U.S. patent application number 12/692788 was filed with the patent office on 2010-05-27 for cyclic peptide cxcr4 antagonists.
This patent application is currently assigned to ELI LILLY AND COMPANY. Invention is credited to Wayne David Kohn, Sheng-Bin Peng, Liang Zeng Yan.
Application Number | 20100130409 12/692788 |
Document ID | / |
Family ID | 39831600 |
Filed Date | 2010-05-27 |
United States Patent
Application |
20100130409 |
Kind Code |
A1 |
Kohn; Wayne David ; et
al. |
May 27, 2010 |
CYCLIC PEPTIDE CXCR4 ANTAGONISTS
Abstract
Provided are lactam-cyclized peptide CXCR4 antagonists useful in
the treatment of cancers, rheumatoid arthritis, pulmonary fibrosis,
and HIV infection.
Inventors: |
Kohn; Wayne David; (Avon,
IN) ; Peng; Sheng-Bin; (Carmel, IN) ; Yan;
Liang Zeng; (Carmel, IN) |
Correspondence
Address: |
ELI LILLY & COMPANY
PATENT DIVISION, P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Assignee: |
ELI LILLY AND COMPANY
Indianapolis
IN
|
Family ID: |
39831600 |
Appl. No.: |
12/692788 |
Filed: |
January 25, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12123574 |
May 20, 2008 |
7691813 |
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12692788 |
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60940996 |
May 31, 2007 |
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60940802 |
May 30, 2007 |
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Current U.S.
Class: |
514/1.1 ;
530/317 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 29/00 20180101; A61P 31/00 20180101; A61P 11/00 20180101; A61P
31/18 20180101; C07K 7/54 20130101; A61P 35/00 20180101; C07K 7/56
20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/11 ;
530/317 |
International
Class: |
A61K 38/03 20060101
A61K038/03; C07K 4/00 20060101 C07K004/00; A61P 35/00 20060101
A61P035/00; A61P 31/18 20060101 A61P031/18 |
Claims
1-14. (canceled)
15. A lactam-cyclized peptide of formula I: TABLE-US-00004 (I) (SEQ
ID NO: 1)
R.sub.1-cyclo[X.sub.1-Tyr-X.sub.3-DArg-2Nal-Gly-X.sub.7]-X.sub.8-X.sub.9-X-
.sub.10-R.sub.2
or a pharmaceutically acceptable salt thereof, wherein: X.sub.1 is
selected from the group consisting of (D/L)Agl, Ala, .beta.-Ala,
DAla, 5-aminovaleryl, 4-AMB, 4-AMPA, Asp, Dab, Dap, Dap(Ac), Glu,
Gly, Leu, Lys, Lys(Ac), 2Nal, Phe, DPhe, and succinyl, or is
absent, wherein when X.sub.1 is (D/L)Agl, Dab, or Dap and the
.alpha.-amino group of X.sub.1 is not a constituent of the lactam
amide bond, said .alpha.-amino group is substituted with R.sub.1
which is selected from the group consisting of Ac and n-hexanoyl;
wherein when X.sub.1 is Asp or Glu and the .alpha.-amino group of
X.sub.1 is not a constituent of the lactam amide bond, said
.alpha.-amino group is substituted with R.sub.1 which is selected
from the group consisting of Ac and Bz; and wherein when X.sub.1 is
Ala, .beta.-Ala, DAla, 5-aminovaleryl, 4-AMB, 4-AMPA, Dap(Ac), Gly,
Leu, Lys, Lys(Ac), 2Nal, Phe, DPhe or succinyl, R.sub.1 is absent;
X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2); X.sub.7 is selected from the group
consisting of (D/L)Agl, Asp, Dab, Dap, DDap, Glu, DGlu, Lys, and
Orn; X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg, DArg, Gly, Lys, Lys(iPr), and Orn, or is absent; X.sub.9 is
selected from the group consisting of Gly, 2Nal, D2Nal, and DPhe,
or is absent; X.sub.10 is 2Nal, or is absent, wherein when X.sub.8
is absent, X.sub.9 and X.sub.10 are each absent, and when X.sub.9
is absent, X.sub.10 is absent; and R.sub.2 is selected from the
group consisting of NH.sub.2 and NHEt, and further wherein: said
lactam is formed by an amide bond between the side chain amino
group of X.sub.1 and the side chain carboxyl group of X.sub.7, and
X.sub.1 and X.sub.7 are, respectively, a pair selected from the
group consisting of (D/L)Agl/Glu, Dab/Glu, and Dap/Glu, and R.sub.1
is Ac or n-hexanoyl; or said lactam is formed by an amide bond
between the side chain carboxyl group of X.sub.1 and the side chain
amino group of X.sub.7, and X.sub.1 and X.sub.7 are, respectively,
a pair selected from the group consisting of Asp/(D/L)Agl, Asp/Dab,
Asp/Dap, Glu/(D/L)Agl, Glu/Dab, Glu/Dap, Glu/DDap, and Glu/Lys, and
R.sub.1 is Ac or Bz, or wherein X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of
succinyl/(D/L)Agl, succinyl/Dab, succinyl/Dap, succinyl/Lys, and
succinyl/Orn, and R.sub.1 is absent; or said lactam is formed by an
amide bond between the .alpha.-amino group of X.sub.1 and the side
chain carboxyl group of X.sub.7, and X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of Ala/Glu,
Ala/DGlu, DAla/Glu, DAla/DGlu, Dap(Ac)/Glu, Gly/Asp, Gly/Glu,
Gly/DGlu, Leu/Glu, Leu/DGlu, Lys/DGlu, Lys(Ac)/Glu, 2Nal/Glu,
Phe/Glu, Phe/DGlu, DPhe/Glu, and DPhe/DGlu, and R.sub.1 is absent;
or said lactam is formed by an amide bond between a non-.alpha.,
non-side-chain amino group of X.sub.1 and the side chain carboxyl
group of X.sub.7, and X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of .beta.-Ala/Asp,
.beta.-Ala/Glu, 5-amino-valeryl/Asp, 5-aminovaleryl/Glu, 4-AMB/Glu,
4-AMPA/Asp, and 4-AMPA/Glu, and R.sub.1 is absent; or said lactam
is formed by an amide bond between the .alpha.-amino group of Tyr
at X.sub.2 and the side chain carboxyl group of X.sub.7, and
X.sub.7 is selected from the group consisting of Asp, Glu, and
DGlu, and R.sub.1 and X.sub.1 are each absent.
16. The lactam-cyclized peptide or pharmaceutically acceptable salt
thereof of claim 15, wherein: said lactam is formed by an amide
bond between the side chain amino group of X.sub.1 and the side
chain carboxyl group of X.sub.7; R.sub.1 is selected from the group
consisting of Ac and n-hexanoyl; X.sub.1 is selected from the group
consisting of (D/L)Agl, Dab, and Dap; X.sub.3 is selected from the
group consisting of Arg and Lys(iPr); X.sub.7 is Glu; X.sub.8 is
Arg; X.sub.9 is absent; X.sub.10 is absent; and R.sub.2 is
NH.sub.2.
17. The lactam-cyclized peptide or pharmaceutically acceptable salt
thereof of claim 15, wherein: said lactam is formed by an amide
bond between the side chain carboxyl group of X.sub.1 and the side
chain amino group of X.sub.7; R.sub.1 is selected from the group
consisting of Ac and Bz; X.sub.1 is selected from the group
consisting of Asp and Glu; X.sub.3 is selected from the group
consisting of Arg and Lys(Me.sub.2); X.sub.7 is selected from the
group consisting of (D/L)Agl, Dab, Dap, DDap, and Lys; X.sub.8 is
Arg; X.sub.9 is absent; X.sub.10 is absent; and R.sub.2 is
NH.sub.2.
18. The lactam-cyclized peptide or pharmaceutically acceptable salt
thereof of claim 15, wherein: said lactam is formed by an amide
bond between the side chain carboxyl group of X.sub.1 and the side
chain amino group of X.sub.7; R.sub.1 is absent; X.sub.1 is
succinyl; X.sub.3 is Arg; X.sub.7 is selected from the group
consisting of (D/L)Agl, Dab, Dap, Lys, and Orn; X.sub.8 is Arg;
X.sub.9 is absent; X.sub.10 is absent; and R.sub.2 is NH.sub.2.
19. The lactam-cyclized peptide or pharmaceutically acceptable salt
thereof of claim 15, wherein: said lactam is formed by an amide
bond between the .alpha.-amino group of X.sub.1 and the side chain
carboxyl group of X.sub.7; R.sub.1 is absent; X.sub.1 is selected
from the group consisting of Ala, DAla, Gly, Dap(Ac), Leu, Lys,
Lys(Ac), 2Nal, Phe, and DPhe; X.sub.3 is selected from the group
consisting of Arg, Lys, Lys(iPr), and Lys(Me.sub.2); X.sub.7 is
selected from the group consisting of Asp, Glu, and DGlu; X.sub.8
is selected from the group consisting of .beta.-Ala, Arg, Gly, Lys,
Lys(iPr), and Orn, or is absent; X.sub.9 is selected from the group
consisting of Gly, 2Nal, D2Nal, and DPhe, or is absent; X.sub.10 is
2Nal, or is absent; wherein when X.sub.8 is absent, X.sub.9 and
X.sub.10 are each absent; and R.sub.2 is selected from the group
consisting of NH.sub.2 and NHEt.
20. The lactam-cyclized peptide or pharmaceutically acceptable salt
thereof of claim 15, wherein: said lactam is formed by an amide
bond between a non-.alpha., non-side-chain amino group of X.sub.1
and the side chain carboxyl group of X.sub.7; R.sub.1 is absent;
X.sub.1 is selected from the group consisting of .beta.-Ala, 4-AMB,
5-aminovaleryl, and 4-AMPA; X.sub.3 is Arg; X.sub.7 is selected
from the group consisting of Asp and Glu; X.sub.8 is selected from
the group consisting of Arg and DArg; X.sub.9 is absent; X.sub.10
is absent; and R.sub.2 is NH.sub.2.
21. The lactam-cyclized peptide or pharmaceutically acceptable salt
thereof of claim 15, wherein: said lactam is formed by an amide
bond between the .alpha.-amino group of X.sub.2 and the side chain
carboxyl group of X.sub.7; R.sub.1 is absent; X.sub.1 is absent;
X.sub.3 is Arg; X.sub.7 is selected from the group consisting of
Asp, Glu, and DGlu; X.sub.8 is Arg; X.sub.9 is absent; X.sub.10 is
absent; and R.sub.2 is NH.sub.2.
22. A pharmaceutical composition, comprising a lactam-cyclized
peptide or pharmaceutically acceptable salt thereof of claim 15,
and a pharmaceutically acceptable carrier, diluent, or
excipient.
23. A method of treating rheumatoid arthritis, pulmonary fibrosis,
HIV infection, or a cancer selected from the group consisting of
breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney
cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian
cancer, colorectal cancer, multiple myeloma, glioblastoma
multiforme, and chronic lymphocytic leukemia, comprising
administering to a patient in need thereof an effective amount of a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of claim 15.
Description
[0001] The present invention relates to novel cyclic peptide CXCR4
antagonist compounds and their use in treating diseases in which
pathogenesis is mediated by CXCR4 and SDF-1.
[0002] CXCR4, a G-protein-coupled receptor, and its naturally
occurring ligand, stromal cell-derived factor-1 (SDF-1; CXCL12),
are a chemokine receptor-ligand pair. CXCR4 is constitutively- or
over-expressed in a wide variety of human cancers. SDF-1, the only
known ligand of CXCR4, is highly expressed in tumor
microenvironments, as well as in bone marrow, lung, liver, and
lymph nodes, i.e., organ sites most commonly involved in tumor
metastasis. CXCR4/SDF-1 interaction plays important roles in
multiple stages of tumorigenesis, including tumor growth, invasion,
angiogenesis, and metastasis, as well as in rheumatoid arthritis,
pulmonary fibrosis, and HIV infection (Tsutsumi et al. (2006)
Peptide Science 88(2):279-289).
[0003] In view of the involvement of CXCR4/SDF-1 in these serious
diseases, CXCR4 is an attractive therapeutic target.
[0004] AMD3100, a bicyclam CXCR4 antagonist, is currently in Phase
III clinical trials for stem cell mobilization for transplantation
of stern cells in patients with multiple myeloma and non-Hodgkins
lymphoma. AMD070, another small molecule CXCR4 antagonist, is
currently in Phase II clinical trials for HIV infection. CTCE9908,
a bivalent (dimeric) peptide CXCR4 antagonist, is currently in
Phase Ib/II clinical trials for cancer. FC131, a cyclic
pentapeptide CXCR4 antagonist, inhibits .sup.125I-SDF-1 binding to
CXCR4 transfectants with an IC.sub.50 of 4 nM (Fujii et al. (2003)
Angew. Chem. Int. Ed. 42:3251-3253; Araki et al. (2003) Peptide
Science. The Japanese Peptide Society (2004):207-210).
[0005] There exists a need for improved CXCR4 antagonists that are
potent and selective, exhibiting little or no activity at other
chemokine receptors. The compounds of the present invention are
such potent and selective CXCR4 antagonists. Their high potency
permits the use of low doses in therapeutic regimens, while their
high selectivity minimizes non-target related adverse side effects.
In addition, compounds disclosed herein possess other highly
desirable pharmacologic properties, such as high bioavailability
when administered subcutaneously, good in vivo metabolic stability,
and pharmacokinetic/pharmacodynamic properties that permit
convenient dosing.
[0006] Accordingly, in a first aspect, the present invention
provides a lactam-cyclized peptide of formula I:
TABLE-US-00001 (I) (SEQ ID NO: 1)
R.sub.1-cyclo[X.sub.1-Tyr-X.sub.3-DArg-2Nal-Gly-X.sub.7]-X.sub.8-X.sub.9-X-
.sub.10-R.sub.2
wherein:
[0007] a) said lactam is formed by an amide bond between the side
chain amino group of X.sub.1 and the side chain carboxyl group of
X.sub.7, wherein X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of (D/L)Agl/Glu, Dab/Glu, and
Dap/Glu, and R.sub.1 is Ac or n-hexanoyl; or
[0008] b) said lactam is formed by an amide bond between the side
chain carboxyl group of X.sub.1 and the side chain amino group of
X.sub.7, wherein X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of Asp/(D/L)Agl, Asp/Dab,
Asp/Dap, Glu/(D/L)Agl, Glu/Dab, Glu/Dap, Glu/DDap, and Glu/Lys, and
R.sub.1 is Ac or Bz, or wherein X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of
succinyl/(D/L)Agl, succinyl/Dab, succinyl/Dap, succinyl/Lys, and
succinyl/Orn, and R.sub.1 is absent; or
[0009] c) said lactam is formed by an amide bond between the
.alpha.-amino group of X.sub.1 and the side chain carboxyl group of
X.sub.7, wherein X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of Ala/Glu, Ala/DGlu, DAla/Glu,
DAla/DGlu, Dap(Ac)/Glu, Gly/Asp, Gly/Glu, Gly/DGlu, Leu/Glu,
Leu/DGlu, Lys/DGlu, Lys(Ac)/Glu, 2Nal/Glu, Phe/Glu, Phe/DGlu,
DPhe/Glu, and DPhe/DGlu, and R.sub.1 is absent; or
[0010] d) said lactam is formed by an amide bond between a
non-.alpha., non-side-chain amino group of X.sub.1 and the side
chain carboxyl group of X.sub.7, wherein X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of
.beta.-Ala/Asp, .beta.-Ala/Glu, 5-amino-valeryl/Asp,
5-aminovaleryl/Glu, 4-AMB/Glu, 4-AMPA/Asp, and 4-AMPA/Glu, and
R.sub.1 is absent; or
[0011] e) said lactam is formed by an amide bond between the
.alpha.-amino group of X.sub.2 and the side chain carboxyl group of
X.sub.7, wherein X.sub.2 and X.sub.7 are, respectively, a pair
selected from the group consisting of Tyr/Asp, Tyr/Glu, and
Tyr/DGlu, and R.sub.1 and X.sub.1 are each absent;
[0012] R.sub.1 is a substituent on the .alpha.-amino group of
X.sub.1 when X.sub.1 contains an .alpha.-amino group and said
.alpha.-amino group is not a constituent of said lactam amide bond,
selected from the group consisting of Ac, Bz, and n-hexanoyl, or is
absent, wherein X.sub.1 is selected from the group consisting of
(D/L)Agl, Asp, Dab, Dap, and Glu;
[0013] X.sub.1 is selected from the group consisting of (D/L)Agl,
Ala, .beta.-Ala, DAla, 5-aminovaleryl, 4-AMB, 4-AMPA, Asp, Dab,
Dap, Dap(Ac), Glu, Gly, Leu, Lys, Lys(Ac), 2Nal, Phe, DPhe, and
succinyl, or is absent;
[0014] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0015] X.sub.7 is selected from the group consisting of (D/L)Agl,
Asp, Dab, Dap, DDap, Glu, DGlu, Lys, and Orn;
[0016] X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg, DArg, Gly, Lys, Lys(iPr), and Orn, or is absent;
[0017] X.sub.9 is selected from the group consisting of Gly, 2Nal,
D2Nal, and DPhe, or is absent;
[0018] X.sub.10 is 2Nal, or is absent;
[0019] wherein when X.sub.8 is absent, X.sub.9 and X.sub.10 are
each absent, and when X.sub.9 is absent, X.sub.10 is absent,
and
[0020] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt, or
[0021] a pharmaceutically acceptable salt thereof.
[0022] Expressed alternatively, this is equivalent to a
lactam-cyclized peptide of formula I:
TABLE-US-00002 (I) (SEQ ID NO: 1)
R.sub.1-cyclo[X.sub.1-Tyr-X.sub.3-DArg-2Nal-Gly-X.sub.7]-X.sub.8-X.sub.9-X-
.sub.10-R.sub.2
wherein:
[0023] R.sub.1 is a substituent on the .alpha.-amino group of
X.sub.1 when X.sub.1 contains an .alpha.-amino group and said
.alpha.-amino group is not a constituent of said lactam amide bond,
selected from the group consisting of Ac, Bz, and n-hexanoyl, or is
absent, wherein X.sub.1 is selected from the group consisting of
(D/L)Agl, Asp, Dab, Dap, and Glu;
[0024] X.sub.1 is selected from the group consisting of (D/L)Agl,
Ala, .beta.-Ala, DAla, 5-aminovaleryl, 4-AMB, 4-AMPA, Asp, Dab,
Dap, Dap(Ac), Glu, Gly, Leu, Lys, Lys(Ac), 2Nal, Phe, DPhe, and
succinyl, or is absent;
[0025] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0026] X.sub.7 is selected from the group consisting of (D/L)Agl,
Asp, Dab, Dap, DDap, Glu, DGlu, Lys, and Orn;
[0027] X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg, DArg, Gly, Lys, Lys(iPr), and Orn, or is absent;
[0028] X.sub.9 is selected from the group consisting of Gly, 2Nal,
D2Nal, and DPhe, or is absent;
[0029] X.sub.10 is 2Nal, or is absent;
[0030] wherein when X.sub.8 is absent, X.sub.9 and X.sub.10 are
each absent, and when X.sub.9 is absent, X.sub.10 is absent,
and
[0031] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt,
wherein:
[0032] a) said lactam is formed by an amide bond between the side
chain amino group of X.sub.1 and the side chain carboxyl group of
X.sub.7, when X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of (D/L)Agl/Glu, Dab/Glu, and
Dap/Glu, and R.sub.1 is Ac or n-hexanoyl; or
[0033] b) said lactam is formed by art amide bond between the side
chain carboxyl group of X.sub.1 and the side chain amino group of
X.sub.7, when X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of Asp/(D/L)Agl, Asp/Dab,
Asp/Dap, Glu/(D/L)Agl, Glu/Dab, Glu/Dap, Glu/DDap, and Glu/Lys, and
R.sub.1 is Ac or Bz, or when X.sub.1 and X.sub.7 are, respectively,
a pair selected from the group consisting of succinyl/(D/L)Agl,
succinyl/Dab, succiny/Dap, succinyl/Lys, and succinyl/Orn, and
R.sub.1 is absent; or
[0034] c) said lactam is formed by an amide bond between the
.alpha.-amino group of X.sub.1 and the side chain carboxyl group of
X.sub.7, when X.sub.1 and X.sub.7 are, respectively, a pair
selected from the group consisting of Ala/Glu, Ala/DGlu, DAla/Glu,
DAla/DGlu, Dap(Ac)/Glu, Gly/Asp, Gly/Glu, Gly/DGlu, Leu/Glu,
Leu/DGlu, Lys/DGlu, Lys(Ac)/Glu, 2Nal/Glu, Phe/Glu, Phe/DGlu,
DPhe/Glu, and DPhe/DGlu, and R.sub.1 is absent; or
[0035] d) said lactam is formed by an amide bond between a
non-.alpha., non-side-chain amino group of X.sub.1 and the side
chain carboxyl group of X.sub.7, when X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of
.beta.-Ala/Asp, .beta.-Ala/Glu, 5-amino-valeryl/Asp,
5-aminovaleryl/Glu, 4-AMB/Glu, 4-AMPA/Asp, and 4-AMPA/Glu, and
R.sub.1 is absent; or
[0036] e) said lactam is formed by an amide bond between the
.alpha.-amino group of X.sub.2 and the side chain carboxyl group of
X.sub.7, when X.sub.2 and X.sub.7 are, respectively, a pair
selected from the group consisting of Tyr/Asp, Tyr/Glu, and
Tyr/DGlu, and R.sub.1 and X.sub.1 are each absent, or
[0037] a pharmaceutically acceptable salt thereof.
[0038] In another aspect, the present invention provides a
lactam-cyclized peptide of formula I:
TABLE-US-00003 (I) (SEQ ID NO: 1)
R.sub.1-cyclo[X.sub.1-Tyr-X.sub.3-DArg-2Nal-Gly-X.sub.7]-X.sub.8-X.sub.9-X-
.sub.10-R.sub.2
or a pharmaceutically acceptable salt thereof,
[0039] wherein:
[0040] X.sub.1 is selected from the group consisting of (D/L)Agl,
Ala, .beta.-Ala, DAla, 5-aminovaleryl, 4-AMB, 4-AMPA, Asp, Dab,
Dap, Dap(Ac), Glu, Gly, Leu, Lys, Lys(Ac), 2Nal, Phe, DPhe, and
succinyl, or is absent, [0041] wherein when X.sub.1 is (D/L)Agl,
Dab, or Dap and the .alpha.-amino group of X.sub.1 is not a
constituent of the lactam amide bond, said .alpha.-amino group is
substituted with R.sub.1 which is selected from the group
consisting of Ac and n-hexanoyl; [0042] wherein when X.sub.1 is Asp
or Glu and the .alpha.-amino group of X.sub.1 is not a constituent
of the lactam amide bond, said .alpha.-amino group is substituted
with R.sub.1 which is selected from the group consisting of Ac and
Bz; and [0043] wherein when X.sub.1 is Ala, .beta.-Ala, DAla,
5-aminovaleryl, 4-AMB, 4-AMPA, Dap(Ac), Gly, Leu, Lys, Lys(Ac),
2Nal, Phe, DPhe or succinyl, R.sub.1 is absent:
[0044] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0045] X.sub.7 is selected from the group consisting of (D/L)Agl,
Asp, Dab, Dap, DDap, Glu, DGlu, Lys, and Orn;
[0046] X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg, DArg, Gly, Lys, Lys(iPr), and Orn, or is absent;
[0047] X.sub.9 is selected from the group consisting of Gly, 2Nal,
D2Nal, and DPhe, or is absent;
[0048] X.sub.10 is 2Nal, or is absent, [0049] wherein when X.sub.8
is absent, X.sub.9 and X.sub.10 are each absent, and when X.sub.9
is absent, X.sub.10 is absent; and
[0050] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt,
[0051] and further wherein: [0052] said lactam is formed by an
amide bond between the side chain amino group of X.sub.1 and the
side chain carboxyl group of X.sub.7, and X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of
(D/L)Agl/Glu, Dab/Glu, and Dap/Glu, and R.sub.1 is Ac or
n-hexanoyl; or [0053] said lactam is formed by an amide bond
between the side chain carboxyl group of X.sub.1 and the side chain
amino group of X.sub.7, and X.sub.1 and X.sub.7 are, respectively,
a pair selected from the group consisting of Asp/(D/L)Agl, Asp/Dab,
Asp/Dap, Glu/(D/L)Agl, Glu/Dab, Glu/Dap, Glu/DDap, and Glu/Lys, and
R.sub.1 is Ac or Bz, or wherein X.sub.1 and X.sub.7 are,
respectively, a pair selected from the group consisting of
succinyl/(D/L)Agl, succinyl/Dab, succinyl/Dap, succinyl/Lys, and
succinyl/Orn, and R.sub.1 is absent; or [0054] said lactam is
formed by an amide bond between the .alpha.-amino group of X.sub.1
and the side chain carboxyl group of X.sub.7, and X.sub.1 and
X.sub.7 are, respectively, a pair selected from the group
consisting of Ala/Glu, Ala/DGlu, DAla/Glu, DAla/DGlu, Dap(Ac)/Glu,
Gly/Asp, Gly/Glu, Gly/DGlu, Leu/Glu, Leu/DGlu, Lys/DGlu,
Lys(Ac)/Glu, 2Nal/Glu, Phe/Glu, Phe/DGlu, DPhe/Glu, and DPhe/DGlu,
and R.sub.1 is absent; or [0055] said lactam is formed by an amide
bond between a non-.alpha., non-side-chain amino group of X.sub.1
and the side chain carboxyl group of X.sub.7, and X.sub.1 and
X.sub.7 are, respectively, a pair selected from the group
consisting of .beta.-Ala/Asp, .beta.-Ala/Glu, 5-amino-valeryl/Asp,
5-aminovaleryl/Glu, 4-AMB/Glu, 4-AMPA/Asp, and 4-AMPA/Glu, and
R.sub.1 is absent; or [0056] said lactam is formed by an amide bond
between the .alpha.-amino group of Tyr at X.sub.2 and the side
chain carboxyl group of X.sub.7, and X.sub.7 is selected from the
group consisting of Asp, Glu, and DGlu, and R.sub.1 and X.sub.1 are
each absent.
[0057] A recurrent sequence motif in all the compounds of formula I
is the presence of Tyr at position X.sub.2, DArg at position
X.sub.4, 2Nal at position X.sub.5, and Gly at position X.sub.6.
[0058] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0059] R.sub.1 is selected from the group consisting of Ac and Bz,
or is absent;
[0060] X.sub.1 is selected from the group consisting of .beta.-Ala,
4-AMB, 4-AMPA, Asp, Dab, Dap, Dap(Ac), Glu, 2Nal, Phe, and
succinyl, or is absent;
[0061] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0062] X.sub.7 is selected from the group consisting of Asp, Dab,
Dap, Glu, DGlu, Lys, and Orn;
[0063] X.sub.8 is selected from the group consisting of Arg and
Lys, or is absent;
[0064] X.sub.9 is absent;
[0065] X.sub.10 is absent; and
[0066] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt.
[0067] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0068] R.sub.1 is selected from the group consisting of Ac and Bz,
or is absent;
[0069] X.sub.1 is selected from the group consisting of DAla,
5-aminovaleryl, 4-AMPA, Asp, Glu, Leu, Lys(Ac), Phe, oPhe, and
succinyl:
[0070] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0071] X.sub.7 is selected from the group consisting of (D/L)Agl,
Asp, Dab, Dap, DDap, Glu, and DGlu;
[0072] X.sub.8 is selected from the group consisting of Arg, DArg,
and Lys, or is absent;
[0073] X.sub.9 is absent;
[0074] X.sub.10 is absent; and
[0075] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt.
[0076] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0077] R.sub.1 is selected from the group consisting of Ac, Bz, and
n-hexanoyl, or is absent;
[0078] X.sub.1 is selected from the group consisting of (D/L)Agl,
Ala, .beta.-Ala, Asp, Dap, Glu, Gly, Lys, and Phe;
[0079] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0080] X.sub.7 is selected from the group consisting of (D/L)Agl,
Asp, Dap, Glu, and DGlu;
[0081] X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg, Gly, Lys, Lys(iPr), and Orn, or is absent;
[0082] X.sub.9 is selected from the group consisting of Gly, 2Nal,
D2Nal, and DPhe, or is absent;
[0083] X.sub.10 is 2Nal, or is absent; and
[0084] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt.
[0085] In a further aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0086] R.sub.1 is selected from the group consisting of Ac and Bz,
or is absent;
[0087] X.sub.1 is selected from the group consisting of Ala,
5-aminovaleryl, Asp, Glu, Gly, Phe, DPhe, and succinyl;
[0088] X.sub.3 is selected from the group consisting of Arg,
Lys(iPr), and Lys(Me.sub.2);
[0089] X.sub.7 is selected from the group consisting of (D/L)Agl,
Asp, Dap, Glu, and DGlu;
[0090] X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg, Gly, Lys, Lys(iPr), and Orn, or is absent;
[0091] X.sub.9 is selected from the group consisting of Gly, D2Nal,
and DPhe, or is absent;
[0092] X.sub.10 is 2Nal, or is absent; and
[0093] R.sub.2 is selected from the group consisting of NH, and
NHEt.
[0094] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0095] X.sub.1 is selected from the group consisting of Gly and
Phe;
[0096] X.sub.3 is Lys(iPr); and
[0097] X.sub.7 is DGlu.
[0098] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0099] R.sub.1 is absent;
[0100] X.sub.1 is selected from the group consisting of Gly and
Phe;
[0101] X.sub.3 is Lys(iPr);
[0102] X.sub.7 is DGlu;
[0103] X.sub.8 is selected from the group consisting of Arg and
Lys(iPr), or is absent;
[0104] X.sub.9 is absent;
[0105] X.sub.10 is absent; and
[0106] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt.
[0107] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0108] said lactam is formed by an amide bond between the side
chain amino group of X.sub.1 and the side chain carboxyl group of
X.sub.7;
[0109] R.sub.1 is selected from the group consisting of Ac and
n-hexanoyl;
[0110] X.sub.1 is selected from the group consisting of (D/L)Agl,
Dab, and Dap;
[0111] X.sub.3 is selected from the group consisting of Arg and
Lys(iPr);
[0112] X.sub.7 is Glu;
[0113] X.sub.8 is Arg;
[0114] X.sub.9 is absent;
[0115] X.sub.10 is absent; and
[0116] R.sub.2 is NH.sub.2.
[0117] In a preferred embodiment of this aspect of the invention,
X.sub.1 is (D/L)Agl or Dap.
[0118] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0119] said lactam is formed by an amide bond between the side
chain carboxyl group of X.sub.1 and the side chain amino group of
X.sub.7;
[0120] R.sub.1 is selected from the group consisting of Ac and
Bz;
[0121] X.sub.1 is selected from the group consisting of Asp and
Glu;
[0122] X.sub.3 is selected from the group consisting of Arg and
Lys(Me.sub.2);
[0123] X.sub.7 is selected from the group consisting of (D/L)Agl,
Dab, Dap, DDap, and Lys;
[0124] X.sub.8 is Arg;
[0125] X.sub.9 is absent;
[0126] X.sub.10 is absent; and
[0127] R.sub.2 is NH.sub.2.
[0128] In a preferred embodiment of this aspect of the invention,
X.sub.7 is (D/L)Agl, Dab, Dap, or DDap. In a more preferred
embodiment, X.sub.7 is (D/L)Agl or Dap.
[0129] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0130] said lactam is formed by an amide bond between the side
chain carboxyl group of X.sub.1 and the side chain amino group of
X.sub.7;
[0131] R.sub.1 is absent;
[0132] X.sub.1 is succinyl;
[0133] X.sub.3 is Arg;
[0134] X.sub.7 is selected from the group consisting of (D/L)Agl,
Dab, Dap, Lys, and Orn;
[0135] X.sub.5 is Arg;
[0136] X.sub.9 is absent;
[0137] X.sub.10 is absent; and
[0138] R.sub.2 is NH.sub.2.
[0139] In a preferred embodiment of this aspect of the invention,
X.sub.7 is (D/L)Agl or Dap.
[0140] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0141] said lactam is formed by an amide bond between the
.alpha.-amino group of X.sub.1 and the side chain carboxyl group of
X.sub.7;
[0142] R.sub.1 is absent;
[0143] X.sub.1 is selected from the group consisting of Ala, DAla,
Gly, Dap(Ac), Leu, Lys, Lys(Ac), 2Nal, Phe, and DPhe;
[0144] X.sub.3 is selected from the group consisting of Arg, Lys,
Lys(iPr), and Lys(Me.sub.2);
[0145] X.sub.7 is selected from the group consisting of Asp, Glu,
and DGlu;
[0146] X.sub.8 is selected from the group consisting of .beta.-Ala,
Arg. Gly, Lys, Lys(iPr), and Orn, or is absent;
[0147] X.sub.9 is selected from the group consisting of Gly, 2Nal,
D2Nal, and DPhe, or is absent;
[0148] X.sub.10 is 2Nal, or is absent;
[0149] wherein when X.sub.s is absent, X.sub.9 and X.sub.10 are
each absent; and
[0150] R.sub.2 is selected from the group consisting of NH.sub.2
and NHEt.
[0151] In a preferred embodiment of this aspect of the invention,
X.sub.1 is Ala, DAla, Gly, Leu, Lys, Lys(Ac), Phe, or DPhe. In a
more preferred embodiment, X.sub.1 is Ala, Gly, Lys, or Phe.
[0152] In a preferred embodiment of this aspect of the invention,
X.sub.3 is Arg, Lys, Lys(iPr), or Lys(Me.sub.2). In a more
preferred embodiment, X.sub.3 is Arg.
[0153] In a preferred embodiment of this aspect of the invention,
X.sub.7 is Asp, Glu, or DGlu. In a more preferred embodiment,
X.sub.7 is Asp.
[0154] In a preferred embodiment of this aspect of the invention,
X.sub.8 is .beta.-Ala, Arg, Gly, Lys, Lys(iPr), Orn, or is absent.
In a more preferred embodiment. X.sub.8 is .beta.-Ala, Gly, Lys,
Lys(iPr), Orn, or is absent.
[0155] In a preferred embodiment of this aspect of the invention,
X.sub.9 is Gly, 2Nal, D2Nal, DPhe, or is absent. In a more
preferred embodiment, X.sub.9 is Gly, 2Nal, D2Nal, or DPhe.
[0156] In a preferred embodiment of this aspect of the invention,
X.sub.10 is 2Nal, or is absent. In a more preferred embodiment,
X.sub.10 is 2Nal.
[0157] In a preferred embodiment of this aspect of the invention,
R.sub.2 is NHEt.
[0158] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0159] said lactam is formed by an amide bond between a
non-.alpha., non-side-chain amino group X.sub.1 and the side chain
carboxyl group of X.sub.7;
[0160] R.sub.1 is absent;
[0161] X.sub.1 is selected from the group consisting of .beta.-Ala,
4-AMB, 5-aminovaleryl, and 4-AMPA:
[0162] X.sub.3 is Arg;
[0163] X.sub.7 is selected from the group consisting of Asp and
Glu;
[0164] X.sub.8 is selected from the group consisting of Arg and
DArg;
[0165] X.sub.9 is absent;
[0166] X.sub.10 is absent; and
[0167] R.sub.2 is NH.sub.2.
[0168] In a preferred embodiment of this aspect of the invention,
X.sub.1 is .beta.-Ala, 5-amino-valeryl, or 4-AMPA. In a more
preferred embodiment, X.sub.1 is .beta.-Ala.
[0169] In a preferred embodiment of this aspect of the invention,
X.sub.7 is Asp.
[0170] In a preferred embodiment of this aspect of the invention
X.sub.8 is Arg.
[0171] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
of formula I (SEQ ID NO:1), wherein:
[0172] said lactam is formed by an amide bond between the
.alpha.-amino group of X.sub.2 and the side chain carboxyl group of
X.sub.7:
[0173] R.sub.1 is absent;
[0174] X.sub.1 is absent:
[0175] X.sub.3 is Arg;
[0176] X.sub.7 is selected from the group consisting of Asp, Glu,
and DGlu;
[0177] X.sub.8 is Arg;
[0178] X.sub.9 is absent;
[0179] X.sub.10 is absent; and
[0180] R.sub.2 is NH.sub.2.
[0181] In another aspect, the present invention provides a
lactam-cyclized peptide of the formula:
##STR00001##
or a pharmaceutically acceptable salt thereof. The lactam is formed
by an amide bond between the .alpha.-amino group of Phe and the
side chain carboxyl group of DGlu. The pharmaceutically acceptable
salt can be an acetic acid salt.
[0182] In another aspect, the present invention provides a
pharmaceutical composition, comprising a lactam-cyclized peptide or
pharmaceutically acceptable salt thereof as variously described
above, and a pharmaceutically acceptable carrier, diluent, or
excipient.
[0183] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
as variously described above, for use in therapy.
[0184] In another aspect, the present invention provides a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
as variously described above, for the treatment of rheumatoid
arthritis, pulmonary fibrosis, HIV infection, or a cancer selected
from the group consisting of breast cancer, pancreatic cancer,
melanoma, prostate cancer, kidney cancer, neuroblastoma,
non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal
cancer, multiple myeloma, glioblastoma multiforme, and chronic
lymphocytic leukemia.
[0185] In another aspect, the present invention provides the use of
a lactam-cyclized peptide or pharmaceutically acceptable salt
thereof as variously described above, for the manufacture of a
medicament for the treatment of rheumatoid arthritis, pulmonary
fibrosis, HIV infection, or a cancer selected from the group
consisting of breast cancer, pancreatic cancer, melanoma, prostate
cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung
cancer, ovarian cancer, colorectal cancer, multiple myeloma,
glioblastoma multiforme, and chronic lymphocytic leukemia.
[0186] In another aspect, the present invention provides a method
of treating rheumatoid arthritis, pulmonary fibrosis, HIV
infection, or a cancer selected from the group consisting of breast
cancer, pancreatic cancer, melanoma, prostate cancer, kidney
cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian
cancer, colorectal cancer, multiple myeloma, glioblastoma
multiforme, and chronic lymphocytic leukemia, comprising
administering to a patient in need thereof an effective amount of a
lactam-cyclized peptide or pharmaceutically acceptable salt thereof
as variously described above.
[0187] In the macrocyclic peptidic compounds of the present
invention (SEQ ID NO:1), amino acids X.sub.1 through X.sub.10 are
referred to herein by their commonly employed three letter symbols,
shown left to right from amino-terminal end to carboxy-terminal
end. D- and L- (small capital letters) refer to absolute
stereochemistry. Where neither designation is indicated in a
particular formula, the L-form of the amino acid is present.
X.sub.1 can also be a dicarboxylic acid residue, i.e., a succinyl
group. Amino acid or carboxylic acid residues within the brackets
"[ ]" are within the cyclic structure; groups external to the
brackets are outside the cyclized ring. In all cases, cyclization
is via a lactam (amide) bond between X.sub.1 (or X.sub.2, i.e.,
Tyr) and X.sub.7, which can be formed in several different ways,
depending on the structures of X.sub.1, X.sub.2, and X.sub.7.
[0188] When the lactam bond is formed between the side chain amino
group of X.sub.1 and the side chain carboxyl group of X.sub.7
(Schemes 1 and 2; Examples 1-5), the .alpha.-amino group of X.sub.1
is capped with Ac or n-hexanoyl.
[0189] When the lactam bond is formed between the side chain
carboxyl group of X.sub.1 and the side chain amino group of
X.sub.7, the .alpha.-amino group of X.sub.1 is capped with Ac or Bz
(Schemes 3 and 4; Examples 6-19). X.sub.1 also can be a
bifunctional residue other than an .alpha.-amino acid, for example
one with two carboxyl groups, i.e., a succinyl residue. In this
case, one carboxyl group forms an amide bond with the .alpha.-amino
group of Tyr, and the other forms the cyclic lactam structure
through an amide bond with the side chain amino group of X.sub.7
(Schemes 3 and 4; Examples 20-24). When X.sub.1 is succinyl,
R.sub.1 is absent.
[0190] In most of the lactam-cyclized peptides disclosed herein
(Schemes 5-15; Examples 25-28, 32-66, and 75-89), the .alpha.-amino
group of X.sub.1 forms the lactam structure via an amide bond with
the side chain carboxyl group of X.sub.7, and R.sub.1 is absent.
Also included in the synthetic schemes of this category are cyclic
peptides containing X.sub.1 residues, i.e., .beta.-Ala, 4-AMB,
5-aminovaleryl, and 4-AMPA, wherein the amino group is a non-side
chain, non-.alpha.-amino group (Schemes 5 and 6; Examples 67-74).
R.sub.1 is also absent in these cases.
[0191] When both R.sub.1 and X.sub.1 are absent, the lactam
structure is formed through an amide bond between the .alpha.-amino
group of Tyr (X.sub.2) and the side chain carboxyl group of X.sub.7
(Schemes 5 and 6; Examples 29-31).
[0192] Structures of common amino acids, e.g., alanine, glycine,
etc., are well known in the art. Structures of non-standard and
substituted amino acids present in the instant invention compounds
are shown below.
##STR00002##
[0193] The lactam-cyclized peptides of the present invention can be
prepared as pharmaceutically acceptable salts. Such salts, and
common methodology for preparing them, are well known in the art.
See, e.g., P. Stahl et al. (2002) Handbook of Pharmaceutical Salts
Properties, Selection and Use, VCHA/Wiley-VCH; Berge et al. (1977)
"Pharmaceutical Salts," Journal of Pharmaceutical Sciences
66(1):1-19.
[0194] The compounds of the present invention are potent
antagonists of CXCR4/SDF-1 interaction. Compounds of formula (I)
and their pharmaceutically acceptable salts specifically
exemplified herein exhibit an average K.sub.i value of about 7.5 nM
or less as determined by the CXCR4/.sup.125I-SDF-1.alpha. binding
assay described below. More preferred compounds of formula (I) and
their pharmaceutically acceptable salts exhibit an average K.sub.i
value in the range of from about 0.2 nM to about 1 nM in this
assay. Especially preferred compounds of formula (I) and their
pharmaceutically acceptable salts exhibit an average K.sub.i value
less than about 0.2 nM in this assay.
[0195] In addition, the compounds and pharmaceutically acceptable
salts of the present invention are preferably highly selective for
the CXCR4 receptor, exhibiting little or no inhibitory activity
against other chemokine receptors, including CCR1, CCR2, CXCR2,
CXCR3, and other G-protein coupled receptors at the concentrations
tested, and no significant activity against serotonin, dopamine,
and opioid receptors. They also preferably exhibit good stability
in blood and plasma, good subcutaneous bioavailability, desirable
pharmacokinetic/pharmacodynamic properties, and potent in vivo
efficacy in tumor growth inhibition, with a wide safety margin.
[0196] In view of these pharmacological properties, the compounds
of the present invention are indicated for the treatment of
disorders involving CXCR4/SDF-1 interaction, or receptor activity
of CXCR4, such as in HIV infection. In particular, the present
compounds are useful in treating malignancies in which angiogenic,
growth, survival, and metastatic pathways mediated by CXCR4 and
SDF-1 are implicated in pathogenesis, including breast cancer,
pancreatic cancer, melanoma, prostate cancer, kidney cancer,
neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer,
colorectal cancer, multiple myeloma, glioblastoma multiforme, and
chronic lymphocytic leukemia, as well as in rheumatoid arthritis,
pulmonary fibrosis, and HIV infection (Tsutsumi et al. (2006)
Peptide Science 88(2):279-289).
[0197] Agl (aminoglycine) is a pro-chiral building block. When this
residue appears in a peptide formula herein, the .alpha.-carbon
becomes a chiral center at which the two associated .alpha.-amino
groups are each individually bonded to different moieties. In this
case, the final peptide product contains two diastereomers that are
unresolved, and that may be present in other than a 1:1 ratio.
"(D/L)Agl" in a peptide formula denotes such a mixture of
diastereomers. "(DL)Agl" denotes an Agl derivative that is racemic,
for example Fmoc-(DL)Agl(Boc).
[0198] "K.sub.i" values are calculated using IC.sub.50 values
determined in the CXCR4/.sup.125I-SDF-1.alpha. binding assay
described below by employing equation 7.22 of Enzymes, A Practical
Introduction to Structure, Mechanism, and Data Analysis, Robert A.
Copeland, Wiley-VCH, New York, 1996, page 207.
[0199] The term "SDF-1" includes two isoforms, SDF-1.alpha. and
SDF-1.beta., currently understood to exhibit similar
functionality.
[0200] "Treatment" as used herein refers to curative treatment of
disorders associated with CXCR4/SDF-1 interaction or CXCR4 receptor
activity. Curative treatment refers to processes involving a
slowing, interrupting, arresting, controlling, or stopping of
disease progression, but does not necessarily involve a total
elimination of all disease-related symptoms, conditions, or
disorders.
[0201] The compounds of the present invention can be used as
medicaments in human or veterinary medicine, administered by a
variety of routes. Most preferably, such compositions are for
parenteral administration. Such pharmaceutical compositions can be
prepared by methods well known in the art. See, e.g., Remington:
The Science and Practice of Pharmacy, 19.sup.th ed. (1995), A.
Gennaro et al., Mack Publishing Co., and comprise one or more
compounds of formula (I) or a pharmaceutically acceptable salt(s)
thereof, and a pharmaceutically acceptable carrier, diluent, or
excipient.
[0202] The effective amount of the present compounds is in the
range of from about 1 mg to about 300 mg, more preferably from
about 1 mg to about 200 mg, more preferably from about 1 mg to
about 100 mg, and even more preferably from about 1 mg to about 50
mg, on a daily basis.
[0203] All lactam-cyclized peptides of the present invention can be
synthesized either by solid-phase synthesis or solution phase
synthesis, or a combination of both, with peptide chain assembly on
solid phase and cyclization or other modifications on resin or in
solution. Such methods are well known in the art.
[0204] The following abbreviations used herein have the indicated
meanings:
[0205] Ac: acetyl; Agl: aminoglycine; AMB: aminomethyl benzoic
acid; AMPA: aminomethyl phenyl acetic acid; Bn: benzyl; Boc:
tent-butyloxycarbonyl; BOP:
(benzotriazol-1-yloxy)-tris(dimethylamino)phosphoniumhexafluorophosp-
hate; 2-Br-Z: 2-bromobenzyloxycarbonyl; Bz: benzoyl; Bzl: benzyl;
2-Cl-Z: 2-chlorobenzyl-oxycarbonyl; Dab: 2,4-diaminobutyric acid;
Dap: 2,3-diamino-propionic acid; DCC: dicyclohexyl-carbodiimide;
DCM: dichloromethane; DIC: diisopropyl carbodiimide; DIEA:
diisopropyl-ethylamine; DMF: N,N-dimethyl formamide; DMSO:
dimethyl-sulfoxide; EDT: 1,2-ethane-dithiol; Et: ethyl; Fm:
9-fluorenylmethyl; Fmoc: 9-fluor-enyl methoxy carbonyl; HATU:
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methy-
lmethanaminium hexafluorophosphate N-oxide; HBTU:
O-benzo-triazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate;
HCTU: 1H-benzotriazo-lium
1-[bis(dimethylamino)methylene]-5-chloro-3-oxide
hexafluorophosphate; HF: hydrogen fluoride; HOBt:
hydroxybenzotriazole; IBCF: isobutyl chloroformate; iPr: isopropyl;
IPA: isopropyl alcohol; Me: methyl; 2Nal: 2-naphthylalanine; NMM:
N-methylmorpholine; NMP: N-methyl-pyrrolidone; OtBu: tert-butyl
ester; Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl:
PBS: phosphate buffered saline; PyBOP:
(benzotriazol-1-yloxy)-tris(pyrrolidino)-phosphonium
hexafluoro-phosphate; PyBrOP: bromotris(pyrrolidino)phosphonium
hexafluorophosphate; tBu: tert-butyl; TFA: trilluoroacetic acid;
THF: tetrahydrofuran; TIS: triisopropyl silane; Tos:
p-toluene-sulfonyl; Z: benzyloxycarbonyl; ZOSu:
N-(benzyloxycarbonyl-oxy)succinimide.
[0206] Preparation of Compounds of the Present Invention as
Described in the Following examples is meant to be illustrative
rather than limiting. In each of these examples, the observed
molecular weight is reported as a de-convoluted value. The
de-convoluted value is derived from the formula
MW(observed)=n(m/z)-n, where m/z represents the charged ion
(positive mode) and n is the number of charges of the specific
species. When multiple charged species are present in the mass
spectrum, the observed molecular weight is reported as an
average.
[0207] The synthetic processes disclosed in Examples 85-87, and
isotopic labeling procedures disclosed in Example 89, for cyclic
lactam peptides containing isopropyl lysine side chains are equally
applicable to other peptides disclosed herein containing lysine
alkyl side chains, with appropriate modifications. The synthetic
methods of Examples 86-88, which eliminate the need for toxic
palladium catalysts, are also equally applicable to other peptides
disclosed herein, with appropriate modifications.
EXAMPLE 1
Ac-cyclo[Dap-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:2)
[0208] The sequence
Ac-Dap(Alloc)-Tyr(tBu)-Arg(Pbf)-DArg(Pbf)-2Nal-Gly-Glu(Oallyl)-Arg(Pbf)
(SEQ ID NO:3) is assembled by standard Fmoc chemistry utilizing an
ABI 431 Peptide Synthesizer (Applied Biosystems) as outlined in
Scheme 1 below. The automated assembly is carried out by using the
standard Applied Biosystems DCC/HOBt chemistry protocol or FastMoc
chemistry HBTU/DIEA protocol following the supplier's directions
(PE Applied Biosystems Inc., Foster City, Calif.). The solid
support is Rink amide resin
(4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin) for
C-terminal amides or indole resin
[3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM resin for
C-terminal ethyl amides (NovaBiochem, EMD Biosciences, Inc., San
Diego, Calif.). The stepwise chain assembly starts from the
C-terminal end of the linear peptide and is accomplished in 9 major
steps. In step 1, four equivalents of protected amino acid
Fmoc-Arg(Pbf) are activated with DCC/HOBt (or HBTU/DIEA for FastMoc
chemistry) in NMP, and coupled to deprotected Rink Amide resin
using 20% piperidine. In step 2, four equivalents of
Fmoc-Glu(Oallyl) are activated and coupled to the deprotected
peptide resin from step 1. Appropriate steps are carried out until
step 8, the coupling of Fmoc-Dap(Alloc). Then, Fmoc at the
N-terminal end is removed using 20% piperidine in DMF and
acetylation of the .alpha.-amino group is carried out off-line
using 5 equivalents of acetic anhydride, 10 equivalents of DIEA in
dry DMF or NMP, for 1 h at room temperature.
[0209] The allyl and Alloc side chain protection groups are removed
with 0.1 equivalent of Pd(Ph.sub.3P).sub.4 in the presence of 24
equivalents of phenylsilane in dichloromethane (Scheme 2). This
process is repeated once for complete side chain deprotection. The
deprotected carboxylic acid moiety of Glu is activated with
PyBOP/DIEA and cyclized to the side chain amino group of Dap on the
resin. The cyclized peptide is simultaneously deprotected and
cleaved from the resin using a scavenger cocktail of
TFA/H.sub.2O/TIS/EDT (95/2/1/2, v/v/v/v), or
TFA/H.sub.2O/TIS/anisole (92/2/4/2, v/v/v/v) for 2 hours at room
temperature. The solvents are then evaporated under vacuum, and the
peptide is precipitated and washed three times with cold diethyl
ether to remove the scavengers. Molecular weight calculated (MW
cal.): 1142.30; MW observed (MW obs.): 1142.50.
##STR00003##
[0210] Peptide purification is accomplished using standard
preparative HPLC techniques. Immediately following the cyclization,
the peptide solution is diluted with water containing 0.1% (v/v)
TFA, loaded onto a reversed phase C18 HPLC column, and eluted with
an aqueous 0.1% trilluoroacetic acid/acetonitrile (v/v) gradient
while monitoring at 214 nm. The appropriate fractions are pooled
and lyophilized. Further characterization of the final product is
performed using analytical HPLC and mass spectral analysis by
conventional techniques. For peptides with a basic side chain, the
final lyophilized product is a TFA salt.
##STR00004## ##STR00005##
EXAMPLE 2
Ac-cyclo[Dab-Tyr-Arg-DArg-2Nal-Gly-Clu]-Arg-NH.sub.2 (SEQ ID
NO:6)
[0211] Prepare as in Example 1, except that Fmoc-Dap(Alloc) in step
8 is replaced with Fmoc-Dab(Alloc). MW cal.: 1156.33; MW obs.:
1156.10.
EXAMPLE 3
Ac-cyclo[Dap-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:7)
[0212] Prepare as in Example 1, except that Fmoc-Arg(Pbf) in step 6
is replaced with Fmoc-Lys(iPr)(Boc). MW cal.: 1156.37; MW obs.:
1156.78.
EXAMPLE 4
n-Hexanoyl-cyclo[Dap-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2
(SEQ ID NO:8)
[0213] Prepare as in Example 1, except that Fmoc-Arg(Pbf) in step 6
is replaced with Fmoc-Lys(iPr)(Boc). Additionally, acetic anhydride
in step 9 is replaced with hexanoic acid activated with PyBOP/DIEA.
MW cal.: 1212.47; MW obs.: 1212.92.
EXAMPLE 5
Ac-cyclo[(D/L)Agl-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:9)
[0214] Prepare as in Example 1, except that Fmoc-Glu(Oallyl) in
step 2 is replaced with Fmoc-Glu(OtBu), and Fmoc-Dap(Alloc) in step
8 is replaced with Fmoc-(DL)Agl(Boc). After chain assembly, there
is no Pd(Ph.sub.3P).sub.4 treatment since no allyl protection is
present. Instead, cyclization is carried out in solution after the
linear peptide is cleaved from the solid support and deprotected.
The crude linear peptide (0.25 mmol) from the cleavage is dried
under vacuum and dissolved in 10 mL of dry DMF. This peptide
solution is delivered to the following solution via a syringe pump
during a 2 h period: 15 mL of dry dichloromethane and 15 mL of dry
DMF containing 1.0 mmole of PyBOP and 4.0 mmoles of DIEA. The
reaction is then allowed to proceed at room temperature for 2 h.
Solvents are then evaporated under vacuum, the residue is loaded
onto a preparative reversed phase C18 HPLC column, and target
cyclic peptide is isolated and characterized as described in
Example 1. MW cal.: 1128.27: MW obs.: 1128.26.
EXAMPLE 6
Ac-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-Glu-Dap]-Arg-NH.sub.2 (SEQ ID
NO:10)
[0215] The sequence
Glu(OtBu)-Tyr(tBu)-Arg(Pbf)-DArg(Pb1)-2Nal-Gly-Dap(Boc)-Arg(Pbf)
(SEQ ID NO: 11) is assembled by standard Fmoc chemistry utilizing
an ABI 431 instrument as outlined in Scheme 3 below. The automated
assembly is carried out by using the standard Applied Biosystems
DCC/HOBt chemistry protocol or FastMoc chemistry (HBTU/DIEA)
protocol following the supplier's directions (PE Applied Biosystems
Inc., Foster City, Calif.). The solid support is Rink amide resin
for C-terminal amides or indole resin
[3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM resin for
C-terminal ethyl amides. Stepwise chain assembly starts from the
C-terminal end of the linear peptide and is accomplished in 9 major
steps. In step 1, four equivalents of protected amino acid
Fmoc-Arg(Pbf) are activated with DCC/HOBt (or HBTU/DIEA for FastMoc
chemistry) in NMP, and coupled to deprotected Rink Amide resin. In
step 2, four equivalents of Fmoc-Dap(Boc) are activated and coupled
to the deprotected resin from step 1. Appropriate steps are carried
out until step 8, the coupling of Fmoc-Glu(OtBu). For step 9, Fmoc
at the N-terminal end is removed using 20% piperidine in DMF and
acetylation of the .alpha.-amino group is carried out off-line with
5 equivalents acetic anhydride, 10 equivalents DIEA in dry DMF or
NMP, for 1 h at room temperature. The finished peptide is
simultaneously deprotected and cleaved from the resin using a
scavenger cocktail of TFA/H.sub.2O/TIS/EDT (95/2/1/2, v/v/v/v), or
TFA/H.sub.2O/TIS/anisole (92/2/4/2, v/v/v/v) for 2 hours at room
temperature (Scheme 4). The solvents are then evaporated under
vacuum, and the peptide is precipitated and washed three times with
cold diethyl ether to remove the scavengers. The crude product is
used directly in the cyclization reaction. MW cal.: 1142.30; MW
obs.: 1142.83.
##STR00006##
[0216] Cyclization is carried out in solution after the linear
peptide is cleaved from the solid support with all the side chains
deprotected (Scheme 4). The cleaved crude linear peptide (0.25
mmole) is dried under vacuum and dissolved in 10 mL of dry DMF.
This peptide solution is delivered to the following reaction
mixture via a syringe pump during a 2 h period: 15 mL of dry
dichloromethane and 15 mL of dry DMF containing 1.0 mmole of PyBOP
and 4.0 mmoles of DIEA. The reaction is then allowed to proceed at
room temperature for 2 h. Solvents are evaporated under vacuum, the
residue is loaded onto a preparative reversed phase C18 HPLC
column, and target cyclic peptide is isolated and characterized as
described in Example 1.
##STR00007## ##STR00008##
EXAMPLE 7
Bz-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-Dap]-Arg-NH.sub.2 (SEQ ID
NO:14)
[0217] Prepare as in Example 6, except that acetic anhydride in
step 9 is replaced with benzoic acid anhydride. MW cal.: 1204.37;
MW obs.: 1204.87.
EXAMPLE 8
Ac-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-DDap]-Arg-NH.sub.2 (SEG ID
NO:15)
[0218] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-DDap(Boc). MW cal.: 1142.30; MW obs.:
1142.73.
EXAMPLE 9
Ac-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-Lys]-Arg-NH.sub.2 (SEQ ID
NO:16)
[0219] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-Lys(Boc). MW cal.: 1184.38; MW obs.:
1184.23.
EXAMPLE 10
Ac-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-Dab]-Arg-NH.sub.2 (SEQ ID
NO:17)
[0220] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-Dab(Boc). MW cal.: 1156.33; MW obs.:
1156.07.
EXAMPLE 11
Ac-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-(D/L)Agl]-Arg-NH.sub.2 (SEQ ID
NO:18)
[0221] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-(DL)Agl(Boc). MW cal.: 1128.27; MW obs.:
1128.86.
EXAMPLE 12
Bz-cyclo[Glu-Tyr-Arg-DArg-2Nal-Gly-(D/L)Agl]-Arg-NH.sub.2(SEQ ID
NO:19)
[0222] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-(DL)Agl(Boc). In addition, acetic anhydride
in step 9 is replaced with benzoic acid anhydride. MW cal.:
1190.34; MW obs.: 1190.99.
EXAMPLE 13
Bz-cyclo[Asp-Tyr-Arg-DArg-2Nal-Gly-Dab]-Arg-NH.sub.2 (SEQ ID
NO:20)
[0223] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-Dab(Boc), and Fmoc-Glu(OtBu) in step 8 is
replaced with Fmoc-Asp(OtBu). In addition, acetic anhydride in step
9 is replaced with benzoic acid anhydride. MW cal.: 1204.37; MW
obs.: 1204.87.
EXAMPLE 14
Ac-cyclo[Asp-Tyr-Arg-DArg-2Nal-Gly-Dab]-Arg-NH.sub.2 (SEQ ID
NO:21)
[0224] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-Dab(Boc), and Fmoc-Glu(OtBu) in step 8 is
replaced with Fmoc-Asp(OtBu). MW cal.: 1142.30; MW obs.:
1142.81.
EXAMPLE 15
Ac-cyclo[Asp-Tyr-Arg-DArg-2Nal-Gly-Dap]-Arg-NH.sub.2 (SEQ ID
NO:22)
[0225] Prepare as in Example 6, except that Fmoc-Glu(OtBu) in step
8 is replaced with Fmoc-Asp(OtBu). MW cal.: 1128.27; MW obs.:
1128.78.
EXAMPLE 16
Bz-cyclo[Asp-Tyr-Arg-DArg-2Nal-Gly-Dap]-Arg-NH.sub.2 (SEQ ID
NO:23)
[0226] Prepare as in Example 6, except that Fmoc-Glu(OtBu) in step
8 is replaced with Fmoc-Asp(OtBu). In addition, acetic anhydride in
step 9 is replaced with benzoic acid anhydride. MW cal.: 1190.34;
MW obs.: 1190.69.
EXAMPLE 17
Ac-cyclo[Asp-Tyr-Arg-DArg-2Nal-Gly-(D/L)-Arg]-Arg-NH.sub.2 (SEQ ID
NO:24)
[0227] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-(DL)Agl(Boc), and Fmoc-Glu(OtBu) in step 8 is
replaced with Fmoc-Asp(OtBu). MW cal.: 1114.24; MW obs.:
1114.85.
EXAMPLE 18
Ac-cyclo[Asp-Tyr-Lys(Me.sub.2)-DArg-2Nal-Gly-Dap]-Arg-NH.sub.2 (SEQ
ID NO:25)
[0228] Prepare as in Example 6, except that Fmoc-Arg(Pbf) in step 6
is replaced with Fmoc-Lys(Me.sub.2), and Fmoc-Glu(OtBu) in step 8
is replaced with Fmoc-Asp(OtBu). MW cal.: 1128.31; MW obs.:
1128.92.
EXAMPLE 19
Bz-cyclo[Asp-Tyr-Lys(Me.sub.2)-DArg-2Nal-Gly-Dap]-Arg-NH.sub.2 (SEQ
ID NO:26)
[0229] Prepare as in Example 6, except that Fmoc-Arg(Pbf) in step 6
is replaced with Fmoc-Lys(Me.sub.2), and Fmoc-Glu(OtBu) in step 8
is replaced with Fmoc-Asp(OtBu). In addition, acetic anhydride in
step 9 is replaced with benzoic acid anhydride. MW cal.: 1190.38;
MW obs.: 1191.14.
EXAMPLE 20
cyclo[Succinyl-Tyr-Arg-DArg-2Nal-Gly-(D/L)Agl]-Arg-NH.sub.2 (SEQ ID
NO:27)
[0230] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-(DL)Agl(Boc), Fmoc-Glu(OtBu) in step 8 is not
used, and this step is omitted. In addition, acetic anhydride in
step 9 is replaced with succinic anhydride. MW cal.: 1057.19; MW
obs.: 1057.87.
EXAMPLE 21
cyclo[Succinyl-Tyr-Arg-DArg-2Nal-Gly-Dap]-Arg-NH.sub.2 (SEQ ID
NO:28)
[0231] Prepare as in Example 6, except that Fmoc-Glu(OtBu) in step
8 is not used, and this step is omitted. In addition, acetic
anhydride in step 9 is replaced with succinic anhydride. MW cal.:
1071.22; MW obs.: 1071.85.
EXAMPLE 22
cyclo[Succinyl-Tyr-Arg-DArg-2Nal-Gly-Dab]-Arg-NH.sub.2 (SEQ ID
NO:29)
[0232] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Finoc-Dab(Boc), Fmoc-Glu(OtBu) in step 8 is not
used, and this step is omitted. In addition, acetic anhydride in
step 9 is replaced with succinic anhydride. MW cal.: 1085.25; MW
obs.: 1085.87.
EXAMPLE 23
cyclo[Succinyl-Tyr-Arg-DArg-2Nal-Gly-Orn]-Arg-NH.sub.2 (SEQ ID
NO:30)
[0233] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-Orn(Boc), Fmoc-Glu(OtBu) in step 8 is not
used, and this step is omitted. In addition, acetic anhydride in
step 9 is replaced with succinic anhydride. MW cal.: 1099.27; MW
obs.: 1100.23.
EXAMPLE 24
cyclo[Succinyl-Tyr-Arg-DArg-2Nal-Gly-Lys]-Arg-NH.sub.2 (SEQ ID
NO:31)
[0234] Prepare as in Example 6, except that Fmoc-Dap(Boc) in step 2
is replaced with Fmoc-Lys(Boc), Fmoc-Glu(OtBu) in step 8 is not
used, and this step is omitted. In addition, acetic anhydride in
step 9 is replaced with succinic anhydride. MW cal.: 1113.30; MW
obs.: 1114.25.
EXAMPLE 25
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Arg-NH.sub.2 (SEQ ID
NO:32)
[0235] The sequence
Gly-Tyr(tBu)-Lys(iPr)(Boc)-DArg(Pb0-2Nal-Gly-DGlu(Oallyl)-Arg(Pbf)
(SEQ ID NO:33) is assembled by standard Fmoc chemistry utilizing an
ABI 431 instrument as outlined in Scheme 5 below. The automated
assembly is carried out by using the standard Applied Biosystems
DCC/HOBt chemistry protocol or FastMoc HBTU/DIEA chemistry protocol
following the supplier's directions (PE Applied Biosystems Inc.,
Foster City, Calif.). The solid support is Rink amide resin for
amides or indole resin
[3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM resin for
C-terminal ethyl amides. Stepwise chain assembly starts from the
C-terminal end of the linear peptide and is accomplished in 8 major
steps (Scheme 5). In step 1, four equivalents of protected amino
acid Fmoc-Arg(Pbf) are activated with DCC/HOBt (or HBTU/DIEA for
FastMoc chemistry) in NMP, and are coupled to deprotected Rink
amide resin. In step 2, four equivalents of Fmoc-DGlu(Oallyl) are
activated and coupled to the deprotected peptide resin from step 1.
Appropriate steps are carried out until step 8, the coupling of
Fmoc-Gly.
[0236] The allyl ester side chain protection group is removed with
0.1 equivalent of Pd(Ph.sub.3P).sub.4 in the presence of 24
equivalents of phenylsilane in dichloromethane (Scheme 6). This
process is repeated once for complete side chain deprotection. Then
Fmoc at the N-terminal end is removed using 20% piperidine in DMF.
The deprotected carboxylic acid moiety of DGlu is activated with
PyBOP/DIEA, and cyclized to the .alpha.-amino group of glycine on
the resin. The cyclized peptide is simultaneously deprotected and
cleaved from the resin using a scavenger cocktail or
TFA/H.sub.2O/TIS/EDT (95/2/1/2, v/v/v/v), or
TFA/H.sub.2O/TIS/anisole (92/2/4/2, v/v/v/v) for 2 hours at room
temperature. The solvents are then evaporated under vacuum, and the
peptide is precipitated and washed three times with cold diethyl
ether to remove the scavengers. MW cal.: 1085.29; MW obs.:
1085.32.
EXAMPLE 25a
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Arg-NH.sub.2.acetic acid
salt (SED ID NO:34)
[0237] The TFA salt of the peptide of Example 25 is converted to an
acetic acid salt by adsorbing the material onto a preparative C18
column of suitable size, equilibrated with 2% acetic acid/H.sub.2O
(v/v). The column is then washed with three to live column volumes
of 2% aqueous acetic acid (v/v). The peptide is eluted using 1:1
water/acetonitrile (v/v) containing 2% acetic acid by volume, and
lyophilized. MW cal.: 1085.29; MW obs.: 1085.32.
##STR00009##
[0238] Peptide purification is accomplished using standard
preparative HPLC techniques. Immediately following cyclization, the
peptide solution is diluted with water containing 0.1% (v/v) TFA,
loaded onto a reversed phase C18 HPLC column, and eluted with an
aqueous 0.1% trifluoroacetic acid/acetonitrile (v/v) gradient while
monitoring at 214 nm. The appropriate fractions are pooled and
lyophilized. Further characterization of the final product is
performed using analytical HPLC and mass spectral analysis by
conventional methods. For peptides with a basic side chain, the
final lyophilized product is a TFA salt.
##STR00010## ##STR00011##
EXAMPLE 26
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2 (SEQ ID
NO:37)
[0239] Prepare as in Example 25, except that step 1 is omitted. MW
cal.: 929.10; MW obs.: 929.39.
EXAMPLE 26A
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2.acetic acid
salt (SED ID NO:38)
[0240] The TFA salt of the peptide of Example 26 is converted to an
acetic acid salt by adsorbing the material onto a preparative C18
column of suitable size, equilibrated with 2% acetic acid/H.sub.2O
(v/v). The column is then washed with three to five column volumes
of 2% aqueous acetic acid (v/v). The peptide is eluted using 1:1
water/acetonitrile (v/v) containing 2% acetic acid by volume, and
lyophilized. MW cal.: 929.10; MW obs.: 929.39.
EXAMPLE 27
cyclo[Gly-Tyr-Arg-DArg-2Nal-Gly-DGlu]-Arg-NH.sub.2 (SEQ ID
NO:39)
[0241] Prepare as in Example 25, except that Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf). MW cal.: 1071.22; MW obs.:
1071.02.
EXAMPLE 28
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SEQ
ID NO:40)
[0242] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-Lys(iPr)(Boc). MW cal.: 1099.35; MW obs.:
1099.91.
EXAMPLE 29
cyclo[Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID NO:41)
[0243] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), Fmoc-Gly in step 8 is not
used, and step 8 is omitted. MW cal.: 1014.17; MW obs.:
1014.78.
EXAMPLE 30
cyclo[Tyr-Arg-DArg-2Nal-Gly-DGlu]-Arg-NH.sub.2 (SEQ ID NO:42)
[0244] Prepare as in Example 25, except that Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), Fmoc-Gly in step 8 is not
used, and step 8 is omitted. MW cal.: 1014.17; MW obs.:
1014.65.
EXAMPLE 31
cyclo[Tyr-Arg-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ ID NO:43)
[0245] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(allyl), Fmoc-Lys(iPr)(Boc) in step
6 is replaced with Fmoc-Arg(Pb1), Fmoc-Gly in step 8 is not used,
and step 8 is omitted. MW cal.: 1000.14; MW obs.: 1000.63.
EXAMPLE 32
cyclo[Gly-Tyr-Arg-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ ID
NO:44)
[0246] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(allyl), and Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pb1). MW cal.: 1057.19; MW obs.:
1057.35.
EXAMPLE 33
cyclo[Gly-Tyr-Lys(Me.sub.2)-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ ID
NO:45)
[0247] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(allyl), and Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Lys(Me.sub.2). MW cal.: 1057.23; MW
obs.: 1057.86.
EXAMPLE 34,
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ ID
NO:44)
[0248] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(allyl). MW cal.: 1071.26; MW obs.:
1071.76.
EXAMPLE 35
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Asp]-NH.sub.2 (SEQ ID
NO:47)
[0249] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Asp(allyl). MW
cal.: 915.07; MW obs.: 915.38.
EXAMPLE 36
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Asp]-Lys(iPr)-NH.sub.2 (SEQ ID
NO:48)
[0250] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-Lys(iPr)(Boc), and Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(allyl). MW cal.: 1085.33; MW obs.:
1085.78.
EXAMPLE 37
cyclo[Gly-Tyr-Lys(Me.sub.2)-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:49)
[0251] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), and Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Lys(Me.sub.2). MW cal.: 1071.26; MW
obs.: 1071.05.
EXAMPLE 38
cyclo[Gly-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:50)
[0252] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), and Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf). MW cal.: 1071.22; MW obs.:
1071.50.
EXAMPLE 39
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:51)
[0253] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl). MW cal.: 1085.29; MW
obs.: 1085.91.
EXAMPLE 40
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:52)
[0254] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(Oallyl). MW
cal.: 929.10; MW obs.: 929.39.
EXAMPLE 41
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Asp]-NHEt (SEQ ID NO:53)
[0255] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, step 1 is omitted, and Fmoc-DGlu(Oallyl) in step 2 is
replaced with Fmoc-Asp(allyl). MW cal.: 943.13; MW obs.:
943.36.
EXAMPLE 42
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NHEt (SEQ ID NO:54)
[0256] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, step 1 is omitted, and Fmoc-DGlu(Oallyl) in step 2 is
replaced with Fmoc-Glu(Oallyl). MW cal.: 957.15; MW obs.:
957.50.
EXAMPLE 43
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NHEt (SEQ ID NO:55)
[0257] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, and step 1 is omitted. MW cal.: 957.15: MW obs.: 957.46.
EXAMPLE 44
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Arg-NHEt (SEQ ID
NO:56)
[0258] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin. MW cal.: 1113.34; MW obs.: 1113.81.
EXAMPLE 44a
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Arg-NHEt.acetic acid
salt (SED ID NO:57)
[0259] The TFA salt of the peptide of Example 44 is converted to an
acetic acid salt by adsorbing the material onto a preparative C18
column of suitable size, equilibrated with 2% acetic acid/H.sub.2O
(v/v). The column is then washed with three to live column volumes
of 2% aqueous acetic acid (v/v). The peptide is eluted using 1:1
water/acetonitrile (v/v) containing 2% acetic acid by volume, and
lyophilized. MW cal.: 1113.34; MW obs.: 1113.81.
EXAMPLE 45
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NHEt (SEQ ID
NO:58)
[0260] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, and Fmoc-Arg(Pbf) in step 1 is replaced with
Fmoc-Lys(iPr)(Boc). MW cal.: 1127.41; MW obs.: 1127.35.
EXAMPLE 46
cyclo[Lys(Ac)-Tyr-Lys(Me.sub.2)-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2
(SEQ ID NO:59)
[0261] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(OtBu), Fmoc-Lys(iPr)(Boc) in step
6 is replaced with Fmoc-Lys(Me.sub.2), and Fmoc-Gly in step 8 is
replaced with Boc-Lys(Fmoc). After chain assembly, Fmoc on the side
chain of the N-terminal Lys is removed with 20% piperidine in DMF
and the Lys side chain is then acetylated using 10 equivalents of
acetic anhydride/DIEA at room temperature for one hour. All side
chain protection groups are removed and the linear peptide is
cleaved from the solid support using a mixture of
TFA/water/TIS/anisole (90/5/2.5/2.5, v/v/v/v) for 2 h at room
temperature. Cyclization of the crude linear peptide is carried out
in solution. The cleaved crude linear peptide (-0.25 mmole) is
dried under vacuum and dissolved in 10 mL of dry DMF. This peptide
solution is delivered to the following reaction mixture via a
syringe pump during a 2 h period: 15 mL of dry dichloromethane and
15 mL of dry DMF containing 1.0 mmole of PyBOP and 4.0 mmoles of
DIEA. The reaction is then allowed to proceed at room temperature
for 2 h. Solvents are then evaporated under vacuum, the residue is
loaded onto a reversed phase C18 preparative HPLC column, and the
target cyclic peptide is isolated and characterized as described in
Example 1. MW cal.: 1184.42; MW obs.: 1184.06.
EXAMPLE 47
cyclo[Dap(Ac)-Tyr-Lys(Me.sub.2)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:60)
[0262] Prepare as in Example 25, except that step 1 is omitted,
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(OtBu),
Fmoc-Lys(iPr)(Boc) in step 6 is replaced with Fmoc-Lys(Me.sub.2),
and Fmoc-Gly in step 8 is replaced with Boc-Dap(Fmoc). After chain
assembly, Fmoc on the side chain of the N-terminal Dap is removed
using 20% piperidine in DMF, and the Dap side chain is then
acetylated with 10 equivalents of acetic anhydride/DIEA at room
temperature for one hour. All side chain protection groups are then
removed and the linear peptide is cleaved from the solid support
using a mixture of TFA/water/TIS/anisole (90/5/2.5/2.5, v/v/v/v)
for 2 h at room temperature. Cyclization of the crude linear
peptide is carried out in solution. The cleaved crude linear
peptide (-0.25 mmole) is dried under vacuum and dissolved in 10 mL
of dry DMF. This peptide solution is delivered to the following
reaction mixture via a syringe pump during a 2 h period: 15 mL of
dry dichloromethane and 15 mL of dry DMF containing 1.0 mmole of
PyBOP and 4.0 mmoles of DIEA. The reaction is allowed to proceed at
room temperature for 2 h. Solvents are then evaporated under
vacuum, the residue is loaded onto a preparative HPLC column, and
the target cyclic peptide is isolated and characterized as
described in Example 1. MW cal.: 986.15; MW obs.: 985.97.
EXAMPLE 48
cyclo[Ala-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:61)
[0263] Prepare as in Example 25, except that step 1 is omitted,
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(Oallyl), and
Fmoc-Gly in step 8 is replaced with Fmoc-Ala. MW cal.: 943.13; MW
obs.: 942.92.
EXAMPLE 49
cyclo[DAla-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:62)
[0264] Prepare as in Example 25, except that step 1 is omitted,
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(Oallyl), and
Fmoc-Gly in step 8 is replaced with Fmoc-DAla. MW cal.: 943.13; MW
obs.: 943.44.
EXAMPLE 50
cyclo[DAla-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2 (SEQ ID
NO:63)
[0265] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-Gly in step 8 is replaced with Fmoc-DAla. MW cal.: 943.13; MW
obs.: 943.42.
EXAMPLE 51
cyclo[Ala-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2 (SEQ ID
NO:64)
[0266] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-Gly in step 8 is replaced with Fmoc-Ala. MW cal.: 943.13; MW
obs.: 943.48.
EXAMPLE 52
cyclo[Leu-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2 (SEQ ID
NO:65)
[0267] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-Gly in step 8 is replaced with Fmoc-Leu. MW cal.: 985.21; MW
obs.: 985.56.
EXAMPLE 53,
cyclo[Leu-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:66)
[0268] Prepare as in Example 25, except that step 1 is omitted,
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(Oallyl), and
Fmoc-Gly in step 8 is replaced with Fmoc-Leu. MW cal.: 985.21; MW
obs.: 985.49.
EXAMPLE 54
cyclo[DPhe-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:67)
[0269] Prepare as in Example 25, except that step 1 is omitted,
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(Oallyl), and
Fmoc-Gly in step 8 is replaced with Fmoc-DPhe. MW cal.: 1019.22; MW
obs.: 1019.52.
EXAMPLE 55
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NH.sub.2 (SEQ ID
NO:68)
[0270] Prepare as in Example 25, except that step 1 is omitted,
Fmoc-DGlu(Oallyl) in step 2 is replaced with Fmoc-Glu(Oallyl), and
Fmoc-Gly in step 8 is replaced with Fmoc-Phe. MW cal.: 1019.22; MW
obs.: 1019.53.
EXAMPLE 56
cyclo[DPhe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2 (SEQ ID
NO:69)
[0271] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-Gly in step 8 is replaced with Fmoc-DPhe. MW cal.: 1019.22; MW
obs.: 1019.50.
EXAMPLE 57
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SEQ
ID NO:70)
[0272] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-Lys(iPr)(Boc), and Fmoc-Gly in step 8 is
replaced with Fmoc-Phe. MW cal.: 1189.48; MW obs.: 1189.92.
EXAMPLE 57a
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2.acetic
acid salt (SEQ ID NO:71)
[0273] The TFA salt of the peptide of Example 57 is converted to an
acetic acid salt by adsorbing the material onto a preparative C18
column of suitable size, equilibrated with 2% acetic acid/H.sub.2O
(v/v). The column is then washed with three to five column volumes
of 2% aqueous acetic acid (v/v). The peptide is eluted using 1:1
water/acetonitrile (v/v) containing 2% acetic acid by volume, and
lyophilized. MW cal.: 1189.48; MW obs.: 1189.92.
EXAMPLE 58
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Arg-NH.sub.2 (SEQ ID
NO:72)
[0274] Prepare as in Example 25, except that Fmoc-Gly in step 8 is
replaced with Fmoc-Phe. MW cal.: 1175.41; MW obs.: 1175.81.
EXAMPLE 59
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-NH.sub.2 (SEQ ID
NO:73)
[0275] Prepare as in Example 25, except that step 1 is omitted, and
Fmoc-Gly in step 8 is replaced with Fmoc-Phe. MW cal.: 1019.22; MW
obs.: 1019.56.
EXAMPLE 60
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NHEt (SEQ ID
NO:74)
[0276] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, Fmoc-Arg(Pbf) in step 1 is replaced with Fmoc-Lys(iPr)(Boc),
and Fmoc-Gly in step 8 is replaced with Fmoc-Phe. MW cal.: 1217.53;
MW obs.: 1217.97.
EXAMPLE 60a
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NHEt.acetic
acid salt (SEQ ID NO:75)
[0277] The TFA salt of the peptide of Example 60 is converted to an
acetic acid salt by adsorbing the material onto a preparative C18
column of suitable size, equilibrated with 2% acetic acid/H.sub.2O
(v/v). The column is then washed with three to five column volumes
of 2% aqueous acetic acid (v/v). The peptide is eluted using 1:1
water/acetonitrile (v/v) containing 2% acetic acid by volume, and
lyophilized. MW cal.: 1217.53; MW obs.: 1217.97.
EXAMPLE 61
cyclo[2Nal-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NHEt (SEQ ID NO:77)
[0278] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, step 1 is omitted, Fmoc-DGlu(Oallyl) in step 2 is replaced
with Fmoc-Glu(Oallyl), and Fmoc-Gly in step 8 is replaced with
Fmoc-DAla. MW cal.: 971.18; MW obs.: 971.49.
EXAMPLE 62
cyclo[DPhe-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NHEt (SEQ ID NO:78)
[0279] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, step 1 is omitted, Fmoc-DGlu(Oallyl) in step 2 is replaced
with Fmoc-Glu(Oallyl), and Fmoc-Gly in step 8 is replaced with
Fmoc-2Nal. MW cal.: 1097.34; MW obs.: 1097.53.
EXAMPLE 63
cyclo[DPhe-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NHEt (SEQ ID NO:78)
[0280] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, step 1 is omitted, Fmoc-DGlu(Oallyl) in step 2 is replaced
with Fmoc-Glu(Oallyl), and Fmoc-Gly in step 8 is replaced with
Fmoc-DPhe. MW cal.: 1047.28; MW obs.: 1047.51.
EXAMPLE 64
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-NHEt (SEQ ID NO:79)
[0281] Prepare as in Example 25, except that Rink amide resin is
replaced with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM
resin, step 1 is omitted, Fmoc-DGlu(Oallyl) in step 2 is replaced
with Fmoc-Glu(Oallyl), and Fmoc-Gly in step 8 is replaced with
Fmoc-Phe. MW cal.: 1047.28; MW obs.: 1047.57.
EXAMPLE 65
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Gly-2Nal-NH.sub.2 (SEQ
ID NO:80)
[0282] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-2Nal, and one step is added between steps 1
and 2 using Fmoc-Gly. MW cal.: 1183.39; MW obs.: 1183.26.
EXAMPLE 66
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-Glu]-.beta.-Ala-D2Nal-NH.sub.2
(SEQ ID NO:81)
[0283] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-D2Nal, one step is added between steps 1
and 2 using Fmoc-.beta.-Ala, and Fmoc-DGlu(Oallyl) in step 2 is
replaced with Fmoc-Glu(Oallyl). MW cal.: 1197.40; MW obs.:
1196.70.
EXAMPLE 67
cyclo[.beta.-Ala-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:82)
[0284] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-(3-Ala. MW cal.: 1085.25; MW obs.: 1085.05.
EXAMPLE 68
cyclo[.beta.-Ala-Tyr-Arg-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ ID
NO:83)
[0285] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(Oallyl), Fmoc-Lys(iPr)(Bcc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-.beta.-Ala. MW cal.: 1071.22; MW obs.:
1071.15.
EXAMPLE 69
cyclo[5-aminovaleryl-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ
ID NO:84)
[0286] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-5-aminovaleric acid. MW cal.: 1113.30; MW obs.:
1113.40.
EXAMPLE 70
cyclo[5-aminovaleryl-Tyr-Arg-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ
ID NO:85)
[0287] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-5-amino valeric acid. MW cal.: 1099.27; MW obs.:
1100.25.
EXAMPLE 71
cyclo[(4-AMPA)-Tyr-Arg-DArg-2Nal-Gly-Asp]-Arg-NH.sub.2 (SEQ ID
NO:86)
[0288] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Asp(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-4-aminomethyl phenylacetic acid (4-AMPA). MW
cal.: 1147.32; MW obs.: 1148.20.
EXAMPLE 72
cyclo[(4-AMPA)-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:87)
[0289] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-4-aminomethyl phenylacetic acid (4-AMPA). MW
cal.: 1161.34; MW obs.: 1161.99.
EXAMPLE 73
cyclo[(4-AMPA)-Tyr-Arg-DArg-2Nal-Gly-Glu]-DArg-NH.sub.2 (SEQ ID
NO:88)
[0290] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-DArg(Pbf), Fmoc-DGlu(Oallyl) in step 2 is
replaced with Fmoc-Glu(Oallyl), Fmoc-Lys(iPr)(Boc) in step 6 is
replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is replaced
with Fmoc-4-aminomethyl phenylacetic acid (4-AMPA). MW cal.:
1161.34; MW obs.: 1161.83.
EXAMPLE 74
cyclo[(4-AMPA)-Tyr-Arg-DArg-2Nal-Gly-Glu]-Arg-NH.sub.2 (SEQ ID
NO:89)
[0291] Prepare as in Example 25, except that Fmoc-DGlu(Oallyl) in
step 2 is replaced with Fmoc-Glu(Oallyl), Fmoc-Lys(iPr)(Boc) in
step 6 is replaced with Fmoc-Arg(Pbf), and Fmoc-Gly in step 8 is
replaced with Fmoc-4-aminomethyl benzoic acid (4-AMB). MW cal.:
1147.32; MW obs.: 1147.66.
EXAMPLE 75
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-Gly-2Nal-NH.sub.2
(SED ID NO:90)
[0292] Prepare as in Example 25, except that step 1 is replaced
with three sequential residue couplings: first Fmoc-2Nal, then
Fmoc-Gly, and then Fmoc-Lys(iPr)(Boc). MW cal.: 1353.69; MW obs.:
1354.03.
EXAMPLE 76
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-Gly-2Nal-NH.sub.2
(SED ID NO:91)
[0293] Prepare as in Example 25, except that step 1 is replaced
with three sequential residue couplings: first Fmoc-2Nal, then
Fmoc-Gly, and then Fmoc-Lys(iPr)(Boc). In addition, Fmoc-Gly in
step 8 is replaced with Fmoc-Phe. MW cal.: 1443.82; MW obs.:
1444.13.
EXAMPLE 77
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Gly-DPhe-NH.sub.2 (SED
ID NO:92)
[0294] Prepare as in Example 25, except that step 1 is replaced
with two sequential residue couplings: first Fmoc-DPhe, then
Fmoc-Gly. MW cal.: 1133.36; MW obs.: 1133.73.
EXAMPLE 78
cyclo[Gly-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-DPhe-NH.sub.2
(SED ID NO:93)
[0295] Prepare as in Example 25, except that step 1 is replaced
with two sequential residue couplings: first Fmoc-DPhe, then
Fmoc-Lys(iPr)(Boc). MW cal.: 1246.58; MW ohs.: 1246.88.
EXAMPLE 79
cyclo[Lys-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SED
ID NO:94)
[0296] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-Lys(iPr)(Boc), and Fmoc-Gly in step 8 is
replaced with Fmot-Lys(Boc). MW cal.: 1170.50; MW obs.:
1169.80.
EXAMPLE 80
cyclo[Phe-Tyr-Lys-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SED ID
NO:95)
[0297] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-Lys(iPr)(Boc), Fmoc-Lys(iPr)(Boc) in step 6
is replaced with Fmoc-Lys(Boc), and Fmoc-Gly in step 8 is replaced
with Fmoc-Phe. MW cal.: 1147.40; MW obs.: 1146.70.
EXAMPLE 81
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SED
ID NO:96)
[0298] Prepare as in Example 25, except that Fmoc-Aig(Pbf) in step
1 is replaced with Fmoc-Lys(Boc), and Fmoc-Gly in step 8 is
replaced with Fmoc-Phe. MW cal.: 1147.40; MW obs.: 1146.70.
EXAMPLE 82
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Orn-NH.sub.2 (SED ID
NO:97)
[0299] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 is replaced with Fmoc-Orn(Boc), and Fmoc-Gly in step 8 is
replaced with Fmoc-Phe. MW cal.: 1133.40; MW obs.: 1132.70.
EXAMPLE 83
cyclo[Phe-Tyr-Lys-DArg-2Nal-Gly-DGlu]-Lys-NH.sub.2 (SED ID
NO:98)
[0300] Prepare as in Example 25, except that Fmoc-Arg(Pbf) in step
1 and Fmoc-Lys(iPr)(Boc) in step 6 are each replaced with
Fmoc-Lys(Boc), and Fmoc-Gly in step 8 is replaced with Fmoc-Phe. MW
cal.: 1105.37; MW obs.: 1105.40.
EXAMPLE 84
cyclo[Phe-Tyr-Lys-DArg-2Nal-Gly-DGlu]-Lys-NHEt (SED ID NO:99)
[0301] Prepare as in Example 25, except that Rink resin is replaced
with [3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM resin,
Fmoc-Arg(Pbf) in step 1 and Fmoc-Lys(iPr)(Boc) in step 6 are each
replaced with Fmoc-Lys(Boc), and Fmoc-Gly in step 8 is replaced
with Fmoc-Phe. MW cal.: 1133.36; MW obs.: 1133.82.
EXAMPLE 85
Alternative Synthesis I of
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SED
ID NO:70)
[0302] Example 57 discloses the synthesis of SEQ ID NO:70 via Fmoc
solid phase peptide synthesis chemistry employing the commercially
available building block Fmoc-Lys(iPr)Boc, which is expensive and
difficult to obtain in large quantity. The process described in
this example permits the synthesis of SEQ ID NO:70 using less
expensive Fmoc-Lys(Boc), solution cyclization, and lysine
alkylation through reductive amination using sodium
cyanoboronhydride, providing a more economical route to this end
product. Additional advantages are that the reaction media (acetic
acid, acetone, and methanol) are relatively inexpensive, the
reaction conditions are easily controlled, the ratio of solvents
can vary significantly without affecting the alkylation reaction,
and the recovery yield is 90% or higher.
[0303] The sequence
Phe-Tyr(tBu)-Lys(Boc)-DArg(Pbf)-2Nal-Gly-DGlu(Oallyl)-Lys(Boc) (SEQ
ID NO:100) is assembled on Rink Amide Resin by standard Fmoc
chemistry utilizing an ABI 431 Peptide Synthesizer as outlined in
Scheme 7. The automated assembly is carried out using the standard
Applied Biosystems DCC/HOBt chemistry protocol or FastMoc chemistry
(HBTU/DIEA) protocol following the supplier's directions (PE
Applied Biosystems Inc., Foster City, Calif.). The side chain
protecting group scheme is: Lys(Boc), DGlu(Oallyl), DArg(Pbf),
Tyr(tBu). The stepwise chain assembly starts from the C-terminal
end of the linear peptide and is accomplished in 8 steps. In step
1, four equivalents of protected amino acid Fmoc-Lys(Boc) are
activated with DCC/HOBt (or HBTU/DIEA for FastMoc chemistry) in
NMP, and coupled to deprotected Rink amide resin. In step 2, four
equivalents of Fmoc-DGlu(Oallyl) are activated and coupled to the
deprotected peptide resin from step 1. These steps are repeated
appropriately until step 8, the coupling of Fmoc-Phe.
[0304] The allyl ester side chain protecting group is removed with
0.1 equivalent of Pd(Ph.sub.3P).sub.4 in the presence of 24
equivalents of phenylsilane in dichloromethane (Scheme 8). This
process is repeated once for complete side chain deprotection. Fmoc
at the N-terminal end is then removed using 20% piperidine in DMF.
The deprotected carboxylic acid moiety of 1)Glu is activated with
PyBOP/DIEA and cyclized to the .alpha.-amino group of Phe on the
resin. The cyclized peptide is simultaneously deprotected and
cleaved from the resin using a scavenger cocktail of
TFA/H.sub.2O/TIS/EDT (95/2/1/2, v/v/v/v) or
TFA/H.sub.2O/TIS/anisole (92/2/4/2, v/v/v/v) for 2 hours at room
temperature. The solvents are then evaporated under vacuum, and the
peptide is precipitated and washed three times with cold diethyl
ether to remove the scavengers.
##STR00012##
##STR00013## ##STR00014##
[0305] Purification of the cyclic precursor peptide is accomplished
using standard preparative HPLC techniques. The crude cleavage
product is dissolved in a minimum amount of DMSO, loaded onto a
reversed phased C18 HPLC column, and eluted with an aqueous 0.1%
trifluoroacetic acid/acetonitrile gradient (v/v) while monitoring
at 214 nm. The appropriate fractions are pooled and lyophilized.
Further characterization of the intermediate precursor cyclic
peptide is performed using analytical HPLC and mass spectral
analysis by conventional techniques.
[0306] The lyophilized precursor cyclic peptide is then alkylated
in a solution of acetic acid/acetone/methanol (1:1:4, v/v/v)
through reductive amination using sodium cyanoboronhydride (Scheme
9). Peptide concentration is about 10 mg/mL, and can vary
significantly without affecting the results. Three to 5 equivalents
of the reducing reagent sodium cyanoboronhydride are used, and the
reaction is normally completed within 2 h at room temperature. The
recovery yield is 90% or higher. For example, 20 mg of the
precursor cyclic peptide are dissolved in 2 mL of methanol, to
which 0.5 mL of acetic acid and 0.5 mL of acetone are added, mixed
well, and then 5.6 mg of sodium cyanoboronhydride (2.5 equivalents
in methanol) are added under stirring. The reaction mixture is
stirred at room temperature for 30 min, upon which another 5.6 mg
of sodium cyanoboronhydride are added. The reaction is monitored by
HPLC and mass spectral analysis. After the reaction is complete,
desalting of the reaction mixture and lyophilization yields 18.9 mg
of final product (SEQ ID NO:70) with a purity of 97.5%. MW cal.:
1189.48; MW obs.: 1189.6.
##STR00015##
[0307] The compound of Example 60 (SEQ ID NO:74) can also be
prepared in an analogous manner, with appropriate modifications
based on its constituent amino acids, using
[3-({ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetyl AM resin. First,
the precursor peptide (SEQ ID NO:99) is prepared as described in
Example 84, and then reductive amination is carried out as shown in
Scheme 9. This affords a final cyclic C-terminal ethyl amide
peptide with a purity of 99.16%. MW cal.: 1217.53; MW obs.:
1217.84.
EXAMPLE 86
Alternative Synthesis II of
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SED
ID NO:70)
[0308] Example 85 discloses a cost effective synthesis of SEQ ID
NO:70 via Fmoc solid phase peptide synthesis chemistry employing
relatively inexpensive Fmoc-Lys(Boc) and alkylation of lysine
through reductive amination using sodium cyanoboronhydride in
relatively inexpensive solvents (acetic acid, acetone, and
methanol). However, this process involves the use of the heavy
metal catalyst palladium for the removal of the allyl ester side
chain protective group. Palladium is highly toxic, and quality
control to insure complete removal of this element is complicated
and difficult. The Boc solid phase peptide synthesis process with
cyclization on the resin described in this example permits the
production of SEQ ID NO:70 using relatively inexpensive
Boc-Lys(2-Cl-Z) without the need for a toxic and expensive
palladium catalyst, providing an even more economical, easily
upscalable, less toxic route to an end product requiring a simpler
quality control process.
[0309] The sequence
Fmoc-Phe-Tyr(2-Br-Z)-Lys(2-Cl-Z)-DArg(Tos)-2Nal-Gly-DGlu(OFm)-Lys(2-Cl-Z)
(SEQ ID NO:102) is manually assembled on MBHA
(4-methyl-benzhydryl-amine) resin (Cat. No. D-2095, BaChem
California Inc., Torrance, Calif.) using established solid phase
peptide synthesis Boc chemistry (Schnolzer et al. (1992) Int. J.
Pept. Protein Res. 40:180-193) as outlined in Scheme 10. The chain
assembly is carried out using an in situ neutralization/HBTU/DIEA
activation procedure as described in this reference. The side chain
protecting group scheme is: Lys(2-Cl-Z), DGlu(OFm), DArg(Tos), and
Tyr(2-Br-Z). The alpha-amino group of all the amino acid building
blocks is protected with tert-butoxycarbonyl (Boc) except the
N-terminal residue Phe, which is protected with Fmoc for efficiency
of synthesis. The stepwise chain assembly starts from the
C-terminal end of the linear peptide and is accomplished in 8
steps. In step 1, five equivalents of protected amino acid
Boc-Lys(2-Cl-Z) are activated with HBTU/DIEA in DMF, and coupled to
MBHA resin. In step 2, five equivalents of Boc-DGlu(OFm) are
activated and coupled to the deprotected peptide resin from step 1
using neat TFA. These steps are repeated appropriately until step
8, the coupling of Fmoc-Phe.
[0310] The Fm side chain protecting group of DGlu, together with
the Fmoc at the N-terminal end, is removed using 20% piperidine in
DMF. The deprotected carboxylic acid moiety of DGlu is activated
with PyBOP/DIEA, HCTU/DIEA, or other appropriate activation
reagents, and cyclized to the .alpha.-amino group of Phe on the
resin. The cyclic peptide is simultaneously deprotected and cleaved
from the resin using HF with 5% m-cresol or p-cresol as a scavenger
for 1 hour at 0.degree. C. The solvents are then evaporated and the
crude peptide is precipitated and washed three times with cold
diethyl ether.
##STR00016##
[0311] Purification of the cyclic precursor peptide (SEQ ID NO:98
as shown in Scheme 10) is accomplished using standard preparative
HPLC techniques. The crude cleavage product is dissolved in a
minimum amount of DMSO, loaded onto a reversed phased C18 HPLC
column, and eluted with an aqueous 0.1% trifluoroacetic
acid/acetonitrile gradient (v/v) while monitoring at 214 nm. The
appropriate fractions are pooled and lyophilized. Further
characterization of the intermediate precursor cyclic peptide is
performed using analytical HPLC and mass spectral analysis by
conventional techniques. For SEQ ID NO:98, MW cal.: 1105.29; MW
obs.: 1105.4.
[0312] The lyophilized precursor cyclic peptide (SEQ ID NO:98) is
then alkylated in a solution of acetic acid/acetone/methanol
(1:1:4, v/v/v) through reductive amination using sodium
cyanoboronhydride as in Scheme 9. Peptide concentration is about 10
mg/mL, and can vary significantly without affecting the results.
Five equivalents of the reducing reagent sodium cyanoboronhydride
are used, and the reaction is normally completed within 2 h at room
temperature. The recovery yield is 90% or higher. For example, 6.6
mg of the precursor cyclic peptide are dissolved in 0.8 mL of
methanol, to which 0.2 mL of acetic acid and 0.2 mL of acetone are
added, mixed well, and then 1.9 mg of sodium cyanoboronhydride (2.5
equivalents in methanol) are added under stirring in two equal
portions. The reaction mixture is stirred at room temperature for
30 min, upon which another 1.9 mg of sodium cyanoboronhydride are
added. The reaction is monitored by HPLC and mass spectral
analysis. After the reaction is complete, desalting of the reaction
mixture and lyophilization yields the final product (SEQ ID NO:70)
with a purity of 96.5%. MW cal.: 1189.45; MW obs.: 1189.6.
EXAMPLE 87
Alternative Synthesis III of
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SED
ID NO:70)
[0313] The compound of Example 57 (SEQ ID NO:70) can also be
prepared without the use of a palladium catalyst via solution
cyclization, facilitating scaleup, as follows.
[0314] The sequence
Boc-Phe-Tyr(2-Br-Z)-Lys(Fmoc)-DArg(Tos)-2Nal-Gly-DGlu(OBzl)-Lys(Fmoc)
(SEQ ID NO:103) is manually assembled on MBHA resin using solid
phase peptide synthesis Boc chemistry (Schnolzer et al. (1992) Int.
J. Pept. Protein Res. 40:180-193) as outlined in Scheme 11. The
chain assembly is carried out using the in situ
neutralization/HBTU/DIEA activation procedure as described in
Schnolzer et al. The side chain protecting group scheme is:
Lys(Fmoc), DGlu(OBzl), nArg(Tos), Tyr(2-Br-Z). The alpha-amino
group of all the amino acid building blocks is protected with
tert-butoxycarbonyl (Boc). The stepwise chain assembly starts from
the C-terminal end of the linear peptide and is accomplished in 8
steps as shown in Scheme 11. In step 1, five equivalents of
protected amino acid Boc-Lys(2-Cl-Z) are activated with HBTU (4
eq)/DIEA (10 eq) in DMF, and coupled to MBHA resin. In step 2, five
equivalents of Boc-DGlu(OBzl) are activated and coupled to the
deprotected peptide resin from step 1 using neat TFA. These steps
are repeated appropriately until step 8, the coupling of Boc-Phe.
The Boc protecting group is removed with neat TFA, the resin is
neutralized with DIEA, and washed with DMF and methanol and dried
in air before HF cleavage. The linear peptide is simultaneously
deprotected and cleaved from the resin using HF with 5% m-cresol or
p-cresol as a scavenger for 1 hour at 0.degree. C. The solvents are
then evaporated and the crude peptide is precipitated and washed
three times with cold diethyl ether.
##STR00017##
[0315] Purification of the linear precursor peptide (SEQ ID NO:104)
is accomplished using standard preparative HPLC techniques. The
crude cleavage product is dissolved in a minimum amount of DMSO,
loaded onto a reversed phased C18 HPLC column, and eluted with an
aqueous 0.1% trifluoroacetic acid/acetonitrile gradient (v/v) while
monitoring at 214 nm. The appropriate fractions are pooled and
lyophilized. Further characterization of the intermediate precursor
cyclic peptide is performed using analytical HPLC and mass spectral
analysis by conventional techniques. For SEQ ID NO:104, MW cal.:
1567.78; MW obs.: 1567.6.
[0316] Cyclization of the lyophilized precursor linear peptide (SEQ
ID NO:104) is carried out in solution (Scheme 12). The linear
peptide is dissolved in a small amount of dry DMF (.about.10
mg/mL). This peptide solution is slowly delivered via a syringe
pump to the reaction mixture of PyBOP (2 eq, or other appropriate
activation reagents, such as HCTU, BOP, HBTU, etc.) and DIEA (10
eq) in dry DMF under magnetic stirring. The reaction is then
allowed to proceed at room temperature for 2 h. Neat piperidine is
then added to the reaction mixture to a final concentration of 25%
(v/v). The reaction mixture is kept under stirring for another 20
min to completely remove Fmoc protection. Solvents are evaporated
under vacuum and the residue is loaded onto a preparative reversed
phase C18 HPLC column, and eluted with an aqueous 0.1%
trifluoroacetic acid/acetonitrile gradient (v/v) while monitoring
at 214 nm. The appropriate fractions are pooled and lyophilized,
and afford the cyclic precursor peptide (SEQ ID NO:98). Further
characterization of the intermediate precursor cyclic peptide is
performed using analytical HPLC and mass spectral analysis by
conventional techniques. For SEQ ID NO:98, MW cal.: 1105.29; MW
obs.: 1105.4.
[0317] Alkylation of cyclic peptide SEQ ID NO:98 is carried out in
a solution of acetic acid/acetone/methanol (1:1:4, v/v/v) through
reductive amination using sodium cyanoboronhydride as in Scheme 9
to generate the final product (SEQ ID NO:70). MW cal.: 1189.45: MW
obs.: 1189.6.
##STR00018##
EXAMPLE 88
Alternative Synthesis IV of
cyclo[Phe-Tyr-Lys(iPr)-DArg-2Nal-Gly-DGlu]-Lys(iPr)-NH.sub.2 (SEQ
ID NO:70)
[0318] The compound of Example 57 (SEQ ID NO:70) can also be
prepared without the use of a palladium catalyst by the synthetic
process summarized in Scheme 13 below.
[0319] The dipeptide Fmoc-DGlu-Lys(iPr,Z)-NH.sub.2 is first
prepared in solution with an exposed DGlutamic acid side chain. The
dipeptide is linked to a hyper-acid labile CTC
(2-chlorotritylchloride PS resin, 1% DVB (100-200 mesh) resin (Senn
Chemicals USA Inc., San Diego, Calif.; catalog number 40207), and
the final peptide product is then synthesized on this resin via
standard Fmoc synthesis as described above. Selective removal of
the peptide from the CTC resin allows only the DGlutamic acid side
chain to react with the N-terminus of the peptide in solution and
generate the cyclic peptide product. Subsequently, the remaining
side chains are cleaved with 95% TFA or other strong acid.
##STR00019##
[0320] The dipeptide Fmoc-DGlu-Lys(iPr,Z)-NH.sub.2 is first
prepared as shown below (Scheme 14):
##STR00020##
[0321] Boc-Lys(iPr,Z)-OH is reacted with NMM and IBCF in THF. After
addition of NH.sub.4OH, the solvents are removed by rotary
evaporation and the product is taken up in ethyl acetate. The ethyl
acetate phase is washed extensively with 5% NaHCO.sub.3 and then
with 0.1 N HCl, and then dried over anhydrous sodium sulfate.
Sodium sulfate is removed by filtration and the ethyl acetate is
removed by evaporation at reduced pressure. The resulting
Boc-Lys(iPr,Z)-NH.sub.2 is dissolved in DCM, and TFA is added. Once
the reaction has proceeded to completion, the solvents are removed
by rotary evaporation. H-Lys(iPr,Z)-NH.sub.2 is then dissolved in
DMF. The pH is adjusted to 8 with DIEA. In a separate vessel,
Fmoc-DGlu(OtBu)-OH, HBTU, and HOBt are dissolved in DMF; DIEA is
added to adjust the pH to 8. The two solutions are mixed, and the
reaction is monitored by C18 reversed phase HPLC. The pH is
monitored and adjusted, where necessary, with DIEA. The solvents
are removed by rotary evaporation, and the product dissolved in
ethyl acetate. The ethyl acetate phase is washed extensively with
5% NaHCO.sub.3 and then with 0.1 N HCl, and then dried over
anhydrous sodium sulfate. The sodium sulfate is removed by
filtration and the ethyl acetate is removed by evaporation at
reduced pressure. Rotary evaporation is continued until a dry
residue is formed. The resulting
Fmoc-DGlu(OtBu)-Lys(iPr,Z)-NH.sub.2 is dissolved in DCM, and TFA is
added. Once the reaction has proceeded to completion, the solvents
are removed by rotary evaporation. The solid product is obtained by
trituration with diethyl ether. After the precipitate is washed
with ether, the product is dried in a vacuum oven.
[0322] Solid phase peptide synthesis of the final product is
performed as follows. Fmoc-DGlu(OtBti)-Lys(iPr,Z)-NH.sub.2 is
dissolved in DCM and reacted with CTC resin in the presence of DIEA
in a reaction vessel. After 3 h, the peptide-resin is washed free
of reagents with DCM and Z-OSu is added. The pH is monitored and
adjusted to pH 8-9 by adding DIEA if necessary. After 8 h, the
peptide-resin is washed free of reagents with DCM, transferred to a
polypropylene container, and dried in a vacuum oven.
[0323] The protected peptide-resin is assembled using
Fmoc-chemistry as follows. The coupling cycle used is: 1)
De-blocking: treatment with 25% piperidine in DMF; 2) Washing
cycles with DMF, IPA and DMF again; 3) Ninhydrin test (qualitative:
if positive, proceed to coupling Step 4); 4) Coupling with 2
equivalents Fmoc-amino acid in presence of HOBt/DIC in DMF; 5)
Washing cycles with DMF; 6) Ninhydrin test (qualitative: if
negative, proceed to next de-blocking/coupling cycle; if positive,
proceed to re-coupling Step 7; if slightly positive, proceed to
acetylation Step 10); 7) Re-coupling (if required), with 1
equivalent Fmoc-amino acid in the presence of HOBt, HBTU/DIEA in
DMF; 8) Washing cycles with DMF; 9) Ninhydrin test (qualitative: if
negative, proceed to next de-blocking/coupling cycle; if positive,
proceed to acetylation Step 10); 10) Acetylation (if required) with
2% acetic anhydride in 4% DIEA in DMF; 11) Washing cycles (with
DMF, IPA, and DMF again); 12) Ninhydrin test (qualitative: if
poSitive, proceed to next de-blocking/coupling cycle). After the
final coupling cycle, the peptide-resin (SEQ ID NO:105) is washed
with ether and dried under vacuum.
[0324] The protected peptide-resin is washed with DCM. Cleavage of
the fully protected linear peptide from the resin is performed with
2% TFA in DCM followed by filtration. The solvents are removed by
rotary evaporation, and the fully linear protected peptide (SEQ ID
NO:106) is precipitated by trituration with ether. The fully
protected linear peptide is transferred to a polypropylene
container and dried in a vacuum oven.
[0325] The fully protected linear peptide is cyclized in the
presence of PyBOP, HOBt, and DIEA in DMF. The pH is maintained
between pH 7-8 by addition of DIEA, if necessary. After the
reaction has proceeded to completion, the solvents are removed by
rotary evaporation, and the product is taken up in ethyl acetate.
The ethyl acetate phase is washed extensively with 5% NaHCO.sub.3
and then with 0.1 N HCl and saturated NaCl solution. It is then
dried over anhydrous sodium sulfate. The sodium sulfate is removed
by filtration and the ethyl acetate is removed by rotary
evaporation at reduced pressure. The protected cyclic peptide (SEQ
ID NO:107) is precipitated by trituration with ether and dried in a
vacuum oven.
[0326] Deprotection is performed in TFA:H.sub.2O:TIS. When the
reaction is complete, the solvents are removed by rotary
evaporation and the cyclic peptide (SEQ ID NO:70) is precipitated
by trituration with ether and dried in a vacuum oven. MW cal.:
1189.48; MW obs.: 1189.50.
EXAMPLE 89
Incorporation of Isotopic Labels: Synthesis of
cyclo[Phe-Tyr-Lys(iPr-d.sub.6)-DArg-2Nal-Gly-DGlu]-Lys(iPr-d.sub.6)-NH.su-
b.2 (SED ID NO:108)
[0327] Starting with isotopically labeled acetone such as
.sup.13C-, .sup.14C-deuterium-, or tritium-labeled acetones as
shown below, the processes of Examples 85-87 permit site-specific
isotopic labeling of cyclic peptide CXCR4 antagonists for various
pharmaco-logical and imaging studies. The isotopically labeled
acetones are commercially available from various sources. An
example is given below using acetone-d.sub.6 to prepare a peptide
containing 12 deuterium atoms. The resulting compound, differing in
molecular weight by 12 Da compared to the non-labeled counterpart,
is easily differentiated in mass spectra and exhibits identical
target receptor affinity.
##STR00021##
[0328] Using any of the methods in Examples 85-87, one can prepare
and purify cyclic peptide precursors (SEQ ID NO:98, SEQ ID NO:99,
etc.). Alkylation is carried out in a solution of acetic
acid/acetone/methanol (1:1:4, v/v/v) through reductive amination
using sodium cyanoboronhydride as in Scheme 9, with the exception
that standard acetone is replaced with the desired isotopically
labeled acetone. In the present example, deuterium acetone-d6 is
used.
[0329] Peptide (97 mg) is dissolved in 15 mL of acetic
acic/acetone-d.sub.6/methanol (1:1:4, v/v/v). Peptide concentration
can vary significantly without affecting the results. Five
equivalents of sodium cyanoboronhydride are used, and the reaction
is normally completed within 2 h at room temperature. The reaction
is monitored by HPLC and mass spectral analysis. After the reaction
is complete, desalting of the reaction mixture and lyophilization
yields 90.5 mg of final product (SEQ ID NO:108) with a purity of
99.9% MW cal.: 1201.48; MW obs.: 1201.7.
##STR00022##
[0330] Use of isotopically labeled sodium cyanoboronhydride (such
as NaBD3CN, NaBT3C N, etc.) permits incorporation of additional
variations to the site-specific labeling patterns.
[0331] The pharmacological properties of the present compounds can
be determined by employing the assays described below.
Human CXCR4/.sup.125I-SDF-1.alpha. Binding Inhibition Assay
[0332] SDF-1 binding to CXCR4 is the first step in activating the
CXCR4 intracellular signalling pathway. To determine if a compound
can block the interaction of SDF-1 and CXCR4, human leukemia
CCRF-CEM cells (ATCC CCL 119) expressing endogenous CXCR4 are
employed in an .sup.125I-labeled SDF-1.alpha. binding assay. The
assay is performed in a 96-well U-bottom, non-treated polystyrene
plate (Corning Incorporated, Costar, No. 3632). The binding assay
buffer is prepared with RPMI 1640 medium (Gibco, Grand Island,
N.Y.) containing 10 mM HEPES, pH 7.5, and 0.2% BSA. Briefly, 200
.mu.L reaction mixtures containing 300 pM SDF ligand (60 pM
.sup.125I-SDF-1.alpha. (Perkin Elmer) and 240 pM cold SDF-1.alpha.
(R&D Systems), different concentrations of the test compound in
assay buffer, 100,000 human CCRF-CEM cells, and 0.5 mg SPA beads
(Wheatgerm agglutinin beads; Amersham) are incubated at room
temperature for 2 hr. Plates are then counted in a 1450 Microbeta
Liquid Scintillation and Luminescence Counter (Wallac) in SPA mode.
CXCR4 antagonists decrease the bound radioactivity in this assay in
a dose-dependent manner. The inhibitory potency (K.sub.i or
IC.sub.50) of a test compound is calculated using GraphPad Prism
software, based on the dose-dependent decrease of bound
radioactivity.
[0333] All compounds exemplified above exhibit an average K.sub.i
value of about 7.5 nM or less in this assay. For example, the
compound of Example 1 exhibits an average K.sub.i of 3.45 nM in
this assay. Many of these compounds exhibit an average K.sub.i
value between about 0.2 nM and about 1 nM. For example, the
compound of Example 50 exhibits an average K.sub.i value of 0.285
nM in this assay. Other compounds exhibit an average K.sub.i value
less than about 0.2 nM. For example, the compound of Example 75
exhibits an average K.sub.i value of 0.096 nM in this assay.
Chemotaxis Assay
[0334] CXCR4/SDF-1 interaction regulates migration (chemotaxis) of
cells bearing CXCR4 on their surface. To determine the antagonist
and cellular activities of a test compound, a chemotaxis assay
using human histiocytic lymphoma U937 cells (ATCC CRL 1593) that
express endogenous CXCR4 are employed. Briefly, U937 cells, grown
in DMEM medium (Gibco, Grand Island, N.Y.) containing 10% FBS, 1%
MEM sodium pyruvate (Gibco), 1% MEM nonessential amino acids
(Gibco), and 1% GlutaMAX 1 (Gibco), are harvested and washed once
with chemotaxis assay buffer prepared with 1.times.RPMI medium
(Gibco) containing 10 mM HEPES, pH 7.5, and 0.3% BSA. After
washing, cells are resuspended in assay buffer at a concentration
of 5.times.10.sup.6 cells/mL. The assay is performed in a 96-well
ChemoTx plate (NeuroProbe) according to the manufacturer's
directions. Generally, 50 .mu.L of cell mixture with or without
test compound are plated on the upper chamber, and 30 .mu.L of
SDF-1.alpha. (R&D Systems, 10 ng/mL) prepared in 1.times.
chemotaxis buffer are added to the lower chamber. After assembly,
the plate is incubated for 2.5 hr at 37.degree. C. under 5%
CO.sub.2. Following the incubation, 5 .mu.L of CellTiter 96 AQ
(Promega, Madison, Wis.) are added into the lower chamber. The
plate is then incubated for 60 min at 37.degree. C., and the
migrated cells are detected by measuring the absorbance at 492 nm
with a Tecan Spectrafluor Plus Microplate Reader (Salzburg,
Austria). CXCR4 antagonists inhibit cell migration, reducing the
absorbance reading. The inhibitory potency (IC.sub.50) of a test
compound in this assay is calculated using GraphPad Prism software,
based on the dose-dependent decrease of absorbance at 492 nm.
[0335] Most of the compounds exemplified above exhibit an average
IC.sub.50 value of about 60 nM or less in this assay. Many of these
compounds exhibit an average IC.sub.50 value of about 6 nM or less,
e.g., the compound of Example 19 exhibits an average IC.sub.50
value of 2.05 nM in this assay. Many of these compounds exhibit an
average IC.sub.50 value of about 0.6 nM or less, e.g., the compound
of Example 50 exhibits an average IC.sub.50 value of 0.171 nM in
this assay.
Chemokine Receptor Binding Selectivity Assays
[0336] The binding selectivity of the present compounds for the
CXCR4 receptor compared to that for other chemokine receptors, such
as human CCR1, CCR2, CXCR2, or CXCR3, and other G-protein-coupled
receptors, can be assessed in cells transfected with nucleic acid
encoding and expressing such receptors, or in cells in which such
receptors are endogenously expressed. Whole cells or membrane
fragments can be used to assess competition of test compounds with
the respective ligands for these receptors in a manner similar to
that described above for the CXCR4/.sup.125I-SDF-1.alpha. binding
inhibition assay.
[0337] For example, the compound of Example 57a exhibits a K; value
greater than 73,000 nM in a ligand binding assay using human
chemokine receptor CXCR2.
Compound-Induced White Blood Cell and Neutrophil Mobilization in
C57BL/6 Mice
[0338] Stem cells within the bone marrow actively maintain
continuous production of all mature blood cell lineages throughout
life. Bone marrow is the primary site for white blood cell
(WBC)/neutrophil production and release into the circulation. The
CXCR4/SDF-1 axis appears to be critical for the retention and
release of WBCs, neutrophils, and hematopoietic progenitor cells in
the bone marrow, and interruption of CXCR4/SDF-1 interaction in
bone marrow leads to an increase of these cells in peripheral
blood. A short-term mouse WBC/neutrophil mobilization model can be
used to determine the in vivo target-modulating activity of a test
compound. Briefly, pathogen-free 5-6 week old female C57BL/6 mice
(Taconic) are housed for at least one week prior to assay. Animals
are allowed continuous access to sterilized rodent chow and
acidified water. Groups of 5 mice are injected subcutaneously with
test compounds in saline, or with saline control, and then
sacrificed by CO.sub.2 asphyxiation and cervical dislocation at
various time points post compound administration. Peripheral blood
is collected by cardiac puncture using EDTA-coated syringes and
tubes. Complete blood cell analysis is performed on a Hernavet
Mascot hematology analyzer (Drew Scientific Group, Dallas, Tex.).
Total WBCs, neutrophils, and lymphocytes in the peripheral blood
are recorded. Effective CXCR4 antagonists administered
subcutaneously to mice increase the neutrophil and WBC counts in
peripheral blood compared to saline control.
[0339] A significant number of compounds exemplified above exhibit
an average neutrophil ratio (ratio of neutrophil increase in
treatment group vs. neutrophil increase in saline control group),
measured 3 hours after compound administration, greater than about
2 in this assay. For example, the compound of Example 39 exhibits
an average neutrophil ratio of 4.6 at a dose of 5 mg/kg in this
assay.
Anti-Tumor Activity in a SCID/Namalwa Xenograft Model
[0340] SDF-1/CXCR4 interaction appears to play an important role in
multiple stages of tumorigenesis, including tumor growth, invasion,
angiogenesis, and metastasis. To evaluate in vivo anti-tumor
activity of a test compound, a tumor xenograft model using NOD/SCID
mice (Jackson Laboratories) and human non-Hodgkin's lymphoma
Namalwa cells (ATCC CRL 1432) are employed. Briefly, 200,000
Namalwa cells mixed with matrigel (1:1) are implanted
subcutaneously into the rear flank of the animals. The implanted
tumor cells grow as solid tumors, the dimensions of which can be
continuously monitored and measured using a caliper. To determine
the in vivo efficacy of a test compound in this model, one can
treat animals (10/group) with different doses of test compounds
dissolved in saline or PBS, beginning 48 hours post tumor cell
implantation. Compounds are dosed subcutaneously, and tumor volume
and body weight are determined every 2 or 3 days. Studies generally
last 3-4 weeks, depending on tumor growth. The anti-tumor growth
activity of a test compound is determined by the percent reduction
in tumor volume in treatment groups compared to tumor volume in
control groups treated with vehicle alone.
[0341] Several compounds exemplified above, for example the
compound of Example 26, significantly inhibit tumor growth in this
assay when administered at 1 mg/kg BID.
[0342] Pharmacologic properties such as compound bioavailability,
in vivo metabolic stability, and pharmacokinetic/pharmacodynamic
properties can be determined by methods well known in the art of
drug development. Preferred compounds of the present invention
exhibit high bioavailability when administered subcutaneously. Some
compounds exemplified herein exhibit bioavailability near 100% in
rats, for example the compound of Example 44. Preferred compounds
also exhibit good in vivo metabolic stability. For example, no
detectable metabolites are observed in dog and monkey plasma and
urine up to 24 hours after administration of the compound of
Example 57a. Preferred compounds also exhibit favorable
pharmacokinetic/pharmacodynamic properties that permit convenient
dosing. For example, in mice, the half-life (T1/2) of the compound
of Example 58 is about 3 hours. With respect to pharmacodynamic
properties, preferred compounds induce prolonged neutrophil and
white blood cell mobilization in mice. For example, the compound of
Example 25 induces a significant increase of neutrophils and white
blood cells in peripheral blood for at least 6 hours after single
dose subcutaneous administration at 5 mg/kg in mice.
Sequence CWU 1
1
108110PRTArtificialSynthetic construct 1Xaa Tyr Xaa Arg Xaa Gly Xaa
Xaa Xaa Xaa1 5 1028PRTArtificialSynthetic construct 2Xaa Tyr Arg
Arg Xaa Gly Glu Arg1 538PRTArtificialSynthetic construct 3Xaa Xaa
Xaa Xaa Xaa Gly Xaa Xaa1 548PRTArtificialSynthetic construct 4Xaa
Xaa Xaa Xaa Xaa Gly Xaa Xaa1 558PRTArtificialSynthetic construct
5Xaa Xaa Xaa Xaa Xaa Gly Glu Xaa1 568PRTArtificialSynthetic
construct 6Xaa Tyr Arg Arg Xaa Gly Glu Arg1
578PRTArtificialSynthetic construct 7Xaa Tyr Xaa Arg Xaa Gly Glu
Arg1 588PRTArtificialSynthetic construct 8Xaa Tyr Xaa Arg Xaa Gly
Glu Arg1 598PRTArtificialSynthetic construct 9Xaa Tyr Arg Arg Xaa
Gly Glu Arg1 5108PRTArtificialSynthetic construct 10Glu Tyr Arg Arg
Xaa Gly Xaa Arg1 5118PRTArtificialSynthetic construct 11Xaa Xaa Xaa
Xaa Xaa Gly Xaa Xaa1 5128PRTArtificialSynthetic construct 12Xaa Xaa
Xaa Xaa Xaa Gly Xaa Xaa1 5138PRTArtificialSynthetic construct 13Glu
Tyr Arg Arg Xaa Gly Xaa Arg1 5148PRTArtificialSynthetic construct
14Glu Tyr Arg Arg Xaa Gly Xaa Arg1 5158PRTArtificialSynthetic
construct 15Glu Tyr Arg Arg Xaa Gly Xaa Arg1
5168PRTArtificialSynthetic construct 16Glu Tyr Arg Arg Xaa Gly Lys
Arg1 5178PRTArtificialSynthetic construct 17Glu Tyr Arg Arg Xaa Gly
Xaa Arg1 5188PRTArtificialSynthetic construct 18Glu Tyr Arg Arg Xaa
Gly Xaa Arg1 5198PRTArtificialSynthetic construct 19Glu Tyr Arg Arg
Xaa Gly Xaa Arg1 5208PRTArtificialSynthetic construct 20Asp Tyr Arg
Arg Xaa Gly Xaa Arg1 5218PRTArtificialSynthetic construct 21Asp Tyr
Arg Arg Xaa Gly Xaa Arg1 5228PRTArtificialSynthetic construct 22Asp
Tyr Arg Arg Xaa Gly Xaa Arg1 5238PRTArtificialSynthetic construct
23Asp Tyr Arg Arg Xaa Gly Xaa Arg1 5248PRTArtificialSynthetic
construct 24Asp Tyr Arg Arg Xaa Gly Xaa Arg1
5258PRTArtificialSynthetic construct 25Asp Tyr Xaa Arg Xaa Gly Xaa
Arg1 5268PRTArtificialSynthetic construct 26Asp Tyr Xaa Arg Xaa Gly
Xaa Arg1 5277PRTArtificialSynthetic construct 27Tyr Arg Arg Xaa Gly
Xaa Arg1 5287PRTArtificialSynthetic construct 28Tyr Arg Arg Xaa Gly
Xaa Arg1 5297PRTArtificialSynthetic construct 29Tyr Arg Arg Xaa Gly
Xaa Arg1 5307PRTArtificialSynthetic construct 30Tyr Arg Arg Xaa Gly
Xaa Arg1 5317PRTArtificialSynthetic construct 31Tyr Arg Arg Xaa Gly
Lys Arg1 5328PRTArtificialSynthetic construct 32Gly Tyr Xaa Arg Xaa
Gly Glu Arg1 5338PRTArtificialSynthetic construct 33Gly Xaa Xaa Xaa
Xaa Gly Xaa Xaa1 5348PRTArtificialSynthetic construct 34Gly Tyr Xaa
Arg Xaa Gly Glu Arg1 5358PRTArtificialSynthetic construct 35Gly Xaa
Xaa Xaa Xaa Gly Xaa Xaa1 5368PRTArtificialSynthetic construct 36Gly
Xaa Xaa Xaa Xaa Gly Glu Xaa1 5377PRTArtificialSynthetic construct
37Gly Tyr Xaa Arg Xaa Gly Glu1 5387PRTArtificialSynthetic construct
38Gly Tyr Xaa Arg Xaa Gly Glu1 5398PRTArtificialSynthetic construct
39Gly Tyr Arg Arg Xaa Gly Glu Arg1 5408PRTArtificialSynthetic
construct 40Gly Tyr Xaa Arg Xaa Gly Glu Xaa1
5417PRTArtificialSynthetic construct 41Tyr Arg Arg Xaa Gly Glu Arg1
5427PRTArtificialSynthetic construct 42Tyr Arg Arg Xaa Gly Glu Arg1
5437PRTArtificialSynthetic construct 43Tyr Arg Arg Xaa Gly Asp Arg1
5448PRTArtificialSynthetic construct 44Gly Tyr Arg Arg Xaa Gly Asp
Arg1 5458PRTArtificialSynthetic construct 45Gly Tyr Xaa Arg Xaa Gly
Asp Arg1 5468PRTArtificialSynthetic construct 46Gly Tyr Xaa Arg Xaa
Gly Asp Arg1 5477PRTArtificialSynthetic construct 47Gly Tyr Xaa Arg
Xaa Gly Asp1 5488PRTArtificialSynthetic construct 48Gly Tyr Xaa Arg
Xaa Gly Asp Xaa1 5498PRTArtificialSynthetic construct 49Gly Tyr Xaa
Arg Xaa Gly Glu Arg1 5508PRTArtificialSynthetic construct 50Gly Tyr
Arg Arg Xaa Gly Glu Arg1 5518PRTArtificialSynthetic construct 51Gly
Tyr Xaa Arg Xaa Gly Glu Arg1 5527PRTArtificialSynthetic construct
52Gly Tyr Xaa Arg Xaa Gly Glu1 5537PRTArtificialSynthetic construct
53Gly Tyr Xaa Arg Xaa Gly Asp1 5547PRTArtificialSynthetic construct
54Gly Tyr Xaa Arg Xaa Gly Glu1 5557PRTArtificialSynthetic construct
55Gly Tyr Xaa Arg Xaa Gly Glu1 5568PRTArtificialSynthetic construct
56Gly Tyr Xaa Arg Xaa Gly Glu Arg1 5578PRTArtificialSynthetic
construct 57Gly Tyr Xaa Arg Xaa Gly Glu Arg1
5588PRTArtificialSynthetic construct 58Gly Tyr Xaa Arg Xaa Gly Glu
Xaa1 5598PRTArtificialSynthetic construct 59Xaa Tyr Xaa Arg Xaa Gly
Glu Arg1 5607PRTArtificialSynthetic construct 60Xaa Tyr Xaa Arg Xaa
Gly Glu1 5617PRTArtificialSynthetic construct 61Ala Tyr Xaa Arg Xaa
Gly Glu1 5627PRTArtificialSynthetic construct 62Ala Tyr Xaa Arg Xaa
Gly Glu1 5637PRTArtificialSynthetic construct 63Ala Tyr Xaa Arg Xaa
Gly Glu1 5647PRTArtificialSynthetic construct 64Ala Tyr Xaa Arg Xaa
Gly Glu1 5657PRTArtificialSynthetic construct 65Leu Tyr Xaa Arg Xaa
Gly Glu1 5667PRTArtificialSynthetic construct 66Leu Tyr Xaa Arg Xaa
Gly Glu1 5677PRTArtificialSynthetic construct 67Phe Tyr Xaa Arg Xaa
Gly Glu1 5687PRTArtificialSynthetic construct 68Phe Tyr Xaa Arg Xaa
Gly Glu1 5697PRTArtificialSynthetic construct 69Phe Tyr Xaa Arg Xaa
Gly Glu1 5708PRTArtificialSynthetic construct 70Phe Tyr Xaa Arg Xaa
Gly Glu Xaa1 5718PRTArtificialSynthetic construct 71Phe Tyr Xaa Arg
Xaa Gly Glu Xaa1 5728PRTArtificialSynthetic construct 72Phe Tyr Xaa
Arg Xaa Gly Glu Arg1 5737PRTArtificialSynthetic construct 73Phe Tyr
Xaa Arg Xaa Gly Glu1 5748PRTArtificialSynthetic construct 74Phe Tyr
Xaa Arg Xaa Gly Glu Xaa1 5758PRTArtificialSynthetic construct 75Phe
Tyr Xaa Arg Xaa Gly Glu Xaa1 5767PRTArtificialSynthetic construct
76Ala Tyr Xaa Arg Xaa Gly Glu1 5777PRTArtificialSynthetic construct
77Xaa Tyr Xaa Arg Xaa Gly Glu1 5787PRTArtificialSynthetic construct
78Phe Tyr Xaa Arg Xaa Gly Glu1 5797PRTArtificialSynthetic construct
79Phe Tyr Xaa Arg Xaa Gly Glu1 5809PRTArtificialSynthetic construct
80Gly Tyr Xaa Arg Xaa Gly Glu Gly Xaa1 5819PRTArtificialSynthetic
construct 81Gly Tyr Xaa Arg Xaa Gly Glu Xaa Xaa1
5828PRTArtificialSynthetic construct 82Xaa Tyr Arg Arg Xaa Gly Glu
Arg1 5838PRTArtificialSynthetic construct 83Xaa Tyr Arg Arg Xaa Gly
Asp Arg1 5847PRTArtificialSynthetic construct 84Tyr Arg Arg Xaa Gly
Glu Arg1 5857PRTArtificialSynthetic construct 85Tyr Arg Arg Xaa Gly
Asp Arg1 5867PRTArtificialSynthetic construct 86Tyr Arg Arg Xaa Gly
Asp Arg1 5877PRTArtificialSynthetic construct 87Tyr Arg Arg Xaa Gly
Glu Arg1 5887PRTArtificialSynthetic construct 88Tyr Arg Arg Xaa Gly
Glu Arg1 5897PRTArtificialSynthetic construct 89Tyr Arg Arg Xaa Gly
Glu Arg1 59010PRTArtificialSynthetic construct 90Gly Tyr Xaa Arg
Xaa Gly Glu Xaa Gly Xaa1 5 109110PRTArtificialSynthetic construct
91Phe Tyr Xaa Arg Xaa Gly Glu Xaa Gly Xaa1 5
10929PRTArtificialSynthetic construct 92Gly Tyr Xaa Arg Xaa Gly Glu
Gly Phe1 5939PRTArtificialSynthetic construct 93Gly Tyr Xaa Arg Xaa
Gly Glu Xaa Phe1 5948PRTArtificialSynthetic construct 94Lys Tyr Xaa
Arg Xaa Gly Glu Xaa1 5958PRTArtificialSynthetic construct 95Phe Tyr
Lys Arg Xaa Gly Glu Xaa1 5968PRTArtificialSynthetic construct 96Phe
Tyr Xaa Arg Xaa Gly Glu Lys1 5978PRTArtificialSynthetic construct
97Phe Tyr Xaa Arg Xaa Gly Glu Xaa1 5988PRTArtificialSynthetic
construct 98Phe Tyr Lys Arg Xaa Gly Glu Lys1
5998PRTArtificialSynthetic construct 99Phe Tyr Lys Arg Xaa Gly Glu
Lys1 51008PRTArtificialSynthetic construct 100Phe Xaa Xaa Xaa Xaa
Gly Xaa Xaa1 51018PRTartificialSynthetic construct 101Phe Xaa Xaa
Xaa Xaa Gly Glu Xaa1 51028PRTartificialsynthetic construct 102Phe
Xaa Xaa Xaa Xaa Gly Xaa Xaa1 51038PRTartificialsynthetic construct
103Phe Xaa Xaa Xaa Xaa Gly Xaa Xaa1 51048PRTartificialsynthetic
construct 104Phe Tyr Xaa Arg Xaa Gly Glu Xaa1
51058PRTartificialsynthetic construct 105Phe Xaa Xaa Xaa Xaa Gly
Xaa Xaa1 51068PRTartificialsynthetic construct 106Phe Xaa Xaa Xaa
Xaa Gly Xaa Xaa1 51078PRTartificialsynthetic construct 107Phe Xaa
Xaa Xaa Xaa Gly Glu Xaa1 51088PRTartificialsynthetic construct
108Phe Tyr Xaa Arg Xaa Gly Glu Xaa1 5
* * * * *