is Lys Asn, Asn Lys, Lys-NNBR Asn, Asn Lys-NHC-R, Lys-NH--R 360 Ala, Ala Lys-NH'-R where R is Lys, Arg, straight chain or branched aJlkanoyi or cycloalkylalkanoy.

Z, is -OH, 0 -NH 2 Gly-Z,, Gly Gly-Z 2 1 S Gly Gly Xaa 3 l-Z 2 Gly Gly Xaa 31 Ser-Z 2 Gly Gly Xaa 31 Ser Ser-Z., Gly Gly Xaa 31 Ser Ser Gly-Z 2 GlIlND 32 SrSrGl l- 2 0Gly Gly Xaa 31 Ser Ser Gly Ala-Xa 36

Z

2 0Gly Gly Xaa 31 Ser Ser Gly Ala Xaa 36 Xa-- 2 o Gly Gly Xaa 31 Ser Ser Gly Ala Xaa 36 Xaa 3 7-Z or 3 8

Z

wherein S' Xaa 31 Xaa 3 1, Xaa 3 and Xaa 28 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and

Z

2 is -OH or -NH,; provided that no more than three of Xaa 3 Xaa., Xaa 6 Xaa, Xaa 10 Xaajj, Xaa 12 Xaa 13 Xaa 14 Xaa 1 Xaa 16 Xaal., Xaa 19 Xaa 20 Xaa 2 l, Xaa 24 Xaa 25 and Xaa 26 are Ala. Also within the scope of the present invention are pharmaceutically acceptable salts of the compounds of formula (II) and pharmaceutical compositions including said compounds and salts thereof.

Preferred exendin agonist compounds of formula (II) include those wherein Xaa, is His, Tyr or 4-imidazopropionyl.

More preferably Xaa, is His. Also, preferred are those compounds of formula (II) wherein Xaa, is 4-imidazopropionyl.

'NO Preferred are those compounds of formula (II) wherein Xaa 2 is Gly.

Preferred compounds of formula (II) are those wherein Xaa 14 is Leu, pentyiglycine or Met.

D11 SPreferred compounds of formula (II) are those wherein O Xaa 2 is Trp or Phe.

According to one aspect, preferred are compounds of formula (II) wherein Xaa 6 is Phe or naphthylalanine; Xaa.. is Phe or naphthylalanine; and Xaa23 is Ile or Val. More O preferably, Z, is -NH 2 According to one aspect, especially Spreferred are such compounds of formula (II) wherein Xaa 3 1 CXaa 36 Xaa 3 7 and Xaa3, are independently selected from the group Sconsisting of Pro, homoproline, thioproline and N- C \o alkylalanine. More preferably,

Z

2 is -NH,.

Preferred compounds of formula (II) include those wherein X, is Lys Asn, Lys-NHC-R Asn, or Lys-NHC-R Ala where R is Lys, Arg, Ci-C,, straight chain or branched alkanoyl.

Especially preferred compounds of formula (II) include

I

5 Compound Nos. 62-69 (SEQ. ID. NOS. 67-74].

Definitions In\accordance with the present invention and as used herein, the following terms are defined to have the following meanings, unless explicitly stated otherwise.

The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers if their structure allow such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), typtophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2aminoadipic acid, 3 -aminoadipic acid, beta-alanine, aminopropionic acid, 2 -aminobutyric acid, 4 -aminobutyric

IO

8 12 acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2- 0 aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic 0C acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, Shydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4- VB hydroxyproline, isodesmosine, allo-isoleucine, N- CA methylalanine, N-methylglycine, N-methylisoleucine, N- Smethylpentylglycine, N-methylvaline, naphthalanine, o norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, Is methionine sulfoxide, methionine sulfone, S-(carboxymethyl)cysteine, S-(carboxymethyl)-cysteine sulfoxide and S- (carboxymethyl)-cysteine sulfone.

The term "amino acid analog" refers to an amino acid wherein either the C-terminal carboxy group, the N-terminal o2 amino group or side-chain functional group has been chemically codified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine.

The term "amino acid residue" refers to radicals having the structure: wherein R typically is wherein R' is an amino acid side chain, typically H or a carbon containing substitutent; or (2)

ID

13 c

H

2 0 0 wherein p is 1, 2 or 3 representing the azetidinecarboxylic Cs acid, proline or pipecolic acid residues, respectively.

N0 The term "lower" referred to herein in connection with O organic radicals such as alkyl groups defines such groups with up to and including about 6, preferably up to and including 4 and advantageously one or two carbon atoms. Such groups may be straight chain or branched chain.

"Pharmaceutically acceptable salt" includes salts of the compounds of the present invention derived from the o combination of such compounds and an organic or inorganic acid. In practice the use of the salt form amounts to use of the base form. The compounds of the present invention are useful in both free base and salt form, with both forms being considered as being within the scope of the present invention.

In addition, the following abbreviations stand for the following: "ACN" or "CH 3 CN" refers to acetonitrile.

2 "Boc", "tBoc" or "Tboc" refers to t-butoxy carbonyl.

-O "DCC" refers to N,N'-dicyclohexylcarbodiimide.

"Fmoc" refers to fluorenylmethoxycarbonyl.

"HBTU" refers to 2 -(lH-benzotriazol-l-yl)- 1,1,3,3,-tetramethyluronium hexaflurophosphate.

"HOBt" refers to l-hydroxybenzotriazole monohydrate.

"homoP" or hPro" refers to homoproline.

"MeAla" or "Nme" refers to N-methylalanine.

"naph" refers to naphthylalanine.

ID

14 "pG" or pGly" refers to pentylglycine.

0 "tBuG" refers to tertiary-butylglycine.

"ThioP" or tPro" refers to thioproline.

"3Hyp" refers to 3-hydroxyproline "4Hyp" refers to 4-hydroxyproline "NAG" refers to N-alkylglycine V' "NAPG" refers to N-alkylpentylglycine

(N

(C BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the amino acid sequence for exendin-3 [SEQ. ID. NO. 1].

Figure 2 depicts the amino acid sequence for exendin-4 [SEQ. ID. NO. 2].

Figure 3 depicts the amino acid sequence for GLP-1[7- 36]NH 2 (GLP-1) [SEQ. ID. NO. 3].

Figure 4 depicts the amino acid sequences for certain compounds of the present invention, Compounds 1-61 [SEQ. ID.

NOS. 5 to Figure 5 depicts the effect on lowering blood glucose of various concentrations of Compound 3 [SEQ. ID. NO. 7].

2 Figure 6 depicts the effect on gastric emptying of various concentrations of Compound 2 [SEQ. ID. NO. 6].

Figure 7 depicts a comparison of effects on gastric emptying of various concentrations of Compound 3 [SEQ. ID.

NO. 7].

Figure 8 depicts a comparison of effects on gastric emptying of various concentrations of Compound 10 [SEQ. ID.

NO. 14].

Figure 9 depicts a comparison of effects on gastric emptying of various concentrations of Compound 13 [SEQ. ID.

NO. 17].

Figure 10 depicts the amino acid sequences for certain U compounds of the present invention, Compound Nos. 62 69 [SEQ. ID. NOS. 67 74].

DETAILED DESCRIPTION OF THE INVENTION £According to the present invention, provided are compounds of the formula [SEQ. ID. NO. 4): Xaa, Xaa, Xaa 3 Gly Xaa5 Xaa, Xaa, Xaa~, Xaa, Xaa 1 Xaa 1 1 Xaa 12 Xaa, 3 Xaa 1 4 Xaa,. Xaa 1 Xaa., Ala Xaa 1 9 Xaa 2 Xaa 2 l Xaa,, Xaa,, Xaa 2 4 Xaa, 5 Xaa 2 Xaa,7 Xaa 28 -Zl; wherein Xaaj is H-is, Arg or Tyr; Xaa 2 is Ser, Gly, Ala or Thr; Xaa, is Asp or Glu; Xaa, is Ala or Thr; Xaa, is Ala, Phe, Tyr or naphthylalanine; Xaa 7 is Thr or Ser; Xaa, is Ala, Ser or Thr; Xaa, is Asp or Glu; Xaa 10 is Ala, Leu, Ile, Val, pentyiglycine or Met; Xaaj, is Ala or Ser; Xaa 12 is Ala or Lys; Xaa 13 is Ala or Gin; Xaaj, is Ala, Leu, Ile, pentylglycine, Val or Met; Xaal 15 is Ala or Glu; Xaaj, is Ala or Glu; Xaa 17 is Ala or Giu; Xaal 9 is Ala or Val; Xaa 2 0 is Ala or Arg; Xaa 21 is Ala or Leu; Xaa.

2 i S Phe, Tyr or naphthylalanine; 16 o Xaa 23 is Ile, Val, Leu, pentyliycine, tert-butyiglycine 0or Met; Xaa 24 is Ala, Glu or Asp; Xaa 2 is Ala, Trp, Phe, Tyr or naphthylalanine; 2 6 is Ala or Leu; Xaa 2 is Ala or Lys; Xaa 2 is Ala or Asn; Z, is-OH,

-NH

2 tO Gly-Z 2 Gly Gly-Z 2 Gly Gly Xaa,,-Z 2 Gly Gly Xaa 3 1 Ser-Z 2 1 Gly Gly Xaa 31 Ser Ser-Z, Is l aalSrSe l-, Gly Gly Xaa 3 1 Ser Ser Gly-Z 2 Gly Gly Xaa 3 l Ser Ser Gly Ala-Z 2 Gly Gly Xaa 21 Ser Ser Gly Ala Xaa 36

-Z

2 o Gly Gly Xaa 3 l Ser Ser Gly Ala Xaa 36 Xaa~ 7 -Z or3,Z Xaa.

1 Xaa 36 Xaa 3 and Xaa 38 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and

Z

2 is -OH or -NH 2 21S provided that no more than three of Xaa 3 Xaa,, Xaa 6 Xaa., Xaaj 0 Xaa 11 Xaa 12 Xaa 13 Xaa 14 Xaa 15 Xaal., Xaa 1 Xaaj 9 Xaa 2 Xaa 21 Xaa.4 Xaa 25 Xaa 26 Xaa 2 and Xaa 2 8 are Ala. Also within the scope of the present invention are pharmaceutically acceptable salts of the compounds for formula and %W pharmaceutical compositions including said compounds and salts thereof.

Preferred N-alkyl groups for N-alkylglycine, Nalkylpentylglycine and N-alkylalanine include lower alkyl

IO

0 17 groups preferably of 1 to about 6 carbon atoms, more o preferably of 1 to 4 carbon atoms. Suitable compounds of formula include those identified in Examples 1-.61 ("Compounds 1-61," respectively) [SEQ. ID. NOS. 5 to 65], as s well as those corresponding compounds identified in Examples and 71.

l Preferred such exendin agonist compounds include those wherein Xaa, is His or Tyr. More preferably Xaa, is His.

I Preferred are those such compounds wherein Xaa, is Gly.

0 Preferred are those such compounds wherein Xaa 1 is Leu, pentylglycine or Met.

Preferred such compounds are those wherein Xaa2s is Trp or Phe.

Preferred compounds of formula are those where Xaa 6 is Phe or naphthylalanine; Xaa,, is Phe of naphthylalanine and Xaa,, is lie or Val.

Preferred are compounds of formula wherein Xaan, Xaa 3 Xaa., and Xaa, 3 are independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z, is -NH,.

Preferable Z, is -NH,.

According to one aspect, preferred are compounds of formula wherein Xaa, is His or Tyr, more preferably His; Xaa, is Gly; Xaa, is Phe or naphthylalanine; Xaa 1 is Leu, pentylglycine or Met; Xaa 22 is Phe or naphthylalanine; Xaa 23 is Ile or Val; Xaa,, Xaa 6 Xaa,, and Xaa 3 are independently selected from Pro, homoproline, thioproline or Nalkylalanine. More preferably Z, is -NH,.

According to an especially preferred aspect, especially o preferred compounds include those of formula wherein: Xaa, is His or Arg-;--Xaa---i-s Gly or Ala; Xaa 3 is Asp or Glu; Xaa, is Ala or Thr; Xaa, is Ala, Phe or nephthylalaine; Xaa, is Thr or Ser; Xaa, is Ala, Ser or Thr.; Xaa, is Asp or Glu; 18 Xaao is Ala, Leu or pentylglycine; Xaa,, is Ala or Ser; Xaa12 0 is Ala or Lys; Xaa 13 is Ala or Gin; Xaa, is Ala, Leu or pentylglycine; Xaa,, is Ala or Glu; Xaal, is Ala or Glu; Xaa,, is Ala or Glu; Xaa, 9 is Ala or Val; Xaa 20 is Ala or Arg; Xaa 21 is Ala or Leu; Xaa, 2 is Phe or naphthylalanine; Xaa 3 is Ile, Val or tert-butylglycine; Xaa 24 is Ala, Glu or Asp; Xaa 2 s is Ala, Trp or Phe; Xaa,, is Ala or Leu; Xaa 2 is Ala or Lys; Xaa, I is Ala or Asn; Zi is -OH, Gly-Z,, Gly Gly-Z,, Gly Gly Xaa 2 1

-Z

2 Gly Gly Xaa 3 l Ser-Z,, Gly Gly Xaa 3 l Ser Ser-Z,, Gly Gly C 1 oXaaal Ser Ser Gly-Z,, Gly Gly Xaa 31 Ser Ser Gly Ala-Z,, Gly Gly Xaa 1 Ser Ser Gly Ala Xaa 3 6 Gly Gly Xaa 3 Ser Ser Gly Ala Xaa 3 Xaa,-Z,, Gly Gly Xaa,, Ser Ser Gly Ala Xaa36 Xaa, Xaa3,-z,; Xaa 3 1 Xaa,,, Xaa 3 and Xaa3, being independently Pro homoproline, thioproline or N-methylalanine; and Z being -OH or provided that no more than three of Xaa 3 Xaa,, Xaa 6 Xaa 8 Xaa 10 Xaa,,f Xaa 12 Xaa 1 3 Xaa 4 Xaa 1 Xaa 1 6 Xaa,, Xaa.

9 Xaa 20 Xaa,, 2 1 Xaa,,, Xaa 25 Xaa,,, Xaa 2 and Xaa 2 are Ala.

Especially preferred compounds of formula include those having the amino acid sequence of SEQ. ID. NOS. 6-27 o 2-o (Compounds 2-23).

According to an especially preferred aspect, provided are compounds of compound where Xaa 14 is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa 2 is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will be less susceptive to oxidative degration, both in vitro and in vivo, as well as during synthesis of the compound.

Also within the scope of the present invention are narrower genera of compounds having peptides of various lengths, for example genera of compounds which do not include peptides having a length of 28, 29 or 30 amino acid residues, respectively. Additionally, the present invention includes narrowe-r genera of compounds having particular amino acid 19 sequences, for example, compounds of the formula

(SEQ.

ID. NO. 4]: Xaa, Xaa,, Xaa 3 Gly Xaa 5 Xaa 6 Xaa, Xaa 8 Xaa 9 Xaaj 0 Xaal 1 Xaa 12 Xaa 13 Xaa 14 Xaa 15 Xaa 16 Xaa, 1 Ala Xaaj, Xaal 9 Xaa 90 Xaa 2 l Xaa 22 Xaa 23 Xaa 24 Xaa 2 5 Xaa 26 Xaa 2 7 Xaa,,-Zi; wherein Xaa 1 i s His or Arg; Xaa, is Gly or Ala; Xaa 3 is Asp or Glu; Xaa 5 is Ala or Thr; Xaa 6 is Ala, Phe or naphthylalanine; Xaa 1 is Thr or Ser; Xaa 8 is Ala, Ser or Thr; Xaa 9 is Asp or Giu; Xaa 10 is Ala, Leu or pentylglycine; Xaall is Ala or-Ser; Xaa 12 is Ala or Lys; Xaa 13 is Ala or Gin; Xaa 14 is Ala, Leu or pentylglycine; Xaa 15 is Ala or Glu; Xaa 16 is Ala or Glu; Xaa,, is Ala or Glu; Xaal 9 is Ala or Val; Xaa 20 is Ala or Arg; Xaa 21 is Ala or Leu; Xaa 22 iS Phe or naphthylalanine; Xaa 2 3 iS Ile, Val or tert-butylglycine; Xaa 2 is Ala, Glu or Asp; Xaa 2 5 is Ala, Trp, or Phe; Xaa 26 is Ala or Leu; o Xaa 2 is Ala or Lys; 0Xaa, 8 is Ala or Asn; Z' is -OH,

-NH

2 GlY-Z 2 Gly Gly -Z 2 1 Gly Gly Xaa,,-Z, Gly Gly Xaa 3 l Serr-Z 2 Gly Gly Xaa,, Ser Sery-Z, Gtol a 32 SrSrGl l- 2 Gly Gly Xaa31 Ser Ser Gly Aa aa,-, Gly Gly Xaa 3 l Ser Ser Gly Ala Xaa 3 6 Xaa 3 2 or Gly Gly Xaa 3 Ser Ser Gly Ala Xaa 36 Xaa 3 7 Xaa 3

,-Z

2 K Xaa 31 Xaa 36 Xaa 3 and Xaa 3 are independently selected from the group consisting of Pro, homoproline, thioproline and N-methylylalanine; and

Z

2 iS -OH or -NH 2 provided that no more than three of Xaa 3 Xaa 5 Xaa 6 Xaa., Xaa 30 Xaall, Xaa1 2 1 Xaa, 3 Xaa1 4 Xaa, 5 Xaa1 6 Xaa 17 Xaa, 9 Xaa 20 Xaa,,, Xaa 2 Xaa 2 Xaa 2 6 Xaa 2 anXa 8 arAl;ad pharmaceutically acceptable salts thereof.

Also provided are compounds of the formula (II) [SEQ.

ID. NO. 66]: 2,S Xaaj Xaa 2 Xaa 3 Gly Xaa 5 Xaa, Xaa., Xaae, Xaa 9 Xaaj 0 Xaa, 1 Xaa,., Xaa1 3 Xaa1 4 Xaaj, Xaa1 6 Xaa,., Ala Xaa 19 Xaa 20 Xaa 2 Xaa 22 Xaa 23 Xaa 2 4 Xaa 2 Xaa 26 X1 wherein Xaa, is His, Arg or Tyr or 4-imidazopropionyl; Xaa 2 is Ser, Gly, Ala or Thr; so Xaa 3 is As'p or Glu; 21 Xaa 5 is Ala or Thr; 0 Xaa 6 is Ala, Phe, Tyr or naphth yla la nine; Xaa, is Thr or Ser; Xaa 8 is Ala, Ser or Thr; SXaa 9 is Asp or Glu; Xaa 1 O is Ala, Leu, Ile, Val, pentyiglycine or Met; Xa a, is Ala or Ser; Xaa 1 is Ala or Lys; IND Xaa 13 is Ala or Gin; 0 Xaa,, is Ala, Leu, Ile, pentyiglycine, Val. or Met; Xaa, 5 is Ala or Glu; Xaaj, is Ala or Glu; Xaa., is Ala or Glu; Xaa 19 is Ala or Val; Xaa 2 is Ala or Arg; Xaa 2 1 is Ala, Leu or Lys-NHc-R where R is Lys, Arg, C 1

-C

10 straight chain or branched alkanoyl or cycloalkylalkanoyi; Xaa 2 is Phe, Tyr or naphthylalanine; Xaa 23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met; Xaa 2 4 is Ala, Glu or Asp; Xaa 25 is Ala, Trp, Phe, Tyr or naphthylalanine; Xaa 2 is Ala or Leu; X, is Lys Asn, Asn Lys, Lys-NHrE-R Asn, Asn Lys-NHc-R, Lys-NI,-R 2-S Ala, Ala Lys-NHe-R where R is Lys, Arg, straight chain or branched alkanoyl or cycloalkylalkanoyl Z, is -OH,

-NH

2 GlY-Z 2 3" Gly Gly-Z 2 Gly Gly Xaa 31

-Z

2 Gly Gly Xaa 3 l Ser-Z 2

IND

LO

22 Gly Gly Xaa 3 l Ser Ser-Z 2 Gly Gly Xaa., Ser Ser Gly-Z 2 1 Gly Gly Xaa 3 1 Ser Ser Gly Ala-Z 2 Gly Gly Xaa 3 1 Ser Ser Gly Ala Xaal 6

-Z

2 Gly Gly Xaa 3 l Ser Ser Gly Ala Xaa, 6 Xaa 3 7

-Z

2 or Gly Gly Xaa~l Ser Ser Gly Ala Xaa 36 Xaa 3 7 Xaa 3 wherein Xaa 3 l, Xaa 36 Xaa 3 7 and Xaa 3 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and

Z

2 is, -OH or -NH 2 provided that no more than three of Xaa., Xaa., Xaa,, Xaa 8 Xaa,,, Xaal 1 Xaa 1 2 Xaa 13 Xaa 1 4 Xaal,, Xaa 16 Xaa 1 Xaalg, Xaa 20 Xaa 2 1 Xaa 24 Xaa 2 and Xaa 2 are Ala. Also within the scope of the present invention are pharmaceutically acceptable salts of the compound of formula (II) and pharmaceutical compositions including said compounds and salts thereof.

Preferred exendin agonist compounds of formula

(II)

include those wherein Xaa, is His, Tyr or 4 -imidazopropionyl.

More preferably Xaa, is His.

Preferred are those compounds of formula (II) wherein Xaa, is 4-imidazopropionyl.

Preferred are those compounds of formula (II) wherein Xaa 2 is Gly.

Preferred compounds of formula (II) are those wherein Xaa, 4 is Leu, pentylglycine or Met.

Preferred compounds of formula (II) are those wherein Xaa 2 is Trp or Phe.

According to one aspect, preferred are compounds of formula (II) wherein Xaa 6 is Phe or naphthylalanine; and Xaa 22 is Phe or naphthylalanine; and Xaa 23 is Ile or Val. More O 23 preferably, Z is According to one aspect, especially preferred are such compounds of formula (II) wherein Xaa 3 Xaa3,, Xaa 3 and Xaa 3 8 are independently selected from the group consisting of Pro, homoproline, thioproline and N- S alkylalanine. More preferds, Z, is -NH,.

SPreferred compounds of formula (II) include those wherein X, is Lys Asn, Lys-NH'-R Asn, or Lys-NH-R Ala where

R

is Lys, Arg, straight chain or branched alkanoyl.

I Preferred compounds of formula (II) include Compound Nos. 62- 0 69 [SEQ. ID. NOS. 67-74] The compounds referenced above form salts with various inorganic and organic acids and bases. Such salts include salts prepared with organic and inorganic acids, for example, HC1, HBr, H 2

SO

4

H

3

PO

4 trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonic acid. Salts prepared with bases include ammonium salts, alkali metal salts, e.g. sodium and potassium salts, and alkali earth salts, e.g. calcium and magnesium salts. Acetate, hydrochloride, and trifluoroacetate salts are preferred. The salts may be formed by conventional means, as by reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the ions of an existing salt for another ion on a suitable ion exchange resin.

Utility The compounds described above are useful in view of 0their pharmacological properties. In particular, the compounds of the invention are exendin agonists, and possess 24 activity as agents to regulate gastric motility and to slow 0 gastric emptying, as evidenced by the ability to reduce post- Oh prandial glucose levels in mammals.

The compounds of the present invention are useful in in S vitro and in vivo scientific methods for investigation of C exendins and exendin agonists for example in methods such as |n those described in Examples A-E below.

(O

SPreparation of Compounds C\ The compounds of the present invention may be prepared 10 using standard solid-phase peptide synthesis techniques and preferably an automated or semiautomated peptide synthesizer.

Typically, using such techniques, an a-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert ts solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and l-hydroxybenzotriazole in the presence of a base such as diisopropylethylamine. The a-Ncarbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, with t-butyloxycarbonyl (tBoc) and 2 fluorenylmethoxycarbonyl (Fmoc) being preferred herein.

The solvents, amino acid derivatives and 4 -methylbenzhydryl-amine resin used in the peptide synthesizer may be purchased from Applied Biosystems Inc.

(Foster City, CA). The following side-chain protected amino so acids may be purchased from Applied Biosystems, Inc.: Boc- Arg(Mts), -Fmoc-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc- Ser(Bzl), Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc- Lys(Cl-Z), Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc- O His(Trt), Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may C be purchased from Applied Biosystems, Inc. or Bachem Inc.

(Torrance, CA). Anisole, dimethylsulfide, phenol, S ethanedithiol, and thioanisole may be obtained from Aldrich O Chemical Company (Milwaukee, WI). Air Products and Chemicals ID (Allentown, PA) supplies HF. Ethyl ether, acetic acid and methanol may be purchased from Fisher Scientific (Pittsburgh,

PA).

Solid phase peptide synthesis may be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, CA) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (see, Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B July 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, CA) with capping. Boc-peptide-resins may be cleaved with HF to 0 C, 1 hour). The peptide may be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized. The Fmoc-peptide resins may be cleaved according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pp. 6- 12). Peptides may be also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Kentucky).

Peptides may be purified by RP-HPLC (preparative and 1 analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10 i, 2.2 x 25 cm; Vydac, Hesperia, CA) may be used to isolate peptides, and purity may be determined using a C4, C8 or C18 analytical column (5 p, 0.46 x 25 cm; Vydac). Solvents TFA/water and B=0.1%

TFA/CH

3 CN) may be delivered to the analytical column at a flowrate of 1.0 ml/min and to the preparative column at ml/min. Amino acid analyses may be performed on the Waters

NO

8 26

(N

Pico Tag system and processed using the Maxima program.

o Peptides may be hydrolyzed by vapor-phase acid hydrolysis S(115 0 C, 20-24 Hydrolysates may be derivatized and analyzed by standard methods (Cohen, et al., The Pico Tac Method: A Manual of Advanced Techniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation, Milford, MA (1989)). Fast atom bombardment analysis may be carried out g by M-Scan, Incorporated (West Chester, PA). Mass calibration 0 may be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection may be carried out on an Applied Biosystems Bio-Ion 20 mass spectrometer. Electrospray mass spectroscopy may be carried and on a VG-Trio machine.

Peptide compounds useful in the invention may also be prepared using recombinant DNA techniques, using methods now known in the art. See, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor (1989). Non-peptide compounds useful in the present invention may be prepared by art-known methods.

Formulation and Administration Compounds of the invention are useful in view of their exendin-like effects, and may conveniently be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous) or nasal, sublingual, buccal or oral administration. In some cases, it will be convenient to provide an exendin agonist and another anti-gastric-emptying agent, such as glucagon, an amylin, or an amylin agonist, in a single composition or solution for administration together. In other cases, it may be more advantageous to administer another anti-emptying agent separately from said exendin agonist. In yet other cases, it may be beneficial to provide an exendin agonist either co- I N D 2 7 (N formulated or separately with other glucose lowering agents .O such as insulin. A suitable administration format may best be 0 determined by a medical practitioner for each patient individually. Suitable pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, Remington's Pharmaceutical Sciences by E.W.

C Martin. See also Wang, Y.J. and Hanson, M.A. "Parenteral (N Formulations of Proteins and Peptides: Stability and IND Stabilizers," Journal of Parenteral Science and Technology, LO Technical Report No. 10, Supp. 42:2S (1988).

Compounds useful in the invention can be provided as parenteral compositions for injection or infusion. They can, for example, be suspended in an inert oil, suitably a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier. Preferably, they are suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 5.6 to 7.4. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include for example, sodium acetate/acetic acid buffers. A form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or other form of delivery.

The desired isotonicity may be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

IO

28

(N

The claimed compounds can also be formulated as O pharmaceutically acceptable salts acid addition salts) h and/or complexes thereof. Pharmaceutically acceptable salts are non-toxic salts at the concentration at which they are administered. The preparation of such salts can facilitate O the pharmacological use by altering the physical-chemical V/ characteristics of the composition without preventing the C composition from exerting its physiological effect. Examples 0of useful alterations in physical properties include lowering C O the melting point to facilitate transmucosal administration and increasing the solubility to facilitate the administration of higher concentrations of the drug.

Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, S phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.

Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, 2o sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared by, for example, reacting the free acid or base 2S forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the ions of an existing salt for another ion on a suitable o ion exchange resin.

Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate,

ID

29 various sugars such as lactose, glucose, or sucrose, or types 0 of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.

The compositions or pharmaceutical composition can be administered by different routes including intravenously, Sintraperitoneal, subcutaneous, and intramuscular, orally, C/ topically, transdermally, buccally or transmucosally.

N fIf desired, solutions of the above compositions may be 0 thickened with a thickening agent such as methyl cellulose.

c N They may be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, a Triton).

Compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.

For use by the physician, the compounds will be provided in dosage unit form containing an amount of an exendin agonist, with or without another anti-emptying agent.

Therapeutically effective amounts of an exendin agonist for use in the control of gastric emptying and in conditions in which gastric emptying is beneficially slowed or regulated are those that decrease post-prandial blood glucose levels, preferably to no more than about 8 or 9 mM or such that blood glucose levels are reduced as desired. In diabetic or glucose intolerant individuals, plasma glucose levels are

\O

IND

c, o higher than in normal individuals. In such individuals, 0 beneficial reduction or "smoothing" of post-prandial blood 0 glucose levels, may be obtained. As will be recognized by those in the field, an effective amount of therapeutic agent will vary with many factors including the patient's physical M condition, the blood sugar level or level of inhibition of gastric emptying to be obtained, and other factors.

NO Such pharmaceutical compositions are useful in causing gastric hypomotility in a subject and may be used as well in io other disorders where gastric motility is beneficially reduced.

The effective daily anti-emptying dose of the compounds will typically be in the range of 0.001 or 0.005 to about mg/day, preferably about 0.01 or 0.05 to 2 mg/day and more 1 preferably about 0.05 or 0.1 to 1 mg/day, for a 70 kg patient. The exact dose to be administered is determined by the attending clinician and is dependent upon where the particular compound lies within the above quoted range, as well as upon the age, weight and condition of the individual.

Administration should begin at the first sign of symptoms or shortly after diagnosis of diabetes mellitus. Administration may be by injection, preferably subcutaneous or intramuscular, or by other routes for example, by oral, nasal, sublingual or buccal administration. For oral, nasal 2 or buccal dosages should be increased to about 5-10 fold over injection dosages.

Generally, in treating or preventing elevated, inappropriate, or undesired post-prandial blood glucose levels, the compounds of this invention may be administered to patients in need of such treatment in a dosage ranges similar to those given above, however, the compounds are administered more frequently, for example, one, two, or three times a day.

ID

31 The optimal formulation and mode of administration of 0 compounds of the present application to a patient depend on factors known in the art such as the particular disease or disorder, the desired effect, and the type of patient. While Sthe compounds will typically be used to treat human patients, O they may also be used to treat similar or identical diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sports animals and 0 pets such as horses, dogs and cats.

C ,O To assist in understanding the present invention the following Examples are included which describe the results of a series of experiments. The experiments relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, r1 now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.

EXAMPLE 1 Preparation of Compound 1 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly-

NH

2 [SEQ. ID. NO. The above amidated peptide was assembled on 2 5 dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.). In general, single-coupling cycles were used throughout the synthesis and Fast Moc (HBTU activation) chemistry was

ID

0 32 o employed. Deprotection (Fmoc group removal)of the growing o peptide chain was achieved using piperidine. Final deprotection of the completed peptide resin was achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol f- 5 (0.2 mL), anisole (0.2 mL), water (0.2 mL) and Cc trifluoroacetic acid (15 mL) according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, N0 Inc.) The peptide was precipitated in ether/water (50 mL) Sand centrifuged. The precipitate was reconstituted in C 1 I glacial acetic acid and lyophilized. The lyophilized peptide was dissolved in water). Crude purity was about Used in purification steps and analysis were Solvent A TFA in water) and Solvent B TFA in ACN).

The solution containing peptide was applied to a preparative C-18 column and purified (10% to 40% Solvent B in Solvent

A

over 40 minutes). Purity of fractions was determined isocratically using a C-18 analytical column. Pure fractions were pooled furnishing the above-identified peptide.

Analytical RP-HPLC (gradient 30% to 50% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 18.9 minutes.

Electrospray Mass Spectrometry calculated 3408.0; found 3408.9.

EXAMPLE 2 130 Preparation of Compound 2 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH, [SEQ.

ID. NO. 6] 33 The above amidated peptide was assembled on O dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

g (gradient 30% to 40% Solvent B in Solvent A over 30 minutes) Sof the lyophilized peptide gave product peptide having an observed retention time of 17.9 minutes. Electrospray Mass Spectrometry calculated 3294.7; found 3294.8.

EXAMPLE 3 Preparation of Compound 3 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu

I

s Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH,

[SEQ.

ID. NO. 7] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoco protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 29% to 36% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 20.7 minutes. Electrospray Mass Spectrometry calculated 3237.6; found 3240.

ID

0 34 cN o EXAMPLE 4 N S -ID. NO. 8] C1 The above amidated peptide was assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved o from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 15.2 minutes. Electrospray Mass Spectrometry calculated 3251.6; found 3251.5.

EXAMPLE Preparation of Compound His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH, [SEQ. ID. NO. 9] The above amidated peptide was assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved

IO

from the resin, deprotected and purified in a similar way to o Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 13.1 minutes. Electrospray Mass m Spectrometry calculated 3207.6; found 3208.3.

SEXAMPLE 6 Preparation of Compound 6 His Gly Glu Gly Thr Ala Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH, [SEQ.

ID. NO. The above amidated peptide was assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

2 (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 12.8 minutes. Electrospray Mass Spectrometry calculated 3161.5; found 3163.

ID

36 o EXAMPLE 7 Preparation of Compound 7 His Gly Glu Gly Thr Phe Thr Ala Asp Leu Ser Lys Gin Leu Glu SGlu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH 2

[SEQ.

n ID. NO. 11] The above-identified amidated peptide was assembled on C<1 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 15.2 minutes. Electrospray Mass Spectrometry calculated 3221.6; found 3222.7.

EXAMPLE 8 Preparation of Compound 8 His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gin Leu Glu 2o Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH,

[SEQ.

ID. NO. 12] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in O water) and Solvent B TFA in ACN). Analytical

RP-HPLC

S g(gradient 34% to 44% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.3 minutes. Electrospray Mass C Spectrometry calculated 3195.5; found 3199.4.

1c EXAMPLE 9 C Preparation of Compound 9 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Leu Glu ia Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH,

[SEQ.

ID. NO. 13] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 38% to 48% Solvent B in Solvent A over 30 minutes) 0 of the lyophilized peptide gave product peptide having an observed retention time of 15.7 minutes. Electrospray Mass Spectrometry calculated 3221.6; found 3221.6.

EXAMPLE Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH 2

[SEQ.

ID

0 38 O ID. NO. 14] 0 The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- S protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to \0 Compound 1. Used in analysis were Solvent A TFA in Swater) and Solvent B TFA in ACN) Analytical RP-HPLC (c s (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) 1 o of the lyophilized peptide gave product peptide having an observed retention time of 18.1 minutes. Electrospray Mass Spectrometry calculated 3180.5; found 3180.9.

EXAMPLE 11 Preparation of Compound 11

L

S His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH, [SEQ.

ID. NO. The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide 2 L o norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 17.0 minutes. Electrospray Mass Spectrometry calculated 3180.6; found 3182.8.

IO

8 39

C)

0 EXAMPLE 12 Preparation of Compound 12 C His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Ala Glu V Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH, [SEQ.

Ng 5 ID. NO. 16] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved ,o from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 32% to 42% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.9 minutes. Electrospray Mass Spectrometry calculated 3195.5; found 3195.9.

EXAMPLE 13 Preparation of Compound 13 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Ala Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH,

[SEQ.

ID. NO. 17] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to o Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) s of the lyophilized peptide gave product peptide having an observed retention time of 17.9 minutes. Electrospray Mass f Spectrometry calculated 3179.6; found 3179.0.

IN

SEXAMPLE 14 Preparation of Compound 14 to His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Ala Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH 2

[SEQ.

ID. NO. 18] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.3 minutes. Electrospray Mass Spectrometry calculated 3179.6; found 3180.0.

41 SEXAMPLE

O

Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu O Glu Ala Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH 2

[SEQ.

n c S ID. NO. 19]

IO

SThe above-identified peptide was assembled on C- dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved ao from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 13.7 minutes. Electrospray Mass Spectrometry calculated 3179.6; found 3179.0.

EXAMPLE 16 Preparation of Compound 16 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu zo Glu Glu Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH 2

[SEQ.

ID. NO. The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected, amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to 42 Compound 1. Used in analysis were Solvent A TFA in o water) and Solvent B TFA in ACN). Analytical RP-HPLC g (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.0 minutes. Electrospray Mass Spectrometry calculated 3209.6; found 3212.8.

C EXAMPLE 17 0

C

Preparation of Compound 17 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Ala Leu Phe Ile Glu Phe Leu Lys Asn-NH [SEQ.

ID. NO. 21] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.3 minutes. Electrospray Mass Spectrometry calculated 3152.5; found 3153.5.

EXAMPLE 18 Preparation of Compound 18 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Ala Phe Ile Glu Phe Leu Lys Asn-NH 2

[SEQ.

ID

43 ID. NO. 22] 0 The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) ,o of the lyophilized peptide gave product peptide having an observed retention time of 12.1 minutes. Electrospray Mass Spectrometry calculated 3195.5; found 3197.7.

EXAMPLE 19 Preparation of Compound 19 s His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Ala Phe Leu Lys Asn-NH 2

[SEQ.

ID. NO. 23] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide '0 norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 10.9 minutes. Electrospray Mass Spectrometry calculated 3179.6; found 3180.5.

ID

44 c-I 0 EXAMPLE Preparation of Compound pp His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu 3 Glu Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn-NH, [SEQ.

c- 5 ID. NO. 24] CN| The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved t' from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 17.5 minutes. Electrospray Mass Spectrometry calculated 3161.5; found 3163.0.

EXAMPLE 21 Preparation of Compound 21 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu 2° Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Ala Lys Asn-NH 2

[SEQ.

ID. NO. The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to o Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 32% to 42% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 19.5 minutes. Electrospray Mass ID Spectrometry calculated 3195.5; found 3199.

SEXAMPLE 22 Preparation of Compound 22 tO His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Ala Asn-NH 2

[SEQ.

ID. NO. 26] The above-identified amidated peptide was assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide

IS

norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

o (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.5 minutes. Electrospray Mass Spectrometry calculated 3180.5; found 3183.7.

ID

46 o EXAMPLE 23 0 Preparation of Compound 23 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu 9 Glu Glu Ala Val Arg Leu Phe lie Glu Phe Leu Lys Ala-NH, [SEQ.

SID. NO. 27]

IN

0 The above-identified amidated peptide was assembled on CA 4 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis were Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 34% to 44% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 22.8 minutes. Electrospray Mass Spectrometry calculated 3194.6; found 3197.6.

EXAMPLE 24 Preparation of Compound 24 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu 0 Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NH, [SEQ. ID. NO. 28] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to

\O

47 Compound 1. Used in analysis are Solvent A TFA in

O

O water) and Solvent B TFA in ACN). Analytical RP-HPLC h (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine p the retention time of the product peptide. Electrospray Mass SSpectrometry calculated 4099.6.

C-I EXAMPLE eC\ Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NH, [SEQ. ID. NO. 29] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 2o of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 4042.5.

EXAMPLE 26 Preparation of Compound 26 'I His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly

ID

48 Pro Ser Ser Gly Ala Pro Pro-NH, [SEQ. ID. NO. 0 The above-identified peptide is assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Pmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to N Compound 1. Used in analysis are Solvent A TFA in 0 water) and Solvent B TFA in ACN). Analytical RP-HPLC C (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) o of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 4002.4 EXAMPLE 27 Preparation of Compound 27 S His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro-NH, [SEQ. ID. NO. 31] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3945.4.

IO

49 0 EXAMPLE 28 Preparation of Compound 28 O His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met r Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly

S

5 Gly Pro Ser Ser Gly Ala Pro-NH 2 [SEQ. ID. NO. 32] C The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved 0 from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3905.3.

EXAMPLE 29 Preparation of Compound 29 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu 2 Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro-NH, [SEQ. ID. NO. 33] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved

ID

0%

CI

from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3848.2.

EXAMPLE Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala-NH 2 [SEQ. ID. NO. 34) The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3808.2.

51 EXAMPLE 31 Ch Preparation of Compound 31 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe lie Glu Phe Leu Lys Asn Gly Gly V 5 Pro Ser Ser Gly Ala-NH 2 [SEQ. ID. NO. D The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved I0 from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3751.1.

EXAMPLE 32 Preparation of Compound 32 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu o0 Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly-NHA [SEQ. ID. NO. 36] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to 52 Compound 1. Used in analysis are Solvent A TFA in O water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine S the retention time of the product peptide. Electrospray Mass O Spectrometry calculated 3737.1.

EXAMPLE 33 CA Preparation of Compound 33 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu i Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly-NH 2 [SEQ. ID. NO. 37] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- 5 protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 2o of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3680.1.

EXAMPLE 34 Preparation of Compound 34 26 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly 53 Pro Ser Ser-NH, [SEQ. ID. NO. 38) C The above-identified amidated peptide is assembled on 4- 2 4 '-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- S protected amino acids (Applied Biosystems, Inc.), cleaved m from the resin, deprotected and purified in a similar way to q Compound 1. Used in analysis are Solvent A TFA in Swater) and Solvent B TFA in ACN). Analytical RP-HPLC S(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 6 of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3680.1 EXAMPLE Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser-NH, [SEQ. ID. NO. 39] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3623.0.

ID

54 0 EXAMPLE 36 Preparation of Compound 36 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu V, Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser-NH, [SEQ. ID. NO. 0C The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3593.0 EXAMPLE 37 Preparation of Compound 37 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu o2 Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser-NH 2 [SEQ. ID. NO. 41] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- 2 s protected amino acids (Applied Biosystems, Inc.), cleaved

ID

from the resin, deprotected and purified in a similar way to o Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass l Spectrometry calculated 3535.9

IO

SEXAMPLE 38 Preparation of Compound 38 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro-NH, [SEQ. ID. NO. 42] The above-identified peptide is assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3505.9.

ID

56 o EXAMPLE 39 0 Preparation of Compound 39 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly '3 5 Pro-NH, [SEQ. ID. NO. 43]

(O

g The above-identified amidated peptide is assembled on 4- C1 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved

O

1 from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3448.8.

EXAMPLE Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu 0 o Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly- NH, [SEQ. ID. NO. 44] The above-identified peptide is assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to

ID

57 Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

0 (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass SSpectrometry calculated 3351.7.

C< EXAMPLE 41

\O

Preparation of Compound 41 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu o0 Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly-NH, [SEQ. ID. NO. The above-identified peptide is assembled on dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 0 of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3351.8.

EXAMPLE 42 Preparation of Compound 42 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly-NH, NO 58 C [SEQ. ID. NO. 46] 0 The above-identified amidated peptide is assembled on 4- 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoct 5 protected amino acids (Applied Biosystems, Inc.), cleaved Sfrom the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

S(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) o of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3294.7.

EXAMPLE 43 Preparation of Compound 43 £His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly tPro Ser Ser Gly Ala tPro tPro tPro-NH, [SEQ. ID. NO. 47] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide 2o norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 37,36 and 31. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass 59 Spectrometry calculated 4197.1.

O

C EXAMPLE 44 Preparation of Compound 44 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly SPro Ser Ser Gly Ala tPro tPro tPro-NH, [SEQ. ID. NO. 48] The above-identified amidated peptide is assembled on 4- 2 4 '-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 37, 36 and 31. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 4179.1.

EXAMPLE 20 Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly NMeala Ser Ser Gly Ala Pro Pro-NH, [SEQ. ID. NO. 49] The above-identified amidated peptide is assembled on 4- 2 5 (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide

NO

0 norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- O protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 36 and S 31. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the J lyophilized peptide is then carried out to determine the IN retention time of the product peptide. Electrospray Mass o Spectrometry calculated 3948.3.

EXAMPLE 46 Preparation of Compound 46 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly NMeala Ser Ser Gly Ala NMeala Nmeala-NH, [SEQ. ID. NO. The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3840.1.

\D

EXAMPLE 47 0 Preparation of Compound 47 0\ 0 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly S hPro Ser Ser Gly Ala hPro hPro-NH 2 [SEQ. ID. NO. 51] 0 The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide Snorleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved o from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 4050.1.

EXAMPLE 48 Preparation of Compound 48 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly hPro Ser Ser Gly Ala hPro-NH, [SEQ. ID. NO. 52] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide -L norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved

IND

0 from the resin, deprotected and purified in a similar way to Compound 1. A double coupling is required at residue 31. Used 0 in analysis are Solvent A TFA in water) and Solvent B Ch TFA in ACN). Analytical RP-HPLC (gradient 30% to S Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time 0 of the product peptide. Electrospray Mass Spectrometry n3 calculated 3937.1 0 EXAMPLE 49 Preparation of Compound 49 Arg Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala-NH, [SEQ. ID. NO. 53] The above-identified amidated peptide is assembled on 4- 2 4 '-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3827.2.

0 EXAMPLE

C)

o Preparation of Compound His Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly- 0 5 NH, [SEQ. ID. NO. 54] ci The above-identified amidated peptide is assembled on 4- 2 4 '-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide Snorleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved 0 from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine S the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3394.8.

EXAMPLE 51 Preparation of Compound 51 His Gly Glu Gly Thr Naphthylala Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn- NH, [SEQ. ID. NO. The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- 2 protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to 64 Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine 0 S the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3289.5.

qq EXAMPLE 52 Preparation of Compound 52 His Gly Glu Gly Thr Phe Ser Ser Asp Leu Ser Lys Gin Met Glu *o Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH, [SEQ.

ID. NO. 56] The above-identified amidated peptide is assembled on 4- 2 4 '-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) LO of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3280.7.

EXAMPLE 53 Preparation of Compound 53 His Gly Glu Gly Thr Phe Ser Thr Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH 2

[SEQ.

ID

0 ID. NO. 57] o The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- S protected amino acids (Applied Biosystems, Inc.), cleaved 0 from the resin, deprotected and purified in a similar way to V] Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 1 'O of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3294.7.

EXAMPLE 54 Preparation of Compound 54 His Gly Glu Gly Thr Phe Thr Ser Glu Leu Ser Lys Gin Met Ala Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH 2

[SEQ.

ID. NO. 58] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3250.7.

ID

EXAMPLE 0 Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp pentylgly Ser Lys Gin Leu 0 Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH 2 I 5 [SEQ. ID. NO. 59] I The above-identified amidated peptide is assembled on 4- 0 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine 1 5 the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3253.5.

EXAMPLE 56 Preparation of Compound 56 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Naphthylala Ile Glu Phe Leu Lys Asn- NH, [SEQ. ID. NO. The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved

D

0 from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in O water) and Solvent B TFA in ACN). Analytical RP-HPLC C (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass SSpectrometry calculated 3289.5.

EXAMPLE 57 eC Preparation of Compound 57 0 o His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe tButylgly Glu Trp Leu Lys Asn-NH, [SEQ. ID. NO. 61] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide 6 norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3183.4.

00 EXAMPLE 58 0 Preparation of Compound 58 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Asp Phe Leu Lys Asn-NH, [SEQ.

0S ID. NO. 62] (Nc C The above-identified amidated peptide is assembled on 4-

\O

I 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide C4 norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved o from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine 1 5 the retention time of the product peptide. Electrospray Mass Spectrometry calculated '3237.6.

EXAMPLE 59 Preparation of Compound 59 His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gin Leu Glu 2 0 Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser-NH 2 [SEQ. ID. NO. 63] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc- 1 S protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to

ID

0 Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC O (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3637.9.

I3n EXAMPLE Preparation of Compound His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gin Met Glu io Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly-NH, [SEQ. ID. NO. 64] The above-identified amidated peptide is assembled on 4- (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 2 0 of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3309.7.

EXAMPLE 61 Preparation of Compound 61 His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly ShPro Ser Ser Gly Ala hPro hPro-NH, [SEQ. ID. NO. 0 O The above-identified amidated peptide is assembled on 4- C (2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmocprotected amino acids (Applied Biosystems, Inc.), cleaved 0 from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 36 and C 31. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 0o to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3711.1.

EXAMPLE 62 Preparation of Peptide having SEQ. ID. NO. 67 Compound 62, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys-NH'octanoyl Asn-NH, [SEQ. ID. NO. 67], is assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Fmoc-Lys-NHcoctanoyl acid is used for coupling at position 27. Instead of using a 5 protected amino acid for the final coupling at position 1, 4imidazolylpropionic acid is coupled directly to the Nterminus of residues 2-28 on the resin. Used in analysis are Solvent A. TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in

IO

0 Solvent A over 30 minutes) of the lyophilized peptide is then c_ carried out to determine the retention time of the product o peptide. Electrospray Mass Spectrometry calculated C 3405.0

S

EXAMPLE 63 l~ Preparation of Peptide having SEO. ID. NO. 68 Co Compound 63, 4 -imidazolylpropionyl-Gly Glu Gly Thr Phe SThr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys-NH'octanoyl Asn-NH, [SEQ. ID. NO. 68], 0 is assembled on 4 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Fmoc-Lys-NHcoctanoyl acid is used for coupling at position 27. Instead of using a protected amino acid for the final coupling at position 1, 4imidazolylpropionic acid is coupled directly to the Nterminus of residues 2-28 on the resin. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3347.9 EXAMPLE 64 Preparation of Pentide having SE. ID. NO. 69 Compound 64, 4 -imidazolylpropionyl-Gly Glu Gly Thr Phe

ID

0 Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys-NH'octanoyl Asn Gly Gly-NH, [SEQ. ID.

O NO. 69], is assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1.

Fmoc-Lys-NHcoctanoyl acid is used for coupling at position 27.

O Instead of using a protected amino acid for the final lo coupling at position 1, 4-imidazolylpropionic acid is coupled C directly to the N-terminus of residues 2-30 on the resin.

Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3519.0 EXAMPLE Preparation of Peptide having SEQ. ID. NO. Compound 65, 4 -imidazolylpropionyl-Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys-NHoctanoyl Asn Gly Gly-NH, [SEQ. ID.

NO. 70], is assembled on 4 2 '-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin 2 (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1.

Fmoc-Lys-NH'octanoyl acid is used for coupling at position 27.

Instead of using a protected amino acid for the final

IO

0 coupling at position 1, 4-imidazolylpropionic acid is coupled c_ directly to the N-terminus of residues 2-30 on the resin.

o Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient S to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass l^ Spectrometry calculated 3451.9 SEXAMPLE 66 Preparation of Peptide having SEO. ID. NO. 71 Compound 66, 4 -imidazolylpropionyl-Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Lys-NHoctanoyl-NH, [SEQ. ID. NO. 71], is assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Fmoc-Lys-NH'octanoyl acid is used for the initial coupling onto the resin at position 28. Instead of using a protected amino acid for the final coupling at position 1, 4 -imidazolylpropionic acid is coupled directly to the N-terminus of protected residues 2-28 on the resin. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical

RP-HPLC

2 (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3405.0

ID

EXAMPLE 67 0 Preparation of Peptide having SEQ. ID. NO. 72 Compound 67, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu 3 Phe Ile Glu Phe Leu Asn Lys-NH'octanoyl-NH 2 [SEQ. ID. NO. 72], is assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl C phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 0 mmole/g) using Fmoc-protected amino acids (Applied C-i Biosystems, Inc.), cleaved from the resin, deprotected and to purified in a similar way to Example 1. Fmoc-Lys-NHcoctanoyl acid is used for the initial coupling onto the resin at position 28. Instead of using a protected amino acid for the final coupling at position 1, 4-imidazolylpropionic acid is coupled directly to the N-terminus of residues 2-28 on the LS resin. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3347.9 EXAMPLE 68 Preparation of Peptide having SEQ. ID. NO. 73 Compound 68, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu I2 Phe Ile Glu Trp Leu Asn Lys-NHcoctanoyl Gly Gly-NH [SEQ. ID.

NO. 73], is assembled on 4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin

ID

0 (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, o deprotected and purified in a similar way to Example 1.

0 Fmoc-Lys-NHcoctanoyl acid is used for coupling at position 28.

Instead of using a protected amino acid for the final coupling at position 1, 4-imidazolylpropionic acid is coupled 0 directly to the N-terminus of protected residues 2-30 on the S resin. Used in analysis are Solvent A TFA in water) and C Solvent B TFA in ACN). Analytical RP-HPLC (gradient 0 o to 60% Solvent B in Solvent A over 30 minutes) of the C- lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry calculated 3519.0 EXAMPLE 69 Preparation of Peptide having SEQ. ID. NO. 74 Compound 69, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Lys-NHCoctanoyl Gly Gly-NH, [SEQ. ID.

NO. 74], is assembled on 4 -(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1.

Fmoc-Lys-NH'octanoyl acid is used for coupling at position 28.

v s Instead of using a protected amino acid for the final coupling at position 1, 4-imidazolylpropionic acid is coupled directly to the N-terminus of residues 2-30 on the resin.

Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient

D

O to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the 0 retention time of the product peptide. Electrospray Mass SSpectrometry calculated 3451.9 SEXAMPLE C 5 Preparation of C-terminal carboxylic acid peptides

\O

Scorresponding to the above C-terminal amide sequences for C, Compounds 1-3, 30-37, 40-42, 49-58 and 62-69 Compounds 1-23, 30-37, 40-42, 49-58 and 62-69 [SEQ. ID.

NOS. 5-27, 34-41, 44-46, 53-62 and 67-74] are assembled on o the so called Wang resin (p-alkoxybenzylalacohol resin (Bachem, 0.54 mmole/g)) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B ls TFA in ACN). Analytical RP-HPLC (gradient 30% to Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry provides an experimentally determined EXAMPLE 71 Preparation of C-terminal carboxylic acid peptides corresponding to the above C-terminal amide sequences for Compounds 24-29, 38, 39 and 43-48 Compounds 24-29, 38, 39, and 43-48 [SEQ. ID. NOS. 28-33, 1-6 42, 43 and 47-52] are assembled on the 2-chlorotritylchloride resin (200-400 mesh), 2% DVB (Novabiochem, 0.4-1.0 mmole/g)) c- using Fmoc-protected amino acids (Applied Biosystems, Inc.), O cleaved from the resin, deprotected and purified in a similar 0 way to Compound 1. Used in analysis are Solvent A TFA in water) and Solvent B TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) 0 of the lyophilized peptide is then carried out to determine 3 the retention time of the product peptide. Electrospray Mass C Spectrometry provides an experimentally determined \O ID q EXAMPLES A TO E Reagents Used GLP-1(7-36)[NH 2 was purchased from Bachem (Torrance, CA). All other peptides were prepared using synthesis methods such as those described therein. All chemicals were IS of the highest commercial grade. The cAMP SPA immunoassay was purchased from Amersham. The radioligands were purchased from New England Nuclear (Boston, MA). RINm5f cells (American Type Tissue Collection, Rockville, MD) were grown in DME/F12 medium containing 10% fetal bovine serum and 2mM L-glutamine. Cells were grown at 37 0 C and 5% C0 2 humidified air and medium was replaced every 2 to 3 days.

Cells were grown to confluence then harvested and homogenized using on a Polytron homogenizer. Cell homogenates were stored frozen at -70 0 C until used.

2 EXAMPLE A GLP-1 Receptor Binding Studies Receptor binding was assessed by measuring displacement of 2 I]GLP-1 or 125 I]exendin(9-39) from RINm5f membranes.

Assay buffer contained 5 pg/ml bestatin, 1 ug/ml S0 phosphoramidon, 1 mg/ml bovine serum albumin (fraction 1 0/0 mg/ml bacitracin, and 1 mM MgC1, in 20 mM HEPES, pH 7.4. To measure binding, 30 pg membrane protein (Bradford protein o assay) was resuspended in 200 pl assay buffer and incubated with 60 pM 125 I]GLP-1 or [12 5 I]exendin(9-39) and unlabeled peptides for 120 minutes at 23 0 C in 96 well plates (Nagle Nunc, Rochester, NY). Incubations were terminated by rapid filtration with cold phosphatebuffered saline, pH 7.4, through polyethyleneimine-treated GF/B glass fiber filters ri (Wallac Inc., Gaithersburg, MD) using a Tomtec Mach II plate SL0 harvester (Wallac Inc., Gaithersburg, MD). Filters were C dried, combined with scintillant, and radioactivity determined in a Betaplate liquid scintillant counter (Wallac Inc.).

Peptide samples were run in the assay as duplicate points at 6 dilutions over a concentration range of 10- 6 M to 2 M to generate response curves. The biological activity of a sample is expressed as an IC 50 value, calculated from the raw data using an iterative curve-fitting program using a 4parameter logistic equation (PrizmTM, GraphPAD Software). The results are shown in Table I.

IO

TABLE I O Compound ICL (nM) Exendin-4 (SEQ. ID. NO. 2] 0.70 Compound 1 [SEQ. ID. NO. 5] 0.67 Compound 2 [SEQ. ID. NO. 6] 1.21 V Compound 3 [SEQ. ID. NO. 7] 0.67 C. Compound 4 [SEQ. ID. NO. 8] 0.42 Compound 5 [SEQ. ID. NO. 9] 1.91 Compound 6 [SEQ. ID. NO. 10] 59.05 Compound 7 [SEQ. ID. NO. 11] 5.44 Compound 8 [SEQ. ID. NO. 12] 1.75 Compound 9 [SEQ. ID. NO. 13] 0.88 Compound 10 [SEQ. ID. NO. 14] 1.96 Compound 11 [SEQ. ID. NO. 15] 0.69 Compound 12 [SEQ. ID. NO. 16] 2.94 Compound 13 [SEQ. ID. NO. 17] 7.82 Compound 14 [SEQ. ID. NO. 18) 0.04 Compound 15 [SEQ. ID. NO. 19] 0.48 Compound 16 [SEQ. ID. NO. 20] 1.10 Compound 17 [SEQ. ID. NO. 21] 21.6 Compound 18 [SEQ. ID. NO. 22] 0.63 Compound 19 [SEQ. ID. NO. 23] 0.63 Compound 20 [SEQ. ID. NO. 24] 0.94 Compound 21 [SEQ. ID. NO. 25] 9.91 Compound 22 [SEQ. ID. NO. 26] 8.24 Compound 23 [SEQ. ID. NO. 27] 0.82

\D

C)

0 0,

(N

EXAMPLE B Cyclase Activation Study Assay buffer contained 10 pM GTP, 0.75 mM ATP, 2.5 mM MgCl 2 0.5mM phosphocreatine, 12.5 U/ml creatine kinase, 0.4 mg/ml aprotinin, 1 pM IBMX in 50 mM HEPES, pH 7.4. Membranes and peptides were combined in 100 ml of assay buffer in 96 well filter-bottom plates (Millipore Corp., Bedford, MA).

After 20 minutes incubation at 37 0 C, the assay was terminated by transfer of supernatant by filtration into a fresh 96 well plate using a Millipore vacuum manifold. Supernatant cAMP contents were quantitated by SPA immunoassay.

Peptide samples were run in the assay as triplicate points at 7 dilutions over a concentration range of 10- 6 M to 2 M to generate response curves. The biological activity of a particular sample was expressed as an EC,, value, calculated as described above. Results are tabulated in Table II.

TABLE II Compound Exendin-4 Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 Compound 14 Compound 15 Compound 16 Compound 17 Compound 18 Compound 19 Compound 20 Compound 21 Compound 22 Compound 23 [SEQ. ID. NO. 2] [SEQ. ID. NO. 5] [SEQ. ID. NO. 6] [SEQ. ID. NO. 7] [SEQ. ID. NO. 8] [SEQ. ID. NO. 9] [SEQ. ID. NO. 10] [SEQ. ID. NO. 11] [SEQ. ID. NO. 12] [SEQ. ID. NO. 13] EC, (nM) 0.23 0.3 0.79 2.35 0.22 9.85 79.4 63.6 6.8 1.68

[SEQ.

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ID.

ID.

ID.

ID.

ID.

ID.

NO.

NO.

NO.

NO.

NO.

NO.

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NO.

NO.

NO.

NO.

NO.

NO.

NO.

14] 15] 16] 17] 18] 19] 20] 21] 22] 23] 24] 25] 26] 27] 5.37 0.48 15.55 79.6 1.11 1.05 5.12 43.6 0.76 3.68 5.25 45.1 20.43 3.05 OoL EXAMPLE C Determination of Blood Glucose Levels in db/db Mice o C57BLKS/J-m-db mice at least 3 months of age were C utilized for the study. The mice were obtained from The Jackson Laboratory and allowed to acclimate for at least one week before use. Mice were housed in groups of ten at 220° 1°C with a 12:12 light:dark cycle, with lights on at 6 a.m.

V' All animals were deprived of food for 2 hours before taking N baseline blood samples. Approximately 70 pl of blood was drawn from each mouse via eye puncture, after a light (Cl anesthesia with metophane. After collecting baseline blood samples, to measure plasma glucose concentrations, all animals receive subcutaneous injections of either vehicle (10.9% NaC1), exendin-4 or test compound (1 pg) vehicle.

Blood samples were drawn again, using the same procedure, after exactly one hour from the injections, and plasma glucose concentrations were measured.

For each animal, the change in plasma value, from baseline value, was calculated. The percent decrease in i0 plama glucose after one hour is shown in Table III.

TABLE III Test Compound Exendin-4 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 Compound 14 Compound 15 Compound 16 Compound 17 Compound 18 Compound 19 Compound 20 Compound 21 Compound 22 Compound 23 [SEQ. ID. NO. 2] [SEQ. ID. NO. 6] [SEQ. ID. NO. 7] [SEQ. ID. NO. 8] [SEQ. ID. NO. 9] [SEQ. ID. NO. 10] [SEQ. ID. NO. 111 drop in glucose 39% 38% 49% 27% 47% 40% 31%

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12] 13] 14] 15] 16] 17] 18] 19] 20] 21] 22] 23] 24] 25] 26] 27] 44% 41% 46% 40% 53% 45% 54% 45% 54% 45% 50% 48% 37% 30% 46% 42% (n =78) (n =4) (n =4) (n =4) (n =4) (n (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) (n =4) EXAMPLE D Dose Response Determination of Blood Glucose Levels in 0 db/db Mice C C57BLKS/J-m-db/db mice, at least 3 months of age were utilized for the study. The mice were obtained from The Jackson Laboratory and allowed to acclimate for at least one week before use. Mice were housed in groups of ten at 22 0

C

1 0 C with a 12:12 light:dark cycle, with lights on at 6 a.m.

All animals were deprived of food for 2 hours before go taking baseline blood samples. Approximately 70 pl of blood was drawn from each mouse via eye puncture, after a light anesthesia with metophane. After collecting baseline blood samples, to measure plasma glucose concentrations, all animals receive subcutaneous injections of either vehicle, exendin-4 or test compound in concentrations indicated.

Blood samples were drawn again, using the same procedure, after exactly one hour from the injections, and plasma glucose concentrations were measured.

For each animal, the change in plasma value, from 2 baseline value, was calculated and a dose dependent relationship was evaluated using Graphpad Prizm TM software.

Figure 5 depicts the effects of varying doses of exendin-4 [SEQ. ID. NO. 2] and Compound 3 [SEQ. ID. NO. 7] on plasma glucose levels. Exendin-4 had an ED, 0 of 0.01 pg per mouse and Compound 3 had an ED, 0 of 0.04 pg per mouse.

EXAMPLE E Gastric Emptying The following study was carried out to examine the effects of exendin-4 and exendin agonist compounds of the ,0 present invention on gastric emptying in rats. This experiment followed a modification of the method of

NO

SScarpignato, et al., Arch. Int. Pharmacodyn. Ther.

246:286-94, 1980.

o Male Harlan Sprague Dawley (HSD) rats were used. All animals were housed at 22.7 0.8 C in a 12:12 hour light:dark cycle (experiments being performed during the light cycle) and were fed and watered ad libitum (Diet LM-485, Teklad, Madison, WI). Exendin-4 was synthesized V/ according to standard peptide synthesis methods. The preparation of Compounds 2, 3, 10 and 13 [SEQ. ID. NOS. 6, 7, C 1 o 14 and 17] is described in Examples 2, 3, 10 and 13, O respectively.

The determination of gastric emptying by the method described below was performed after a fast of -20 hours to ensure that the stomach contained no chyme that would interfere with spectrophotometric absorbance measurements.

Conscious rats received by gavage, 1.5ml of an acaloric gel containing 1.5% methyl cellulose (M-0262, Sigma Chemical Co, St Louis, MO) and 0.05% phenol red indicator. Twenty minutes after gavage, rats were anesthetized using halothane, the stomach exposed and clamped at the pyloric and lower esophageal sphincters using artery forceps, removed and opened into an alkaline solution which was made up to a fixed volume. Stomach content was derived from the intensity of the phenol red in the alkaline solution, measured by absorbance at a wavelength of 560 nm. In separate experiments on 7 rats, the stomach and small intestine were both excised and opened into an alkaline solution. The quantity of phenol red that could be recovered from the upper gastrointestinal tract within 20 minutes of gavage was 89±4%; dye which appeared to bind irrecoverably to the gut luminal surface may have accounted for the balance. To account for a maximal dye recovery of less than 100%, percent of stomach contents remaining after 20 min were expressed as a fraction of the gastric contents recovered from control rats sacrificed immediately after gavage in the same experiment.

Percent gastric contents remaining (absorbance at min)/(absorbance at 0 mm) x 100.

In baseline studies, with no drug treatment, gastric emptying over 20 min was determined. In dose-response studies, rats were treated with 0.01, 0.1, 0.3, 1, 10 and 100 pg of exendin-4, and 0.1, 0.3, 1, 10 and 100 pg of Compounds 2, 3, 10 and 13 [SEQ. ID. NOS. 6, 7, 14 and 17].

The results, shown in Figures 6-9, show that the exendin agonists, Compounds 2, 3, 10 and 13, are potent inhibitors of gastric emptying. The EC, 0 for exendin-4 was 0.27 pg. The EC,, for Compound 2 was 0.79 pg, for Compound 3 was 0.34 pg, for Compound 10 was 0.27 pg, and for Compound 13 was 60 pg.

Claims (12)

1. 2. A compound according to claim 1 wherein Xaal is His.
2. 3. A compound according to claim 1 wherein Xaal is 4-imidazopropionyl.
3. 4. A compound according to claim 1 wherein Zi is -NH 2 A compound according to claim 1 wherein Z 2 is -NH 2
4. 6. A compound according to claim 1 wherein Xaa 2 1 is Lys-NH'-R where R is Lys, Arg, Ci-Clo straight chain or branched alkanoyl.
5. 7. The peptide of claim 1, substantially as hereinbefore described with reference to any one of the examples.
6. 8. A composition comprising a compound of any one of claims 1-7 in a pharmaceutically acceptable carrier.
7. 9. A method for the treatment of diabetes mellitus comprising administering a therapeutically effective amount of a compound of any one of claims 1-7. The method according to claim 9, wherein the diabetes mellitus is type I diabetes mellitus.
8. 11. The method according to claim 9, wherein the diabetes mellitus is type II diabetes mellitus. 1837776 I:LNB IN 89 S12. The use of any one of the compounds of claims 1-7 in the manufacture of a medicament for the treatment of diabetes mellitus. O 13. The use according to claim 12, wherein the diabetes mellitus is type I diabetes Smellitus.
9. 14. The use according to claim 12, wherein the diabetes mellitus is type II diabetes mellitus. n
10. 15. The method according to any one of claims 9-11, further comprising C administration of a therapeutically effective amount of insulin. I 16. The use of any one of claims 12-14, wherein said medicament further comprises insulin.
11. 17. A method for treating hyperglycemia comprising administering a therapeutically effective amount of a compound of any one of claims 1-7.
12. 18. The use of the compound of any one of claims 1-7 for the manufacture of a medicament for the treatment of hyperglycemia. Dated 9 October, 2006 Amylin Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\IA L Specifications\506672] D2spec.doc:gcc