U.S. patent application number 14/736971 was filed with the patent office on 2015-10-01 for novel gh-rh analogs with potent agonistic effects.
The applicant listed for this patent is UNITED STATES OF AMERICA, REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS, UNIVERSITY OF MIAMI. Invention is credited to Ren Zhi CAI, Andrew V. SCHALLY, Marta ZARANDI.
Application Number | 20150274798 14/736971 |
Document ID | / |
Family ID | 48669348 |
Filed Date | 2015-10-01 |
United States Patent
Application |
20150274798 |
Kind Code |
A1 |
SCHALLY; Andrew V. ; et
al. |
October 1, 2015 |
NOVEL GH-RH ANALOGS WITH POTENT AGONISTIC EFFECTS
Abstract
There are provided a novel series of peptide analogs of
hGH-RH(1-29)NH.sub.2 and hGH-RH(1-30)NH.sub.2 which show high
activities in stimulating the release of pituitary GH in animals.
They retain their physiological activity in solution for extended
periods of time and resist enzymic degradation in the body. These
novel and useful properties appear to be due to novel substitution
patterns ant at the 1, 15, 27 and 29 positions on the peptide.
Inventors: |
SCHALLY; Andrew V.; (Miami
Beach, FL) ; CAI; Ren Zhi; (Miami, FL) ;
ZARANDI; Marta; (Szeged, HU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MIAMI
UNITED STATES OF AMERICA, REPRESENTED BY THE DEPARTMENT OF VETERANS
AFFAIRS |
Miami
Washington |
FL
DC |
US
US |
|
|
Family ID: |
48669348 |
Appl. No.: |
14/736971 |
Filed: |
June 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13332624 |
Dec 21, 2011 |
9079974 |
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14736971 |
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Current U.S.
Class: |
514/11.4 ;
530/324 |
Current CPC
Class: |
A61P 5/06 20180101; A61P
9/00 20180101; A61P 3/00 20180101; A61K 38/00 20130101; C07K 14/60
20130101; A61P 43/00 20180101; C07K 14/61 20130101 |
International
Class: |
C07K 14/60 20060101
C07K014/60 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made in part with Government support. The
Government has certain rights in this application.
Claims
1. A GH-RH agonist peptide having the formula: [R.sub.1-A.sup.1,
A.sup.2, A.sup.6, A.sup.8, A.sup.11, A.sup.12, A.sup.15, A.sup.20,
A.sup.21, A.sup.22, Nle.sup.27, A.sup.28,
Agm.sup.29]hGH-RH(1-29)NH.sub.2, wherein R.sub.1 is Ac, Tfa, or is
absent, A.sup.1 is Tyr, Dat, or N-Me-Tyr, A.sup.2 is Ala, D-Ala,
Abu, or D-Abu, A.sup.6 is Phe or Fpa5, A.sup.8 is Asn, Ala, Gln,
Thr, or N-Me-Ala, A.sup.11 is Arg, His, or Har, A.sup.12 is Orn, or
Lys(Me).sub.2, A.sup.15 is His, Abu, Aib, Aah, Aap, Ala, or D-Ala
A.sup.20 is Arg, His, or Har, A.sup.21 is Orn, or Lys(Me).sub.2,
A.sup.22 is Leu, or Orn, A.sup.28 is Ser, or Asp, and
pharmaceutically acceptable salts thereof.
2. The GH-RH agonist peptide of claim 1, wherein: R.sub.1 is Ac or
is absent, A.sup.1 is Tyr, Dat, or N-Me-Tyr, A.sup.2 is Ala, D-Ala,
Abu, or D-Abu, A.sup.6 is Phe or Fpa5, A.sup.8 is Asn, Ala, Gln, or
Thr, A.sup.12 is Orn, A.sup.15 is Abu, A.sup.21 is Orn, A.sup.28 is
Ser, or Asp, and pharmaceutically acceptable salts thereof.
3. The GH-RH agonist peptide of claim 1, wherein: A.sup.1 is Dat or
N-Me-Tyr, A.sup.2 is Ala or D-Ala, A.sup.6 is Phe or Fpa5, A.sup.8
is Asn, Gln, or Thr, A.sup.12 is Orn, A.sup.15 is Abu, A.sup.21 is
Orn, A.sup.28 is Ser, or Asp, and pharmaceutically acceptable salts
thereof.
4. The GH-RH agonist peptide of claim 1, wherein the GH-RH agonist
peptide is selected from the group consisting of: P-20103
[N-Me-Tyr.sup.1, Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 2); P-20105 [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20107 [N-Me-Tyr.sup.1,
Fpa5.sup.6, Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 3);
P-20109 [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29); P-20110 [N-Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29); P-20111 [N-Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20113
[N-Me-Tyr.sup.1, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 4);
P-20115 [N-Me-Tyr.sup.1, Fpa5.sup.6, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 5); P-20117 [N-Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29); P-20350
[Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29);
P-20351 [Ac-N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29); P-20356 [N-Me-Tyr.sup.1, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 6); P-20357 [Dat.sup.1,
D-Ala.sup.2, N-Me-Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20358
[N-Me-Tyr.sup.1, D-Ala.sup.2, N-Me-Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29); P-20359 [N-Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20360 [N-Me-Tyr.sup.1,
D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20361
[N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29);
P-20367 [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29);
P-20370 [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 7);
P-20371 [N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 8); P-20372 [N-Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 9); P-20373 [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 10);
P-20374 [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 11); P-20375
[N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 12); and P-20376
[N-Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 13).
5. The GH-RH agonist peptide of claim 1, wherein the GH-RH peptide
is: P-20356 [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 6).
6. The GH-RH agonist peptide of claim 1, wherein the GH-RH peptide
is: P-20350 [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGHRH(1-29).
7. The GH-RH agonist peptide of claim 1, wherein the GH-RH peptide
is: P-20367 [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGHRH(1-29).
8. A pharmaceutical composition comprising a therapeutically
effective amount of GH-RH agonist peptide having the formula:
[R.sub.1-A.sup.1, A.sup.2, A.sup.6, A.sup.8, A.sup.11, A.sup.12,
A.sup.15, A.sup.20, A.sup.21, A.sup.22, Nle.sup.27, A.sup.28,
Agm.sup.29]hGH-RH(1-29)NH.sub.2, wherein R.sub.1 is Ac, Tfa, or is
absent, A.sup.1 is Tyr, Dat, or N-Me-Tyr, A.sup.2 is Ala, D-Ala,
Abu, or D-Abu, A.sup.6 is Phe or Fpa5, A.sup.8 is Asn, Ala, Gln,
Thr, or N-Me-Ala, A.sup.11 is Arg, His, or Har, A.sup.12 is Orn, or
Lys(Me).sub.2, A.sup.15 is His, Abu, Aib, Aah, Aap, Ala, or D-Ala
A.sup.20 is Arg, His, or Har, A.sup.21 is Orn, or Lys(Me).sub.2,
A.sup.22 is Leu, or Orn, A.sup.28 is Ser, or Asp, and
pharmaceutical salts thereof, and a pharmaceutically acceptable
excipient.
9. The pharmaceutical composition of claim 8, wherein the GH-RH
peptide is selected from the group consisting of: P-20103
[N-Me-Tyr.sup.1, Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 2); P-20105 [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20107 [N-Me-Tyr.sup.1,
Fpa5.sup.6, Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 3);
P-20109 [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29); P-20110 [N-Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29); P-20111 [N-Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20113
[N-Me-Tyr.sup.1, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 4);
P-20115 [N-Me-Tyr.sup.1, Fpa5.sup.6, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 5); P-20117 [N-Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29); P-20350
[Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29);
P-20351 [Ac-N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29); P-20356 [N-Me-Tyr.sup.1, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 6); P-20357 [Dat.sup.1,
D-Ala.sup.2, N-Me-Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20358
[N-Me-Tyr.sup.1, D-Ala.sup.2, N-Me-Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29); P-20359 [N-Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20360 [N-Me-Tyr.sup.1,
D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29); P-20361
[N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29);
P-20367 [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29);
P-20370 [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 7);
P-20371 [N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 8); P-20372 [N-Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 9); P-20373 [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 10);
P-20374 [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 11); P-20375
[N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 12); and P-20376
[N-Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 13).
10. The pharmaceutical composition of claim 8, wherein the GH-RH
peptide is: P-20356 [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 6).
11. The pharmaceutical composition of claim 8, wherein the GH-RH
peptide is: P-20350 [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGHRH(1-29).
12. The pharmaceutical composition of claim 8, wherein the GH-RH
peptide is: P-20367 [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGHRH(1-29).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of application Ser. No.
13/332,624 filed on Dec. 21, 2011, which is incorporated herein by
reference in its entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 13, 2011, is named SHAL3041.txt and is 35,007 bytes in
size.
FIELD OF THE INVENTION
[0004] The present invention relates to growth hormone-releasing
hormone (GH-RH) analogous peptides having high binding affinity to
GH-RH receptor in vitro and having influence on the function of the
pituitary gland in vivo. In particular, the present invention is
directed to synthetic peptides which promote the release of growth
hormone by the pituitary gland. More particularly, the present
invention relates to hGH-RH analogs of 29 or 30 amino acids that
show much higher affinity to the GH-RH receptor than the native
hGH-RH(1-29)NH.sub.2.
[0005] This invention also relates to a pharmaceutical composition
comprising any one of said GH-RH agonists and to the use of these
agonistic peptides in the treatment or prevention of disorders.
BACKGROUND OF THE INVENTION
[0006] In 1981 human pancreatic growth hormone releasing hormone
(hGH-RH) was first isolated from extracts of human pancreatic
tumors and subsequently from the hypothalamus of various mammals.
This peptide was found to promote the release of growth hormone
(GH) by the pituitary. The human hypothalamic GH-RH was found to
have the same amino acid sequence as the pancreatic one. Human
GH-RH (hGH-RH) contains 44 amino acids with an amidated carboxyl
terminus. The structure of hGH-RH was reproduced by synthesis.
Several analogs of hGH-RH have been synthesized and their
biological activity studied. These studies revealed that:
[0007] a) a fragment of GH-RH containing at least 29 amino acid
residues has at least 50% of the potency of natural GH-RH; further
deletion of amino acid residues results in a marked decrease in
bioactivity [Cambell R M et al. Peptides 12: 569-574 (1991)];
[0008] b) replacement of Arg in position 29 by Agm (agmatine,
4-guanidino-butylamine) is said to provide resistance to enzymatic
degradation from C-terminus (Bajusz S et al. in Peptides, 1982,
Blaha and Melon, Eds., Walter de Gruyter, Berlin-N.Y., 1983, pp.
643-647);
[0009] c) replacement of Tyr in position 1 by des-aminotyrosine
(Dat) is said to lead to analogs with increased biological
activities as a result of the resistance of N-terminus to enzymatic
degradation [Felix A et al. Int. J. Peptide Protein Res. 32:
441-454 (1988), Kovacs M et al. Life Sci. 42: 27-35 (1988)]. U.S.
Pat. Nos. 4,622,312, 4,649,131 and 4,784,987 disclose hGH-RH(1-29)
agonists with Ala at position 15, as well as Dat at position 1.
Several of these agonists are said to have potency four-fold that
of hGH-RH(1-29);
[0010] d) several analogs containing Dat in position 1 and Agm in
position 29 are said to exhibit enhanced GH releasing ability, and
hence potency greater than that of hGH-RH(1-29) and in vivo
[Zarandi M et al. Int. J. Peptide Protein Res. 36: 499-505 (1990),
Zarandi M et al. Int. J. Peptide Protein Res. 39: 211-217
(1992)];
[0011] e) hydrophobic groups at the C-terminal of a peptides
sequence can result in significantly increased specific activity.
In terms of hydrophobicity, these results are contradicted by works
of other groups, e.g. Muranichi [Muranichi S et al. Pharm. Res. 8:
649-652 (1991)];
[0012] Similarly, U.S. Pat. No. 4,914,189 discloses agonists having
Dat at position 1, D-Ala at position 2, Lys or Orn at position 12,
Gly at position 15, Lys at position 21 and Agm at position 29. It
should be noted however that those agonists said to have had
greater potency in inducing GH release than hGH-RH(1-29) had Lys at
position 12 and D-Ala at position 2.
[0013] In U.S. Pat. No. 4,689,318 analogs of hGH-RH(1-29) may have
Lys.sup.12 or Lys.sup.21 replaced by Orn and in which position 8
could be Ser (rat GH-RH sequence), Asn (human GH-RH sequence), Thr
(mouse GH-RH sequence) or Gln (not naturally occurring in GH-RH);
and position 27 could be Nle. In these analogs, position 1 is never
Dat, position 15 never Abu, position 28 never Asp, and position 29
never Agm, Arg-NH--CH.sub.3, or Arg-NH--CH.sub.2--CH.sub.3. Those
analogs said to have potency as strong as hGH-RH(1-40) had
substitutions by Arg at positions 12 and 21.
[0014] Other hGH-RH(1-29) agonists are taught in PCT patent
applications numbers WO 94/1396 and 94/1397, where at position 12,
Lys.sup.12 is to be replaced by D-Lys, Arg or Orn. These analogs
may also contain Dat as R.sup.1; Asn, D-Asn, Ser, D-Ser as R.sup.8;
Abu as R.sup.15; Lys, D-Lys, Arg or D-Arg as R.sup.21; Nle as
R.sup.27; Asp or Ser as R.sup.28; and Agm as R.sup.29. Those
agonists which are said to induce GH at levels exceeding those
induced by hGH-RH(1-29) had Lys at positions 12. The teachings of
these two publications however are considered open to question
since, some time after the filing of these applications, it was
discovered that the compounds believed to have been synthesized
could not with certainty be said to correspond with the formulae
they were originally paired with. Moreover, it was further
discovered after filing these applications that the compounds could
not release GH at the levels originally asserted.
[0015] European Patent Application 0 413 839 discloses further
hGH-RH analogs in which positions 12 and 21 may both be Lys or Orn,
and where position 15 is Ala. However, those analogs tested for GH
releasing ability and said to have greater potency than
hGH-RH(1-29) had Lys at positions 12 and 21.
[0016] U.S. Pat. No. 5,262,519 discloses agonists having the
following substitutions: N-MeTyr at position 1, Ser at position 8,
Ala at position 15, Ala or Aib at positions 16, 24, and 25, Asn at
position 28, and --NHR at the C-terminus with R being H or lower
alkyl. It should be noted however that these agonists said to have
had greater potency in inducing GH release than hGH-RH(1-29) never
had Gln or Thr at position 8, Orn at positions 12 and 21, Abu at
position 15, and Asp at position 28.
[0017] U.S. Pat. No. 5,792,747 discloses GH-RH agonists having Dat
at position 1; Glu at position 3; Ser, Gln, or Thr at position 8;
Orn at positions 12 and 21; Ile at position 13; Ala or Abu at
positions 15, 22, and 23; Glu at position 25; Nle, Ile, or Leu at
position 27; Asn or Asp at position 28; and Agm at position 29. It
should be noted however that those agonists said to have had
greater potency in inducing GH release than hGH-RH(1-29) never had
N-Me-Tyr.sup.1, D-Ala.sup.2, and/or --NH--CH.sub.3 or
--NH--CH.sub.2--CH.sub.3 at the C-terminus.
[0018] U.S. Pat. No. 7,928,063 discloses GH-RH agonists having the
following substitutions: Dat at position 1; 6-guanidino-2-caproic
acid (hArg), 4-guanidine-2-aminobutyric acid (Gab), or
3-guanidino-2-aminopropionic acid (Gap) at positions 11 and 20;
Orn, hArg, Gab, or Gap at positions 12 and 21; Ala at position 15,
Nle at position 27; and D-Arg, hArg, Gab, or Gap at position 29.
These peptides are said to exhibit high resistance to enzymatic
actions and are potent and selective GH release stimulators. It
should be noted however that these agonists have been tested and
showed high stability only in trypsin digestion test.
[0019] Up to now, most of the GH-RH analogs described have been
tested in rat models, either in vitro or in vivo. Since human and
rat GRF(1-29)NH.sub.2 are markedly different, the
structure-activity relationships of GH-RH are different in both
species. Therefore, it is not possible to extrapolate results
obtained in rats to humans. (Brazeau et al. U.S. Pat. No.
6,458,764).
[0020] Other hGH-RH(1-29) analogs are taught in US published
application 20090023646 A1 and WO 2009009727 A2, where the most
potent compound has Ala.sup.2, Tyr.sup.10, Gly.sup.15, and
Leu.sup.22 replaced by D-Ala.sup.2, D-Tyr.sup.10, D-Ala.sup.15, and
Lys.sup.22, respectively, showed binding to GH-RH receptor on
somatotrophs in rat and dog pituitaries and was at least two times
more potent in vivo than the natural GH-RH(1-44).
[0021] An analog of hGH-RH(1-44)NH.sub.2 (tesamorelin) that was
modified by trans-3-hexenoyl group at the N-terminus showed
resistance against DPP-IV catalyzed deactivation [Ferninandi E S et
al. Basic Clin Pharmacol Toxicol. 100: 49-58 (2007) and Falutz J et
al. Acquir Immune Defic Syndr 53: 311-322, (2010)]. It should be
mentioned however that this agonist was not protected against
endopeptidases and chemical degradation in aqueous solution and was
only about twice as active as GH-RH itself.
[0022] Native hGH-RH(1-44) and its analogs are subject to
biological inactivation by both enzymatic and chemical routes. In
plasma, hGH-RH is rapidly degraded via dipeptidylpeptidase IV
(DPP-IV) cleavage between residues 2 and 3 [Frohman et al., J.
Clin. Invest. 83, 1533-1540 (1989), Kubiak et al. Drug Met. Disp.
17, 393-397 (1989)]. the major cleavage site in plasma. In
pituitary and hypothalamus, the major cleavage sites are between
Leu.sup.14-Gly.sup.15 (chymotrypsin-like enzymes) and between
Lys.sup.21-Leu.sup.22 (trypsin-like enzymes) [Boulanger et al.
Brain Res. 616, 39-47 (1993)]. Other trypsin specific cleavages at
basic amino acid residues are also observed. The hGH-RH(1-44) is
also subject to chemical rearrangement [forming Asp.sup.8 or
beta-Asp.sup.8 from Asn.sup.8 via aminosuccinimide formation] and
oxidation [Met(O).sup.27 from Met.sup.27] in aqueous environment
that greatly reduce its bioactivity.
[0023] It is therefore advantageous to develop long-acting GH-RH
analogues using specific amino acid replacements at the
amino-terminus (to prevent enzymatic degradation), at residue 8 (to
reduce isomerization), and residue 27 (to prevent oxidation).
Inclusion of Ala15 or Abu15 substitutions for Gly 15, previously
demonstrated to enhance receptor binding affinity, also improves
GH-RH potency.
[0024] It would be desirable to produce hGH-RH analogs by multiple
amino acid substitutions that have elevated binding affinities to
the pituitary receptors in vitro, and increased potencies in vivo
as compared to the native hGH-RH(1-29)NH.sub.2.
[0025] Since one change in the amino acid sequence of a peptide
could cause a big change in the three dimensional structure of the
peptide which has influence on the binding property as well as the
biological potency of the peptide, it is impossible to predict
which one or more amino acid replacements or combinations of
substitutions in hGH-RH analogs might result in improved binding
affinity or high in vivo potency.
[0026] Although the invention has been described with regard to its
preferred embodiments, which constitute the best mode presently
known to the inventors, it should be understood that various
changes and modifications as would be obvious to one having the
ordinary skill in this art may be made without departing from the
scope of the invention which is set forth in the claims appended
hereto. For example, modifications in the peptide chain,
particularly deletions beginning at the carboxyl terminus of the
peptide and extending to about position-29, can be made in
accordance with the known experimental practices to date to create
peptides or peptide fragments that retain all or very substantial
portions of the biological potency of the peptide, and such
peptides are considered as being within the scope of the invention.
Moreover, additions may be made to either terminus, or to both
terminals, and/or generally equivalent residues can be substituted
for naturally occurring residues, as is well-known in the overall
art of peptide chemistry, to produce other analogs having at least
a substantial portion of the potency of the claimed polypeptide
without deviating from the scope of the invention. Moreover,
modifications may be made to the preferred --NH.sub.2 group at the
C-terminus in accordance with the state of this art today; for
example, the carboxyl moiety of the amino acid residue at the
C-terminus can be the radical --COOR, --CRO, --CONHNHR,
--CON(R)(R') or --CH.sub.2--OR, with R and R' being lower alkyl,
fluoro lower alkyl or hydrogen, without deviating from the
invention, for such modifications result in equivalent synthetic
peptides. (Rivier U.S. Pat. No. 5,262,519).
SUMMARY OF THE INVENTION
[0027] There is provided a novel series of synthetic peptide
analogs of hGH-RH(1-29)NH.sub.2 or hGH-RH(1-30)NH.sub.2 The novel
synthetic peptides of this invention exhibit high activities in
stimulating the release of pituitary GH in animals, including
humans. They also show extremely high binding capacity to the
hGH-RH receptor. These synthetic hGH-RH analogs also retain their
physiological activity in solution for an extended period of time
and resist enzymatic degradation in the body. The stronger GH
releasing potencies of the new analogs in vivo, as compared to
previously described ones, results from combination of replacements
in hGH-RH(1-29)NH.sub.2 or hGH-RH(1-30)NH.sub.2 and from resistance
to in vivo degradation. Without in any way limiting the invention
or its scope, applicants wish to express their understanding that
the retention of activity in vitro and resistance to in vivo
degradation are due to multiple substitutions in the molecule:
incorporation of N-Me-Tyr or des-amino-Tyr (Dat) in position 1
which protect peptides from the degradation at the N-terminus;
incorporation of agmatine (Agm) or --NH--CH.sub.3 or
--NH--CH.sub.2--CH.sub.3 at position 29 or extension of the
C-terminus with an omega-amino acid which protects peptides from
degradation at the C-terminus; and also the replacements of both
lysines in the synthetic peptides with ornithine (Orn), which is a
poor substrate for trypsin-like enzymes; Gly at residue 15 by Abu.
To increase chemical stability, Asn at position 8 is replaced by
Gln, Thr, or Ala. And Met in position 27 is replaced by norleucine
(Nle). Replacement of other residues in the peptides and the
combination of these replacements also are found to promote
biological activity.
Synthetic Peptides
[0028] The synthetic hGH-RH analogs which may be expressed as
[R.sub.1-A.sup.1, A.sup.2, A.sup.6, A.sup.8, A.sup.11, A.sup.12,
A.sup.15, A.sup.20, A.sup.21, A.sup.22, Nle.sup.27, A.sup.28,
A.sup.29, A.sup.30]hGH-RH(1-30)-R.sub.2 or alternatively as
[R.sub.1-A.sup.1-A.sup.2-Asp-Ala-Ile A.sup.6
Thr-A.sup.8-Ser-Tyr-A.sup.11-A.sup.12-Val-Leu-A.sup.15-Gln-Leu-Ser-Ala-A.-
sup.20, A.sup.21-A.sup.22-Leu-Gln-Asp-Ile-Nle-A.sup.28, A.sup.29,
A.sup.30]-R.sub.2 (SEQ ID NO: 1) wherein R.sub.1 is Ac, Tfa, or is
absent,
A.sup.1 is Tyr, Dat, or N-Me-Tyr,
A.sup.2 is Ala, D-Ala, Abu, or D-Abu,
A.sup.6 is Phe or Fpa5,
A.sup.8 is Asn, Ala, Gln, Thr, or N-Me-Ala,
A.sup.11 is Arg, His, or Har,
A.sup.12 is Orn, or Lys(Me).sub.2,
A.sup.15 is Abu or Ala
A.sup.20 is Arg, His, or Har,
A.sup.21 is Orn, or Lys(Me).sub.2,
A.sup.22 is Leu, or Orn,
A.sup.28 is Ser, or Asp,
A.sup.29 is Arg, Har, Agm, D-Arg, or D-Har,
[0029] A.sup.30 is Arg, Agm, Ada, Amc, Aha, Apa, Har, D-Arg, D-Har,
Gab, Gln, D-Gln, Gln-Gab, D-Gln-Gab, or is absent, R.sub.2 is
--NH.sub.2, --OH, --NHR.sub.3, --N(R.sub.3).sub.2, or --OR.sub.3,
in which R.sub.3 is any of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, or
C.sub.2-12 alkinyl, preferably provided that if A.sup.29 is Agm
then A.sup.30 and R.sub.2 are absent and suitably A.sup.1 is
N-Me-Tyr only, and further preferably provided that where A.sup.30
is Agm then R.sub.2 is absent, and pharmaceutically acceptable
salts thereof.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Synthetic Peptides 1. Nomenclature
[0030] The nomenclature used to define the amino acid residues and
synthetic peptides is that specified by the IUPAC-IUB Commission on
Biochemical Nomenclature (European J. Biochem., 1984, 138, 9-37).
By natural amino acid is meant one of the common, naturally
occurring amino acids found in proteins comprising Gly, Ala, Val,
Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met Phe,
Tyr, Pro, Trp and His. By Nle is meant norleucine, by Abu is meant
alpha amino butyric acid, and by Orn is meant ornithine. Other
abbreviations used are:
[0031] Abbreviations [0032] Aah alpha-amino-hexanoic acid [0033]
Aap alpha-amino-pentanoic acid [0034] Abu alpha-aminobutyric acid
[0035] Ac acetyl [0036] AcOH acetic acid [0037] Ac.sub.2O acetic
anhydride [0038] Ada 12-aminododecanoyl [0039] Agm agmatine [0040]
Aha 6-aminohexanoyl [0041] AM aminomethyl [0042] Amc
8-Aminocaprylyl [0043] Apa 5-Aminopentanoyl [0044] Aib
alpha-aminoisobutyroyl [0045] Boc tert-butyloxycarbonyl [0046] Bom
benzyloxymethyl [0047] 2BrZ 2-bromo-benzyloxycarbonyl [0048]
Bu.sup.t tertiary butyl (ester) [0049] Bzl benzyl [0050] cHx
cyclohexyl [0051] 2CIZ 2-chloro-benzyloxycarbonyl [0052] 2CITrt
2-chlorotrityl [0053] Cpa para-chlorophenylalanine [0054] Dat
des-amino-tyrosine [0055] DCM dichloromethane [0056] DIC
N,N'-diisopropylcarbodiimide [0057] DIEA diisopropylethylamine
[0058] DMF dimethylformamide [0059] Et ethyl [0060] Fm
fluorenylmethyl [0061] Fmoc fluorenylmethoxycarbonyl [0062] For
formyl [0063] Fpa mono- or poly-fluorinated Phe (fluorine
substitution on the aromatic ring) [0064] Fpa5 pentafluoro-Phe
[0065] Gab gamma-amino butanoyl [0066] GH growth hormone [0067]
GH-RH GH releasing hormone [0068] Har homoarginine [0069] HBTU
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium [0070]
hexaflourophosphate [0071] hGH-RH human GH-RH [0072] HOBt
1-hydroxybenzotriazole [0073] HPLC high performance liquid
chromatography [0074] Ibu isobutyryl [0075] MBHA
para-methylbenzhydrylamine [0076] Me methyl [0077] MeOH methanol
[0078] MeCN acetonitrile [0079] Mmt 4-methoxytrityl [0080] Mtr
4-methoxy-2,3,6-trimethylbenzenesulphonyl [0081] N-Me-Ala
N-methyl-Ala [0082] N-Me-Tyr N-methyl-Tyr [0083] Nle norleucine
[0084] NMM N-methylmorpholine [0085] Oaa omega-amino acid [0086]
Orn ornithine [0087] PAM phenylacetamidomethyl [0088] Pbf
2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl [0089] Ph
phenyl [0090] PS polystyrene [0091] rGH-RH rat GH-RH [0092] RP-HPLC
reversed phase HPLC [0093] SPA para-sulfonyl-phenoxyacetyl [0094]
tBu tertiary butyl (ether) [0095] TFA trifluoroacetic acid [0096]
Tfa trifluoroacetyl [0097] Tos para-toluenesulfonyl [0098] Trt
trityl(triphenylmethyl) [0099] Z benzyloxycarbonyl
[0100] The amino acid sequences of the synthetic peptides are
numbered in correspondence with the amino acid residues in
hGH-RH(1-29); thus, for example, the Ala.sup.4 and R.sup.8 in the
synthetic peptides occupy the same position in the sequence as the
Ala.sup.4 and R.sup.8 residues in hGH-RH(1-29).
[0101] The convention under which the N-terminal of a peptide is
placed to the left, and the C-terminal to the right is also
followed herein.
2. Preferred Embodiments
[0102] The hGH-RH agonists of the present invention were designed
to increase Gh release at the pituitary level. Particularly
preferred embodiments of the synthetic peptides of the present
invention are shown in Table 1:
TABLE-US-00001 P-20103 [N--Me-Tyr.sup.1, Fpa5.sup.6, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH- RH(1-29) (SEQ ID NO: 2) P-20105 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) P-20107
[N--Me-Tyr.sup.1, Fpa5.sup.6, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH- RH(1-29) (SEQ
ID NO: 3) P-20109 [N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6,
Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Agm.sup.29]hGH-RH(1-29) P-20110 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1- 29) P-20111 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) P-20113
[N--Me-Tyr.sup.1, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1- 29) (SEQ ID NO: 4)
P-20115 [N--Me-Tyr.sup.1, Fpa5.sup.6, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-
RH(1-29) (SEQ ID NO: 5) P-20117 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH- RH(1-29) P-20350 [Dat.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1- 29) P-20351
[Ac--N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) P-20356 [N--Me-Tyr.sup.1, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1- 29) (SEQ ID NO: 6) P-20357 [Dat.sup.1,
D-Ala.sup.2, N--Me-Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH- RH(1-29) P-20358
[N--Me-Tyr.sup.1, D-Ala.sup.2, N--Me-Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) P-20359 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Agm.sup.29]hGH-RH(1-29) P-20360 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH- RH(1-29) P-20361
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH- RH(1-29)
P-20367 [N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1- 29)
P-20370 [N--Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 7)
P-20371 [N--Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1- 29) (SEQ
ID NO: 8) P-20372 [N--Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1- 29) (SEQ ID NO: 9) P-20373 [N--Me-Tyr.sup.1,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 10) P-20374 [N--Me-Tyr.sup.1,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 11) P-20375 [N--Me-Tyr.sup.1,
Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 12) P-20376 [N--Me-Tyr.sup.1,
Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 13) P-21300 [Dat.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Amc.sup.30]hGH-RH(1- 30)NH.sub.2 P-21301
[N--Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Amc.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 14) P-21303
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-RH(1- 30)NH.sub.2 P-21304
[Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-RH(1-
30)NH.sub.2 P-21305 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH- RH(1-30)NH.sub.2 P-21306 [Dat.sup.1, D-Ala.sup.2,
Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Amc.sup.30]hGH-RH(1- 30)NH.sub.2 P-21307
[N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-
RH(1-30)NH.sub.2 P-21308 [Dat.sup.1, D-Ala.sup.2, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1- 30)NH.sub.2 P-21309 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Orn.sup.12, Ala.sup.8, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH- RH(1-30)NH.sub.2 P-21310
[Dat.sup.1, D-Ala.sup.2, His.sup.11, Orn.sup.12, Abu.sup.15,
His.sup.20, Orn.sup.21, Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-
RH(1-30)NH.sub.2 P-21311 [N--Me-Tyr.sup.1, D-Ala.sup.2, His.sup.11,
Orn.sup.12, Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Amc.sup.30]hGH-RH(1-30)NH.sub.2 P-22325
[N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID
NO: 15) P-22326 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH- RH(1-30)NH.sub.2 P-22327 [Dat.sup.1, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 16) P-22328
[Ac-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-
RH(1-30)NH.sub.2 P-22329 [N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Apa.sup.30]hGH-RH(1-30)NH.sub.2
P-22330 [N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-
RH(1-30)NH.sub.2 P-22331 [N--Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH- RH(1-30)NH.sub.2 P-22332 [N--Me-Tyr.sup.1,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 17) P-22334
[N--Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Apa.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 18) P-22335
[N--Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID
NO: 19) P-22336 [N--Me-Tyr.sup.1 Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1-
30)NH.sub.2 (SEQ ID NO: 20) P-22337 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Cpa.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1-30)NH.sub.2 P-23250
[Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-
30)NH.sub.2 P-23251 [Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2
(SEQ ID NO: 21) P-23252 [Dat.sup.1, D-Ala.sup.2, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2 P-23253 [Dat.sup.1, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 22) P-23254 [Dat.sup.1,
D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1- 30)NH.sub.2 P-23255
[Dat.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
23) P-23256 [Dat.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1- 30)NH.sub.2 P-23257 [Dat.sup.1, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 24) P-23258
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-
RH(1-30)NH.sub.2 P-23259 [N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID NO: 25) P-23260
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1- 30)NH.sub.2 P-23261
[N--Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 26) P-23262
[N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-
RH(1-30)NH.sub.2 P-23263 [N--Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID NO: 27) P-23264
[N--Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-
RH(1-30)NH.sub.2 P-23265 [N--Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID NO: 28) P-24340
[N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Aha.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID
NO: 29) P-24341 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH- RH(1-30)NH.sub.2 P-24342 [Dat.sup.1, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 30) P-24344 [Dat.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Aha.sup.30]hGH-RH(1- 30)NH.sub.2 P-24345
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Aha.sup.30]hGH-RH(1- 30)NH.sub.2 P-24346
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Aha.sup.30]hGH-RH(1- 30)NH.sub.2 P-24347
[N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Aha.sup.30]hGH-
RH(1-30)NH.sub.2 P-24348 [N--Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH- RH(1-30)NH.sub.2 P-25501 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1- 30)NH.sub.2 P-25502 [Dat.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1-30)NH.sub.2 P-25503 [N--Me-Tyr.sup.1,
Abu.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 31) P-25504 [Dat.sup.1,
D-Abu.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1-30)NH.sub.2 P-25506 [N--Me-Tyr.sup.1,
D-Abu.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1- 30)NH.sub.2 P-25508 [Tfa-Tyr.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1-30)NH.sub.2 P-25516 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Gab.sup.30]hGH-RH(1- 30)NH.sub.2 P-26802 [Dat.sup.1,
D-Ala.sup.2, Thr.sup.8, His.sup.11, Orn.sup.12, Abu.sup.15,
His.sup.20, Orn.sup.21, Nle.sup.27, Ada.sup.30]hGH-
RH(1-30)NH.sub.2 P-26803 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8,
His.sup.11, Orn.sup.12, Abu.sup.15, His.sup.20, Orn.sup.21,
Nle.sup.27, Ada.sup.30]hGH-RH(1-30)NH--CH.sub.3 P-26804
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Ada.sup.30]hGH-RH(1- 30)NH.sub.2 P-27400
[Dat.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 P-27401 [Dat.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, D-Arg.sup.29]hGH- RH(1-29)NH--CH.sub.3
P-27403 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-
29)NH--CH.sub.3
P-27404 [N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-
29)NH--CH.sub.3 P-27405 [N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID
NO: 32) P-27406 [N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 33) P-27407
[Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 34)
P-27408 [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-30)NH--CH.sub.3 P-27409
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 P-27410
[N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.3
P-27411 [N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 P-27412
[N--Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.3
P-27413 [Dat.sup.1, Gln.sup.8, His.sup.11, Orn.sup.12, Abu.sup.15,
His.sup.20, Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-
29)NH--CH.sub.3 (SEQ ID NO: 35) P-27414 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Gab.sup.30]hGH-RH(1- 30)NH--CH.sub.3 P-27415
[N--Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1-30)NH--CH.sub.3 (SEQ ID NO: 36) P-27416
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, His.sup.11, Orn.sup.12,
Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 P-27417 [Ac-Tyr.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.3 P-27418
[Ac-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.3
P-27419 [Ac-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 37)
P-27422 [N--Me-D-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 P-27423
[N--Me-D-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.3
P-27424 [Dat.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 38) P-27425
[N--Me-D-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH- RH(1-29)NH--CH.sub.3
P-27440 [Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3
P-27441 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, D-
Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3 P-27442 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, D- Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3
P-27443 [N--Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3 P-27444
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, D-Arg.sup.29]hGH- RH(1-29)NH--CH.sub.3
P-27445 [N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, D-
Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3 P-27446 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, D- Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3
P-27447 [N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-
RH(1-30)NH--CH.sub.3 P-27448 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Aha.sup.30]hGH- RH(1-30)NH--CH.sub.3 P-27449
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-
RH(1-30)NH--CH.sub.3 P-27450 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Har.sup.30]hGH- RH(1-30)NH--CH.sub.3 P-27451
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, His.sup.11, Orn.sup.12,
Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH--CH.sub.3 P-28420 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.2--CH.sub.3 P-28421
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28430
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28431
[N--Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-
29)NH--CH.sub.2--CH.sub.3 P-28460 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH- RH(1-29)NH--CH.sub.2--CH.sub.3 P-28461
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28462
[N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28463
[N--Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28464
[N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27]hGH-RH(1-
29)NH--CH.sub.2--CH.sub.3 P-28465 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1- 29)NH--CH.sub.2--CH.sub.3 P-28466
[N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-
29)NH--CH.sub.2--CH.sub.3 P-28467 [N--Me-Tyr.sup.1, D-Ala.sup.2,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28468
[N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-
RH(1-29)NH--CH.sub.2--CH.sub.3 P-28469 [Dat.sup.1, D-Ala.sup.2,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28470 [Dat.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28471 [Dat.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28472
[Dat.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-
29)NH--CH.sub.2--CH.sub.3 P-28473 [Dat.sup.1, D-Ala.sup.2,
Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28474 [Dat.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 P-28475
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-
RH(1-30)NH--CH.sub.2--CH.sub.3 P-28476 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Aha.sup.30]hGH-
RH(1-30)NH--CH.sub.2--CH.sub.3 P-28477 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-
RH(1-30)NH--CH.sub.2--CH.sub.3 P-28478 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-
RH(1-30)NH--CH.sub.2--CH.sub.3 P-28479 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Gln.sup.8, His.sup.11, Orn.sup.12, Abu.sup.15,
His.sup.20, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3 P-29701
[N--Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Gln-Gab.sup.30]hGH-
RH](1-30)NH.sub.2 P-29702 [Dat.sup.1, D-Ala.sup.2, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2 P-29703 [N--Me-Tyr.sup.1,
Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gln-Gab.sup.30]hGH-RH(1- 30)NH.sub.2 (SEQ ID NO: 39). P-29704
[Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, 22, Nle.sup.27, Gln-Gab.sup.30]hGH-RH(1- 30)NH.sub.2
P-29706 [Tfa-Tyr.sup.1, D-Abu.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Gln-Gab.sup.30]hGH-
RH(1-30)NH.sub.2 P-29708 [N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Gln-Gab.sup.30]hGH-
RH(1-30)NH.sub.2 P-29710 [N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6,
Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gln-Gab.sup.30]hGH- RH(1-30)NH.sub.2 P-29720 [Dat.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Gln-Gab.sup.30]hGH-RH(1- 30)NH.sub.2 P-29721
[Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, 22, Nle.sup.27, Asp.sup.28, Gln-Gab.sup.30]hGH-
RH(1-30)NH.sub.2 P-29722 [Tfa-Tyr.sup.1, D-Abu.sup.2, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30] hGH-RH(1-30)NH.sub.2 P-29723 [N--Me-Tyr.sup.1,
D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Gln-Gab.sup.30] hGH-RH(1-30)NH.sub.2
P-29724 [N--Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, Gln-
Gab.sup.30] hGH-RH(1-30)NH.sub.2
B. Method of Preparation
1. Overview of Synthesis
[0103] The peptides are synthesized by suitable methods such as by
exclusive solid phase techniques, by partial solid-phase
techniques, by fragment condensation or by classical solution phase
synthesis. For example, the techniques of exclusive solid-phase
synthesis are set forth in the textbook "Solid Phase Peptide
Synthesis", J. M. Stewart and J. D. Young, Pierce Chem. Company,
Rockford, Ill., 1984 (2nd. ed.), and M. Bodanszky, "Principles of
Peptide Synthesis", Springer Verlag, 1984. The hGH-RH agonist
peptides are preferably prepared using solid phase synthesis, such
as that generally described by Merrifield, J. Am. Chem. Soc, 85 p.
2149 (1963), although other equivalent chemical syntheses known in
the art can also be used as previously mentioned.
[0104] The synthesis is carried out with amino acids that are
protected at their alpha amino group. Urethane type protecting
groups (Boc or Fmoc) are preferably used for the protection of the
alpha amino group. In certain cases, protected omega-amino acids
are also used during the synthesis. Boc or Fmoc protecting groups
are also appropriate for the protection of omega-amino groups.
[0105] In solid phase synthesis, the N-alpha-protected or
N-omega-protected amino acid moiety which forms the aminoacyl group
of the final peptide at the C-terminus is attached to a polymeric
resin support via a chemical link. After completion of the coupling
reaction, the alpha (or omega) amino protecting group is
selectively removed to allow subsequent coupling reactions to take
place at the amino-terminus, preferably with 50% TFA in DCM when
the N-alpha-(N-omega-) protecting group is Boc, or by 20%
piperidine in DMF when the N-alpha-(N-omega-) protecting group is
Fmoc. The remaining amino acids with similarly Boc or
Fmoc-protected alpha (or omega) amino groups are coupled stepwise
to the free amino group of the preceding amino acid on the resin to
obtain the desired peptide sequence. Because the amino acid
residues are coupled to the alpha (or omega) amino group of the
C-terminus residue, growth of the synthetic hGH-RH analogue
peptides begins at the C terminus and progresses toward the
N-terminus. When the desired sequence has been obtained, the
peptide is acylated, or the amino group is left free at the
N-terminus, and the peptide is removed from the support
polymer.
[0106] Each protected amino acid is used in excess (2.5 or 3
equivalents) and the coupling reactions are usually carried out in
DCM, DMF or mixtures thereof. The extent of completion of the
coupling reaction is monitored at each stage by the ninhydrin
reaction. In cases where incomplete coupling is determined, the
coupling procedure is repeated, or a capping by acetylation of
unreacted amino groups is carried out, before removal of the alpha
(or omega) amino protecting group prior to the coupling of the next
amino acid.
[0107] Typical synthesis cycles are shown in Table 2 and Table
3.
TABLE-US-00002 TABLE 2 Protocol for a Typical Synthetic Cycle Using
Boc-strategy Mixing Step Reagent Time (min) 1. 50% TFA in DCM 5 +
25 Deprotection DCM wash 1 2-propanol wash 1 2. 5% DIEA in DCM 1
Neutralization DCM wash 1 MeOH wash 1 5% DIEA in DCM 3 MeOH wash 1
DCM wash (3 times) 1 3. 3 eq. Boc-amino acid in DCM or DMF + 60
Coupling 3 eq. DIC or the preformed HOBt ester of the Boc-amino
acid MeOH wash (3 times) 1 DCM wash (3 times) 1 4. Acetylation
Ac.sub.2O in pyridine (30%) 10 + 20 (if appropriate) MeOH wash (3
times) 1 DCM wash (3 times) 1
TABLE-US-00003 TABLE 3 Protocol for a Typical Synthetic Cycle Using
Fmoc-strategv Mixing Step Reagent Time (min) 1. 20% piperidine in
DMF 5 + 15 Deprotection DMF wash (3 times) 1 2. 3 eq. Fmoc-amino
acid in DMF + 60 Coupling 3 eq. DIC or + 3 eq. HBTU + 3 eq. HOBt +
6 eq. DIEA DMF wash (3 times) 1 3. Acetylation 3 eq.
1-acetylimidazole in DMF 30 (if appropriate) DMF wash (3 times)
1
After completion of the synthesis, the cleavage of the peptide from
the resin can be effected using procedures well known in peptide
chemistry.
2. Choice of the Support Polymer
[0108] The hGH-RH agonist peptides may be synthesized on a variety
of support polymers, i.e. MBHA, Merrifield, PAM, Rink amide or Wang
resins. The peptides can also be synthesized on aminomethyl, MBHA,
or other resins that have been previously derivatized with suitable
linkers. Examples of such linkers are the base-labile
4-hydroxymethyl benzoic acid (HMBA) linker for the attachment of
C-terminal carboxyl groups, the acid-labile
para-sulfonyl-phenoxyacetyl (SPA) linker which permits the
attachment of agmatine through its guanidino group, or the
acid-labile [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl
linker which allows the synthesis of peptides with C-terminal
methylamide (--NH--CH.sub.3).
[0109] When peptides with an amidated C-terminus are synthesized by
using Boc strategy, the preferred resin is MBHA. Attachment of the
C-terminal amino acid to this resin can be accomplished by the
standard DIC-mediated coupling method described in Table 2.
[0110] In order to prepare peptides with C-terminal methylamide
(--NH--CH.sub.3) or ethylamide (--NH--CH.sub.2--CH.sub.3)
modification, two methods can be used: a) the Merrifield resin is
loaded with the Boc-protected C-terminal amino acid by coupling
mediated by potassium fluoride (KF) or cesium salt at elevated
temperature; a) [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetylAm
or b) 3-[(Ethyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl Am resins are
used, respectively for the synthesis of peptides having C-terminal
methylamide (--NH--CH.sub.3) or ethylamide
(--NH--CH.sub.2--CH.sub.3) modification. Using these resins, the
Fmoc protecting group has to be removed from the resin with the
method described in Table 3 before the synthesis.
[0111] For the synthesis of peptides having Agm at the C-terminus,
two methods have been used. In one case, the support phase is MBHA
resin or an aminomethyl resin, and the guanidino group of Boc-Agm
is joined to the support polymer through a stable, but readily
cleavable linker such as the para-sulfonyl-phenoxyacetyl (SPA)
moiety. The alpha-amino-Boc-protected Agm is reacted with the
chlorosulfonyl phenoxyacetic acid
Cl--SO.sub.2--C.sub.6H.sub.4--O--CH.sub.2--COOH to form
Boc-Agm-SO.sub.2--C.sub.6H.sub.4--O--CH.sub.2--COOH. This compound
is then coupled to the support polymer e.g. to MBHA resin using DIC
or HBTU-HOBt-DIEA as activating reagent to yield Boc-Agm-SPA-MBHA.
In another case, Agm-SO.sub.2--PS resin is used for the synthesis
(1% DVB, 100-200 mesh, 2.5 mmol/g, Advanced ChemTech (Louisville,
Ky.)) at pH 10-13 to from Boc-Agm-SO.sub.2-resin
3. Amino Acid Derivatives Used
[0112] Bifunctional amino acids, i.e. those not having side chain
functional groups, are mostly used in the form of their N-alpha
Boc- or Fmoc-derivatives for synthesis. Bifunctional omega-amino
acids are also typically used in the form of their N-omega Boc- or
Fmoc-derivatives. Thus, Boc-Gly-OH or Fmoc-Gly-OH is typically used
for incorporating the Gly residue. The naturally occurring
bifunctional amino acids are Gly, Ala, Val, Leu, Ile, Phe, and Pro,
and some well-known non-coded bifunctional amino acids used in this
invention are Abu, Aib, Gab, Nle, Aah, and Aap.
[0113] Some of the amino acid residues of the peptides have side
chain functional groups which are reactive with reagents used in
coupling or deprotection. When such side chain groups are present,
suitable protecting groups are joined to these functional groups to
prevent undesirable chemical reactions occurring during the
coupling reactions.
[0114] The following general rules are followed in selecting a
particular side chain protecting group: (a) the protecting group
preferably retains its protecting properties and is not split off
under coupling conditions, (b) the protecting group should be
stable under conditions for removing the alpha amino protecting
group at each step of the synthesis, (c) the side chain protecting
group must be removable upon the completion of the synthesis of the
desired amino acid sequence, under reaction conditions that will
not undesirably alter the peptide chain.
[0115] When Boc-amino acids are used in the synthesis, the reactive
side chain functional groups can be protected as follows: Tos or
nitro (NO.sub.2) for Arg and Har; cHx or Fm for Asp and Glu; Bom
for His; 2CIZ or Fmoc for Lys and Orn; Bzl for Ser and Thr; and
2BrZ for Tyr. The side chains of Asn and Gln are unprotected.
[0116] In the case of Fmoc synthesis, the reactive side chain
functional groups can be protected by other appropriate protective
groups as follows:
2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl (Pbf),
4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), or bis-Boc for Arg
and Har; tert-butyl (Bu.sup.t) for Asp and Glu; no protective group
or trityl (Trt) protection for Asn and Gln; Trt for His; Boc or
4-methoxytrityl (Mmt) for Lys and Orn; tBu or Trt for Ser and Thr;
and tBu or 2-chlorotrityl (2CITrt) for Tyr. In addition to the
widely known coded and non-coded amino acids mentioned above, some
of the peptides of this application contain less common non-coded
amino acids such as homoarginine (Har); ornithine (Orn);
IM-methyl-alanine [N-Me-Ala]; N-methyl-tyrosine [N-Me-Tyr];
pentafluoro-phenylalanine [Phe(F).sub.5, Fpa5]. These amino acid
residues are incorporated into the peptides by coupling the
suitable protected amino acid derivatives. A non-exclusive list of
such protected amino acid derivatives that can be used is as
follows: Boc-Har(Tos)-OH, Boc-Orn(2CIZ)-OH, Boc-N-Me-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH, Boc-Fpa5-OH, Fmoc-Har(Pbf)-OH,
Fmoc-Orn(Boc)-OH, Fmoc-N-Me-Ala-OH, and Fmoc-N-Me-Tyr(2CITrt)-OH.
The protected derivatives of noncoded amino acids mentioned above
are commonly available from several commercial suppliers, including
Novabiochem (San Diego, Calif.), Advanced ChemTech (Louisville,
Ky.), Bachem (King of Prussia, Pa.), Peptides International
(Louisville, Ky.), RSP Amino Acid Analogues DBA (Worcester, Mass.),
and AnaSpec (San Jose, Calif.).
4. Stepwise Coupling of Amino Acid Residues
[0117] Utilizing the above mentioned support polymers and after
loading of the protected C-terminal amino acid or Agm residue, the
peptide itself may suitably be built up by solid phase synthesis in
the conventional manner. Each protected amino acid is coupled in
about a three-fold molar excess, with respect to resin-bound free
amino residues, and the coupling may be carried out in a medium
such as DMF-DCM (1:1) or in DMF or DCM alone. The selection of an
appropriate coupling reagent is within the skill of the art.
Particularly suitable as coupling reagents are N,N'-diisopropyl
carbodiimide (DIC), or HBTU combined with HOBt in the presence of
DIEA. The success of the coupling reaction at each stage of the
synthesis is preferably monitored by the ninhydrin reaction. In
cases where incomplete coupling occurs, either the coupling
procedure is repeated, or the resin-bound unreacted amino residues
are acetylated using a capping reagent, before removal of the alpha
(or omega) amino protecting group. Suitable capping reagents are
1-acetylimidazole and Ac.sub.2O in pyridine
5. Cleavage of the Peptide from the Support Polymer and Removal of
the Side-Chain Protecting Groups
[0118] When the synthesis is complete, the peptide is cleaved from
the support phase and its side-chain protecting groups are
removed.
[0119] In cases where peptides with an amidated C-terminus
(--CONH.sub.2) or with a C-terminal carboxyl group (--COOH) are
prepared by Boc strategy on an MBHA, Merrifield, or PAM resin, the
removal of the peptide from the resin is performed by treatment
with a reagent such as liquid hydrogen fluoride (HF). This is also
the case for peptides synthesized on the Boc-Agm-SPA-MBHA or
Bos-Agm-tosyl-resin. In some instances, the liquid HF also cleaves
all the remaining side chain protecting groups. However, if side
chain protecting groups resistant to HF treatment are present on
the peptide, additional cleavage steps should be performed in order
to remove these protecting groups. Thus, Fm and Fmoc protecting
groups are removed by treatment with 20% piperidine in DMF, prior
to or after the HF treatment.
[0120] Suitably, the dried and protected peptide-resin is treated
with a mixture consisting of 1.0 mL m-cresol and 10 mL anhydrous
hydrogen fluoride per gram of peptide-resin for 60-120 min at
0-.degree. C. to cleave the peptide from the resin as well as to
remove the HF-labile side chain protecting groups. After the
removal of the hydrogen fluoride under a stream of nitrogen and
vacuum, the free peptides are precipitated with ether, filtered,
washed with ether and ethyl acetate, extracted with 50% acetic
acid, and lyophilized.
[0121] In cases where peptides with a methyl-(--NH--CH.sub.3), or
ethyl-amide (--NH--CH.sub.2--CH.sub.3) C-terminus are prepared by
Boc strategy on the Merrifield or HMBA-MBHA resin, the protected
peptides are first cleaved from the resin by methylamine
(CH.sub.3NH.sub.2) or ethylamine (CH.sub.3CH.sub.2NH.sub.2 mediated
aminolysis. Suitably, liquid CH.sub.3NH.sub.2 or
CH.sub.3CH.sub.2NH.sub.2 is transferred into a cooled, heavy-walled
glass flask that contains the dried and protected peptide-resin.
The quantity of liquid CH.sub.3NH.sub.2 or CH.sub.3CH.sub.2NH.sub.2
should be sufficient to cover the peptide-resin. The flask is
stoppered, and shaken with the liquid CH.sub.3NH.sub.2 or
CH.sub.3CH.sub.2NH.sub.2 for 3.5 hours at room temperature in order
to allow for the reaction to take place. After this, the flask is
cooled in a dry ice bath, opened, and the liquid CH.sub.3NH.sub.2
or CH.sub.3CH.sub.2NH.sub.2 is filtered off the solid residue that
contains a mixture of resin and cleaved peptide, the peptide still
having the protecting groups attached.
[0122] The solid residue is dried and subjected to HF treatment as
described above, in order to remove the side chain protecting
groups of the peptide.
[0123] In cases when peptides with a methyl-(--NH--CH.sub.3), or
ethyl-amide (--NH--CH.sub.2--CH.sub.3) C-terminus are prepared by
Fmoc strategy on [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl
AM or 3-[(Ethyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resins,
respectively, the protected peptides are cleaved from the resin
with a cleavage cocktail. Since no single cleavage and deprotection
procedure is optimal for all peptides due to the nature of the
linker and the amino acid composition of the peptide, the following
cleavage cocktail proved to be the most suitable for cleavage and
deprotection of GHRH agonists: 94% TFA, 3% H.sub.2O, 1.5% m-cresol,
and 1.5% phenol. Cleavage cocktail must be prepared fresh and have
to use high quality TFA and scavengers. The amount of cleavage
cocktail used depends on both the amount of the peptide-resin and
its properties. Enough cocktail solution should be used to saturate
and swell the resin during the reaction, with about 2-3 mm of clear
solution below the floating beads. Generally 5 mL of cleavage
cocktail is used for 0.5 g of resin. The choice of reaction time
depends on the linker and the side-chain protecting groups of the
peptide. Preferably, 3-hour reaction time is used for the cleavage
and deprotection of GHRH agonists. To induce peptide precipitation,
the cleavage mixture is added dropwise to cold (preferably
-20.degree. C.) ether. The non-peptide products remain in the ether
solution. The precipitated material is collected by filtration or
centrifugation and is washed three times with cold ether to remove
any residual scavengers. The cleaved and deprotected peptide is
dissolved in 50% acetic acid and separated from the resin by
filtration and, after dilution with water, the solution is
lyophilized.
6. Purification
[0124] The purification of the crude peptides can be effected using
procedures well known in peptide chemistry. For example,
purification may be performed on a Beckman System Gold HPLC system
(Beckman Coulter, Inc., Brea, Calif.) equipped with 127P solvent
Module; UV-VIS Detector, model 166P; Computer workstation with CPU
Monitor and printer, and 32-Karat software, version 3.0 using an
XBridge Prep OBD.TM. reversed phase column (4.6.times.250 mm,
packed with C.sub.18 silica gel, 300 .ANG. pore size, 5 .mu.m
particle size) (Waters Co., Milford, Mass.). The column is eluted
with a solvent system consisting of (A) 0.1% aqueous TFA and (B)
0.1% TFA in 70% aqueous MeCN in a linear gradient mode (e.g.,
30-55% B in 120 min); flow rate of 12 mL/min. The eluent is
monitored at 220 nm, and fractions are examined by analytical HPLC
using a Hewlett-Packard Model HP-1090 liquid chromatograph and
pooled to give maximum purity. Analytical HPLC is carried out on a
Supeico Discovery HS C18 reversed-phase column (2.1.times.50 mm,
C18, 300 .ANG. pore size, 3 .mu.m particle size) (Supeico,
Bellefonte, Pa.) using isocratic elution with a solvent system
consisting of (A) and (B) defined above with a flow rate of 0.2
mL/min. The peaks are monitored at 220 and 280 nm. The peptides are
judged to be substantially (>95%) pure by analytical HPLC.
Molecular masses are checked by electrospray mass spectrometry
(Agilent Technologies 6210 Time-of-Light LCMS, Santa Clara, Calif.)
and the expected amino acid compositions are confirmed by amino
acid analysis
C. Pharmaceutical Compositions and Mode of Administration
[0125] The peptides of the invention may be administered in the
form of pharmaceutically acceptable, nontoxic salts, such as acid
addition salts. Illustrative of such acid addition salts are
hydrochloride, hydrobromide, sulphate, phosphate, fumarate,
gluconate, tannate, maleate, acetate, trifluoroacetate, citrate,
benzoate, succinate, alginate, pamoate, malate, ascorbate,
tartarate, and the like. Particularly preferred agonists are salts
of low solubility, e.g., pamoate salts and the like. These exhibit
long duration of activity.
[0126] The compounds of the present invention are suitably
administered to subject humans or animals subcutaneously (s.c),
intramuscularly (i.m.), or intravenously (i.v); intranasally or by
pulmonary inhalation; by transdermal delivery; or in a depot form
(e.g., microcapsules, microgranules, or cylindrical rod like
implants) formulated from a biodegradable suitable polymer (such as
D,L-lactide-coglycolide), the former two depot modes being
preferred. Other equivalent modes of administration are also within
the scope of this invention, i.e., continuous drip, cutaneous
patches, depot injections, infusion pump and time release modes
such as microcapsules and the like. Administration is in any
physiologically acceptable injectable carrier, physiological saline
being acceptable, though other carriers known to the art may also
be used.
[0127] The peptides are preferably administered parenterally,
intramuscularly, subcutaneously or intravenously with a
pharmaceutically acceptable carrier such as isotonic saline.
Alternatively, the peptides may be administered as an intranasal
spray with an appropriate carrier or by pulmonary inhalation. One
suitable route of administration is a depot form formulated from a
biodegradable suitable polymer, e.g., poly-D,L-lactide-coglycolide
as microcapsules, microgranules or cylindrical implants containing
dispersed antagonistic compounds.
[0128] The amount of peptide needed depends on the type of
pharmaceutical composition and on the mode of administration. In
cases where human subjects receive solutions of GH-RH agonists,
administered by i.m. or s.c. injection, or in the form of
intranasal spray or pulmonary inhalation, the typical doses are
between 2-20 mg/day/patient, given once a day or divided into 2-4
administrations/day. When the GH-RH agonists are administered
intravenously to human patients, typical doses are in the range of
8-80 .mu.g/kg of body weight/day, divided into 1-4 bolus
injections/day or given as a continuous infusion. When depot
preparations of the GH-RH agonists are used, e.g. by i.m. injection
of pamoate salts or other salts of low solubility, or by i.m. or
s.c. administration of microcapsules, microgranules, or implants
containing the antagonistic compounds dispersed in a biodegradable
polymer, the typical doses are between 1-10 mg
agonist/day/patient
D. Medical Applications of hGH-RH Agonists
[0129] The products of the present invention may be utilized to
promote the growth of warm-blooded animals (e.g., humans) and also
enhance the milk production of females of milk producing mammals,
suitably but not exclusively goats and cows, preferably cows.
[0130] The peptides of the invention may be administered in the
form of pharmaceutically acceptable, nontoxic salts, such as acid
addition salts. Illustrative of such acid addition salts are
hydrochloride, hydrobromide, sulphate, phosphate, fumarate,
gluconate, tannate, maleate, acetate, citrate, benzoate, succinate,
alginate, pamoate, malate, ascorbate, tartrate, and the like.
[0131] The compounds of the present invention are suitably
administered to the subject humans or animals s.c, i.m., or i.v;
intranasally or by pulmonary inhalation; or in a depot form (e.g.,
microcapsules, microgranules, or cylindrical rod like implants)
formulated from a biodegradable suitable polymer (such as
D,L-lactide-co-glycolide), the former two depot modes being
preferred. Other equivalent modes of administration are also within
the scope of this invention, i.e., continuous drip, depot
injections, infusion pump and time release modes such as
microcapsules and the like. Administration is in any
physiologically acceptable injectable carrier, physiological saline
being acceptable, though other carriers known to the art may also
be used.
[0132] The dosage level is suitably between 0.01 .mu.g and 2
.mu.g/kg body weight per injection, except for depot form where the
amount injected would be calculated to last from about 15 to about
30 days or longer. These dosage ranges are merely preferred.
Administration of non-depot forms may be between 1 and 4 times per
day, or in the case of lactating mammals, after each milking.
[0133] Until the production of growth hormone by recombinant-DNA
methods began, the small supply of pituitary-derived human growth
hormone limited its use to the treatment of children with growth
hormone deficiency. The wide availability of synthetic human growth
hormone has made possible long-term studies of other potentially
beneficial uses of growth hormone and its more physiologic actions.
Although synthetic GH is currently approved in the USA only for
treatment of growth failure due to lack of endogenous growth
hormone, this therapy has also been used to treat short children
not classically GH deficient. However the cost of long-term
treatment with hGH and the need of daily s.c. administration are
important considerations. Currently, the cost of growth hormone
therapy for a child with growth deficiency ranges from $10,000 to
30,000 a year depending on body weight. Treatment of a 70-kg adult
with hGH three times a week costs $13,800 a year. Vance, M. L, N.
Eng. J. Med 323:52-54 (1990). Thus, long-term growth hormone
treatment in elderly adults with diminished growth hormone
secretion would require a considerable personal and financial
investment. In addition there are many children all over the world
with growth retardation due to the lack of GH who cannot be treated
with hGH because of the cost of this therapy. Consequently there is
an urgent need to develop a drug that releases GH and with an
affordable price. This alternative method to increase endogenous
growth hormone secretion is through the administration of agonistic
analogs of growth hormone-releasing hormone. The therapy with GH-RH
agonistic analogs should be much less expensive than that utilizing
hGH. In addition, the development of long-acting delivery systems
for these analogs can make this new modality of treatment more
practical and convenient.
[0134] The ability to produce synthetic growth hormone by
recombinant DNA technology has enabled the manufacture of GH in
potentially unlimited quantities. This greatly facilitated the
treatment of GH-deficient children. As stated above, synthetic hGH
is currently approved only for the treatment of growth failure due
to a lack of adequate endogenous growth hormone, but hGH has also
been used to treat short children who are not classically
GH-deficient such as girls with Turner's syndrome; prepubertal
children with chronic renal insufficiency and severe growth
retardation; and children with non-GH deficient short stature.
[0135] The production of GH by genetic engineering is very
expensive for clinical use. In particular, there are risks of
contamination of these commercial preparations with material from
the bacterial strain used. These bacterial contaminants may be
pyrogens or may result in immunogenic reactions in patients. The
purification of the recombinant product is carried out by following
a plurality of successive chromatography steps. The drastic purity
criteria imposed by regulatory agencies necessitate multiple
quality control steps. U.S. Pat. No. 6,458,764.
[0136] Of the 3 million children born in the USA annually, 90,000
will be below the third percentile for height. These children may
be labeled as having short stature and may be candidates for GH
treatment. Therapy with human growth hormone currently costs about
$20,000 per year and the average length of treatment is about 10
years. The treatment will usually be stopped when the patient
reaches an acceptable adult size (a height of well over five feet)
or when the patient matures sexually and the epiphyses close, at
which time linear growth eases, or if the patient fails to respond
to the treatment. If all children who are less than the third
percentile for height receive a five year course of hGH therapy,
hGH for height augmentation therapy will cost at least $8 billion
to 10 billion per year. Lantos J. et al., JAMA 261:1020-1024,
(1989).
[0137] It is desirable to ascertain the endogenous physiological
ability of children having short stature to produce hGH. This may
be done with a diagnostic test using a 50-100 .mu.g dose of GH-RH;
a 50-100 .mu.g dose of a GH-RH analog which is a synthetic peptide
of Formula I; and assaying the GH response evoked by each dose.
[0138] The assay means may be any conventional means which will
indicate the quantitative amount of hGH present in a blood sample
drawn from the patient. The concentration of GH in serum is
determined using standard radioimmunoassay ("RIA") procedures as
set forth in e.g., Miles I. E. M. et al., Lancet ii, 492-493 (1968)
or O'Dell W et al., J. Lab. Clin. Med. 70, 973-80 (1967).
[0139] The test is used as follows. First, the GH-RH dose is
administered. Thirty minutes later, a blood sample is taken for RIA
of GH. Various commercially available kits (e.g., Nichols Institute
of Diagnostics, San Juan Capistrano, Calif.) or reference
preparations of hGH (e.g., NIAMDD-hGH-RP-1) can be used for RIA of
GH. After waiting 6-24 hours for the effect of GH-RH to wear off,
the dose of the synthetic peptide GH-RH analog is administered.
Blood again is drawn for radioimmunoassay of GH.
[0140] The presence of a normal hGH response in the first assay
reveals that endogenous hGH producing ability is present. This
result also suggests a short, mild course of GH-RH therapy, if any,
may be suitable. A low GH response, or no response, to the first
dose reveals only that GH-RH must be evaluated in view of the
second test result. If a good hGH response follows the second dose,
there is clear physiological hGH producing ability which is not
evoked by GH-RH. This indicates that a therapy with the GH-RH
analog may be desirable. Finally, no or low response to both tests
reliably reveals lack of physiological ability to produce hGH, and
so suggests therapy with hGH is probably needed.
[0141] As indicated above, short stature in children may result
from many causes, none of which are immediately apparent. Use of
the diagnostic test on all children with this condition would
greatly clarify the cause of short stature. Such a widespread
screening test would also provide earlier indications for desirable
treatment.
[0142] Glucocorticoids are potent inhibitors of linear growth in
man and growth suppression is a well known risk of long term
treatment of asthmatic children with steroids. Thus stunted growth
is an important consequence of chronic administration of
glucocorticoids in childhood. The inhibition of GH secretion is due
in some extent to the fact that chronic administration of
glucocorticoids suppresses GHRH. This inhibition occurs at the
level of the hypothalamus or above and in this situation only the
treatment with GH-RH agonists will stimulate linear growth.
[0143] Growth hormone tends to decline with the aging process and
may lead to decrease in muscle mass and adiposity. Studies have
shown that healthy older men and women with growth hormone
deficiency had increases in lean body mass and decreases in the
mass of adipose tissue after six months of hGH administration.
Other effects of long-term administration of hGH on body
composition included increase in vertebral-bone density and
increase in skin-fold thickness. In addition, it has been reported
that daily GH-RH injection to menopausal women, for 8 days augments
GH responses and IGF-I levels and raises serum osteocalcin levels.
Thus the therapy with GH-RH agonistic analogs reduces the loss of
muscle, bone and skin mass and lessen the increase of body fat that
normally accompanies the aging process.
[0144] Growth hormone is a potent anabolic hormone that enhances
protein synthesis and nitrogen retention. Chronic administration of
agonistic analogs of GH-RH increases the endogenous growth hormone
secretion. The therapy with GH-RH agonistic analogs has uses in
other areas of medicine such as catabolic states causing
accelerated weight loss; tissue repair in patients with severe body
surface burn, accelerating healing of nonunion fractures; and in
some cases of cardiac failure.
[0145] Although long term follow-up is necessary before all
treatment responses can be ascribed to GH, there has been
improvement in cardiac mass and an increase in both cardiac mass
and contractility. The therapy with hGH interrupts the
cardiac-cachexia cycle. This response is in keeping with other
observations that the therapy with GH has a major role in catabolic
states in adults. An alternative method to increase endogenous
growth hormone secretion in these conditions is the administration
of GH-RH agonistic analogs [Korpas et al., J. Clin. Endoc. Metabol.
75, 530-535, (1992)].
[0146] These agonistic analogs of GH-RH can replace hGH for many
applications. GH-deficient children respond to GH-RH(1-40),
GH-RH(1-29) or GH-RH(1-44), with an increase in growth. Thorner M.
0. et al., supra; Ross et al., supra; Takano K et al., supra; and
Kirk et al., supra. Most children who respond to hGH, will respond
to GH-RH. This is because most GH-deficient children have a
hypothalamic defect in GH release, and will show a GH response
after the administration of analogs of the hypothalamic hormone
GH-RH. Thus repeated administration of GH-RH promotes linear
growth. GH-RH(1-29)NH.sub.2 given subcutaneously twice a day
promoted linear growth in approximately 50% of a group of
GH-deficient children (Ross et al, cited above). A small group of
severely GH-deficient children will respond to GH-RH after 6 (six)
months of treatment.
[0147] Further Clinical Applications of Agonistic Analogs of GH-RH
in Children with Growth Retardation
1. As a screening test for growth hormone deficiency. 2. Treatment
of Hypothalamic GH-RH deficiency. 3. Constitutional growth
delay.
4. Turner Syndrome.
[0148] 5. Familial short stature. 6. Prepubertal children with
chronic renal insufficiency and severe growth retardation. 7.
Infants and children with intrauterine growth retardation. 8.
Children with GH deficiency following radiotherapy for pituitary or
hypothalamic lesions. 9. Children on long-term treatment with
glucocorticoids and growing at subnormal rate.
[0149] Further Clinical Applications of Agonistic Analogs of GH-RH
in Adults
1. Geriatric Patients: To reduce the loss of muscle, bone and skin
mass and lessen the increase of body fat that normally accompanies
the aging process. 2. Catabolic states 3. Wound healing 4. Delayed
healing of fractures
5. Osteoporosis
6. Obesity
[0150] 7. As an adjunct to total parenteral nutrition in
malnourished patients with chronic obstructive pulmonary disease 8.
Cardiac failure 9. GH-RH agonists could be used during and after
space flights to counteract the decrease in GH secretion.
Weightlessness of space flight significantly decreases the release
of growth hormone, which could explain the bone loss and muscle
weakness many astronauts experience after prolonged space
flights.
Therapeutic Uses of GH-RH Agonists
[0151] Successful treatment of growth hormone deficiency using
hGH-RH and hGH-RH(1-40) has been reported in Takano K et al.,
Endocrinol. Japan 35; 775-781 (1988) and Thorner M. O. et al., N.
Engl. J. Med., 312, 4-9 (1985) respectively. Therapeuticic
treatments using hGH-RH(1-29) have also been reported against human
growth hor-mone deficiency, Ross R. J. M. et al., Lancet 1:5-8,
(1987); decreased GH in elderly males, Corpas et al., J. Clin.
Endocrin. Metabol. 75, 530-535 (1992); and idiopathic short
stature, Kirk J. M. W. et al., Clinical Endocrinol. 41, 487-493
(1994). Since earlier analogs of hGH-RH have successfully treated
conditions associated with low levels of GH, it is not surprising
that the novel synthetic hGH-RH peptides described herein also
induce release of GH and are novel therapeutic treatments for these
conditions.
[0152] Indeed, this suitability as a therapeutic agent is confirmed
by the in vivo testing reported below. This testing is considered
reasonably predictive of the results which one could expect in
treating higher mammals, including humans. From the results below,
one would expect that the novel synthetic hGH-RH analogs to be
useful in therapeutically treating humans for growth hormone
deficiency, as well as for a number of other conditions growing out
of very low levels of GH. Thus, the invention further comprises a
method of treating human growth hormone deficiency comprising
administering from 0.01.mu.g to 2.mu.g of a peptide per day per kg
body weight.
[0153] The synthetic peptides may be formulated in a pharmaceutical
dosage form with an excipient and administered to humans or animals
for therapeutic or diagnostic purposes. More particularly, the
synthetic peptides may be used to promote the growth of
warm-blooded animals, as, in humans, to treat human growth
deficiency by stimulating in vivo synthesis and/or release of
endogenous GH; to treat certain physiological conditions such as
severe growth retardation due to chronic renal in-sufficiency; to
offset certain effects of aging, e.g., reducing loss of muscle and
bone loss; to accelerate healing and tissue repair; to improve feed
utilization, thereby increasing lean/fat ratio favoring muscle gain
at the cost of fat; and also to enhance milk production in
lactating cattle. Further, the synthetic peptides may be used in a
method to ascertain endogenous physiological ability to produce
hGH.
EXAMPLES
[0154] The present invention is described in connection with the
following examples which are set forth for the purposes of
illustration only. In the examples, optically active protected
amino acids in the L-configuration are used except where
specifically noted. The following Examples set forth suitable
methods of synthesizing the novel GH-RH antagonists by the
solid-phase technique.
Example I
N-Me-Tyr.sup.1-Ala.sup.2-Asp.sup.3-Ala.sup.4-Ile.sup.5-Fpa5.sup.6-Thr.sup.-
7-Gln.sup.8-Ser.sup.9-Tyr.sup.10-Arg.sup.11-Orn.sup.12-Val.sup.13-Leu.sup.-
14-Abu.sup.15-Gln.sup.16-Leu.sup.17-Ser.sup.18-Ala.sup.19-Arg.sup.20-Orn.s-
up.21-Leu.sup.22-Leu.sup.23-Gln.sup.24-Asp.sup.25-Ile.sup.26-Nle.sup.27-As-
p.sup.28-Agm.sup.29 (Peptide 20103)
[0155] [N-Me-Tyr.sup.1, Fpa5.sup.6, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 2)
[0156] The synthesis is conducted in a stepwise manner using manual
solid phase peptide synthesis equipment. Two methods have been used
for the synthesis of peptides having Agm at the C-terminus. In one
case, the starting material of the synthesis is
Boc-agmatine-N.sup.G-sulfonyl-phenoxyacetyl-MBHA (Boc-Agm-SPA-MBHA)
resin with a substitution of 0.3 mmol/g, which was obtained
commercially from California Peptide Research, Inc. (Napa, Calif.).
The synthesis of this resin has been described in U.S. Pat. No.
4,914,189 and in the scientific literature (Zarandi M, Serfozo P,
Zsigo J, Bokser L, Janaky T, Olsen D B, Bajusz S, Schally A V, Int.
J. Peptide Protein Res. 39: 211-217, 1992), hereby incorporated by
reference. Briefly, Boc-Agm-SPA-MBHA resin (1.67 g, 0.50 mmol) is
pre-swollen in DCM and then the deprotection and neutralization
protocols described in Table 2 are performed in order to remove the
Boc protecting group and prepare the peptide-resin for coupling of
the next amino acid. In another case, Agm-sulfonyl-polystyrene (PS)
resin is used [1% DVB, 100-200 mesh, 0.74 mmol/g, American Peptide
Company (Sunnyvale, Calif.)]. Briefly, Agm-sulfonyl-PS resin (680
mg, 0.50 mmol) is neutralized with 5% DIEA in DCM and washed
according to the protocol described in Table 2. The solution of
Boc-Asp(OcHx)-OH (475 mg, 1.5 mmol) in DMF-DCM (1:1) is shaken with
the neutralized resin and DIC (235 .mu.L, 1.5 mmol) in a manual
solid phase peptide synthesis apparatus for 1 hour. Then, the
deprotection and neutralization protocols described in Table 2 are
performed in order to remove the Boc protecting group and prepare
the peptide-resin for coupling of the next amino acid. The
synthesis is continued in a stepwise manner using manual solid
phase peptide synthesis equipment in both cases, and the peptide
chain is built stepwise by coupling the following protected amino
acids in the indicated order on the resin to obtain the desired
peptide sequence: Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0157] These protected amino acid residues (also commonly available
from NovaBiochem, Advanced Chemtech, Bachem, and Peptides
International) are represented above according to a well accepted
convention. The suitable protecting group for the side chain
functional group of particular amino acids appears in parentheses.
The OH groups in the above formulae indicate that the carboxyl
terminus of each residue is free.
[0158] The protected amino acids (1.5 mmol each) are coupled with
DIC (235 .mu.L, 1.5 mmol) with the exceptions of Boc-Asn-OH and
Boc-Gln-OH which are coupled with their preformed HOBt esters.
[0159] In order to cleave the peptide from the resin and deprotect
it, a portion of 250 mg of the dried peptide resin is stirred with
0.5 mL m-cresol and 5 mL hydrogen fluoride (HF) at 0-.degree. C.
for 2 hours. After evaporation of the HF under a stream of nitrogen
and in vacuo, the residue is washed with dry diethyl ether and
ethyl acetate. The cleaved and deprotected peptide is dissolved in
50% acetic acid and separated from the resin by filtration. After
dilution with water and lyophilization, 68 mg crude product is
obtained.
[0160] The crude peptide is checked by analytical HPLC using a
Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a
Supelco Discovery HS C18 reversed-phase column (2.1 mm.times.5 cm,
packed with C18 silica gel, 300 .ANG. pore size, 3 .mu.m particle
size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g.,
40-70% B) is used with a solvent system consisting of (A) 0.1%
aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate
is 0.2 mL/min. Purification is performed on a Beckman System Gold
HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with
127P solvent Module; UV-VIS Detector, model 166P; Computer
workstation with CPU Monitor and printer, and 32-Karat software,
version 3.0. 68 mg of crude peptide is dissolved in AcOH/H.sub.2O,
stirred, filtered and applied on an XBridge Prep OBD.TM. reversed
phase column (4.6.times.250 mm, packed with C.sub.18 silica gel,
300 .ANG. pore size, 5 .mu.m particle size) (Waters Co., Milford,
Mass.). The column is eluted with a solvent system described above
in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12
mL/min. The eluent is monitored at 220 nm, and fractions are
examined by analytical HPLC. Fractions with purity higher than 95%
are pooled and lyophilized to give 18 mg pure product. The
analytical HPLC is carried out on a Supelco Discovery C18
reversed-phase column described above using isocratic elution with
a solvent system described above with a flow rate of 0.2 mL/min.
The peaks are monitored at 220 and 280 nm. The product is judged to
be substantially (>95%) pure by analytical HPLC. Molecular mass
is checked by electrospray mass spectrometry, and the expected
amino acid composition is confirmed by amino acid analysis.
[0161] In accordance with the above procedure Peptide 20105,
Peptide 20107, Peptide 20109, Peptide 20110, Peptide 20111, Peptide
20113, Peptide 20115, Peptide 20350, Peptide 20351, Peptide Peptide
20356, Peptide 20357, Peptide 20358, Peptide 20359, Peptide 20360,
Peptide 20361, Peptide 20363, Peptide 20367, Peptide 20370, Peptide
20371, Peptide 20372, Peptide 20373, Peptide 20374, Peptide 20375,
Peptide 20376, are synthesized in the same manner as Peptide 20103,
except that these peptides also contain other amino acid
substitutions in the peptide sequence, and acyl moieties at their
N-termini. The details for these syntheses are set forth below.
[0162] For the synthesis of Peptide 20105, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0163] For the synthesis of Peptide 20107, the chemical structure
of which is [[N-Me-Tyr.sup.1, Fpa5.sup.6, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 3), the following protected
amino acids are coupled in the indicated order on the
Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0164] For the synthesis of Peptide 20109, the chemical structure
of which is [N-Me-T\ D-Ala.sup.2, Fpa5.sup.6, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Ala-OH,
Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0165] For the synthesis of Peptide 20110, the chemical structure
of which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29), the
following protected amino acids are coupled in the indicated order
on the Agm-SO.sub.2--PS resin: Boc-Ser(Bzl)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0166] For the synthesis of Peptide 20111, the chemical structure
of which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Thr.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the resin: Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0167] For the synthesis of Peptide 20113, the chemical structure
of which is [N-Me-Tyr.sup.1, Fpa5.sup.6, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 4), the following protected amino acids are coupled in the
indicated order on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0168] For the synthesis of Peptide 20115, the chemical structure
of which is [N-Me-Tyr.sup.1, Fpa5.sup.6, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 5), the following protected
amino acids are coupled in the indicated order on the
Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0169] For the synthesis of Peptide 20117 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Ser(Bzl)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0170] For the synthesis of Peptide 20350 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29), the
following protected amino acids are coupled in the indicated order
on the resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0171] For the synthesis of Peptide 20351 the chemical structure of
which [Ac-N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Ac-N-Me-Tyr(2BrZ)--OH.
[0172] For the synthesis of Peptide 20356, the chemical structure
of which [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 6), the following protected amino acids are coupled in the
indicated order on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0173] For the synthesis of Peptide 20357 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, N-Me-Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH,
Boc-N-Me-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0174] For the synthesis of Peptide 20358 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, N-Me-Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH,
Boc-N-Me-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH, Boc-Ile-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0175] For the synthesis of Peptide 20359, the chemical structure
of which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm]hGH-RH(1-29),
the following protected amino acids are coupled in the indicated
order on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0176] For the synthesis of Peptide 20360, the chemical structure
of which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH,
Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0177] For the synthesis of Peptide 20361, the chemical structure
of which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29), the following protected amino acids are
coupled in the indicated order on the Agm-SO.sub.2--PS resin:
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0178] For the synthesis of Peptide 20367, the chemical structure
of which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29), the
following protected amino acids are coupled in the indicated order
on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0179] For the synthesis of Peptide 20370, the chemical structure
of which is [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 7), the
following protected amino acids are coupled in the indicated order
on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0180] For the synthesis of Peptide 20371, the chemical structure
of which is [N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID
NO: 8), the following protected amino acids are coupled in the
indicated order on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0181] For the synthesis of Peptide 20372, the chemical structure
of which is [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 9), the
following protected amino acids are coupled in the indicated order
on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0182] For the synthesis of Peptide 20373, the chemical structure
of which is [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 10),
the following protected amino acids are coupled in the indicated
order on the Agm-SO.sub.2--PS resin: Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0183] For the synthesis of Peptide 20374, the chemical structure
of which is [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 11), the following
protected amino acids are coupled in the indicated order on the
Agm-SO.sub.2--PS resin: Boc-Ser(Bzl)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0184] For the synthesis of Peptide 20375, the chemical structure
of which is [N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 12),
the following protected amino acids are coupled in the indicated
order on the Agm-SO.sub.2--PS resin: Boc-Ser(Bzl)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0185] For the synthesis of Peptide 20376, the chemical structure
of which is [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Agm.sup.29]hGH-RH(1-29) (SEQ ID NO: 13), the following
protected amino acids are coupled in the indicated order on the
Agm-SO.sub.2--PS resin: Boc-Ser(Bzl)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0186] HF cleavage and deprotection, and subsequent purification by
semipreparative HPLC of Peptide 20105, Peptide 20107, Peptide
20109, Peptide 20110, Peptide 20111, Peptide 20113, Peptide 20115,
Peptide 20350, Peptide 20351, Peptide 20356, Peptide 357, Peptide
20358, Peptide 20359, Peptide 20360, Peptide 20361, Peptide 20363,
Peptide 20367, Peptide 20370, Peptide 20371, Peptide 20372, Peptide
20373, Peptide 20374, Peptide 20375, Peptide 20376 are done as
described in the case of Peptide 20103. The purified compounds are
judged to be substantially (>95%) pure by analytical HPLC. Their
molecular masses are checked by electrospray mass spectrometry, and
the expected amino acid compositions are confirmed by amino acid
analysis.
Example II
Dat.sup.1-D-Ala.sup.2-Asp.sup.3-Ala.sup.4-Ile.sup.5-Phe.sup.6-Thr.sup.7-As-
n.sup.8-Ser.sup.9-Tyr.sup.10-Arg.sup.11-Orn.sup.12-Val.sup.13-Leu.sup.14-A-
bu.sup.15-Gln.sup.16-Leu.sup.17-Ser.sup.18-Ala.sup.19-Arg.sup.20-Orn.sup.2-
1-Leu.sup.22-Leu.sup.23-Gln.sup.24-Asp.sup.25-Ile.sup.26-Nle.sup.27-Asp.su-
p.28-Arg.sup.29-Amc.sup.30-NH.sub.2 (Peptide 21300) [Dat.sup.1,
D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Amc.sup.30]hGH-RH(1-30)NH.sub.2
[0187] The synthesis is conducted in a stepwise manner using manual
solid phase peptide synthesis equipment. Briefly,
para-methylbenzhydrylamine (MBHA) resin (100-200 mesh, 1% DVB, 0.7
mmol/g, Advanced Chemtech, Louisville, Ky.) (350 mg, 0.50 mmol) is
neutralized with 5% DIEA in DCM and washed according to the
protocol described in Table 2. The solution of Boc-Amc-OH (390 mg,
1.5 mmol) in DMF-DCM (1:1) is shaken with the neutralized MBHA
resin and DIC (235 .mu.L, 1.5 mmol) in a manual solid phase peptide
synthesis apparatus for 1 hour. After the completion of the
coupling reaction is proved by negative ninhydrin test, the
deprotection and neutralization protocols described in Table 2 are
performed in order to remove the Boc protecting group and prepare
the peptide-resin for coupling of the next amino acid. The
synthesis is continued and the peptide chain is built stepwise by
coupling the following protected amino acids in the indicated order
on the resin to obtain the desired peptide sequence:
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH. These protected amino acid residues (also
commonly available from Novabiochem, Advanced Chemtech, Bachem, and
Peptides International) are represented above according to a well
accepted convention. The suitable protecting group for the side
chain functional group of particular amino acids appears in
parentheses. The OH groups in the above formulae indicate that the
carboxyl terminus of each residue is free. The protected amino
acids (1.5 mmol each) are coupled with DIC (235 .mu.L, 1.5 mmol)
with the exceptions of Boc-Asn-OH and Boc-Gln-OH which are coupled
with their preformed HOBt esters.
[0188] In order to cleave the peptide from the resin and deprotect
it, a portion of 250 mg of the dried peptide resin is stirred with
0.5 mL m-cresol and 5 mL hydrogen fluoride (HF) at 0-.degree. C.
for 2 hours. After evaporation of the HF under a stream of nitrogen
and in vacuo, the residue is washed with dry diethyl ether and
ethyl acetate. The cleaved and deprotected peptide is dissolved in
50% acetic acid and separated from the resin by filtration. After
dilution with water and lyophilization, 130 mg crude product is
obtained.
[0189] The crude peptide is checked by analytical HPLC using a
Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a
Supelco Discovery HS C18 reversed-phase column (2.1 mm.times.5 cm,
packed with C18 silica gel, 300 .ANG. pore size, 3 .mu.m particle
size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g.,
40-70% B) is used with a solvent system consisting of (A) 0.1%
aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate
is 0.2 mL/min. Purification is performed on a Beckman System Gold
HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with
127P solvent Module; UV-VIS Detector, model 166P; Computer
workstation with CPU Monitor and printer, and 32-Karat software,
version 3.0. 130 mg of crude peptide is dissolved in AcOH/H.sub.2O,
stirred, filtered and applied on an XBridge Prep OBD.TM. reversed
phase column (4.6.times.250 mm, packed with C.sub.18 silica gel,
300 .ANG. pore size, 5 .mu.m particle size) (Waters Co., Milford,
Mass.). The column is eluted with a solvent system described above
in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12
mL/min. The eluent is monitored at 220 nm, and fractions are
examined by analytical HPLC. Fractions with purity higher than 95%
are pooled and lyophilized to give 28 mg pure product. The
analytical HPLC is carried out on a Supelco Discovery C18
reversed-phase column described above using isocratic elution with
a solvent system described above with a flow rate of 0.2 mL/min.
The peaks are monitored at 220 and 280 nm. The product is judged to
be substantially (>95%) pure by analytical HPLC. Molecular mass
is checked by electrospray mass spectrometry, and the expected
amino acid composition is confirmed by amino acid analysis.
[0190] In accordance with the above procedure Peptide 21301,
Peptide, Peptide 21304, Peptide 21305, Peptide 21306, Peptide
21307, Peptide 21308, Peptide 21309, Peptide 21310, Peptide 21311,
Peptide 22325, Peptide 22326, Peptide 22327, Peptide 22328, Peptide
22329, Peptide 22330, Peptide 22331, Peptide 22332, Peptide 22334,
Peptide 22335, Peptide 22336, Peptide 22337, Peptide 23250, Peptide
23251, Peptide 23252, Peptide 23253, Peptide 23254, Peptide 23255,
Peptide 23256, Peptide 23257, Peptide 23258, Peptide 23259, Peptide
23260, Peptide 23261, Peptide 23262, Peptide 23263, Peptide 23264,
Peptide 23265, Peptide 24340, Peptide 24341, Peptide 24342, Peptide
24344, Peptide 24345, Peptide 24346, Peptide 24347, Peptide 24348,
Peptide 25501, Peptide 25502, Peptide 25503, Peptide 25504, Peptide
25506, Peptide 25508, Peptide 25516, Peptide 26802, Peptide 26803,
Peptide 2680, are synthesized in the same manner as Peptide 20300,
except that these peptides also contain other amino acid
substitutions in the peptide sequence, and/or different alpha- or
omega-amino acid moieties at their C-termini. The details for these
syntheses are set forth below.
[0191] For the synthesis of Peptide 21301, the chemical structure
of which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Amc.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
14), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Amc-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Asn-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0192] For the synthesis of Peptide 21303, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0193] For the synthesis of Peptide 21304, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0194] For the synthesis of Peptide 21305, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0195] For the synthesis of Peptide 21306, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH), the following protected amino acids are
coupled in the indicated order on the MBHA resin: Boc-Amc-OH,
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0196] For the synthesis of Peptide 21307, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0197] For the synthesis of Peptide 21308, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0198] For the synthesis of Peptide 21309, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0199] For the synthesis of Peptide 21310, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, His.sup.11, Orn.sup.12,
Abu.sup.15, HiS.sup.20, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0200] For the synthesis of Peptide 21311, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, His.sup.11, Orn.sup.12,
Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Amc-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0201] For the synthesis of Peptide 22325, the chemical structure
of which [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2(SEQ ID NO: 15), the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0202] For the synthesis of Peptide 22326, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0203] For the synthesis of Peptide 22327, the chemical structure
of which [Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
16), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Apa-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Dat-OH.
[0204] For the synthesis of Peptide 22328, the chemical structure
of which [Ac-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Ac-Tyr(2BrZ)--OH.
[0205] For the synthesis of Peptide 22329, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0206] For the synthesis of Peptide 22330, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0207] For the synthesis of Peptide 22331, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0208] For the synthesis of Peptide 22332, the chemical structure
of which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
17), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Apa-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Asn-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0209] For the synthesis of Peptide 22334, the chemical structure
of which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Apa.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 18), the
following protected amino acids are coupled in the indicated order
on the MBHA resin: Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0210] For the synthesis of Peptide 22335 the chemical structure of
which [N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1-30)NH.sub.2
(SEQ ID NO: 19), the following protected amino acids are coupled in
the indicated order on the MBHA resin: Boc-Apa-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH,
Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0211] For the synthesis of Peptide 22336 the chemical structure of
which [N-Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Apa.sup.30]hGH-RH(1-30)NH.sub.2
(SEQ ID NO: 20), the following protected amino acids are coupled in
the indicated order on the MBHA resin: Boc-Apa-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Ala-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0212] For the synthesis of Peptide 22337 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Cpa.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Apa-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Cpa-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0213] For the synthesis of Peptide 23250, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0214] For the synthesis of Peptide 23251, the chemical structure
of which [Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
21), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Har(Tos)-OH,
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH, Dat-OH.
[0215] For the synthesis of Peptide 23252, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nile.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0216] For the synthesis of Peptide 23253, the chemical structure
of which [Dat.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
22), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Har(Tos)-OH,
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH, Dat-OH.
[0217] For the synthesis of Peptide 23254, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0218] For the synthesis of Peptide 23255, the chemical structure
of which [Dat.sup.1 Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
23), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Har(Tos)-OH,
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH, Dat-OH.
[0219] For the synthesis of Peptide 23256, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0220] For the synthesis of Peptide 23257, the chemical structure
of which [Dat.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
24), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Har(Tos)-OH,
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH, Dat-OH.
[0221] For the synthesis of Peptide 23258, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0222] For the synthesis of Peptide 23259, the chemical structure
of which [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2(SEQ ID NO: 25), the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0223] For the synthesis of Peptide 23260, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0224] For the synthesis of Peptide 23261, the chemical structure
of which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Har.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
26), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Har(Tos)-OH,
Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH,
Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0225] For the synthesis of Peptide 23262, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0226] For the synthesis of Peptide 23263, the chemical structure
of which [N-Me-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2(SEQ ID NO: 27), the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0227] For the synthesis of Peptide 23264, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0228] For the synthesis of Peptide 23265, the chemical structure
of which [N-Me-Tyr.sup.1, Ala.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH.sub.2(SEQ ID NO: 28), the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Har(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0229] For the synthesis of Peptide 24340 the chemical structure of
which [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aba.sup.30]hGH-RH(1-30)NH.sub.2(SEQ ID NO: 29), the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0230] For the synthesis of Peptide 24341 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH.sub.2) the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0231] For the synthesis of Peptide 24342 the chemical structure of
which [Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Aha.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
30), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Aha-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Dat-OH.
[0232] For the synthesis of Peptide 24344 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0233] For the synthesis of Peptide 24345 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0234] For the synthesis of Peptide 24346 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Aha.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0235] For the synthesis of Peptide 24347 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0236] For the synthesis of Peptide 24348 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Aha-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0237] For the synthesis of Peptide 25501 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH.sub.2, the
following protected amino acids are coupled in the indicated order
on the MBHA resin: Boc-Gab-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0238] For the synthesis of Peptide 25502 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH.sub.2, the
following protected amino acids are coupled in the indicated order
on the MBHA resin: Boc-Gab-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0239] For the synthesis of Peptide 25503 the chemical structure of
which [N-Me-Tyr.sup.1, Abu.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO:
31), the following protected amino acids are coupled in the
indicated order on the MBHA resin: Boc-Gab-OH, Boc-Arg(Tos)-OH,
Boc-Ser(Bzl)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Asn-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Abu-OH, Boc-N-Me-Tyr(2BrZ)--OH.
[0240] For the synthesis of Peptide 25504 the chemical structure of
which [Dat.sup.1, D-Abu.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Gab-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Abu-OH, Dat-OH.
[0241] For the synthesis of Peptide 25506 the chemical structure of
which [N-Me-Tyr.sup.1, D-Abu.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH.sub.2, the
following protected amino acids are coupled in the indicated order
on the MBHA resin: Boc-Gab-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Abu-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0242] For the synthesis of Peptide 25508 the chemical structure of
which [Tfa-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH.sub.2, the
following protected amino acids are coupled in the indicated order
on the MBHA resin: Boc-Gab-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0243] For the synthesis of Peptide 25516 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0244] For the synthesis of Peptide 26802 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Thr.sup.8, His.sup.11, Orn.sup.12,
Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Ada.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Ada-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0245] For the synthesis of Peptide 26803 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, His.sup.11,
Orn.sup.12, Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Ada.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Ada-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-His(Bom)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0246] For the synthesis of Peptide 26804 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Ada.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Ada-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Nle-OH,
Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0247] HF cleavage and deprotection, and subsequent purification by
semipreparative HPLC of Peptide 21301, Peptide 21303, Peptide
21304, Peptide 21305, Peptide 21306, Peptide 21307, Peptide 21308,
Peptide 21309, Peptide 21310, Peptide 21311, Peptide 22325, Peptide
22326, Peptide 22327, Peptide 22328, Peptide 22329, Peptide 22330,
Peptide 22331, Peptide 22332, Peptide 22334, Peptide 22335, Peptide
22336, Peptide 22337, Peptide 23250, Peptide 23251, Peptide 23252,
Peptide 23253, Peptide 23254, Peptide 23255, Peptide 23256, Peptide
23257, Peptide 23258, Peptide 23259, Peptide 23260, Peptide 23261,
Peptide 23262, Peptide 23263, Peptide 23264, Peptide 23265, Peptide
24340, Peptide 24341, Peptide 24342, Peptide 24344, Peptide 24345,
Peptide 24346, Peptide 24347, Peptide 24348, Peptide 25501, Peptide
25502, Peptide 25503, Peptide 25504, Peptide 25506, Peptide 25508,
Peptide 25516, Peptide 26802, Peptide 26803, Peptide 2680 are done
as described in the case of Peptide 21300. The purified compounds
are judged to be substantially (>95%) pure by analytical HPLC.
Their molecular masses are checked by electrospray mass
spectrometry, and the expected amino acid compositions are
confirmed by amino acid analysis
Example III
Dat.sup.1-D-Ala.sup.2-Asp.sup.3-Ala.sup.4-Ile.sup.5-Phe.sup.6-Thr.sup.7-As-
n.sup.8-Ser.sup.9-Tyr.sup.10-Arg.sup.11-Orn.sup.12-Val.sup.13-Leu.sup.14-A-
bu.sup.15-Gln.sup.16-Leu.sup.17-Ser.sup.18-Ala.sup.19-Arg.sup.20-Orn.sup.2-
1-Leu.sup.22-Leu.sup.23-Gln.sup.24-Asp.sup.25-Ile.sup.26-Nle.sup.27-Asp.su-
p.28-Arg.sup.29-NH--CH.sub.3 (Peptide 27400)
[0248] [Dat.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 The synthesis is
conducted in a stepwise manner using manual solid phase peptide
synthesis equipment. Briefly,
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin (Nova
Biochem, La Jolla, Calif.) (750 mg, 0.50 mmol) is deprotected with
20% piperidine in DMF for 5 and 15 minutes and washed according to
the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH
(975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC
(235 .mu.L, 1.5 mmol) in a manual solid phase peptide synthesis
apparatus for 1 hour. After washing the resin three times with DMF,
the coupling reaction was repeated as described above. After the
repeated coupling and after the completion of the reaction is
proved by negative ninhydrin test, the deprotection and
neutralization protocols described in Table 3 are performed in
order to remove the Fmoc protecting group and prepare the
peptide-resin for coupling of the next amino acid. The synthesis is
continued and the peptide chain is built stepwise by coupling the
following protected amino acids in the indicated order on the resin
to obtain the desired peptide sequence: Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Dat-OH.
[0249] These protected amino acid residues (also commonly available
from Novabiochem, Advanced Chemtech, Bachem, and Peptides
International) are represented above according to a well accepted
convention. The suitable protecting group for the side chain
functional group of particular amino acids appears in parentheses.
The OH groups in the above formulae indicate that the carboxyl
terminus of each residue is free.
[0250] The protected amino acids (1.5 mmol each) are coupled with
DIC (235 .mu.L, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH
and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent.
[0251] In order to cleave the peptide from the resin and deprotect
it, a portion of 250 mg of the dried peptide resin is stirred with
2.5 mL cleavage cocktail (94% TFA, 3% H.sub.2O, 1.5% m-cresol, and
1.5% phenol) at room temperature for 3 hours. To induce peptide
precipitation, the cleavage mixture is added dropwise to cold
(preferably -20.degree. C.) ether. The precipitated material is
collected by filtration or centrifugation and is washed three times
with cold ether. The cleaved and deprotected peptide is dissolved
in 50% acetic acid and separated from the resin by filtration.
After dilution with water and lyophilization, 118 mg crude product
is obtained.
[0252] The crude peptide is checked by analytical HPLC using a
Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a
Supelco Discovery HS C18 reversed-phase column (2.1 mm.times.5 cm,
packed with C18 silica gel, 300 .ANG. pore size, 3 .mu.m particle
size) (Supeico, Bellefonte, Pa.). Linear gradient elution (e.g.,
40-70% B) is used with a solvent system consisting of (A) 0.1%
aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate
is 0.2 mL/min. Purification is performed on a Beckman System Gold
HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with
127P solvent Module; UV-VIS Detector, model 166P; Computer
workstation with CPU Monitor and printer, and 32-Karat software,
version 3.0. 118 mg of crude peptide is dissolved in AcOH/H.sub.2O,
stirred, filtered and applied on an XBridge Prep OBD.TM. reversed
phase column (4.6.times.250 mm, packed with C.sub.18 silica gel,
300 .ANG. pore size, 5 .mu.m particle size) (Waters Co., Milford,
Mass.). The column is eluted with a solvent system described above
in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12
mL/min. The eluent is monitored at 220 nm, and fractions are
examined by analytical HPLC. Fractions with purity higher than 95%
are pooled and lyophilized to give 19 mg pure product. The
analytical HPLC is carried out on a Supelco Discovery C18
reversed-phase column described above using isocratic elution with
a solvent system described above with a flow rate of 0.2 mL/min.
The peaks are monitored at 220 and 280 nm. The product is judged to
be substantially (>95%) pure by analytical HPLC. Molecular mass
is checked by electrospray mass spectrometry, and the expected
amino acid composition is confirmed by amino acid analysis.
[0253] In accordance with the above procedure Peptide 27401,
Peptide 27403, Peptide 27404, Peptide 27405, Peptide 27406, Peptide
27407, Peptide 27408, Peptide 27409, Peptide 27410, Peptide 27411,
Peptide 412, Peptide 27413, Peptide 27414, Peptide 27415, Peptide
27416, Peptide 27417, Peptide 27418, Peptide 27419, Peptide 27422,
Peptide 27423, Peptide 27424, Peptide 27425, Peptide 27440, Peptide
27441, Peptide 27442, Peptide 27443, Peptide 27444, Peptide 27445,
Peptide 27446, Peptide 27447, Peptide 27448, Peptide 27449, Peptide
27450, Peptide 27451 are synthesized in the same manner as Peptide
27400, except that these peptides also contain other amino acid
substitutions in the peptide sequence. The details for these
syntheses are set forth below.
[0254] For the synthesis of Peptide 27401, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following protected
amino acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl Am resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Dat-OH.
[0255] For the synthesis of Peptide 27403, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0256] For the synthesis of Peptide 27404, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0257] For the synthesis of Peptide 27405, the chemical structure
of which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 32), the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0258] For the synthesis of Peptide 27406, the chemical structure
of which [N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID
NO: 33), the following protected amino acids are coupled in the
indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0259] For the synthesis of Peptide 27407, the chemical structure
of which [Dat.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 34),
the following protected amino acids are coupled in the indicated
order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH,
Dat-OH.
[0260] For the synthesis of Peptide 27408, the chemical structure
of which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Dat-OH.
[0261] For the synthesis of Peptide 27409, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0262] For the synthesis of Peptide 27410, the chemical structure
of which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0263] For the synthesis of Peptide 27411 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0264] For the synthesis of Peptide 27412 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.23]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0265] For the synthesis of Peptide 27413 the chemical structure of
which [Dat.sup.1, Gln.sup.8, His.sup.11, Orn.sup.12, Abu.sup.15,
His.sup.20, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 35), the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-D-Ala-OH, Dat-OH.
[0266] For the synthesis of Peptide 27414 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gab.sup.30]hGH-RH(1-30)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Gab-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0267] For the synthesis of Peptide 27415 the chemical structure of
which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Gab.sup.30]hGH-RH(1-30)NH--CH.sub.3 (SEQ ID NO: 36),
the following protected amino acids are coupled in the indicated
order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Gab-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0268] For the synthesis of Peptide 27416 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, His.sup.11,
Orn.sup.12, Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0269] For the synthesis of Peptide 27417 the chemical structure of
which [Ac-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Ac-Tyr(tBu)-OH.
[0270] For the synthesis of Peptide 27418 the chemical structure of
which [Ac-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Ac-Tyr(tBu)-OH.
[0271] For the synthesis of Peptide 27419 the chemical structure of
which [Ac-Tyr.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 37), the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH, Ac-Tyr(tBu)-OH.
[0272] For the synthesis of Peptide 27422 the chemical structure of
which [N-Me-D-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0273] For the synthesis of Peptide 27423 the chemical structure of
which [N-Me-D-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0274] For the synthesis of Peptide 2742.4 the chemical structure
of which [Dat.sup.1, Thr.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.3 (SEQ ID NO: 38), the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH, Dat-OH.
[0275] For the synthesis of Peptide 27425 the chemical structure of
which [N-Me-D-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0276] For the synthesis of Peptide 27440 the chemical structure of
which [Dat.sup.1 Gln.sup.8, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH,
Dat-OH.
[0277] For the synthesis of Peptide 27441 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following protected
amino acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0278] For the synthesis of Peptide 27442 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following protected
amino acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0279] For the synthesis of Peptide 27443 the chemical structure of
which [N-Me-Tyr.sup.1, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-D-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tfiu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0280] For the synthesis of Peptide 27444 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following protected
amino acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0281] For the synthesis of Peptide 27445 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following protected
amino acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0282] For the synthesis of Peptide 27446 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Ala.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
D-Arg.sup.29]hGH-RH(1-29)NH--CH.sub.3, the following protected
amino acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0283] For the synthesis of Peptide 27447 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Apa-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0284] For the synthesis of Peptide 27448 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Aha-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0285] For the synthesis of Peptide 27449 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Amc-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0286] For the synthesis of Peptide 27450 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH--CH.sub.3, the following protected amino
acids are coupled in the indicated order on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Har(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0287] For the synthesis of Peptide 27451 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, His.sup.11,
Orn.sup.12, Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Aha.sup.30]hGH-RH(1-30)NH--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Apa-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH
Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0288] TFA cleavage and deprotection with the cleavage cocktail,
and subsequent purification by semipreparative HPLC of Peptide
27401, Peptide 27403, Peptide 27404, Peptide 27405, Peptide 27406,
Peptide 27407, Peptide 27408, Peptide 27409, Peptide 27410, Peptide
27411, Peptide 27412, Peptide 27413, Peptide 27414, Peptide 27415,
Peptide 27416, Peptide 27417, Peptide 27418, Peptide 27419, Peptide
27422, Peptide 27423, Peptide 27424, Peptide 27425, Peptide 27440,
Peptide 27441, Peptide 27442, Peptide 27443, Peptide 27444, Peptide
27445, Peptide 27446, Peptide 27447, Peptide 27448, Peptide 27449,
Peptide 27450, Peptide 27451 are done as described in the case of
Peptide 27400. The purified compounds are judged to be
substantially (>95%) pure by analytical HPLC. Their molecular
masses are checked by electrospray mass spectrometry, and the
expected amino acid compositions are confirmed by amino acid
analysis.
Example IV
N-Me-Tyr.sup.1-D-Ala.sup.2-Asp.sup.3-Ala.sup.4-Ile.sup.5-Phe.sup.6-Thr.sup-
.7-Gln.sup.8-Ser.sup.9-Tyr.sup.10-Arg.sup.11-Orn.sup.12-Val.sup.13-Leu.sup-
.14-Abu.sup.15-Gln.sup.16-Leu.sup.17-Ser.sup.18-Ala.sup.19-Arg.sup.20-Orn.-
sup.21-Leu.sup.22-Leu.sup.23-Gln.sup.24-Asp.sup.25-Ile.sup.26-Nle.sup.27-A-
sp.sup.28-Arg.sup.29-NH--CH.sub.2--CH.sub.3 (Peptide 28420)
[0289] N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3 The synthesis is
conducted in a stepwise manner using manual solid phase peptide
synthesis equipment. Briefly,
3-[(Ethyl-Fmoc-amino)methyl]-indol-1-A-acetyl AM resin (Nova
Biochem, La Jolla, Calif.) (610 mg, 0.50 mmmol) is deprotected with
20% piperidine in DMF for 5 and 15 minutes and washed according to
the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH
(975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC
(235 .mu.L, 1.5 mmol) in a manual solid phase peptide synthesis
apparatus for 1 hour. After washing the resin three times with DMF,
the coupling reaction was repeated as described above. After the
repeated coupling and after the completion of the reaction is
proved by negative ninhydrin test, the deprotection and
neutralization protocols described in Table 3 are performed in
order to remove the Fmoc protecting group and prepare the
peptide-resin for coupling of the next amino acid. The synthesis is
continued and the peptide chain is built stepwise by coupling the
following protected amino acids in the indicated order on the resin
to obtain the desired peptide sequence: Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0290] These protected amino acid residues (also commonly available
from Novabiochem, Advanced Chemtech, Bachem, and Peptides
International) are represented above according to a well accepted
convention. The suitable protecting group for the side chain
functional group of particular amino acids appears in parentheses.
The OH groups in the above formulae indicate that the
carboxy-terminus of each residue is free.
[0291] The protected amino acids (1.5 mmol each) are coupled with
DIC (235 .mu.l, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH
and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent.
[0292] In order to cleave the peptide from the resin and deprotect
it, a portion of 250 mg of the dried peptide resin is stirred with
2.5 mL of cleavage cocktail (94% TFA, 3% H.sub.2O, 1.5% m-cresol,
and 1.5% phenol) at room temperature for 3 hours. To induce peptide
precipitation, the cleavage mixture is added dropwise to cold
(preferably -20.degree. C.) ether. The precipitated material is
collected by filtration or centrifugation and is washed three times
with cold ether. The cleaved and deprotected peptide is dissolved
in 50% acetic acid and separated from the resin by filtration.
After dilution with water and lyophilization, 110 mg crude product
is obtained.
[0293] The crude peptide is checked by analytical HPLC using a
Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a
Supelco Discovery HS C18 reversed-phase column (2.1 mm.times.5 cm,
packed with C18 silica gel, 300 .ANG. pore size, 3 .mu.m particle
size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g.,
40-70% B) is used with a solvent system consisting of (A) 0.1%
aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate
is 0.2 mL/min. Purification is performed on a Beckman System Gold
HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with
127P solvent Module; UV-VIS Detector, model 166P; Computer
workstation with CPU Monitor and printer, and 32-Karat software,
version 3.0. 110 mg of crude peptide is dissolved in AcOH/H.sub.2O,
stirred, filtered and applied on an XBridge Prep OBD.TM. reversed
phase column (4.6.times.250 mm, packed with C.sub.i8 silica gel,
300 .ANG. pore size, 5 .mu.m particle size) (Waters Co., Milford,
Mass.). The column is eluted with a solvent system described above
in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12
mL/min. The eluent is monitored at 220 nm, and fractions are
examined by analytical HPLC. Fractions with purity higher than 95%
are pooled and lyophilized to give 16 mg pure product. The
analytical HPLC is carried out on a Supelco Discovery C18
reversed-phase column described above using isocratic elution with
a solvent system described above with a flow rate of 0.2 mL/min.
The peaks are monitored at 220 and 280 nm. The product is judged to
be substantially (>95%) pure by analytical HPLC. Molecular mass
is checked by electrospray mass spectrometry, and the expected
amino acid composition is confirmed by amino acid analysis.
[0294] In accordance with the above procedure Peptide 28421,
Peptide 28430, Peptide 28431, Peptide 28460, Peptide 28461, Peptide
28462, Peptide 28463, Peptide 28464, Peptide 28465, Peptide 28466,
Peptide 28467, Peptide 28468, Peptide 28469, Peptide 28470, Peptide
28471, Peptide 28472, Peptide 28473, Peptide 28474, Peptide 28475,
Peptide 28476, Peptide 28477, Peptide 28478, Peptide 28479 are
synthesized in the same manner as Peptide 28460, except that these
peptides also contain other amino acid substitutions in the peptide
sequence. The details for these syntheses are set forth below.
[0295] For the synthesis of Peptide 28421 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0296] For the synthesis of Peptide 28430 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0297] For the synthesis of Peptide 28431 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Thr.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(tBu)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0298] For the synthesis of Peptide 28460 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0299] For the synthesis of Peptide 28462 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0300] For the synthesis of Peptide 28463 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0301] For the synthesis of Peptide 28464 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0302] For the synthesis of Peptide 28465 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0303] For the synthesis of Peptide 28466 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0304] For the synthesis of Peptide 28467 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0305] For the synthesis of Peptide 28468 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0306] For the synthesis of Peptide 28469 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH,
Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Dat-OH.
[0307] For the synthesis of Peptide 28470 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-D-Arg(Pbf)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-AspfOBuyOH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Dat-OH.
[0308] For the synthesis of Peptide 28471 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-H, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Dat-OH.
[0309] For the synthesis of Peptide 28472 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Fpa5.sup.6, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-D-Ala-OH, Dat-OH.
[0310] For the synthesis of Peptide 28473 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27,
Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Fpa5-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Dat-OH.
[0311] For the synthesis of Peptide 28474 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28]hGH-RH(1-29)NH--CH.sub.2--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Dat-OH.
[0312] For the synthesis of Peptide 28475 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Apa.sup.30]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Apa-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0313] For the synthesis of Peptide 28476 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Aha.sup.30]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Aha-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0314] For the synthesis of Peptide 28477 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Amc.sup.30]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Amc-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0315] For the synthesis of Peptide 28478 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Har.sup.30]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the following
protected amino acids are coupled in the indicated order on the
deprotected [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM
resin: Fmoc-Har(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH,
Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,
Fmoc-Ala-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH,
Fmoc-N-Me-Tyr(tBu)-OH.
[0316] For the synthesis of Peptide 28479 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, His.sup.11,
Orn.sup.12, Abu.sup.15, His.sup.20, Orn.sup.21, Nle.sup.27,
Asp.sup.28, Apa.sup.30]hGH-RH(1-30)NH--CH.sub.2--CH.sub.3, the
following protected amino acids are coupled in the indicated order
on the deprotected
[3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin:
Fmoc-Apa-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Nle-OH,
Fmoc-Ile-OH, Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,
Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH,
Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-His(Trt)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH.
[0317] TFA cleavage and deprotection with the cleavage cocktail,
and subsequent purification by semipreparative HPLC of Peptide
28421, Peptide 28430, Peptide 28431, Peptide 28460, Peptide 28461,
Peptide 28462, Peptide 28463, Peptide 28464, Peptide 28465, Peptide
28466, Peptide 28467, Peptide 28468, Peptide 28469, Peptide 28470,
Peptide 28471, Peptide 28472, Peptide 28473, Peptide 28474, Peptide
28475, Peptide 28476, Peptide 28477, Peptide 28478, Peptide 28479
are done as described in the case of Peptide 28420. The purified
compounds are judged to be substantially (>95%) pure by
analytical HPLC. Their molecular masses are checked by electrospray
mass spectrometry, and the expected amino acid compositions are
confirmed by amino acid analysis
Example V
Dat.sup.1-D-Ala.sup.2-Asp.sup.3-Ala.sup.4-Ile.sup.5-Phe.sup.6-Thr.sup.7-As-
n.sup.8-Ser.sup.9-Tyr.sup.10-Arg.sup.11-Orn.sup.12-Val.sup.13-Leu.sup.14-A-
bu.sup.15-Gln.sup.16-Leu.sup.17-Ser.sup.18-Ala.sup.19-Arg.sup.20-Orn.sup.2-
1-Leu.sup.22-Leu.sup.23-Gln.sup.24-Asp.sup.25-Ile.sup.26-Nle.sup.27-Ser.su-
p.28-Arg.sup.29-Gln-Gab.sup.30-NH.sub.2 (Peptide 29702)
[0318] Dat.sup.1, D-Alu.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Gln-Gab.sup.30]hGH-RH(1-30)-NH.sub.2
[0319] The synthesis is conducted in a stepwise manner using manual
solid phase peptide synthesis equipment. Briefly,
para-methylbenzhydrylamine (MBHA) resin (Bachem, King of Prussia,
Pa.) (720 mg, 0.50 mmol) is pre-swollen in DCM and neutralized with
5% DIEA in DCM and washed according to the protocol described in
Table I. The solution of Boc-Gab-OH (265 mg, 1.5 mmol) in DMF-DCM
(1:1) is shaken with the neutralized resin and DIC (235 .mu.L, 1.5
mmol) in a manual solid phase peptide synthesis apparatus for 1
hour. After the completion of the coupling reaction is proved by
negative ninhydrin test, the deprotection and neutralization
protocols described in Table 2 are performed in order to remove the
Boc protecting group and prepare the peptide-resin for coupling of
the next amino acid. The synthesis is continued and the peptide
chain is built stepwise by coupling the following protected amino
acids in the indicated order on the resin to obtain the desired
peptide sequence: Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH,
Boc-Ser(Bzl)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Asn-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0320] These protected amino acid residues (also commonly available
from Novabiochem, Advanced Chemtech, Bachem, and Peptides
International) are represented above according to a well accepted
convention. The suitable protecting group for the side chain
functional group of particular amino acids appears in parentheses.
The OH groups in the above formulae indicate that the carboxyl
terminus of each residue is free.
[0321] The protected amino acids (1.5 mmol each) are coupled with
DIC (235 .mu.l, 1.5 mmol) with the exceptions of Boc-Asn-OH and
Boc-Gln-OH which are coupled with their preformed HOBt esters.
[0322] In order to cleave the peptide from the resin and deprotect
it, a portion of 250 mg of the dried peptide resin is stirred with
0.5 mL m-cresol and 5 mL hydrogen fluoride (HF) at 0-.degree. C.
for 2 hours. After evaporation of the HF under a stream of nitrogen
and in vacuo, the residue is washed with dry diethyl ether and
ethyl acetate. The cleaved and deprotected peptide is dissolved in
50% acetic acid and separated from the resin by filtration. After
dilution with water and lyophilization, 109 mg crude product is
obtained.
[0323] The crude peptide is checked by analytical HPLC using a
Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a
Supelco Discovery HS C18 reversed-phase column (2.1 mm.times.5 cm,
packed with C18 silica gel, 300 .ANG. pore size, 3 .mu.m particle
size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g.,
40-70% B) is used with a solvent system consisting of (A) 0.1%
aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate
is 0.2 mL/min. Purification is performed on a Beckman System Gold
HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with
127P solvent Module; UV-VIS Detector, model 166P; Computer
workstation with CPU Monitor and printer, and 32-Karat software,
version 3.0. 109 mg of crude peptide is dissolved in AcOH/H.sub.2O,
stirred, filtered and applied on an XBridge Prep OBD.TM. reversed
phase column (4.6.times.250 mm, packed with C.sub.18 silica gel,
300 .ANG. pore size, 5 .mu.m particle size) (Waters Co., Milford,
Mass.). The column is eluted with a solvent system described above
in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12
mL/min. The eluent is monitored at 220 nm, and fractions are
examined by analytical HPLC. Fractions with purity higher than 95%
are pooled and lyophilized to give 27 mg pure product. The
analytical HPLC is carried out on a Supelco Discovery C18
reversed-phase column described above using isocratic elution with
a solvent system described above with a flow rate of 0.2 mL/min.
The peaks are monitored at 220 and 280 nm. The product is judged to
be substantially (>95%) pure by analytical HPLC. Molecular mass
is checked by electrospray mass spectrometry, and the expected
amino acid composition is confirmed by amino acid analysis.
[0324] In accordance with the above procedure Peptide 29701,
Peptide 29703, Peptide 29704, Peptide 29706, Peptide 29708, Peptide
29710, Peptide 29720, Peptide 29721, Peptide 29722, Peptide 29723,
Peptide 29724 are synthesized in the same manner as Peptide 29702,
except that these peptides also contain other amino acid
substitutions in the peptide sequence, and acyl moieties at their
N-termini. The details for these syntheses are set forth below.
[0325] For the synthesis of Peptide 29701 the chemical structure of
which is [N-Me-Tyr.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2; the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-NLe-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(Bzl)-OH.
[0326] For the synthesis of peptide 29703 the chemical structure of
which is N-Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2 (SEQ ID NO: 39) the following
protected amino acids are coupled in the indicated order on the
MBHA resin: Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-NLe-OH, Boc-Ile-OH, Boc-Asp(OcHx),
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2CIZ)-OH, Boc-Ser(Bzl)-OH, Boc-Gln-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-Ala-OH, Boc-N-Me-Tyr(Bzl)-OH.
[0327] For the synthesis of peptide 29704 the chemical structure of
which is [Dat.sup.1, D-Ala.sup.2, Gln.sup.s, Orn.sup.12,
Abu.sup.15, Orn.sup.21,22, Nle.sup.27,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2 the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-NLe-OH, Boc-Asp(OcHx), Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH,
Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH,
Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2CIZ)-OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-Ala-OH, Boc-N-Me-Tyr(Bzl)-OH.
[0328] For the synthesis of Peptide 29706 the chemical structure of
which [Tfa-Tyr.sup.1, D-Abu.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Abu-OH, Tfa-Tyr-OH.
[0329] For the synthesis of Peptide 29708 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0330] For the synthesis of Peptide 29710 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0331] For the synthesis of Peptide 29720 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Orn.sup.12, Abu.sup.15, Orn.sup.21,
Nle.sup.27, Asp.sup.28, Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the
following protected amino acids are coupled in the indicated order
on the MBHA resin: Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH,
Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH,
Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Orn(2CIZ)-OH,
Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH, Boc-Ser(Bzl)-OH, Boc-Asn-OH,
Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-Ala-OH,
Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0332] For the synthesis of Peptide 29721 the chemical structure of
which [Dat.sup.1, D-Ala.sup.2, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-0m(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH, Dat-OH.
[0333] For the synthesis of Peptide 29722 the chemical structure of
which [Tfa-Tyr.sup.1, D-Abu.sup.2, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Abu-OH,
Tfa-Tyr-OH.
[0334] For the synthesis of Peptide 29723 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0335] For the synthesis of Peptide 29724 the chemical structure of
which [N-Me-Tyr.sup.1, D-Ala.sup.2, Fpa5.sup.6, Ala.sup.8,
Orn.sup.12, Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28,
Gln-Gab.sup.30]hGH-RH(1-30)NH.sub.2, the following protected amino
acids are coupled in the indicated order on the MBHA resin:
Boc-Gab-OH, Boc-Gln-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH,
Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH,
Boc-Leu-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH,
Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH,
Boc-Val-OH, Boc-Orn(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)--OH,
Boc-Ser(Bzl)-OH, Boc-Ala-OH, Boc-Thr(Bzl)-OH, Boc-Fpa5-OH,
Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Ala-OH,
Boc-N-Me-Tyr(2BrZ)--OH.
[0336] HF cleavage and deprotection, and subsequent purification by
semipreparative HPLC of Peptide 29701, Peptide 29703, Peptide
29704, Peptide 29706, Peptide 29708, Peptide 29710, Peptide 29720,
Peptide 29721, Peptide 29722, Peptide 29723, Peptide 29724 are done
as described in the case of Peptide 21300. The purified compounds
are judged to be substantially (>95%) pure by analytical HPLC.
Their molecular masses are checked by electrospray mass
spectrometry, and the expected amino acid compositions are
confirmed by amino acid analysis.
Example VI
Aqueous Solution for Intramuscular Injection
TABLE-US-00004 [0337] N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, 500.0 mg
Agm.sup.29]hGH-RH(1-29) (Peptide 20356) (SEQ ID NO: 6) Gelatin,
nonantigenic 5.0 mg Water for injection q.s. ad 100.0 mL
The gelatin and GH-RH antagonist Peptide 11602 are dissolved in
water for injection, and then the solution is sterile filtered.
Example VII
Long Acting Intramuscular Injectable Formulation (Sesame Oil
Gel)
TABLE-US-00005 [0338] [N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12,
Abu.sup.15, Orn.sup.21, Nle.sup.27, Asp.sup.28, 10.0 mg
Agm.sup.29]hGH-RH(1-29) (Peptide 20356) (SEQ ID NO: 6) Aluminum
monostearate, USP 20.0 mg Sesame oil q.s. ad 1.0 mL
The aluminum monostearate is combined with the sesame oil and
heated to 125-.degree. C. with stirring until a clear yellow
solution forms. This mixture is then autoclaved for sterility and
allowed to cool. The GH-RH antagonist Peptide 11604 is then added
aseptically with trituration. Particularly preferred antagonists
are salts of low solubility, e.g., pamoate salts and the like.
These exhibit long duration of activity.
Example VIII
Long Acting Intramuscular (IM) Injectable-Biodearadable Polymer
Microcapsules
[0339] Microcapsules are made from the following:
TABLE-US-00006 25/75 glycolide/lactide copolymer (0.5 intrinsic
viscosity) 99% [N--Me-Tyr.sup.1, Gln.sup.8, Orn.sup.12, Abu.sup.15,
Orn.sup.21, Nle.sup.27, Asp.sup.28, Agm.sup.29]hGH-RH(1-29)
(Peptide 20356) (SEQ ID NO: 6) 1% 25 mg of the above microcapsules
are suspended in 1.0 mL of the following vehicle: Dextrose 5.0%
CMC, sodium 0.5% Benzyl alcohol 0.9% Tween 80 0.1% Water, purified
q.s. ad 100%
Example IX
Growth Hormone Releasing Activity
[0340] Growth hormone releasing is assayed by using a superfused
rat pituitary cell system as described in S. Vigh and A. V.
Schally, Peptides 5, Suppl: 241-347, 1984, which is incorporated by
reference herein. The new synthetic peptide analogs of hGH-RH
P20356 and JI-38 as control are administered for 3 minutes (1 mL
perfusate) at 1 nM concentration as shown below. Fractions of 1 ml
are collected and the GH content in each is determined by ELISA.
Peptide P20356 was about 3 times more potent in vitro than JI-38.
Effect of hGH-RH analogs (1 nM) on the GH release in superfused rat
pituitary cell system
TABLE-US-00007 TABLE 4 GH-releasing effects of GHRH agonists P20356
(MR-356) and JI-38 in superfused rat pituitary cells GH Response
P-20356 vs JI-38 Basal GH (ng/ml) 42.51 GH Response (ng/ml) 1 2 3
Average P-20356 - 1 nM for 3 min 1 49.98 54.99 52.37 52.45 2 310.58
325.76 376.11 337.48 3 491.01 602.1 576.26 556.46 4 399.95 270.02
-- 334.99 5 200.64 195.18 -- 197.91 JI-38 - 1 nM for 3 min 21 42.46
56.07 -- 49.27 22 143.58 119.83 -- 131.71 23 222.13 167.23 --
194.68 24 142.96 131.93 -- 137.45 25 96.34 97.05 -- 96.70
Conclusions: P-20356 is 2-3 times more potent than JI-38
[0341] Pituitary cells from 2 male rates were used for each channel
of the superfusion system. The cells were exposed to 3-min pulses
of the new GHRH agonists or to JI-38 as standard every 30 min.
Outflowing samples of each channel (1 ml) were collected every 3
min, and GH levels were determined by ELISA.
Example X
[0342] Receptor binding assay Ligand competition assay with
.sup.125I-labeled [His.sup.1, Nle.sup.27]hGH-RH(1-32)-NH.sub.2 was
used to determine the binding affinities of the novel hGH-RH
agonists to membrane receptors of rat anterior pituitary cells. The
methods used have been described in detail (Halmos G, et al.
Receptor 3: 87-97, 1993), here by incorporated by reference.
Briefly, radioidonated [His.sup.1, Nle.sup.27]hGH-RH(1-32)-NH.sub.2
is prepared by the chloramines-T method. In competitive binding
analyses, .sup.125I-Iabeled [His.sup.1,
Nle.sup.27]hGH-RH(1-32)-NH.sub.2 (0.2 nM) was displaced by the
GH-RH analogs at 10.sup.-6-10.sup.-12 M. The final binding
affinities were calculated using the LIGAND-PC computerized
curve-fitting program. Relative affinities were compared to
hGH-RH(1-29) and/or analog JI-38 (Izdebski J, et al. Proc. Natl.
Acad. Sci. 92: 4872-4876, 1995) and calculated as the ratio of
IC.sub.50 of the tested peptide to the IC.sub.50 of the standard.
IC.sub.50 is the dose of the tested peptides causing 50% inhibition
of specific binding to receptors.
GHRH Receptor Binding Studies
Binding Affinities
Materials and Methods
[0343] Preparation of human pituitary membrane fraction and
receptor binding of GHRH agonists were performed as previously
described, by using a sensitive in vitro ligand competition assay
based on binding of .sup.125I-labeled JV-1-42 to human pituitary
membrane homogenates. Normal human pituitaries were purchased from
the National Hormone and Peptide Program (A. F. Parlow, Los
Angeles, County Harbor-UCLA Medical Center, Torrance, Calif.).
Briefly, in competitive binding analysis, .sup.125I-labeled JV-1-42
(.about.0.2 nM) was displaced by GHRH agonists at 10.sup.-6 to
10.sup.-12 M.
[0344] The final binding affinities were expressed as IC.sub.50
values and were calculated by using the LIGAND PC computerized
curve-fitting program of Munson and Rodbard as modified by
McPherson.
Results
[0345] The results of these experiments are given in the Table 5.
IC.sub.50 values of the best agonists were in the 0.01-0.09 nM
range. Based on the receptor binding results all these new GHRH
agonists exceeded the binding affinity of our reference peptide
JI-38. Some of these new GHRH agonists tested showed the highest
GHRH receptor binding affinity, their IC.sub.50 values being 45-406
times lower than that of GHRH(1-29). Based on its IC.sub.50 value,
GHRH agonist P20356 showed 171 times higher binding affinity than
our reference compound JI-38.
TABLE-US-00008 TABLE 5 IC.sub.50w values and binding activities of
new GHRH agonistic analogs Relative affinity (Binding potency) GHRH
agonists IC.sub.50 (nM) vs GHRH vs JI-38 GHRH (1-29) 4.06 1 JI-38
1.71 2.4 1 P20303 0.09 45.1 19.0 P20350 0.04 101.5 42.7 P20356 0.01
406.0 171.0 P25502 0.07 58.0 24.4 P29702 0.05 81.2 34.2 * Expressed
relative to GHRH(1-29) = 1 or JI-38 (GHRH agonist) = 1, Values were
calculated from duplicate tubes.
GH-RH Receptor Binding Studies
Binding Affinities
Materials and Methods
[0346] Preparation of human pituitary membrane fraction and
receptor binding of GHRH agonists were performed as previously
described, by using a sensitive in vitro ligand competition assay
based on binding of .sup.125I-labeled [His.sup.1,
Nle.sup.27]hGHRH(1-32)NH.sub.2 to human pituitary membrane
homogenates. Normal human pituitaries were purchased from the
National Hormone and Peptide Program (A. F. Parlow, Los Angeles,
County Harbor--UCLA Medical Center, Torrance, Calif.).
[0347] Briefly, in competitive binding analysis, [His.sup.1,
125I-Tyr.sup.10, Nle.sup.27]hGHRH(1-32)NH.sub.2 (0.2 nM) was
displaced by GHRH agonists at 10.sup.-6 to 10.sup.-12 M. The final
binding affinities were expressed as IC.sub.50 values and were
calculated by using the LIGAND PC computerized curve-fitting
program of Munson and Rodbard as modified by McPherson.
Results
[0348] The results of these experiments are given in the Table
enclosed. IC.sub.50 values of the best agonists were in the
0.04-0.09 nM range. Based on the receptor binding results all new
GHRH agonists exceeded the binding affinity of reference peptides
JI-34, JI-36 and JI-38. Some of these new GHRH agonists showed the
highest GHRH receptor binding affinity, their IC.sub.50 values
being 21-48 times lower than that of the GHRH agonist JI-38.
TABLE-US-00009 TABLE 6 IC.sub.M values and binding activities of
new GHRH agonistic analogs Relative affinity (Binding potency) GHRH
agonists IC.sub.50 (nM) vs GHRH vs JI-38 GH-RH(1-29) 5.92 1 JI-34
1.37 4.32 JI-36 1.82 3.25 JI-38 1.95 3.03 1 P-23252 0.14 42.3 13.9
P-23254 0.07 84.5 27.8 P-23256 0.04 148.0 48.7 P-21304 0.08 74.0
24.4 P-20352 0.07 84.5 27.8 * Expressed relative to GHRH(1-29) = 1
or JI-38 (GHRH agonist) = 1 Values were calculated from duplicate
or triplicate tubes.
GH-RH Receptor Binding Studies
Binding Affinities
Materials and Methods
[0349] Preparation of human pituitary membrane fraction and
receptor binding of GHRH agonists were performed as previously
described, by using a sensitive in vitro ligand competition assay
based on binding of .sup.125I -labeled JV-1-42 to human pituitary
membrane homogenates. Normal human pituitaries were purchased from
the National Hormone and Peptide Program (A. F. Parlow, Los
Angeles, County Harbor--UCLA Medical Center, Torrance, Calif.).
[0350] Briefly, in competitive binding analysis, .sup.125I-labeled
JV-1-42 (.about.0.2 nM) was displaced by GHRH agonists at 10.sup.-6
to 10.sup.-12 M. The final binding affinities were expressed as
IC.sub.50 values and were calculated by using the LIGAND PC
computerized curve-fitting program of Munson and Rodbard as
modified by McPherson
Results
[0351] The results of these experiments are given in the Table
enclosed. IC.sub.50 values of the best agonists were in the
0.16-0.87 nM range. Based on the receptor binding results most of
the new GHRH agonists exceeded the binding affinity of reference
peptides JI-38. Some of these new GHRH agonists showed 5-27 times
higher binding potency than GHRH agonist JI-38. See Table 7.
TABLE-US-00010 TABLE 7 IC.sub.50 values and binding activities of
new GHRH agonistic analogs Relative affinity (Binding potency) GHRH
agonists IC.sub.50 (nM) vs JI-38 JI-38 4.35 1 P-21300 3.61 1.20
P-21301 2.99 1.45 P-21303 1.87 2.32 P-22325 3.80 1.14 P-22326 0.71
6.12 P-22327 1.99 2.18 P-20357 0.86 5.06 P-20350 0.52 8.37 P-20351
3.44 1.26 P-20356 0.27 16.11 P-20359 3.05 1.43 P-20361 0.82 5.30
P-20367 1.70 2.56 P-25501 1.07 4.07 P-25502 0.33 13.18 P-25503 1.18
3.67 P-25504 1.44 3.02 P-27413 2.45 1.78 P-27414 1.56 2.79 P-27415
3.02 1.44 P-29702 0.86 5.06 P-29703 1.22 3.57 P-27400 3.35 1.30
P-27401 2.74 1.59 P-27403 0.16 27.19 P-27404 0.87 5.00 P-27405 1.08
4.03 P-27406 0.30 14.5 P-27407 3.00 1.45 P-27408 0.55 7.91 P-27409
1.06 4.10 P-27410 0.83 5.24 P-28420 0.52 8.37 P-28421 1.47 2.96
Expressed relative to Jl-38 (GHRH agonist) = 1 Values were
calculated from duplicate tubes *reference compound
Example XI
[0352] In vivo tests on endocrine activity of new GHRH
agonists.
Intravenous Administration.
[0353] For in vivo tests based on intravenous administration, adult
male Sprague-Dawley rats are anesthetized with pentobarbital (6
mg/100/g, b.w.), injected i.p.; 20 minutes after the injection of
pentobarbital, blood samples are taken from the jugular vein
(pretreated level) and immediately thereafter hGH-RH(1-29)NH.sub.2
(as a control) or hGH-RH analogs are injected i.v. Blood samples
are taken from the jugular vein 5, 15 and 30 minutes after the
injection. The blood samples are centrifuged, plasma is removed and
the GH level is measured by ELISA. The results expressed as potency
relative to hGH-RH(1-29)NH.sub.2 appear in Table 8.
TABLE-US-00011 TABLE 8 GH releasing potencies of hGH-RH analogs in
vivo relative to JI-38 (=1) in the rat after i.v. injection hGH-RH
Analog After (min) Potency P-20356 5 1.07 15 0.91 30 1.22 P-21300 5
0.39 15 0.51 30 0.81 P-21301 5 0.79 15 0.92 30 1.00 P-21303 5 0.79
15 1.14 30 0.81 P-22326 15 0.28 30 0.94 P-25502 5 6.76 15 5.40 30
5.83 P-25504 5 1.66 15 1.65 30 1.37 P-27403 15 5.01 30 4.01 P-27450
5 0.07 15 0.11 30 0.49 P-28475 5 0.19 15 0.36 30 0.92 P-29702 5
0.98 15 0.99 30 1.22
Example XI
Subcutaneous Administration
[0354] Adult male rats are used and are anesthetized with
pentobarbital (6 mg/100 g, b.w.), injected i.p.; 20 minutes after
the injection of pentobarbital, blood samples are taken from the
jugular vein (pretreated level) and immediately thereafter
hGH-RH(1-29)NH.sub.2 (as a control) or hGH-RH analogs are injected
subcutaneously (s.c.). Blood samples are taken from the jugular
vein; 5, 15 and 30 minutes or only 15 and 30 minutes after the
injection. The blood samples are centrifuged, plasma is removed and
the GH level is measured by ELISA. The results are summarized in
terms of potency in Table 9.
TABLE-US-00012 TABLE 9 GH releasing potencies of hGH-RH analogs
after subcutaneous (s.c.) injection relative to JI-38 (=1) hGH-RH
analog After (min) Potency P-20350 15 1.53 30 1.17 P-20351 15 0.38
30 0.44 P-20353 15 0.26 30 0.31 P-20356 15 1.72 30 1.09 P-20357 5
0.63 15 1.07 30 1.41 P-20360 15 0.24 30 0.39 P-20361 15 1.18 30
1.50 P-20367 15 1.12 30 2.01 P-20373 15 0.23 30 0.88 P-21301 15
0.41 30 0.74 P-221303 15 0.95 30 1.45 P-22325 5 0.33 15 0.68 30
1.03 P-22326 15 1.76 30 2.31 P-22327 15 1.15 30 1.30 P-25501 5 1.40
15 1.36 30 1.63 P-25502 15 1.10 30 0.94 P-25503 5 0.55 15 0.64 30
0.63 P-25504 15 0.78 30 0.98 P-27400 15 0.47 30 0.38 P-27401 15
0.61 30 0.73 P-27403 15 3.60 30 2.57 P-27404 15 2.07 30 1.47
P-27405 15 1.60 30 1.13 P-27406 15 0.47 30 0.50 P-27409 15 1.47 30
1.31 P-27412 15 1.10 30 1.29 P-27413 15 0.36 30 0.57 P-27414 15
1.30 30 1.23 P-27415 15 0.45 30 0.41 P-27425 15 0.49 30 0.31
P-29701 5 0.92 15 1.30 30 1.55 P-29702 15 0.53 30 0.73 P-29703 5
1.18 15 0.96 30 1.04
Analysis of Endocrine Tests
[0355] Following intravenous administration, the new analogs give
growth hormone levels greater than those from hGH-RH(1-29)NH.sub.2
or JI-38. The effect is longer lasting which indicates that the
analogs have not only higher receptor affinity but also increased
peptidase resistance. The most potent analogs i.v. were P-27403 and
P-25502. Following subcutaneous administration, the analogs give
that greater growth hormone levels than hGH-RH or JI-38. Here the
analogs P-22326, P-20350, P-20356, P-27403, P-27404, P-27409,
P-25501, P-25502 produce unusually high responses.
[0356] Results of i.v. and s.c. administration results show
different biological activity pattern. Analogs given i.v. are
subject to degredation in the blood stream. Analogs given s.c. can
be degraded by peptidase at the site of injection. It is believed
that activity depends primarily on binding capacity of the peptide
to its receptor, and from favorable transport properties, suitable
binding to plasma proteins and metabolic stability. The above
findings therefore indicate that the analogs showing better
activity when given subcutaneously are resistant to local
degradation at the injection site and they may also be less
susceptible to enzyme degradation in the blood stream and/or have
more affinity for GH-RH receptors than hGH-RH(1-29)NH.sub.2.
[0357] In conclusion, the most potent analogs i.v. were P-27403 and
P-25502. Following subcutaneous administration, the analogs that
give especially greater growth hormone levels than hGH-RH or JI-38
are P-22326, P-20350, P-20356, P-27403, P-27404, P-27409, P-25501,
P-25502.
Sequence CWU 1
1
42130PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Xaa Xaa Asp Ala Ile Xaa Thr Xaa Ser Tyr Xaa
Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Xaa Xaa Xaa Leu Gln Asp
Ile Xaa Xaa Xaa Xaa 20 25 30 229PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide- P20103 2Xaa Ala Asp Ala
Ile Xaa Thr Gln Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser
Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Xaa 20 25 329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide-P20107
3Xaa Ala Asp Ala Ile Xaa Thr Ala Ser Tyr Arg Xaa Val Leu Xaa Gln 1
5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Xaa 20 25
429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide - P20113 4Xaa Ala Asp Ala Ile Xaa Thr Asn Ser Tyr
Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln
Asp Ile Xaa Asp Xaa 20 25 529PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide - P20115 5Xaa Ala Asp Ala Ile
Xaa Thr Thr Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala
Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Xaa 20 25 629PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide
-P20356 6Xaa Ala Asp Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Xaa 20 25 729PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide - P20370 7Xaa Ala Asp Ala Ile Phe Thr
Asn Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa
Leu Leu Gln Asp Ile Xaa Asp Xaa 20 25 829PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide -
P20371 8Xaa Ala Asp Ala Ile Phe Thr Thr Ser Tyr Arg Xaa Val Leu Xaa
Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Xaa
20 25 929PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide - P20372 9Xaa Ala Asp Ala Ile Phe Thr Ala Ser Tyr
Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln
Asp Ile Xaa Asp Xaa 20 25 1029PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide - P20373 10Xaa Ala Asp Ala
Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser
Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser Xaa 20 25 1129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide -
P20374 11Xaa Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser
Xaa 20 25 1229PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide - P20375 12Xaa Ala Asp Ala Ile Phe Thr
Thr Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa
Leu Leu Gln Asp Ile Xaa Ser Xaa 20 25 1329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide -
P20376 13Xaa Ala Asp Ala Ile Phe Thr Ala Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser
Xaa 20 25 1430PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide - P21301 14Xaa Ala Asp Ala Ile Phe
Thr Asn Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg
Xaa Leu Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30 1530PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P22325 15Xaa Ala Asp Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Arg Xaa 20 25 30 1630PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide - P22327 16Xaa Ala Asp
Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu
Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30
1730PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide - P22332 17Xaa Ala Asp Ala Ile Phe Thr Asn
Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu
Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30 1830PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P22334 18Xaa Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser
Arg Xaa 20 25 30 1930PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide - P22335 19Xaa Ala Asp
Ala Ile Phe Thr Thr Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu
Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30
2030PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide - P22336 20Xaa Ala Asp Ala Ile Phe Thr Ala
Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu
Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30 2130PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P23251 21Xaa Ala Asp Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Arg Xaa 20 25 30 2230PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide - P23253 22Tyr Ala Asp
Ala Ile Phe Thr Asn Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu
Ser Ala Arg Xaa Leu Leu Gln Asp Ile Leu Asp Arg Arg 20 25 30
2330PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide - P23255 23Xaa Ala Asp Ala Ile Phe Thr Thr
Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu
Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30 2430PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P23257 24Xaa Ala Asp Ala Ile Phe Thr Ala Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Arg Xaa 20 25 30 2530PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide - P23259 25Xaa Ala Asp
Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu
Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30
2630PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide - P23261 26Xaa Ala Asp Ala Ile Phe Thr Asn
Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu
Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30 2730PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P23263 27Xaa Ala Asp Ala Ile Phe Thr Thr Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Arg Xaa 20 25 30 2830PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide - P23265 28Xaa Ala Asp
Ala Ile Phe Thr Ala Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu
Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30
2930PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide - P24340 29Xaa Ala Asp Ala Ile Phe Thr Gln
Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu
Leu Gln Asp Ile Xaa Asp Arg Xaa 20 25 30 3030PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P24342 30Xaa Ala Asp Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Arg Xaa 20 25 30 3130PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide - P25503 31Xaa Xaa Asp
Ala Ile Phe Thr Asn Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu
Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser Arg Xaa 20 25 30
3229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide - P27405 32Xaa Ala Asp Ala Ile Phe Thr Gln Ser
Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu
Gln Asp Ile Xaa Ser Arg 20 25 3329PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide - P27406 33Xaa Ala Asp Ala
Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser
Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp Arg 20 25 3429PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide -
P27407 34Xaa Ala Asp Ala Ile Phe Thr Gln Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Asp
Arg 20 25 3529PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide - P27413 35Xaa Ala Asp Ala Ile Phe Thr
Gln Ser Tyr His Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala His Xaa
Leu Leu Gln Asp Ile Xaa Asp Arg 20 25 3630PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide -
P27415 36Xaa Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser
Arg Xaa 20 25 30 3729PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide - P27419 37Tyr Ala Asp Ala
Ile Phe Thr Thr Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser
Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser Arg 20 25 3829PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide -
P27424 38Xaa Ala Asp Ala Ile Phe Thr Thr Ser Tyr Arg Xaa Val Leu
Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Xaa Ser
Arg 20 25 3931PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide - P29703 39Xaa Ala Asp Ala Ile Phe
Thr Gln Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg
Xaa Leu Leu Gln Asp Ile Xaa Ser Arg Gln Xaa 20 25 30
4030PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 40Xaa Xaa Asp Ala Ile Xaa Thr Xaa Ser Tyr Xaa
Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Xaa Xaa Xaa Leu Gln Asp
Ile Leu Xaa Xaa Xaa 20 25 30 4130PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 41Xaa Xaa Asp Ala Ile
Xaa Thr Xaa Ser Tyr Arg Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala
Arg Xaa Leu Leu Gln Asp Ile Xaa Xaa Xaa Xaa 20 25 30
4230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 42Xaa Xaa Asp Ala Ile Xaa Thr Xaa Ser Tyr Arg
Xaa Val Leu Xaa Gln 1 5 10 15 Leu Ser Ala Arg Xaa Leu Leu Gln Asp
Ile Xaa Xaa Xaa Xaa 20 25 30
* * * * *