U.S. patent application number 11/729051 was filed with the patent office on 2009-01-29 for novel exendin agonist compounds.
This patent application is currently assigned to Amylin Pharmaceuticals, Inc.. Invention is credited to Nigel Robert Arnold Beeley, Kathryn S. Prickett.
Application Number | 20090029913 11/729051 |
Document ID | / |
Family ID | 38049537 |
Filed Date | 2009-01-29 |
United States Patent
Application |
20090029913 |
Kind Code |
A1 |
Beeley; Nigel Robert Arnold ;
et al. |
January 29, 2009 |
Novel exendin agonist compounds
Abstract
Novel exendin agonist compounds are provided. These compounds
are useful in treating diabetes and conditions which would be
benefited by lowering plasma glucose or delaying and/or slowing
gastric emptying.
Inventors: |
Beeley; Nigel Robert Arnold;
(San Diego, CA) ; Prickett; Kathryn S.;
(Hillsborough, CA) |
Correspondence
Address: |
Intellectual Property Department;Amylin Pharmaceuticals, Inc.
9360 Towne Centre Drive
San Diego
CA
92121
US
|
Assignee: |
Amylin Pharmaceuticals,
Inc.
San Diego
CA
|
Family ID: |
38049537 |
Appl. No.: |
11/729051 |
Filed: |
March 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09554531 |
Aug 8, 2000 |
7220721 |
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11729051 |
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PCT/US98/24273 |
Nov 13, 1998 |
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09554531 |
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60066029 |
Nov 14, 1997 |
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Current U.S.
Class: |
514/6.9 ;
530/324 |
Current CPC
Class: |
A61P 3/10 20180101; C07K
14/57563 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 ;
530/324 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; A61P 3/10 20060101
A61P003/10 |
Claims
1-73. (canceled)
74. A peptide comprising the amino acid sequence of any one of SEQ
ID NOs. 5-93 and 95-110.
75. The peptide of claim 74, wherein the amino acid sequence is any
one of SEQ ID NOs. 11-93.
76. A pharmaceutical composition comprising the peptide of claim 74
and a pharmaceutically acceptable carrier.
77. A method for treating diabetes in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of the peptide of claim 74.
78. The peptide of claim 74, wherein the amino acid sequence is SEQ
ID NO. 92 or 93.
79. A pharmaceutical composition comprising the peptide of claim 78
and a pharmaceutically acceptable carrier.
80. A method for treating diabetes in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of the peptide of claim 78.
81. A peptide comprising the amino acid sequence of the formula:
Xaa.sub.1 Gly Xaa.sub.3 Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln
Xaa.sub.14 Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Xaa.sub.25 Leu
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-Z.sub.2
wherein: Xaa.sub.1 is Ala or His; Xaa.sub.3 is Glu or Ala;
Xaa.sub.14 is Met or Leu; Xaa.sub.25 Phe or Trp; and Z.sub.2 is
--OH or --NH.sub.2.
82. The peptide of claim 81, wherein Xaa.sub.1 is Ala; Xaa.sub.3 is
Glu; Xaa.sub.14 is Met; and Xaa.sub.25 is Trp (SEQ ID NO: 92).
83. The peptide of claim 81, wherein Xaa.sub.1 is Ala; Xaa.sub.3 is
Ala; Xaa.sub.14 is Leu; and Xaa.sub.25 is Phe (SEQ ID NO: 93).
84. The peptide of claim 81, wherein Xaa.sub.1 is His; Xaa.sub.3 is
Glu; Xaa.sub.14 is Leu; and Xaa.sub.25 is Trp.
85. The peptide of claim 81, wherein: Xaa.sub.1 is Ala; and (i)
Xaa.sub.3 is Glu; Xaa.sub.14 is Met; and Xaa.sub.25 is Phe; (ii)
Xaa.sub.3 is Glu; Xaa.sub.14 is Leu; and Xaa.sub.25 is Trp; (iii)
Xaa.sub.3 is Glu; Xaa.sub.14 is Leu; and Xaa.sub.25 is Phe; (iv)
Xaa.sub.3 is Ala; Xaa.sub.14 is Met; and Xaa.sub.25 is Trp; (v)
Xaa.sub.3 is Ala; Xaa.sub.14 is Met; and Xaa.sub.25 is Phe; or (vi)
Xaa.sub.3 is Ala; Xaa.sub.14 is Leu; and Xaa.sub.25 is Trp.
86. The peptide of claim 81, wherein Xaa.sub.1 is His; and (i)
Xaa.sub.3 is Glu; Xaa.sub.14 is Met; and Xaa.sub.25 is Trp; (ii)
Xaa.sub.3 is Glu; Xaa.sub.14 is Met; and Xaa.sub.25 is Phe; (iii)
Xaa.sub.3 is Glu; Xaa.sub.14 is Leu; and Xaa.sub.25 is Phe; (iv)
Xaa.sub.3 is Ala; Xaa.sub.14 is Met; and Xaa.sub.25 is Trp; (v)
Xaa.sub.3 is Ala; Xaa.sub.14 is Met; and Xaa.sub.25 is Phe; (vi)
Xaa.sub.3 is Ala; Xaa.sub.14 is Leu; and Xaa.sub.25 is Trp; or
(vii) Xaa.sub.3 is Ala; Xaa.sub.14 is Leu; and Xaa.sub.25 is
Phe.
87. A pharmaceutical composition comprising the peptide of claim 81
and a pharmaceutically acceptable carrier.
88. A method for treating diabetes in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of the peptide of claim 81.
89. A peptide comprising the amino acid sequence having the
formula: Xaa.sub.1 Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln
Xaa.sub.14 Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-Z.sub.2 wherein:
Xaa.sub.1 is Ala or His; Xaa.sub.14 is Met or Leu; and Z.sub.2 is
--OH or --NH.sub.2.
90. The peptide of claim 89, wherein Xaa.sub.1 is Ala; and
Xaa.sub.14 is Met (SEQ ID NO: 92).
91. The peptide of claim 89, wherein Xaa.sub.1 is His; and
Xaa.sub.14 is Leu.
92. A pharmaceutical composition comprising the peptide of claim 89
and a pharmaceutically acceptable carrier.
93. A method for treating diabetes in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of the peptide of claim 89.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/066,029, filed Nov. 14, 1997, the contents of
which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel compounds which have
activity as exendin agonists. These compounds are useful in
treatment of Type I and II diabetes, in treatment of disorders
which would be benefited by agents which lower plasma glucose
levels and in treatment of disorders which would be benefited with
agents useful in delaying and/or slowing gastric emptying.
BACKGROUND
[0003] 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
[0004] The exendins are peptides that are found in the venom of the
Gila-monster, a lizard endogenous to Arizona and Northern Mexico.
Exendin-3 [SEQ. ID. NO. 1] is present in the venom of Heloderma
horridum, and exendin-4 [SEQ. ID. NO. 2] is present in the venom of
Heloderma suspectum (Eng, J., et al., J. Biol. Chem., 265:20259-62,
1990; Eng., J., et al., J. Biol. Chem., 267:7402-05, 1992). The
amino acid sequence of exendin-3 is shown in FIG. 1. The amino acid
sequence of exendin-4 is shown in FIG. 2. The exendins have some
sequence similarity to several members of the glucagon-like peptide
family, with the highest homology, 53%, being to
GLP-1[7-36]NH.sub.2 [SEQ. ID. NO. 3] (Goke, et al., J. Biol. Chem.,
268:19650-55, 1993). GLP-1[7-36]NH.sub.2, also known as
proglucagon[78-107] or simply "GLP-1" as used most often herein,
has an insulinotropic effect, stimulating insulin secretion from
pancreatic .beta.-cells; GLP-1 also inhibits glucagon secretion
from pancreatic .alpha.-cells (orsov, et al., Diabetes, 42:658-61,
1993; D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). The
amino acid sequence of GLP-1 is shown in FIG. 3. GLP-1 is reported
to inhibit gastric emptying (Willms B, et al., J Clin Endocrinol
Metab 81 (1): 327-32, 1996; Wettergren A, et al., Dig Dis Sci 38
(4): 665-73, 1993), and gastric acid secretion. Schjoldager B T, et
al., Dig Dis Sci 34 (5): 703-8, 1989; O'Halloran D J, et al., J
Endocrinol 126 (1): 169-73, 1990; Wettergren A, et al., Dig Dis Sci
38 (4): 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 .beta.-cell line (Thorens, Proc. Natl. Acad. Sci. USA
89:8641-45, 1992), hereinafter referred to as the "cloned GLP-1
receptor." Exendin-4 reportedly acts at GLP-1 receptors on
insulin-secreting .beta.TC1 cells, at dispersed acinar cells from
guinea pig pancreas, and at parietal cells from stomach; the
peptide is also reported to stimulate somatostatin release and
inhibit gastrin release in isolated stomachs (Goke, et al., J.
Biol. 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 stimulate cAMP
production in, and amylase release from, pancreatic acinar cells
(Malhotra, R., et al., Regulatory Peptides, 41:149-56, 1992;
Raufman, et al., J. Biol. Chem. 267:21432-37, 1992; Singh, et al.,
Regul. Pept. 53:47-59, 1994).
[0005] 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. Pat.
No. 5,424,286).
[0006] 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. 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-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. J., 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. (J. Am. Coll.
Emergency Physns, 8:228, 1979) described relief of acute esophageal
food obstruction following glucagon therapy. In another study,
glucagon significantly relieved pain and tenderness in 21 patients
with biliary tract disease compared with 22 patients treated with
placebo (M. J. Stower, et al., Br. J. Surg., 69:591-2, 1982).
[0007] Methods for regulating gastrointestinal motility using
amylin agonists are described in International Application No.
PCT/US94/10225, published Mar. 16, 1995.
[0008] Methods for regulating gastrointestinal motility using
exendin agonists are described in U.S. patent application Ser. No.
08/908,867, filed Aug. 8, 1997 entitled "Methods for Regulating
Gastrointestinal Motility," which application is a
continuation-in-part of U.S. patent application Ser. No. 08/694,954
filed Aug. 8, 1996.
[0009] Methods for reducing food intake using exendin agonists are
described in U.S. patent application Ser. No. 09/003,869, filed
Jan. 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 Jan. 7, 1997,
60/055,404 filed Aug. 7, 1997, 60/065,442 filed Nov. 14, 1997 and
60/066,029 filed Nov. 14, 1997.
[0010] Novel exendin agonist compounds are described in PCT
Application Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled
"Novel Exendin Agonist Compounds," which claims the benefit of U.S.
Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997. Other
novel exendin agonists are described in U.S. application Ser. No.
______ filed Nov. 13, 1998, entitled "Novel Exendin Agonist
Compounds," which claims the benefit of U.S. Provisional
Application No. 60/065,442 filed Nov. 14, 1997.
SUMMARY OF THE INVENTION
[0011] According to one aspect, the present invention provides
novel exendin agonist compounds which exhibit advantageous
properties which include effects in slowing gastric emptying and
lowering plasma glucose levels.
[0012] According to the present invention, provided are compounds
of the formula (I) [SEQ. ID. NO. 4]:
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala
Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24
Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1; wherein
Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val
or Norleu;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
Xaa.sub.5 is Ala or Thr;
[0013] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0014] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0015] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu;
Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg;
Xaa.sub.21 is Ala or Leu;
[0016] Xaa.sub.22 is Phe, Tyr or naphthylalanine; Xaa.sub.23 is
Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0017] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa.sub.26 is Ala or Leu;
Xaa.sub.27 is Ala or Lys;
Xaa.sub.28 is Ala or Asn;
Z.sub.1 is --OH,
[0018] --NH.sub.2, [0019] Gly-Z.sub.2, [0020] Gly Gly-Z.sub.2,
[0021] Gly Gly Xaa.sub.31-Z.sub.2, [0022] Gly Gly Xaa.sub.31
Ser-Z.sub.2, [0023] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, [0024] Gly
Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2, [0025] Gly Gly Xaa.sub.31 Ser
Ser Gly Ala-Z.sub.2, [0026] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36-Z.sub.2, [0027] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, [0028] Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 or [0029] Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38
Xaa.sub.39-Z.sub.2; [0030] wherein [0031] Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently Pro, homoproline, 3Hyp,
4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanine; and [0032] Z.sub.2 is --OH or --NH.sub.2; provided
that no more than three of Xaa.sub.3, Xaa.sub.4, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.9, Xaa.sub.10, Xaa.sub.11,
Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16,
Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24,
Xaa.sub.25, Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are Ala; and
provided also that, if Xaa.sub.1 is His, Arg or Tyr, then at least
one of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala.
[0033] Also included within the scope of the present invention are
pharmaceutically acceptable salts of the compounds of formula (I)
and pharmaceutical compositions including said compounds and salts
thereof.
[0034] Also within the scope of the present invention are narrower
genera of peptide compounds 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.
[0035] Additionally, the present invention includes narrower genera
of peptide compounds having particular amino acid sequences, for
example, compounds of the formula [I] [SEQ. ID. NO. 4]:
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala
Xaa.sub.18, Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein
Xaa.sub.1 is His or Ala;
Xaa.sub.2 is Gly or Ala;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala or Gly;
Xaa.sub.5 is Ala or Thr;
[0036] Xaa.sub.6 is Phe or naphthylalanine; [0037] Xaa.sub.7 is Thr
or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Asp or Glu;
[0038] Xaa.sub.10 is Ala, Leu or pentylglycine;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0039] Xaa.sub.14 is Ala, Leu, Met or pentylglycine;
Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu;
Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg;
Xaa.sub.21 is Ala or Leu;
[0040] Xaa.sub.22 is Phe or naphthylalanine; Xaa.sub.23 is Ile, Val
or tert-butylglycine;
Xaa.sub.24 is Ala, Glu or Asp;
Xaa.sub.25 is Ala, Trp or Phe;
Xaa.sub.26 is Ala or Leu;
Xaa.sub.27 is Ala or Lys;
Xaa.sub.28 is Ala or Asn;
Z.sub.1 is --OH,
[0041] --NH.sub.2, [0042] Gly-Z.sub.2, [0043] Gly Gly-Z.sub.2
[0044] Gly Gly Xaa.sub.31-Z.sub.2, [0045] Gly Gly Xaa.sub.31
Ser-Z.sub.2, [0046] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, [0047] Gly
Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2, [0048] Gly Gly Xaa.sub.31 Ser
Ser Gly Ala-Z.sub.2, [0049] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36-Z.sub.2, [0050] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2 [0051] Gly Gly Xaa.sub.31 Ser Ser Gly
Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 [0052] Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38 [0053]
Ser-Z.sub.2; [0054] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are independently Pro, homoproline, thioproline, or
[0055] N-methylylalanine; and [0056] Z.sub.2 is --OH or --NH.sub.2;
provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15 Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20 Xaa.sub.21, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.26, Xaa.sub.27, and Xaa.sub.28 are Ala; and provided that,
if Xaa.sub.1 is His, Arg or Tyr, then at least one of Xaa.sub.3,
Xaa.sub.4 and Xaa.sub.9 is Ala; and pharmaceutically acceptable
salts thereof;
[0057] Also provided are peptide compounds of the formula (II)
[SEQ. ID. NO. 94]: [0058] 5 10
[0058] Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala
Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24
Xaa.sub.25 Xaa.sub.26 X.sub.1-Z.sub.1; wherein
Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val, Norleu or
4-imidazopropionyl;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
Xaa.sub.5 is Ala or Thr;
[0059] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0060] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0061] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu;
Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg;
[0062] Xaa.sub.21 is Ala, Leu or Lys-NH.sup..epsilon.--R where R is
Lys, Arg, C.sup.-c10 straight chain or branched alkanoyl or
cycloalleyl-alkanoyl; Xaa.sub.22 is Phe, Tyr or naphthylalanine;
Xaa.sub.23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or
Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0063] Xaa.sub.2, is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa.sub.26 is Ala or Leu;
[0064] X.sub.1 is Lys Asn, Asn Lys, Lys-NH.sup..epsilon.--R Asn,
Asn Lys-NH.sup..epsilon.--R, Lys-NH.sup..epsilon.--R Ala, Ala
Lys-NHE-R where R is Lys, Arg, C.sub.1-C.sub.10 straight chain or
branched alkanoyl or cycloalkylalkanoyl
Z.sub.1 is --OH,
[0065] --NH.sub.2, [0066] Gly-Z.sub.2, [0067] Gly Gly-Z.sub.2,
[0068] Gly Gly Xaa.sub.31-Z.sub.2, [0069] Gly Gly Xaa.sub.31
Ser-Z.sub.2, [0070] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2 [0071] Gly
Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2, [0072] Gly Gly Xaa.sub.31 Ser
Ser Gly Ala-Z.sub.2, [0073] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36-Z.sub.2, [0074] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, [0075] Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 or [0076] Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38
Xaa.sub.39-Z.sub.2; [0077] wherein [0078] Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from the group
consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and [0079]
Z.sub.2 is --OH or --NH.sub.2; provided that no more than three of
Xaa.sub.3, Xaa.sub.4, Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.9,
Xaa.sub.10, Xaa.sub.11, Xaa.sub.12, Xaa.sub.13, Xaa.sub.14,
Xaa.sub.15, Xaa.sub.16, Xaa.sub.17, Xaa.sub.19, Xaa.sub.20,
Xaa.sub.2., Xaa.sub.24, Xaa.sub.25, Xaa.sub.26, are Ala; and
provided also that, if Xaa.sub.1 is His, Arg, Tyr, or
4-imidazopropionyl then at least one of Xaa.sub.3, Xaa.sub.4 and
Xaa.sub.9 is Ala.
[0080] 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.
[0081] Preferred compounds of formula (II) include those wherein
Xaa.sub.1 is His, Ala, Norval or 4-imidazopropionyl. Preferably,
Xaa.sub.1 is His, or 4-imidazopropionyl or Ala, more preferably His
or 4-imidazopropionyl.
[0082] Preferred compounds of formula (II) include those wherein
Xaa.sub.2 is Gly.
[0083] Preferred compounds of formula (II) include those wherein
Xaa.sub.4 is Ala.
[0084] Preferred compounds of formula (II) include those wherein
Xaa.sub.9 is Ala.
[0085] Preferred compounds of formula (II) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0086] Preferred compounds of formula (II) include those wherein
Xaa.sub.25 is Trp or Phe.
[0087] Preferred compounds of formula (II) include those wherein
Xaa.sub.6 is Ala, Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; and Xaa.sub.23 is Ile or Val.
[0088] Preferred compounds of formula (II) include those wherein
Z.sub.1 is --NH.sub.2.
[0089] Preferred compounds of formula (II) include those wherein
Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently
selected from the group consisting of Pro, homoproline, thioproline
and N-alkylalanine.
[0090] Preferred compounds of formula (II) include those wherein
Xaa.sub.39 is Ser or Tyr, preferably Ser.
[0091] Preferred compounds of formula (II) include those wherein
Z.sub.2 is --NH.sub.2.
[0092] Preferred compounds of formula (II) include those 42 wherein
Z.sub.1 is --NH.sub.2.
[0093] Preferred compounds of formula (II) include those wherein
Xaa.sub.21 is Lys-NH.sup..epsilon.--R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl.
[0094] Preferred compounds of formula (II) include those wherein
X.sub.1 is Lys Asn, Lys-NH.sup..epsilon.--R Asn, or
Lys-NH.sup..epsilon.--R Ala where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl.
[0095] Preferred compounds of formula (II) include those having an
amino acid sequence selected from SEQ. ID. NOS. 95-110.
DEFINITIONS
[0096] In accordance with the present invention and as used herein,
the following terms are defined to have the following meanings,
unless explicitly stated otherwise.
[0097] 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,
2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,
aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,
6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,
3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine,
2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid,
2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,
homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,
N-methylglycine, N-methylisoleucine, N-methylpentylglycine,
N-methylvaline, naphthalanine, 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,
methionine sulfoxide, methionine sulfone,
S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide
and S-(carboxymethyl)-cysteine sulfone.
[0098] The term "amino acid analog" refers to an amino acid wherein
either the C-terminal carboxy group, the N-terminal 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.
[0099] The term "amino acid residue" refers to radicals having the
structure: (1) --C(O)--R--NH--, wherein R typically is --CH(R')--,
wherein R' is an amino acid side chain, typically H or a carbon
containing substitutent; or (2)
##STR00001##
wherein p is 1, 2 or 3 representing the azetidinecarboxylic acid,
proline or pipecolic acid residues, respectively.
[0100] The term "lower" referred to herein in connection with
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.
[0101] "Pharmaceutically acceptable salt" includes salts of the
compounds of the present invention derived from the 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.
[0102] In addition, the following abbreviations stand for the
following:
[0103] "ACN" or "CH.sub.3CN" refers to acetonitrile.
[0104] "Boc", "tBoc" or "Tboc" refers to t-butoxy carbonyl.
[0105] "DCC" refers to N,N'-dicyclohexylcarbodiimide.
[0106] "Fmoc" refers to fluorenylmethoxycarbonyl.
[0107] "HBTU" refers to
2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium
hexafluorophosphate.
[0108] "HOBt" refers to 1-hydroxybenzotriazole monohydrate.
[0109] "homoP" or hPro" refers to homoproline.
[0110] "MeAla" or "Nme" refers to N-methylalanine.
[0111] "naph" refers to naphthylalanine.
[0112] "pG" or pGly" refers to pentylglycine.
[0113] "tBuG" refers to tertiary-butylglycine.
[0114] "ThioP" or tPro" refers to thioproline.
[0115] "3Hyp" refers to 3-hydroxyproline
[0116] "4Hyp" refers to 4-hydroxyproline
[0117] "NAG" refers to N-alkylglycine
[0118] "NAPG" refers to N-alkylpentylglycine
[0119] "Norval" refers to norvaline
[0120] "Norleu" refers to norleucine
BRIEF DESCRIPTION OF THE DRAWINGS
[0121] FIG. 1 depicts the amino acid sequence for exendin-3 [SEQ.
ID. NO. 1].
[0122] FIG. 2 depicts the amino acid sequence for exendin-4 [SEQ.
ID. NO. 2].
[0123] FIG. 3 depicts the amino acid sequence for
GLP-1[7-36]NH.sub.2 (GLP-1) [SEQ. ID. NO. 3].
[0124] FIG. 4 depicts the amino acid sequences for certain
compounds of the present invention, Compounds 1-89 [SEQ. ID. NOS. 5
to 93].
[0125] FIG. 5 depicts the effect on lowering blood glucose of
various concentrations of Compound 1 [SEQ. ID. NO. 5].
[0126] FIG. 6 depicts a comparison of effects on gastric emptying
of various concentrations of Compound 1 [SEQ. ID. NO. 5].
[0127] FIG. 7 depicts the amino acid sequences for certain
compounds of the present invention, Compound Nos. 90-105 [SEQ. ID.
NOS. 95-110].
DETAILED DESCRIPTION OF THE INVENTION
[0128] According to the present invention, provided are compounds
of the formula (I) [SEQ. ID. NO. 4]:
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala
Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24
Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1; wherein
Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val
or Norleu;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
Xaa.sub.5 is Ala or Thr;
[0129] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0130] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0131] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu;
Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg;
[0132] Xaa.sub.21 is Ala, Leu or Lys-NH.sup..epsilon.R where R is
Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl or
cycloalleyl-alkanoyl; Xaa.sub.22 is Phe, Tyr or naphthylalanine;
Xaa.sub.23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or
Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0133] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa.sub.26 is Ala or Leu;
Xaa.sub.27 is Ala or Lys;
Xaa.sub.28 is Ala or Asn;
Z.sub.1 is --OH,
[0134] --NH.sub.2, [0135] Gly-Z.sub.2, [0136] Gly Gly-Z.sub.2,
[0137] Gly Gly Xaa.sub.31-Z.sub.2, [0138] Gly Gly Xaa.sub.31
Ser-Z.sub.2, [0139] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, [0140] Gly
Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2, [0141] Gly Gly Xaa.sub.31 Ser
Ser Gly Ala-Z.sub.2, [0142] Gly Gly xaa 31 Ser Ser Gly Ala
Xaa.sub.36-Z.sub.2, [0143] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, [0144] Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 or [0145] Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38
Xaa.sub.39-Z.sub.2; [0146] wherein [0147] Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from Pro,
homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,
N-alkylpentylglycine or N-alkylalanine; and [0148] Z.sub.2 is --OH
or --NH.sub.2; provided that no more than three of Xaa.sub.3,
Xaa.sub.4, Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.9, Xaa.sub.10,
Xaa.sub.11, Xaa.sub.12, Xaa.sub.13 Xaa.sub.14, Xaa.sub.151
Xaa.sub.16 xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21,
Xaa.sub.24, Xaa.sub.25, Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are
Ala; and provided also that, if Xaa.sub.1 is His, Arg or Tyr, then
at least one of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala. Also
within the scope of the present invention are pharmaceutically
acceptable salts of formula (I) and pharmaceutic compositions
including said compounds and salts thereof.
[0149] Preferred N-alkyl groups for N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine include lower alkyl groups
preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4
carbon atoms. Suitable compounds of formula (I) include those
identified in Examples 1-89 ("Compounds 1-89," respectively) [SEQ.
ID. NOS. 5 to 93], as well as those corresponding compounds
identified in Examples 104 and 105.
[0150] Preferred such exendin agonist compounds include those
wherein Xaa.sub.1 is His, Ala or Norval. More preferably Xaa.sub.1
is His or Ala. Most preferably Xaa.sub.1 is His.
[0151] Preferred are those compounds of formula (I) wherein
Xaa.sub.2 is Gly.
[0152] Preferred are those compounds of formula (I) wherein
Xaa.sub.3 is Ala.
[0153] Preferred are those compounds of formula (I) wherein
Xaa.sub.4 is Ala.
[0154] Preferred are those compounds of formula (I) wherein
Xaa.sub.9 is Ala.
[0155] Preferred are those compounds of formula (I) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0156] Preferred compounds of formula (I) are those wherein
Xaa.sub.25 is Trp or Phe.
[0157] Preferred compounds of formula (I) are those where Xaa.sub.6
is Ala, Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; and Xaa.sub.23 is Ile or Val.
[0158] Preferred are compounds of formula (I) wherein Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently selected
from Pro, homoproline, thioproline and N-alkylalanine.
[0159] Preferably Z.sub.1 is --NH.sub.2.
[0160] Preferably Z.sub.2 is --NH.sub.2.
[0161] According to one aspect, preferred are compounds of formula
(I) wherein Xaa.sub.1 is Ala, His or Tyr, more preferably Ala or
His; Xaa.sub.2 is Ala or Gly; Xaa.sub.6 is Phe or naphthylalanine;
Xaa.sub.4 is Ala, Leu, pentylglycine or Met; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile or Val; Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from Pro,
homoproline, thioproline or N-alkylalanine; and Xaa.sub.39 is Ser
or Tyr, more preferably Ser. More preferably Z.sub.1 is
--NH.sub.2.
[0162] According to an especially preferred aspect, especially
preferred compounds include those of formula (I) wherein: Xaa.sub.1
is His or Ala; Xaa.sub.2 is Gly or Ala; Xaa.sub.3 is Ala, Asp or
Glu; Xaa.sub.4 is Ala or Gly; Xaa.sub.5 is Ala or Thr; Xaa.sub.6 is
Phe or naphthylalanine; Xaa.sub.7 is Thr or Ser; Xaa.sub.8 is Ala,
Ser or Thr; Xaa.sub.9 is Ala, Asp or Glu; Xaa.sub.10 is Ala, Leu or
pentylglycine; Xaa.sub.11 is Ala or Ser; Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln; Xaa.sub.14 is Ala, Leu, Met or
pentylglycine; Xaa.sub.15 is Ala or Glu; Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu; Xaa.sub.19 is Ala or Val; Xaa.sub.20 is
Ala or Arg; Xaa.sub.21 is Ala or Leu; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile, Val or tert-butylglycine;
Xaa.sub.24 is Ala, Glu or Asp; Xaa.sub.25 is Ala, Trp or Phe;
Xaa.sub.26 is Ala or Leu; Xaa.sub.27 is Ala or Lys; Xaa.sub.28 is
Ala or Asn; Z.sub.1 is --OH, --NH.sub.2, Gly-Z.sub.2, Gly
Gly-Z.sub.2, Gly Gly Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31
Ser-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, Gly Gly Xaa.sub.31
Ser Ser Gly-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2
or Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38 Xaa.sub.39-Z.sub.2; Xaa.sub.31, Xaa.sub.36, Xaa.sub.37
and Xaa.sub.38 being independently Pro homoproline, thioproline or
N-methylalanine; and Z.sub.2 being --OH or --NH.sub.2; provided
that no more than three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12, Xaa.sub.13,
Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17, Xaa.sub.19,
Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25, Xaa.sub.26,
Xaa.sub.27 and Xaa.sub.28 are Ala; and provided also that, if
Xaa.sub.1 is His, Arg or Tyr, then at least one of Xaa.sub.3,
Xaa.sub.4 and Xaa.sub.9 is Ala. Especially preferred compounds of
formula (I) include those having the amino acid sequence of SEQ.
ID. NOS. 5-93
[0163] According to an especially preferred aspect, provided are
compounds of formula (I) where Xaa.sub.14 is Ala, Leu, Ile, Val or
pentylglycine, more preferably Leu or pentylglycine, and Xaa.sub.25
is Ala, Phe, Tyr or naphthylalanine, more preferably Phe or
naphthylalanine. These compounds will be less susceptible to
oxidative degration, both in vitro and in vivo, as well as during
synthesis of the compound.
[0164] Also within the scope of the present invention are narrower
genera of peptide compounds 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.
[0165] Additionally, the present invention includes narrower genera
of peptide compounds having particular amino acid sequences, for
example, compounds of the formula [I] [SEQ. ID. NO. 4]:
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.5 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala
Xaa.sub.18 Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein
Xaa.sub.1 is His or Ala;
Xaa.sub.2 is Gly or Ala;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala or Gly;
Xaa.sub.5 is Ala or Thr;
[0166] Xaa.sub.6 is Phe or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
[0167] Xaa.sub.9 is Ala, Asp or Glu; [0168] Xaa.sub.10 is Ala, Leu
or pentylglycine; [0169] Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0170] Xaa.sub.14 is Ala, Leu, Met or pentylglycine;
Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu;
Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg;
Xaa.sub.21 is Ala or Leu;
[0171] Xaa.sub.22 is Phe or naphthylalanine; Xaa.sub.23 is Ile, Val
or tert-butylglycine;
Xaa.sub.24 is Ala, Glu or Asp;
Xaa.sub.25 is Ala, Trp or Phe;
Xaa.sub.26 is Ala or Leu;
Xaa.sub.27 is Ala or Lys;
Xaa.sub.2, is Ala or Asn;
Z.sub.1 is --OH,
[0172] --NH.sub.2, [0173] Gly-Z.sub.2, [0174] Gly Gly-Z.sub.2
[0175] Gly Gly Xaa.sub.31-Z.sub.2, [0176] Gly Gly Xaa.sub.31
Ser-Z.sub.2, [0177] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, [0178] Gly
Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2, [0179] Gly Gly Xaa.sub.31 Ser
Ser Gly Ala-Z.sub.2, [0180] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36-Z.sub.2, [0181] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2 [0182] Gly Gly Xaa.sub.31 Ser Ser Gly
Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 [0183] Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38
Ser-Z.sub.2; [0184] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are independently Pro, homoproline, thioproline, or
N-methylylalanine; and [0185] Z.sub.2 is --OH or --NH.sub.2;
provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.26, Xaa.sub.27, and Xaa.sub.28 are Ala; and provided that,
if Xaa.sub.1 is His, Arg or Tyr, then at least one of Xaa.sub.3,
Xaa.sub.4 and Xaa.sub.9 is Ala; and pharmaceutically acceptable
salts thereof;
[0186] Also provided are peptide compounds of the formula (II)
[SEQ. ID. NO. 94]: [0187] 5 10
[0187] Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.7 Xaa.sub.8
Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13 Xaa.sub.14
Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala Xaa.sub.19 Xaa.sub.20
Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24 Xaa.sub.25 Xaa.sub.26
X.sub.1-Z.sub.1; wherein
Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val, Norleu or
4-imidazopropionyl;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
Xaa.sub.5 is Ala or Thr;
[0188] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0189] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0190] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu;
Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg;
[0191] Xaa.sub.21 is Ala, Leu or Lys-NH.sub..epsilon.--R where R is
Lys, Arg, C.sup.-c10 straight chain or branched alkanoyl or
cycloalleyl-alkanoyl; Xaa.sub.22 is Phe, Tyr or naphthylalanine;
Xaa.sub.23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or
Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0192] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa.sub.26 is Ala or Leu;
[0193] X.sub.1 is Lys Asn, Asn Lys, Lys-NH.sup..epsilon.--R Asn,
Asn Lys-NH.sup..epsilon.--R, Lys-NH.sup..epsilon.--R Ala, Ala
Lys-NH.sup..epsilon.--R where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl or cycloalkylalkanoyl
Z.sub.1 is --OH,
[0194] --NH.sub.2, [0195] Gly-Z.sub.2, [0196] Gly Gly-Z.sub.2,
[0197] Gly Gly Xaa.sub.31-Z.sub.2, [0198] Gly Gly Xaa.sub.31
Ser-Z.sub.2, [0199] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, [0200] Gly
Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2, [0201] Gly Gly Xaa.sub.31 Ser
Ser Gly Ala-Z.sub.2, [0202] Gly Gly Xaa 31 Ser Ser Gly Ala Xaa
36-2, [0203] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2, [0204] Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 or [0205] Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38
Xaa.sub.39-Z.sub.2; [0206] wherein [0207] Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from the group
consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and [0208]
Z.sub.2 is --OH or --NH.sub.2; provided that no more than three of
Xaa.sub.3, Xaa.sub.4, Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.9,
Xaa.sub.10, Xaa.sub.11, Xaa.sub.12, Xaa.sub.13, Xaa.sub.14
Xaa.sub.15, Xaa.sub.16, Xaa.sub.17 Xaa.sub.19, Xaa.sub.20,
Xaa.sub.21, Xaa.sub.24, Xaa.sub.25, Xaa.sub.26, are Ala; and
provided also that, if Xaa.sub.1 is His, Arg, Tyr, or
4-imidazopropionyl then at least one of Xaa.sub.3, Xaa.sub.4 and
Xaa.sub.9 is Ala.
[0209] 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.
[0210] Preferred compounds of formula (II) include those wherein
Xaa.sub.1 is His, Ala, Norval or 4-imidazopropionyl. Preferably,
Xaa.sub.1 is His, or 4-imidazopropionyl or Ala, more preferably His
or 4-imidazopropionyl.
[0211] Preferred compounds of formula (II) include those wherein
Xaa.sub.2 is Gly.
[0212] Preferred compounds of formula (II) include those wherein
Xaa.sub.4 is Ala.
[0213] Preferred compounds of formula (II) include those wherein
Xaa.sub.9 is Ala.
[0214] Preferred compounds of formula (II) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0215] Preferred compounds of formula (II) include those wherein
Xaa.sub.25 is Trp or Phe:
[0216] Preferred compounds of formula (II) include those wherein
Xaa.sub.6 is Ala, Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; and Xaa.sub.23 is Ile or Val.
[0217] Preferred compounds of formula (II) include those wherein
Z.sub.1 is --NH.sub.2.
[0218] Preferred compounds of formula (II) include those wherein
Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently
selected from the group consisting of Pro, homoproline, thioproline
and N-alkylalanine.
[0219] Preferred compounds of formula (II) include those wherein
Xaa.sub.39 is Ser or Tyr, preferably Ser. Preferred compounds of
formula (II) include those wherein Z.sub.2 is --NH.sub.2.
[0220] Preferred compounds of formula (II) include those 42
wherein
[0221] Z.sub.1 is --NH.sub.2.
[0222] Preferred compounds of formula (II) include those wherein
Xaa.sub.21 is Lys-NH.sup..epsilon.--R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl.
[0223] Preferred compounds of formula (II) include those wherein
X.sub.1 is Lys Asn, Lys-NH.sup..epsilon.--R Asn, or
Lys-NH.sup..epsilon.--R Ala where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl.
[0224] Preferred compounds of formula (II) include those having an
amino acid sequence selected from SEQ. ID. NOS. 95-110.
[0225] 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, HCl, HBr,
H.sub.2SO.sub.41H.sub.3PO.sub.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
[0226] The compounds described above are useful in view of their
pharmacological properties. In particular, the compounds of the
invention are exendin agonists, and possess activity as agents to
regulate gastric motility and to slow gastric emptying, as
evidenced by the ability to reduce post-prandial glucose levels in
mammals.
[0227] The compounds of the present invention are useful in in
vitro and in vivo scientific methods for investigation of exendins
and exendin agonists for example in methods such as those described
in Examples A-E below.
Preparation of Compounds
[0228] The compounds of the present invention may be prepared using
standard solid-phase peptide synthesis techniques and preferably an
automated or semiautomated peptide synthesizer. Typically, using
such techniques, an .alpha.-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 solvent such as
dimethylformamide, N-methylpyrrolidinone or methylene chloride in
the presence of coupling agents such as dicyclohexylcarbodiimide
and 1-hydroxybenzotriazole in the presence of a base such as
diisopropylethylamine. The .alpha.-N-carbamoyl 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
fluorenylmethoxycarbonyl (Fmoc) being preferred herein.
[0229] The solvents, amino acid derivatives and
4-methylbenzhydryl-amine resin used in the peptide synthesizer may
be purchased from Applied Biosystems Inc. (Foster City,
Calif.).
[0230] The following side-chain protected amino 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-His(Trt),
Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may be purchased
from Applied Biosystems, Inc. or Bachem Inc. (Torrance, Calif.).
Anisole, dimethylsulfide, phenol, ethanedithiol, and thioanisole
may be obtained from Aldrich Chemical Company (Milwaukee, Wis.).
Air Products and Chemicals (Allentown, Pa.) supplies HF. Ethyl
ether, acetic acid and methanol may be purchased from Fisher
Scientific (Pittsburgh, Pa.).
[0231] Solid phase peptide synthesis may be carried out with an
automatic peptide synthesizer (Model 430A, Applied Biosystems Inc.,
Foster City, Calif.) 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 Jul. 1, 1988, section 6,
pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with
capping. Boc-peptide-resins may be cleaved with HF (-5.degree. C.
to 0.degree. 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, Ky.).
[0232] Peptides may be purified by RP-HPLC (preparative and
analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18
preparative column (10.mu., 2.2.times.25 cm; Vydac, Hesperia,
Calif.) may be used to isolate peptides, and purity may be
determined using a C4, C8 or C18 analytical column (5.mu.,
0.46.times.25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1%
TFA/CH.sub.3CN) may be delivered to the analytical column at a
flowrate of 1.0 ml/min and to the preparative column at 15 ml/min.
Amino acid analyses may be performed on the Waters Pico Tag system
and processed using the Maxima program. Peptides may be hydrolyzed
by vapor-phase acid hydrolysis (115.degree. C., 20-24 h).
Hydrolysates may be derivatized and analyzed by standard methods
(Cohen, et al., The Pico Tag Method: A Manual of Advanced
Techniques for Amino Acid Analysis, pp. 11-52, Millipore
Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis
may be carried out by M-Scan, Incorporated (West Chester, Pa.).
Mass calibration 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.
[0233] Peptide compounds useful in the invention may also be
prepared using recombinant DNA techniques, using methods now known
in the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor (1989).
[0234] Non-peptide compounds useful in the present invention may be
prepared by art-known methods.
Formulation and Administration
[0235] 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, 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-formulated or
separately with other glucose lowering agents such as insulin. A
suitable administration format may best be determined by a medical
practitioner for each patient individually. Suitable
pharmaceutically acceptable carriers and their formulation are
described in standard formulation treatises, e.g., Remington's
Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and
Hanson, M. A. "Parenteral Formulations of Proteins and Peptides:
Stability and Stabilizers," Journal of Parenteral Science and
Technology, Technical Report No. 10, Supp. 42:2 S (1988).
[0236] 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.
[0237] 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.
[0238] The claimed compounds can also be formulated as
pharmaceutically acceptable salts (e.g., acid addition salts)
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 the
pharmacological use by altering the physical-chemical
characteristics of the composition without preventing the
composition from exerting its physiological effect. Examples of
useful alterations in physical properties include lowering the
melting point to facilitate transmucosal administration and
increasing the solubility to facilitate the administration of
higher concentrations of the drug.
[0239] Pharmaceutically acceptable salts include acid addition
salts such as those containing sulfate, hydrochloride, 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, 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 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.
[0240] Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types 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, intraperitoneal, subcutaneous, and
intramuscular, orally, topically, or transmucosally.
[0241] If desired, solutions of the above compositions may be
thickened with a thickening agent such as methyl cellulose. 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, e.g., a Triton).
[0242] 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.
[0243] 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.
[0244] In diabetic or glucose intolerant individuals, plasma
glucose levels are higher than in normal individuals. In such
individuals, beneficial reduction or "smoothing" of post-prandial
blood 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 condition,
the blood sugar level or level of inhibition of gastric emptying to
be obtained, and other factors.
[0245] Such pharmaceutical compositions are useful in causing
gastric hypomotility in a subject and may be used as well in other
disorders where gastric motility is beneficially reduced.
[0246] The effective daily anti-emptying dose of the compounds will
typically be in the range of 0.001 or 0.005 to about 5 mg/day,
preferably about 0.01 or 0.05 to 2 mg/day and more 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. Administration may also be by other routes, for
example, by oral, buccal or nasal routes, however dosages should be
increased about 5-10 fold, over injection doses.
[0247] 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.
[0248] The optimal formulation and mode of administration of
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 the compounds will
typically be used to treat human patients, 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 pets such as horses, dogs and cats.
[0249] 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, 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
Ala 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-NH.sub.2 [SEQ. ID. NO.
5]
[0250] The above amidated peptide was 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.). In general,
single-coupling cycles were used throughout the synthesis and Fast
Moc (HBTU activation) chemistry was employed. Deprotection (Fmoc
group removal) of the growing peptide chain was achieved using
piperidine. Final deprotection of the completed peptide resin was
achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol
(0.2 mL), anisole (0.2 mL), water (0.2 mL) and trifluoroacetic acid
(15 mL) according to standard methods (Introduction to Cleavage
Techniques, Applied Biosystems, Inc.) The peptide was precipitated
in ether/water (50 mL) and centrifuged. The precipitate was
reconstituted in glacial acetic acid and lyophilized. The
lyophilized peptide was dissolved in water). Crude purity was about
75%.
[0251] Used in purification steps and analysis were Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN).
[0252] 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 60%
Solvent B in Solvent A over 30 minutes) of the lyophilized peptide
gave product peptide having an observed retention time of 19.2
minutes. Electrospray Mass Spectrometry (M): calculated 3171.6;
found 3172.
EXAMPLE 2
Preparation of Compound 2
His Gly Ala 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-NH.sub.2 [SEQ. ID. NO.
6]
[0253] The above amidated peptide was 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 Compound 1.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 14.9 minutes.
Electrospray Mass Spectrometry (M): calculated 3179.6; found
3180.
EXAMPLE 3
Preparation of Compound 3
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
7]
[0254] The above amidated peptide was 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 Compound 1.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 12.2 minutes.
Electrospray Mass Spectrometry (M): calculated 3251.6; found
3253.3.
EXAMPLE 4
Preparation of Compound 4
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
8]
[0255] The above amidated peptide was 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 Compound 1.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (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 16.3 minutes.
Electrospray Mass Spectrometry (M): calculated 3193.6; found
3197.
EXAMPLE 5
Preparation of Compound 5
Ala 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.sub.2 [SEQ. ID. NO.
9]
[0256] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3228.6.
EXAMPLE 6
Preparation of Compound 6
His Gly Ala 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.sub.2 [SEQ. ID. NO.
10]
[0257] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3234.7.
EXAMPLE 7
Preparation of Compound 7
His Gly Glu Ala 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.sub.2 [SEQ. ID. NO.
11]
[0258] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3308.7.
EXAMPLE 8
Preparation of Compound 8
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
12]
[0259] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3250.7
EXAMPLE 9
Preparation of Compound 9
His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
13]
[0260] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3252.6.
EXAMPLE 10
Preparation of Compound 10
Ala Ala 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.sub.2 [SEQ. ID. NO.
14]
[0261] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3200.6.
EXAMPLE 11
Preparation of Compound 11
Ala Ala 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-NH.sub.2 [SEQ. ID. NO.
15]
[0262] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3143.5.
EXAMPLE 12
Preparation of Compound 12
Ala Gly Asp 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.sub.2 [SEQ. ID. NO.
16]
[0263] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3214.6.
EXAMPLE 13
Preparation of Compound 13
Ala Gly Asp 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-NH.sub.2 [SEQ. ID. NO.
17]
[0264] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3157.5.
EXAMPLE 14
Preparation of Compound 14
Ala Gly Asp Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
18]
[0265] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3184.6.
EXAMPLE 15
Preparation of Compound 15
Ala Gly Asp 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.sub.2 [SEQ. ID. NO.
19]
[0266] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3127.5.
EXAMPLE 16
Preparation of Compound 16
Ala Gly Asp Gly Thr NaphthylAla Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-H.sub.2 [SEQ.
ID. NO. 20]
[0267] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3266.4.
EXAMPLE 17
Preparation of Compound 17
Ala Gly Asp Gly Thr Naphthylala Thr Ser Asp Leu Ser Lys Gln Leu Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 21]
[0268] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3209.4.
EXAMPLE 18
Preparation of Compound 18
Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
22]
[0269] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3200.6.
EXAMPLE 19
Preparation of Compound 19
Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
23]
[0270] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3143.5.
EXAMPLE 20
Preparation of Compound 20
Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
24]
[0271] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3198.6.
EXAMPLE 21
Preparation of Compound 21
Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
25]
[0272] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3141.5.
EXAMPLE 22
Preparation of Compound 22
Ala Gly Asp Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
26]
[0273] The above-identified peptide 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 Compound 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3170.6.
EXAMPLE 23
Preparation of Compound 23
Ala Gly Asp Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
27]
[0274] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3113.5.
EXAMPLE 24
Preparation of Compound 24
Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
28]
[0275] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3228.6.
EXAMPLE 25
Preparation of Compound 25
Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
29]
[0276] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3171.6.
EXAMPLE 26
Preparation of Compound 26
Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
30]
[0277] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3172.5.
EXAMPLE 27
Preparation of Compound 27
Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
31]
[0278] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3115.4.
EXAMPLE 28
Preparation of Compound 28
Ala Gly Asp Gly Thr Phe Thr Ser Asp Pentylgly Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 32]
[0279] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3230.4.
EXAMPLE 29
Preparation of Compound 29
Ala Gly Asp Gly Thr Phe Thr Ser Asp Pentylgly Ser Lys Gln Leu Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 33]
[0280] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3198.6.
EXAMPLE 30
Preparation of Compound 30
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
34]
[0281] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3141.5.
EXAMPLE 31
Preparation of Compound 31
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
35]
[0282] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3157.5.
EXAMPLE 32
Preparation of Compound 32
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
36]
[0283] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3100.4.
EXAMPLE 33
Preparation of Compound 33
Ala Gly Asp 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.sub.2 [SEQ. ID. NO.
37]
[0284] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3157.6.
EXAMPLE 34
Preparation of Compound 34
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
38]
[0285] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3100.5.
EXAMPLE 35
Preparation of Compound 35
Ala Gly Asp 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.sub.2 [SEQ. ID. NO.
39]
[0286] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3100.5.
EXAMPLE 36
Preparation of Compound 36
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
40]
[0287] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3154.5.
EXAMPLE 37
Preparation of Compound 37
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
41]
[0288] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3115.5.
EXAMPLE 38
Preparation of Compound 38
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Pentylgly Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 42]
[0289] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3212.4.
EXAMPLE 39
Preparation of Compound 39
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Pentylgly Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 43]
[0290] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3173.4.
EXAMPLE 40
Preparation of Compound 40
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Ala Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
44]
[0291] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3156.6.
EXAMPLE 41
Preparation of Compound 41
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Ala Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
45]
[0292] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3099.5.
EXAMPLE 42
Preparation of Compound 42
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Ala Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
46]
[0293] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3156.6.
EXAMPLE 43
Preparation of Compound 43
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Ala Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
47]
[0294] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3099.5.
EXAMPLE 44
Preparation of Compound 44
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Ala
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
48]
[0295] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3156.6.
EXAMPLE 45
Preparation of Compound 45
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Ala
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
49]
[0296] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3099.5.
EXAMPLE 46
Preparation of Compound 46
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Ala Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
50]
[0297] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3186.6.
EXAMPLE 47
Preparation of Compound 47
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2-[SEQ. ID. NO.
51]
[0298] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3129.5.
EXAMPLE 48
Preparation of Compound 48
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Ala Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
52]
[0299] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3129.5.
EXAMPLE 49
Preparation of Compound 49
Ala Gly Asp 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.sub.2 [SEQ. ID. NO.
53]
[0300] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3072.4.
EXAMPLE 50
Preparation of Compound 50
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Ala Phe Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
54]
[0301] The above-identified amidated peptide is assembled on
4-(21-4'-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 Compound 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3172.5.
EXAMPLE 51
Preparation of Compound 51
Ala Gly Asp 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.sub.2 [SEQ. ID. NO.
55]
[0302] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3115.5.
EXAMPLE 52
Preparation of Compound 52
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Naphthylala Ile Glu Trp Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 56]
[0303] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3266.4.
EXAMPLE 53
Preparation of Compound 53
Ala Gly Asp 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.sub.2 [SEQ.
ID. NO. 57]
[0304] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3209.4.
EXAMPLE 54
Preparation of Compound 54
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Val Glu Trp Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
58]
[0305] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3200.6.
EXAMPLE 55
Preparation of Compound 55
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Val Glu Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
59]
[0306] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3143.5.
EXAMPLE 56
Preparation of Compound 56
[0307] Ala Gly Asp 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.sub.2
[SEQ. ID. NO. 60]
[0308] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3216.5.
EXAMPLE 57
Preparation of Compound 57
[0309] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
Glu Glu Ala Val Arg Leu Phe tButylgly Glu Phe Leu Lys Asn-NH.sub.2
[SEQ. ID. NO. 61]
[0310] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3159.4.
EXAMPLE 58
Preparation of Compound 58
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Asp Trp Leu Lys Asn-NH.sub.2-[SEQ. ID. NO.
62]
[0311] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3200.6.
EXAMPLE 59
Preparation of Compound 59
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Asp Phe Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
63]
[0312] The above-identified amidated peptide is assembled on
4-(21-4'-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 Compound 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3143.5.
EXAMPLE 60
Preparation of Compound 60
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
64]
[0313] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3099.5.
EXAMPLE 61
Preparation of Compound 61
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn-NH.sub.2 [SEQ. ID. NO.
65]
[0314] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3081.4.
EXAMPLE 62
Preparation of Compound 62
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Ala Lys Asn-NH.sub.2 [SEQ. ID. NO.
66]
[0315] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3172.5.
EXAMPLE 63
Preparation of Compound 63
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Ala Lys Asn-NH.sub.2 [SEQ. ID. NO.
67]
[0316] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3115.5.
EXAMPLE 64
Preparation of Compound 64
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Ala Asn-NH.sub.2 [SEQ. ID. NO.
68]
[0317] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3157.5.
EXAMPLE 65
Preparation of Compound 65
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Ala Asn-NH.sub.2 [SEQ. ID. NO.
69]
[0318] The above-identified amidated peptide is assembled on
4-(21-4'-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 Compound 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3100.4.
EXAMPLE 66
Preparation of Compound 66
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Ala-NH.sub.2 [SEQ. ID. NO.
70]
[0319] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3171.6.
EXAMPLE 67
Preparation of Compound 67
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Ala-NH.sub.2 [SEQ. ID. NO.
71]
[0320] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3114.5.
EXAMPLE 68
Preparation of Compound 68
Ala 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 Ser Ser Gly
Ala Pro Pro Pro-NH.sub.2 [SEQ. ID. NO. 72]
[0321] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 4033.5.
EXAMPLE 69
Preparation of Compound 69
His Gly Ala 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.sub.2 [SEQ. ID. NO. 73]
[0322] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3984.4.
EXAMPLE 70
Preparation of Compound 70
His Gly Glu Ala 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 Ser Ser Gly
Ala Pro Pro-NH.sub.2 [SEQ. ID. NO. 74]
[0323] The above-identified amidated peptide is assembled on
4-(21-4'-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 Compound 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 4016.5.
EXAMPLE 71
Preparation of Compound 71
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly
Ala Pro-NH.sub.2 [SEQ. ID. NO. 75]
[0324] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3861.3.
EXAMPLE 72
Preparation of Compound 72
Ala Gly Glu Gly Thr Phe Thr Ser Asp Ala 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-NH.sub.2 [SEQ. ID. NO. 76]
[0325] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3746.1.
EXAMPLE 73
Preparation of Compound 73
Ala 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 Ser Ser Gly
Ala-NH.sub.2 [SEQ. ID. NO. 77]
[0326] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3742.1.
EXAMPLE 74
Preparation of Compound 74
His Gly Ala 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-NH.sub.2 [SEQ. ID. NO. 78]
[0327] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3693.1.
EXAMPLE 75
Preparation of Compound 75
His Gly Glu Ala 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 Ser Ser
Gly-NH.sub.2 [SEQ. ID. NO. 79]
[0328] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3751.2.
EXAMPLE 76
Preparation of Compound 76
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
Ser-NH.sub.2 [SEQ. ID. NO. 80]
[0329] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3634.1.
EXAMPLE 77
Preparation of Compound 77
Ala 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
Ser-NH.sub.2 [SEQ. ID. NO. 81]
[0330] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3526.9.
EXAMPLE 78
Preparation of Compound 78
His Gly Ala 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-NH.sub.2 [SEQ. ID. NO. 82]
[0331] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3477.9.
EXAMPLE 79
Preparation of Compound 79
His Gly Glu Ala 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.sub.2
[SEQ. ID. NO. 83]
[0332] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3519.9.
EXAMPLE 80
Preparation of Compound 80
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly-NH.sub.2 [SEQ.
ID. NO. 84]
[0333] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3307.7.
EXAMPLE 81
Preparation of Compound 81
Ala 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-NH.sub.2 [SEQ. ID.
NO. 85]
[0334] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3186.5.
EXAMPLE 82
Preparation of Compound 82
[0335] His Gly Ala 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 tPro
Ser Ser Gly Ala tPro tPro tPro-NH.sub.2 [SEQ. ID. NO. 86]
[0336] 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-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 37, 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 4121.1.
EXAMPLE 83
Preparation of Compound 83
[0337] His Gly Glu Ala 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 Ser
Ser Gly Ala tPro tPro tPro-NH.sub.2 [SEQ. ID. NO. 87]
[0338] 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-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 37, 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 4173.2.
EXAMPLE 84
Preparation of Compound 84
His Gly Glu Gly Thr Phe Thr Ser Ala 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 NMeala Nmeala-NH.sub.2 [SEQ. ID. NO. 88]
[0339] 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-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 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 3796.1.
EXAMPLE 85
Preparation of Compound 85
[0340] Ala 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 hPro
Ser Ser Gly Ala hPro-NH.sub.2 [SEQ. ID. NO. 89]
[0341] 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-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Compound 1.
A double coupling is required at residue 31. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 3871.1.
EXAMPLE 86
Preparation of Compound 86
His Gly Ala 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 Ser Ser Gly
Ala-NH.sub.2 [SEQ. ID. NO. 90]
[0342] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3750.2.
EXAMPLE 87
Preparation of Compound 87
His Gly Asp Ala 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.sub.2 [SEQ.
ID. NO. 91]
[0343] The above-identified amdiated peptide 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 Compound 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3408.8.
EXAMPLE 88
Preparation of Compound 88
Ala 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 Ser Ser Gly
Ala Pro Pro Pro Ser-NH.sub.2 [SEQ. ID. NO. 92]
[0344] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 4120.6.
EXAMPLE 89
Preparation of Compound 89
Ala Gly Ala 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 Ser-NH.sub.2 [SEQ. ID. NO. 93]
[0345] 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-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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 4005.5.
EXAMPLE 90
Preparation of Peptide having SEQ. ID. NO. 95
[0346] Compound No. 90, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys-NH.sup..epsilon.octanoyl Asn-NH.sub.2 [SEQ. ID. NO.
95], 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.sup..epsilon.octanoyl acid is used for
coupling at position 27. 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 resin.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in CAN). 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 (M): calculated 3361.7
EXAMPLE 91
Preparation of Peptide having SEQ. ID. NO. 96
[0347] Compound No. 91, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Phe Leu Lys-NH.sup..epsilon.octanoyl Asn-NH.sub.2 [SEQ. ID. NO.
96], 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.sup..epsilon.octanoyl acid is used for
coupling at position 27. 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 resin.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3304.6
EXAMPLE 92
Preparation of Peptide having SEQ. ID. NO. 97
[0348] Compound No. 92, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys-NH.sup..epsilon.octanoyl Asn Gly Gly-NH.sub.2 [SEQ.
ID. NO. 97], 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.sup..epsilon.octanoyl acid is
used for coupling at position 27. 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 (0.1%
TFA in water) and Solvent B (0.1% 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 (M):
calculated 3475.8
EXAMPLE 93
Preparation of Peptide having SEQ. ID. NO. 98
[0349] Compound No. 93, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Phe Leu Lys-NH.sup..epsilon.octanoyl Asn Gly Gly-NH.sub.2 [SEQ.
ID. NO. 98], 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.sup..epsilon.octanoyl acid is
used for coupling at position 27. 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 (0.1%
TFA in water) and Solvent B (0.1% 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 (M):
calculated 3418.7
EXAMPLE 94
Preparation of Peptide having SEQ. ID. NO. 99
[0350] Compound No. 94, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Asn Lys-NH.sup..epsilon.octanoyl-NH.sub.2 [SEQ. ID. NO.
99], 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.sup..epsilon.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 (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 3361.7
EXAMPLE 95
Preparation of Peptide having SEQ. ID. NO. 100
[0351] Compound No. 95, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Phe Leu Asn Lys-NH.sup..epsilon.octanoyl-NH.sub.2 [SEQ. ID. NO.
100], 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.sup..epsilon.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 residues 2-28 on the resin. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% 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 (M):
calculated 3304.6
EXAMPLE 96
Preparation of Peptide having SEQ. ID. NO. 101
[0352] Compound 96, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe Thr
Ser Ala Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Asn Lys-NH.sup..epsilon.octanoyl Gly Gly-NH.sub.2 [SEQ. ID.
NO. 101], 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.sup..epsilon.octanoyl 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 directly to the N-terminus
of protected residues 2-30 on the resin. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 3475.8
EXAMPLE 97
Preparation of Peptide having SEQ. ID. NO. 102
[0353] Compound No. 97, 4-imidazolylpropionyl-Gly Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Phe Leu Asn Lys-NH.sup..epsilon.octanoyl Gly Gly-NH.sub.2 [SEQ.
ID. NO. 102], 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.sup..epsilon.octanoyl acid is
used for coupling at position 28. Instead of using protected His
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 (0.1% TFA in water) and Solvent B
(0.1% 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 (M): calculated 3418.7
EXAMPLE 98
Preparation of Peptide having SEQ. ID. NO. 103
[0354] Compound No. 98, Ala 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.sup..epsilon.octanoyl Asn-NH.sub.2 [SEQ. ID. NO. 103], 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.sup..epsilon.octanoyl acid is used for coupling at
position 27. Used in analysis are Solvent A (0.1% TFA in water) and
Solvent B (0.1% 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. (M): calculated
3334.6
EXAMPLE 99
Preparation of Peptide having SEQ. ID. NO. 104
[0355] Compound No. 99, Ala 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-NH.sup..epsilon.octanoyl Asn-NH.sub.2 [SEQ. ID. NO. 104], 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.sup..epsilon.octanoyl acid is used for coupling at
position 27. Used in analysis are Solvent A (0.1% TFA in water) and
Solvent B (0.1% 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 (M): calculated
3277.6
EXAMPLE 100
Preparation of Peptide having SEQ. ID. NO. 105
[0356] Compound No. 100, Ala 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.sup..epsilon.octanoyl Asn Gly Gly-NH.sub.2 [SEQ. ID. NO.
105], 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.sup..epsilon.octanoyl acid is used for
coupling at position 27. Used in analysis are Solvent A (0.1% TFA
in water) and Solvent B (0.1% 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 (M):
calculated 3442.8
EXAMPLE 101
Preparation of Peptide having SEQ. ID. NO. 106
[0357] Compound No. 101, Ala 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-NH.sup..epsilon.octanoyl Asn Gly Gly-NH.sub.2 [SEQ. ID. NO.
106], 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.sup..epsilon.octanoyl acid is used for
coupling at position 27. Used in analysis are Solvent A (0.1% TFA
in water) and Solvent B (0.1% 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 (M):
calculated 3391.7
EXAMPLE 102
Preparation of Peptide having SEQ. ID. NO. 107
[0358] Compound No. 102, Ala 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-NH.sup..epsilon.octanoyl-NH.sub.2 [SEQ. ID. NO. 107], 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.sup..epsilon.octanoyl acid is used for the initial
coupling onto the resin at position 28. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 3334.6
EXAMPLE 103
Preparation of Peptide having SEQ. ID. NO. 108
[0359] Compound No. 103, Ala 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-NH.sup..epsilon.octanoyl-NH.sub.2 [SEQ. ID. NO. 108], 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.sup..epsilon.octanoyl acid is used for the initial
coupling onto the resin at position 28. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 (M): calculated 3277.6
EXAMPLE 104
Preparation of Peptide having SEQ. ID. NO. 109
[0360] Compound No. 104, Ala 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-NH.sup..epsilon.octanoyl Gly Gly-1H.sub.2 [SEQ. ID. NO. 109],
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.sub..epsilon.octanoyl acid is used for coupling at
position 28. Used in analysis are Solvent A (0.1% TFA in water) and
Solvent B (0.1% 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 (M): calculated
3442.8
EXAMPLE 105
Preparation of Peptide having SEQ. ID. NO. 110
[0361] Compound No. 105, Ala 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-NH.sup..epsilon.octanoyl Gly Gly-NH.sub.2 [SEQ. ID. NO. 110],
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.sup..epsilon.octanoyl acid is used for coupling at
position 28. Used in analysis are Solvent A (0.1% TFA in water) and
Solvent B (0.1% 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 (M): calculated
3391.7
EXAMPLE 106
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to
the Above C-Terminal Amide Sequences for Compounds 1-67, 73-79,
80-81, 86-89 and 90-105
[0362] C-terminal carboxylic acid peptides corresponding to
amidated Compounds 1-67, 73-79, 80-81, 86-89 and 90-105 are
assembled on 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 that described in Example 1. Used in analysis
are Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 provides an experimentally determined (M).
EXAMPLE 107
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to
the Above C-Terminal Amide Sequences for Compounds 68-72, 79 and
82-85
[0363] C-terminal carboxylic acid peptides corresponding to
amidated Compounds 68-72, 79 and 82-85 are assembled on the
2-chlorotritylchloride resin (200-400 mesh), 2% DVB (Novabiochem,
0.4-1.0 mmole/g)) using Fmoc-protected amino acids (Applied
Biosystems, Inc.), cleaved from the resin, deprotected and purified
in a similar way to that described in Example 1. Used in analysis
are Solvent A (0.1% TFA in water) and Solvent B (0.1% 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 provides an experimentally determined (M).
EXAMPLES A TO E
Reagents Used
[0364] GLP-1[7-36]NH.sub.2 (GLP-1) was purchased from Bachem
(Torrance, Calif.). All other peptides were prepared using
synthesis methods such as those described therein. All chemicals
were of the highest commercial grade. The cAMP SPA immunoassay was
purchased from Amersham. The radioligands were purchased from New
England Nuclear (Boston, Mass.). RINm5f cells (American Type Tissue
Collection, Rockville, Md.) were grown in DME/F12 medium containing
10% fetal bovine serum and 2 mM L-glutamine. Cells were grown at
37.degree. C. and 5% CO.sub.2/95% 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.degree. C. until used.
Example A
GLP-1 Receptor Binding Studies
[0365] Receptor binding was assessed by measuring displacement of
[.sup.125I]GLP-1 or [.sup.125I]exendin(9-39) from RINm5f membranes.
Assay buffer contained 5 .mu.g/ml bestatin, 1 .mu.g/ml
phosphoramidon, 1 mg/ml bovine serum albumin (fraction V), 1 mg/ml
bacitracin, and 1 mM MgCl.sub.2 in 20 mM HEPES, pH 7.4. To measure
binding, 30 .mu.g membrane protein (Bradford protein assay) was
resuspended in 200 .mu.l assay buffer and incubated with 60 pM
[.sup.125I]GLP-1 or exendin (9-39) and unlabeled peptides for 120
minutes at 23.degree. C. in 96 well plates (Nagle Nunc, Rochester,
N.Y.). Incubations were terminated by rapid filtration with cold
phosphatebuffered saline, pH 7.4, through polyethyleneimine-treated
GF/B glass fiber filters (Wallac Inc., Gaithersburg, Md.) using a
Tomtec Mach II plate harvester (Wallac Inc., Gaithersburg, Md.).
Filters were dried, combined with scintillant, and radioactivity
determined in a Betaplate liquid scintillant counter (Wallac
Inc.).
[0366] Peptide samples were run in the assay as duplicate points at
6 dilutions over a concentration range of 10.sup.-6M to 10.sup.-12M
to generate response curves. The biological activity of a sample is
expressed as an IC.sub.50 value, calculated from the raw data using
an iterative curve-fitting program using a 4-parameter logistic
equation (Prizm.TM., GraphPAD Software). The results are shown in
Table I.
TABLE-US-00001 TABLE I Compound IC.sub.50 (nM) Exendin-4 [SEQ. ID.
NO. 2] 0.7 Compound 1 [SEQ. ID. NO. 5] 26.1 Compound 2 [SEQ. ID.
NO. 6] 14.42 Compound 3 [SEQ. ID. NO. 7] 41.65 Compound 4 [SEQ. ID.
NO. 8] 4.96
Example B
Cyclase Activation Study
[0367] Assay buffer contained 10 .mu.M GTP, 0.75 mM ATP, 2.5 mM
MgCl.sub.2, 0.5 mM phosphocreatine, 12.5 U/ml creatine kinase, 0.4
mg/ml aprotinin, 1 .mu.M 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, Mass.). After 20
minutes incubation at 37.degree. 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.
[0368] Peptide samples were run in the assay as triplicate points
at 7 dilutions over a concentration range of 10.sup.-6M to
10.sup.-12M to generate response curves. The biological activity of
a particular sample was expressed as an EC.sub.50 value, calculated
as described above. Results are tabulated in Table II.
TABLE-US-00002 TABLE II Compound EC.sub.50 (nM) Exendin-4 [SEQ. ID.
NO. 2] 0.23 Compound 1 [SEQ. ID. NO. 5] >1,000 Compound 2 [SEQ.
ID. NO. 6] >10,000 Compound 3 [SEQ. ID. NO. 7] >10,000
Compound 4 [SEQ. ID. NO. 8] >10,000
Example C
Determination of Blood Glucose Levels in db/db Mice
[0369] C57BLKS/J-m-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.degree..+-.1.degree. 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 taking
baseline blood samples. Approximately 70 .mu.l 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 (10.9% NaCl), exendin-4 or test
compound (1 .mu.g) in vehicle. Blood samples were drawn again,
using the same procedure, after exactly one hour from the
injections, and plasma glucose concentrations were measured.
[0370] For each animal, the % change in plasma value, from baseline
value, was calculated. The percent decrease in plama glucose after
one hour is shown in Table III.
TABLE-US-00003 TABLE III Test Compound % drop in glucose Exendin-4
[SEQ. ID. NO. 2] 39% (n = 78) Compound 1 [SEQ. ID. NO. 5] 40% (n =
4) Compound 2 [SEQ. ID. NO. 6] 41% (n = 5) Compound 3 [SEQ. ID. NO.
7] 32% (n = 5) Compound 4 [SEQ. ID. NO. 8] 42% (n = 5)
Example D
Dose Response Determination of Blood Glucose Levels in db/db
Mice
[0371] 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.degree. C. 1.degree.
C. with a 12:12 light:dark cycle, with lights on at 6 a.m.
[0372] All animals were deprived of food for 2 hours before taking
baseline blood samples. Approximately 70 .mu.l 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.
[0373] For each animal, the % change in plasma value, from baseline
value, was calculated and a dose dependent relationship was
evaluated using Graphpad Prizm.TM. software.
[0374] FIG. 5 depicts the effects of varying doses of exendin-4
[SEQ. ID. NO. 2] and Compound 1 [SEQ. ID. NO. 5] on plasma glucose
levels. Exendin-4 had an ED.sub.50 of 0.01 .mu.g per mouse and
Compound 1 had an ED.sub.50 of 0.42 .mu.g per mouse.
Example E
Gastric Emptying
[0375] The following study was carried out to examine the effects
of exendin-4 and an exendin agonist compound of the present
invention on gastric emptying in rats. This experiment followed a
modification of the method of Scarpignato, et al., Arch. Int.
Pharmacodyn. Ther. 246:286-94, 1980.
[0376] 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, Wis.).
Exendin-4 was synthesized according to standard peptide synthesis
methods. The preparation of Compound 1 [SEQ. ID. NO. 5] is
described in Example 1.
[0377] The determination of gastric emptying by the method
described below was performed after a fast of .about.20 hours to
ensure that the stomach contained no chyme that would interfere
with spectrophotometric absorbance measurements.
[0378] Conscious rats received by gavage, 1.5 ml 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 5% 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 20
min)/(absorbance at 0 mm).times.100.
[0379] 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 .mu.g of exendin-4,
and 0.1, 0.3, 1, 10 and 100 .mu.g of Compound 1 [SEQ. ID. NO.
5].
[0380] The results, shown in FIG. 6, demonstrate that the exendin
agonists, exendin-4 and Compound 1, are potent inhibitors of
gastric emptying. The EC.sub.50 for exendin-4 was 0.27 .mu.g. The
EC.sub.50 for Compound 1 was 55.9 .mu.g.
Sequence CWU 1
1
110139PRTHeloderma horridumc-term amidation 1His Ser Asp Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser20 25 30Ser Gly Ala
Pro Pro Pro Ser35239PRTHeloderma suspectumc-term amidation 2His Gly
Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser20 25
30Ser Gly Ala Pro Pro Pro Ser35330PRTHomo sapiensc-term amidation
3His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5
10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg20 25
30439PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 4Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Gly Gly Xaa Ser20 25 30Ser Gly Ala Xaa Xaa Xaa
Xaa35528PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 5Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu
Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe
Leu Lys Asn20 25628PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 6His Gly Ala Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 25728PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
7His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5
10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
25828PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 8His Gly Glu Gly Thr Phe Thr Ser Ala Leu
Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe
Leu Lys Asn20 25928PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 9Ala Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 251028PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
10His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
251128PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 11His Gly Glu Ala Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 251228PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 12His Gly Glu Gly Thr Phe Thr
Ser Ala Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 251328PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
13His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
251428PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 14Ala Ala Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 251528PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 15Ala Ala Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 251628PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
16Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
251728PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 17Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn20 251828PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 18Ala Gly Asp Gly Ala Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 251928PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
19Ala Gly Asp Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
252028PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 20Ala Gly Asp Gly Thr Xaa Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 252128PRTArtificial Sequence/note="Description of
Artificial Sequence 7 Syntheticpeptide" 21Ala Gly Asp Gly Thr Xaa
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Phe Leu Lys Asn20 252228PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
22Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
252328PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 23Ala Gly Asp Gly Thr Phe Ser Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn20 252428PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 24Ala Gly Asp Gly Thr Phe Thr
Ala Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 252528PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
25Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
252628PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 26Ala Gly Asp Gly Thr Phe Thr Ser Ala
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 252728PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 27Ala Gly Asp Gly Thr Phe Thr
Ser Ala Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 252828PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
28Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
252928PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 29Ala Gly Asp Gly Thr Phe Thr Ser Glu
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn20 253028PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 30Ala Gly Asp Gly Thr Phe Thr
Ser Asp Ala Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 253128PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
31Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
253228PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 32Ala Gly Asp Gly Thr Phe Thr Ser Asp
Xaa Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 253328PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 33Ala Gly Asp Gly Thr Phe Thr
Ser Asp Xaa Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 253428PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
34Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
253528PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 35Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ala Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn20 253628PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 36Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Ala Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 253728PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
37Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
253828PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 38Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Ala Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 253928PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 39Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Ala Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 254028PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
40Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
254128PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 41Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Ala Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn20 254228PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 42Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Xaa Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 254328PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
43Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
254428PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 44Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Ala Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 254528PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 45Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Ala Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 254628PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
46Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Ala1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn20
254728PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 47Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Ala1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn20 254828PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 48Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Ala Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn20 254928PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
49Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Ala Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn20
255028PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 50Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Ala Arg Leu Phe Ile Glu
Trp Leu Lys Asn20 255128PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 51Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Ala Arg Leu Phe
Ile Glu Phe Leu Lys Asn20 255228PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
52Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Ala Leu Phe Ile Glu Trp Leu Lys Asn20
255328PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 53Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Ala Leu Phe Ile Glu
Phe Leu Lys Asn20 255428PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 54Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Ala Phe
Ile Glu Trp Leu Lys Asn20 255528PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
55Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Ala Phe Ile Glu Phe Leu Lys Asn20
255628PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 56Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Xaa Ile Glu
Trp Leu Lys Asn20 255728PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 57Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Xaa
Ile Glu Phe Leu Lys Asn20 255828PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
58Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Val Glu Trp Leu Lys Asn20
255928PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 59Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Val Glu
Phe Leu Lys Asn20 256028PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 60Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Xaa Glu Trp Leu Lys Asn20 256128PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
61Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Xaa Glu Phe Leu Lys Asn20
256228PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 62Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Asp
Trp Leu Lys Asn20 256328PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 63Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln
Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Asp Phe Leu Lys
Asn20 256428PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 64Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Ala Leu Lys Asn20 256528PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 65Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Ala Leu Lys Asn20 256628PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
66Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Ala Lys Asn20
256728PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 67Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Ala Lys Asn20 256828PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 68Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Ala Asn20 256928PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
69Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Ala Asn20
257028PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 70Ala Gly Asp Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Ala20 257128PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 71Ala Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Ala20 257238PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
72Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro Pro Pro357338PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
73His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro Pro Pro357437PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
74His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro Pro357536PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
75His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro357636PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
76Ala Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro357735PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
77Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala357834PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
78His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly7935PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 79His Gly Glu Ala Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser20 25 30Ser Gly
Ala8033PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 80His Gly Glu Gly Thr Phe Thr Ser Ala
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Pro Ser20 25 30Ser8132PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
81Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser20 25 308232PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 82His Gly Ala Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser20 25 308331PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
83His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
Pro20 25 308430PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 84His Gly Glu Gly Thr Phe Thr
Ser Ala Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly20 25 308529PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
85Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly20
258638PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 86His Gly Ala Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser20 25 30Ser Gly Ala Xaa Xaa
Xaa358738PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 87His Gly Glu Ala Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Pro Ser20 25 30Ser Gly Ala Xaa Xaa
Xaa358837PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 88His Gly Glu Gly Thr Phe Thr Ser Ala
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser20 25 30Ser Gly Ala Xaa
Xaa358936PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 89Ala Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser20 25 30Ser Gly Ala
Xaa359035PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 90His Gly Ala Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Pro Ser20 25 30Ser Gly
Ala359130PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 91His Gly Asp Ala Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly20 25 309239PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
92Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro Pro Pro Ser359339PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
93Ala Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser20 25 30Ser Gly Ala Pro Pro Pro Ser359441PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
94Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
Gly20 25 30Xaa Ser Ser Gly Ala Xaa Xaa Xaa Xaa35
409527PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 95Xaa Glu Gly Thr Phe Thr Ser Ala Leu
Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu Phe Ile Glu Trp
Leu Xaa Asn20 259627PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 96Xaa Glu Gly Thr Phe Thr Ser
Ala Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu Phe Ile
Glu Phe Leu Xaa Asn20 259729PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
97Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1
5 10 15Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn Gly Gly20
259829PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 98Xaa Glu Gly Thr Phe Thr Ser Ala Leu
Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu Phe Ile Glu Phe
Leu Xaa Asn Gly Gly20 259927PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
99Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1
5 10 15Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa20
2510027PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 100Xaa Glu Gly Thr Phe Thr Ser Ala Leu
Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu Phe Ile Glu Phe
Leu Asn Xaa20 2510129PRTArtificial Sequence/note="Description of
Artificial Sequence Syntheticpeptide" 101Xaa Glu Gly Thr Phe Thr
Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu Phe
Ile Glu Trp Leu Asn Xaa Gly Gly20 2510229PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
102Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu1
5 10 15Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa Gly Gly20
2510328PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 103Ala Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Xaa Asn20 2510428PRTArtificial Sequence/note="Description
of Artificial Sequence Syntheticpeptide" 104Ala Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Phe Leu Xaa Asn20 2510530PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
105Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn Gly Gly20 25
3010630PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 106Ala Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Xaa Asn Gly Gly20 25 3010728PRTArtificial
Sequence/note="Description of Artificial Sequence Syntheticpeptide"
107Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa20
2510828PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 108Ala Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Asn Xaa20 2510930PRTArtificial Sequence/note="Description
of Artificial Sequence Synthetic peptidepeptide" 109Ala Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Asn Xaa Gly Gly20 25
3011030PRTArtificial Sequence/note="Description of Artificial
Sequence Syntheticpeptide" 110Ala Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Asn Xaa Gly Gly20 25 30
* * * * *