JP4781621B2 - CXCR4 antagonist and use thereof - Google Patents

Description

  The present invention relates to a compound having a CXCR4 antagonistic action, and a preventive and / or therapeutic agent for cancer and rheumatoid arthritis containing the same.

Many hormones and neurotransmitters regulate biological functions through specific receptors present on cell membranes. Many of these receptors perform intracellular signal transduction through activation of a conjugated guanine nucleotide-binding protein (hereinafter sometimes abbreviated as G protein). In addition, since these receptors have a common structure having seven transmembrane domains, they are collectively referred to as G protein-coupled receptors or seven transmembrane receptors (7TMR).
As one of such G protein-coupled receptor proteins, a human receptor protein [Non-patent Document 1] encoded by the CXCR4 gene is known.
Further, CXCL12 / SDF-1 [Non-patent Document 2] is known as a physiologically active peptide that functions as a ligand for the above-mentioned CXCR4.
Fujii [Patent Document 1] discloses peptidic compounds having an antagonistic action against CXCR4, and describes that these compounds have anti-HIV activity.

  Cancer metastasis is an important factor in determining the life expectancy of a patient. It has been reported that the expression of CXCR4 is increased in breast cancer and the like, and the expression of CXCL12 / SDF-1α, which is its ligand, is further increased in organs (lymph node, lung, liver and bone) of the metastasis destination [ Non-Patent Document 3]. In rheumatoid arthritis, the infiltration of CD4 positive T cells into the joint cavity fluid affects the development of the disease state. It has been reported that CXCR4 gene expression is enhanced in CD4 positive memory T cells in the joint cavity fluid of rheumatoid arthritis patients, and CXCL12 / SDF-1α gene expression is enhanced in joint synovial tissue [Non-Patent Document]. 4].

WO 02/20561 pamphlet (March 14, 2003) Journal of biological chemistry, 273, 4754 (1998) Science, 261, 600-603 (1993) Nature, vol. 410, pp. 50-56 (2001) Journal of Immunology, 165, 6590-98 (2000)

  The object of the present invention is to provide a novel cancer and rheumatoid arthritis prevention and / or treatment means using a compound having a CXCR4 antagonistic action. The present invention also provides novel compounds having preventive and / or therapeutic activities for cancer and rheumatoid arthritis, in particular, various oligopeptides having a common structure.

  As a result of intensive studies to solve the above problems, the present inventors have found that a compound having a CXCR4 antagonistic action, which has been considered effective as a chemotherapeutic agent for AIDS, has been shown to be effective in cancers including metastasis and chronic rheumatoid arthritis. As a result of finding out that it is effective for prevention and / or treatment and conducting further research, the present invention has been completed.

That is, the present invention
(1) The following formula (Ia)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A1-A2-A3-Cys-Tyr-A4-A5-A6-A7-A8-A9-A10-Cys-A11 (Ia)
(Where
A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
A2 represents arginine or glutamic acid residue when A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and A1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
A3 represents an aromatic amino acid residue;
A4, A5 and A9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
A6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
A7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
A8 represents tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
A10 represents citrulline, glutamic acid, arginine or lysine residue;
A11 represents an arginine, glutamic acid, lysine or citrulline residue that may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form A preventive and / or therapeutic agent for cancer or rheumatoid arthritis, comprising the peptide represented by
(2) In the above formula (Ia),
A1 is an arginine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
A2 represents an arginine or glutamic acid residue when A1 is an arginine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, and A1 is N Represents an arginine or glutamic acid residue optionally derivatized at the end;
A4 represents an arginine, citrulline, alanine or glutamic acid residue;
A5 represents an arginine, citrulline, alanine, lysine or glutamic acid residue;
A6 represents a lysine, alanine, citrulline or glutamic acid residue;
A7 represents a proline or alanine residue;
A8 represents a tyrosine, alanine or glutamic acid residue;
A9 represents an arginine, citrulline or glutamic acid residue;
A10 represents citrulline or a glutamic acid residue;
A11 represents an arginine or glutamic acid residue which may be derivatized at the C-terminus, the preventive and / or therapeutic agent according to (1),
(3) The following formula (Ib)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
B1-B2-B3-Cys-Tyr-B4-B5-B6-B7-B8-B9-B10-Cys-B11 (Ib)
(Where
B1 is a glutamic acid residue that may be derivatized at the N-terminus or is deleted;
B2 represents an arginine or glutamic acid residue when B1 is a glutamic acid residue that may be derivatized at the N-terminus, and is derivatized at the N-terminus when B1 is deleted Represents an optional arginine or glutamic acid residue;
B3 represents an aromatic amino acid residue;
B4, B5 and B9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
B6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
B7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
B8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
B10 represents citrulline, glutamic acid, arginine or lysine residue;
B11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(4) B1 is a glutamic acid residue which may be derivatized at the N-terminus, or the peptide or salt thereof according to (3),
(5) The following formula (Ic)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
C1-C2-C3-Cys-Tyr-C4-C5-C6-C7-C8-C9-C10-Cys-C11 (Ic)
(Where
C1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, or is deleted;
C2 represents a glutamic acid residue when C1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue optionally derivatized at the N-terminus, and when C1 is deleted Represents a glutamic acid residue optionally derivatized at the N-terminus;
C3 represents an aromatic amino acid residue;
C4, C5 and C9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
C6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
C7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
C8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
C10 represents citrulline, glutamic acid, arginine or lysine residue;
C11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(6) The following formula (Id)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
D1-D2-D3-Cys-Tyr-D4-D5-D6-D7-D8-D9-D10-Cys-D11 (Id)
(Where
D1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, or is deleted;
D2 represents arginine or glutamic acid residue when D1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and D1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
D3 represents an aromatic amino acid residue;
D4 represents a glutamic acid residue;
D5 and D9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
D6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
D7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
D8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
D10 represents citrulline, glutamic acid, arginine or lysine residue;
D11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(7) The following formula (Ie)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
E1-E2-E3-Cys-Tyr-E4-E5-E6-E7-E8-E9-E10-Cys-E11 (Ie)
(Where
E1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
E2 represents an arginine or glutamic acid residue when E1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, and E1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
E3 represents an aromatic amino acid residue;
E4 and E9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
E5 represents an arginine or glutamic acid residue;
E6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
E7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
E8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
E10 represents citrulline, glutamic acid, arginine or lysine residue;
E11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(8) E5 represents a glutamic acid residue, the peptide according to (7) or a salt thereof,
(9) The following formula (If)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
F1-F2-F3-Cys-Tyr-F4-F5-F6-F7-F8-F9-F10-Cys-F11 (If)
(Where
F1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
F2 represents arginine or glutamic acid residue when F1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and F1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
F3 represents an aromatic amino acid residue;
F4, F5 and F9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
F6 represents a glutamic acid residue;
F7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
F8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
F10 represents citrulline, glutamic acid, arginine or lysine residue;
F11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(10) The following formula (Ig)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
G1-G2-G3-Cys-Tyr-G4-G5-G6-G7-G8-G9-G10-Cys-G11 (Ig)
(Where
G1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
G2 represents arginine or glutamic acid residue when G1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and G1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
G3 represents an aromatic amino acid residue;
G4, G5 and G9 independently represent an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue;
G6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
G7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
G8 represents a glutamic acid residue;
G10 represents citrulline, glutamic acid, arginine or lysine residue;
G11 represents an arginine, glutamic acid, lysine or citrulline residue that may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(11) The following formula (Ih)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
H1-H2-H3-Cys-Tyr-H4-H5-H6-H7-H8-H9-H10-Cys-H11 (Ih)
(Where
H1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
H2 represents arginine or glutamic acid residue when H1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, and H1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
H3 represents an aromatic amino acid residue;
H4 and H5 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
H6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
H7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
H8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
H9 represents a glutamic acid residue;
H10 represents citrulline, glutamic acid, arginine or lysine residue;
H11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(12) The following formula (Ii)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
I1-I2-I3-Cys-Tyr-I4-I5-I6-I7-I8-I9-I10-Cys-I11 (Ii)
(Where
I1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, or is deleted;
I2 represents an arginine or glutamic acid residue when I1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, and I1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
I3 represents an aromatic amino acid residue;
I4, I5 and I9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
I6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
I7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
I8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
I10 represents a glutamic acid, arginine or lysine residue;
I11 represents an arginine, glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(13) The following formula (Ij)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
J1-J2-J3-Cys-Tyr-J4-J5-J6-J7-J8-J9-J10-Cys-J11 (Ij)
(Where
J1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
J2 represents an arginine or glutamic acid residue when J1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, and J1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
J3 represents an aromatic amino acid residue;
J4, J5 and J9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
J6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
J7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
J8 represents tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
J10 represents citrulline, glutamic acid, arginine or lysine residue;
J11 represents a glutamic acid, lysine or citrulline residue which may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
(14) The peptide of any of the following (1) to (58) or a salt thereof:
(1) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(2) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(3) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(4) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH;
(5) Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(6) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(7) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH;
(8) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH;
(9) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(10) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂ ;
(11) Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(12) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(13) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂ ;
(14) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂ ;
(15) H-DGlu-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(16) H-Arg-Glu-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(17) H-Arg-Arg-Nal-Cys-Tyr-Glu-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(18) H-Arg-Arg-Nal-Cys-Tyr-Arg-Glu-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(19) H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(20) H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Glu-Cit-Cys-Arg-OH;
(21) H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Glu-OH;
(22) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(23) H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(24) H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(25) H-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(26) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH ₂ ;
(27) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH ₂ ;
(28) Ac-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(29) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH ₂ ;
(30) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH ₂ ;
(31) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(32) guanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(33) TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(34) TMguanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(35) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(36) 2F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(37) APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(38) desamino-R-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(39) guanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(40) succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(41) glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(42) deaminoTMG-APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(43) nelfinabiryl-succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(44) AZT-glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
_{(45) R-CH 2 -Arg} -Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH 2;
(46) H-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(47) TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(48) ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(49) ACA-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
(50) H-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(51) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(52) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(53) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ ;
(54) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(55) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHMe
(56) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHEt
(57) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHiPr
(58) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-tyramine
(In each sequence, the symbol to the left of the N-terminal amino acid indicates that the amino group is derivatized or not, H indicates that it is not derivatized, Ac indicates an acetyl group, guanyl indicates a guanyl group. Succinyl is a succinyl group, glutaryl is a glutaryl group, TMguanyl is a tetramethylguanyl group, 2F-beozoyl is a 2-fluorobenzoyl group, 4F-benzoyl is a 4-fluorobenzoyl group, and APA is a 5-amino group. Pentanoyl group, ACA is 6-aminohexanoyl group, desamino-R is 2-desamino-arginyl group, deaminoTMG-APA is the following formula (II),

nelfinabiryl-succinyl has the following formula (III):

AZT-glutaryl has the following formula (IV):

R-CH ₂ is represented by the following formula (V)

Arg is an L-arginine residue, Nal is an L-3- (2-naphthyl) alanine residue, Cys is an L-cysteine residue, Tyr is an L-tyrosine residue, and Cit is L -Citrulline residue, Lys is L-lysine residue, DLys is D-lysine residue, Pro is L-proline residue, DCit is D-citrulline residue, DGlu is D-glutamic acid residue. , Glu each represents an L-glutamic acid residue, the two cysteine residues are linked by an intramolecular disulfide bond, and the symbol to the right of the C-terminal amino acid is that the carboxyl group is derivatized or not OH is not derivatized, NH ₂ is an amino group, NMe is a methylamino group, NEt is an ethylamino group, NiPr is an isopropylamino group, and tyramine is a p-hydroxyphenylethylamino group. Amidated at Door are shown, respectively),
(15) A medicament comprising the peptide or salt thereof according to any one of (3) to (14),
(16) The medicament according to (15), which is a CXCR4 antagonist
(17) The medicament according to (15), which is a preventive and / or therapeutic agent for cancer or rheumatoid arthritis,
(18) The medicament according to (17), wherein the cancer type is breast cancer or pancreatic cancer,
(19) A method for preventing / treating cancer or rheumatoid arthritis, comprising administering an effective amount of the peptide or salt thereof according to any of (3) to (14) to a mammal,
(20) Use of the peptide or salt thereof according to any one of (3) to (14) for producing a preventive and / or therapeutic agent for cancer or rheumatoid arthritis,
(21) The following formula (Ia) for mammals
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A1-A2-A3-Cys-Tyr-A4-A5-A6-A7-A8-A9-A10-Cys-A11 (Ia)
(Where
A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
A2 represents arginine or glutamic acid residue when A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and A1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
A3 represents an aromatic amino acid residue;
A4, A5 and A9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
A6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
A7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
A8 represents tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
A10 represents citrulline, glutamic acid, arginine or lysine residue;
A11 represents an arginine, glutamic acid, lysine or citrulline residue that may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form And / or a method for preventing or treating cancer or rheumatoid arthritis, and / or (22) prevention of cancer or rheumatoid arthritis, and / or The following formula (Ia) for producing a therapeutic agent
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A1-A2-A3-Cys-Tyr-A4-A5-A6-A7-A8-A9-A10-Cys-A11 (Ia)
(Where
A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
A2 represents arginine or glutamic acid residue when A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and A1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
A3 represents an aromatic amino acid residue;
A4, A5 and A9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
A6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
A7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
A8 represents tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
A10 represents citrulline, glutamic acid, arginine or lysine residue;
A11 represents an arginine, glutamic acid, lysine or citrulline residue that may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form Or a salt thereof, which may be
Etc.

  The peptidic compound of the present invention has a strong CXCR4 antagonism and inhibits the binding between CXCR4 and CXCL12 / SDF-1, thereby showing a therapeutic effect on cancer and rheumatoid arthritis.

In the peptides described herein, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide designation.
In the present specification and drawings, amino acids and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commision on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when an optical isomer is present with respect to an amino acid, L form is shown unless otherwise specified.
Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamic acid Asp or D: Aspartic acid Lys or K: Lysine Arg or R: Arginine His or H: Histidine Phe or F: Phenylalanine Tyr or Y: Tyrosine Trp or W: Tryptophan Pro or P: Proline Asn or N: Asparagine Gln or Q: Glutamine pGlu: pyroglutamic acid Nal: 3- (2-naphthyl) alanine Cit: citrulline DLys: D-lysine DCit: D-si Toluline DGlu: D-glutamic acid Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
BHA: benzhydrylamine pMBHA: p-methylbenzhydrylamine Tos: p-toluenesulfonyl CHO: formyl HONB: N-hydroxy-5-norbornene-2,3-dicarboximide OcHex: cyclohexyl ester Bzl: benzyl Cl ₂ -Bzl: Dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butyloxycarbonyl DCM: dichloromethane HOBt: 1-hydroxybenztriazole DCC: N, N'-dicyclohexylcarbodiimide TFA: Trifluoroacetic acid DIEA: diisopropylethylamine Fmoc: N-9-fluorenylmethoxycarbonyl DNP: dinitrophenyl Bum: tertiary Butoxymethyl Trt: Trityl Ac: Acetyl guanyl: Guanyl succinyl: Succinyl glutaryl: Glutaryl TMguanyl: Tetramethylguanyl 2F-beozoyl: 2-fluorobenzoyl 4F-benzoyl: 4-fluorobenzoyl APA: 6-aminopentanoyl ACA: 6 Aminohexanoyl desamino-R: 2-desamino-arginyl deamino TMG-APA: the following formula (II)

nerfinabilyl-succinyl: the following formula (III)

AZT-glutaryl: the following formula (IV)

R—CH ₂ : the following formula (V)

  In the N-terminal amino acid of the peptide, “H—” means that the terminal amino group is not derivatized, and in the C-terminal amino acid “—OH” means that the terminal carboxyl group is not derivatized.

The present invention provides a preventive and / or therapeutic agent for cancer and rheumatoid arthritis containing a compound having a CXCR4 antagonistic action. The “compound having CXCR4 antagonistic action” is an anticancer action (for example, migration inhibitory action, invasion inhibitory action and antimetastatic action) by antagonistically inhibiting the binding of CXCR4 and its physiological ligand CXCL12 / SDF-1α. Etc.) or anti-chronic rheumatoid arthritis action (for example, migration inhibitory action), more specifically, the following formula (Ia)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A1-A2-A3-Cys-Tyr-A4-A5-A6-A7-A8-A9-A10-Cys-A11 (Ia)
(Where
A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue that may be derivatized at the N-terminus, or is deleted;
A2 represents arginine or glutamic acid residue when A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue which may be derivatized at the N-terminus, and A1 is deleted Represents an arginine or glutamic acid residue optionally derivatized at the N-terminus;
A3 represents an aromatic amino acid residue;
A4, A5 and A9 independently represent arginine, lysine, ornithine, citrulline, alanine or glutamic acid residues;
A6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
A7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
A8 represents tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
A10 represents citrulline, glutamic acid, arginine or lysine residue;
A11 represents an arginine, glutamic acid, lysine or citrulline residue that may be derivatized at the C-terminus;
In the above formula, Cys represents a cysteine residue, Tyr represents a tyrosine residue, the cysteine residues at positions 4 and 13 may be linked by a disulfide bond, and the amino acid may be L-form or D-form May be)
, Its amide, its ester or its salt (hereinafter may be collectively referred to as “the peptide of the present invention”).

A1 represented by the above formula (Ia) represents an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue (which may be L-form or D-form) which may be derivatized at the N-terminus. Represents or is deleted and preferably represents or is missing an arginine, citrulline, alanine or D-glutamate residue which may be derivatized at the N-terminus. Examples of “derivatized at the N-terminal” include, for example, formyl group; acyl group, for example, C _2-6 alkanoyl group such as acetyl group, propionyl group, butyryl group, pentanoyl group, hexanoyl group, benzoyl group, substituted Benzoyl group (eg 2-fluorobenzoyl, 3-fluorobenzoyl group, 4-fluorobenzoyl group, 2-bromobenzoyl group, 3-bromobenzoyl group, 4-bromobenzoyl group, 2-nitrobenzoyl group, 3-nitrobenzoyl group Arylcarbonyl group such as 4-nitrobenzoyl group), succinyl group, glutaryl group; nicotinyl group; isonicotinyl group; alkylsulfonyl group (eg, methanesulfonyl group, ethanesulfonyl group, propanesulfonyl group, camphorsulfonyl group); aryl A sulfonyl group (eg, p-toluenesulfonyl group, 4 -Fluorobenzene sulfonyl group, mesitylene sulfonyl group, 4-aminobenzene sulfonyl group, dansyl group, 4-bromobenzene sulfonyl group) and the like are exemplified, but not limited thereto. Alternatively, the N-terminal amino group may be deleted.

  A2 represented by the above formula (Ia) may be an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue (L-form or D-form) in which A1 may be derivatized at the N-terminus. ) Represents an arginine or glutamic acid residue (which may be L-form or D-form), and when A1 is deleted, it may be derivatized at the N-terminus. Represents a good arginine or glutamic acid residue (which may be L or D), preferably A1 is an arginine, citrulline, alanine or glutamic acid residue optionally derivatized at the N-terminus In some cases, it represents an arginine or glutamic acid residue, and when A1 is deleted, it represents an arginine or glutamic acid residue that may be derivatized at the N-terminus.Examples of “derivatized at the N-terminus” include, but are not limited to, those similar to A1 above.

  A3 represented by the above formula (Ia) is an aromatic amino acid residue (for example, phenylalanine, tryptophan, 3- (2-naphthyl) alanine, tyrosine, 4-fluorophenylalanine, 3- (1-naphthyl) alanine) (L Or D-form), preferably phenylalanine, tryptophan, 3- (2-naphthyl) alanine.

  A4 represented by the above formula (Ia) represents arginine, lysine, ornithine, citrulline, alanine or a glutamic acid residue (may be L-form or D-form), preferably arginine, citrulline, alanine Or represents an L- or D-glutamic acid residue.

  A5 represented by the above formula (Ia) represents an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue, preferably an arginine, citrulline, alanine, lysine or glutamic acid residue.

  A6 represented by the above formula (Ia) represents proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue (may be L-form or D-form), preferably D -Represents a lysine, D-alanine, D-citrulline or D-glutamic acid residue.

  A7 represented by the above formula (Ia) represents a proline or alanine residue (which may be L-form or D-form), and preferably represents a proline or alanine residue.

  A8 represented by the above formula (Ia) represents tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or a glutamic acid residue (which may be L-form or D-form), preferably tyrosine, alanine or Represents a D-glutamic acid residue.

  A9 represented by the above formula (Ia) represents arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue (may be L-form or D-form), preferably arginine, citrulline or glutamic acid Represents a residue.

  A10 represented by the above formula (Ia) represents citrulline, glutamic acid, arginine or lysine residue (which may be L-form or D-form), preferably citrulline or D-glutamic acid residue. .

A11 represented by the above formula (Ia) represents an arginine, glutamic acid, lysine or citrulline residue (which may be L-form or D-form) which may be derivatized at the C-terminus, preferably Represents an arginine or glutamic acid residue which may be derivatized at the C-terminus. Examples of “C-terminal derivatization” include amidation (—NH ₂ , —NHR, —NRR ′), esterification (—COOR) and the like. Here, R and R ′ in the amide or ester are, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl such as cyclopentyl, cyclohexyl and the like. Groups, for example, C _6-12 aryl groups such as phenyl, α-naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl, phenethyl or α-naphthyl-C _1-2 alkyl groups, such as α-naphthylmethyl In addition to C _7-14 aralkyl groups such as Pivaloyloxymethyl group, which is widely used as an oral ester, is used.

When the peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the peptide of the present invention includes those in which the carboxyl group is amidated or esterified. As the amide and ester in this case, for example, the C-terminal amide and ester exemplified for A11 above are used in the same manner. Furthermore, in the peptide of the present invention, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) is appropriate for the peptide described above. A compound protected by a protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as a formyl group or an acetyl group) or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound included.

  Examples of the salt of the peptide of the present invention include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

The peptide of the present invention is a novel peptide when any one of A1 to A11 represented by the above formula (Ia) is as follows:
(I) when A1 is glutamic acid residue or deleted (ie, in the case of the above formula (Ib));
(Ii) When any one of A2, A4, A6, A8 and A9 is a glutamic acid residue (that is, any one of the above formulas (Ic) to (Ig));
(Iii) A5 is an arginine or glutamic acid residue (that is, in the case of the above formula (Ih));
(Iv) When A10 is a glutamic acid, arginine or lysine residue (ie, in the case of the above formula (Ii));
(V) A11 is a glutamic acid, lysine or citrulline residue (that is, in the case of the above formula (Ij)).
The amino acid residue may be L-form or D-form.

Preferred examples of the novel peptide of the present invention include peptides having the following amino acid sequences (1) to (58) (in each sequence, two cysteine residues are linked by a disulfide bond).
(1) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14003)
(2) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14005)
(3) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14011)
(4) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(AcTC14013)
(5) Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14015)
(6) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14017)
(7) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(AcTC14019)
(8) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(AcTC14021)
(9) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTC14012)
(10) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(AcTC14014)
(11) Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTC14016)
(12) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTC14018)
(13) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(AcTC14020)
(14) Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(AcTC14022)
(15) H-DGlu-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14001)
(16) H-Arg-Glu-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14002)
(17) H-Arg-Arg-Nal-Cys-Tyr-Glu-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14003)
(18) H-Arg-Arg-Nal-Cys-Tyr-Arg-Glu-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14004)
(19) H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14005)
(20) H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Glu-Cit-Cys-Arg-OH
(TE14006)
(21) H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Glu-OH
(TE14007)
(22) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14011)
(23) H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14012)
(24) H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14013)
(25) H-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14014)
(26) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH ₂
(TE14015)
(27) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH ₂
(TE14016)
(28) Ac-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTE14014)
(29) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH ₂
(AcTE14015)
(30) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH ₂
(AcTE14016)
(31) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF1: AcTE14011)
(32) guanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF2: guanyl-TE14011)
(33) TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF3: TMguanyl-TE14011)
(34) TMguanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF4: TMguanyl-TE14011 (2-14))
(35) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF5: 4F-benzoyl-TE14011)
(36) 2F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF6: 2F-benzoyl-TE14011)
(37) APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF7: APA-TE14011 (2-14))
(38) desamino-R-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF8: desamino-R-TE14011 (2-14))
(39) guanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF9: guanyl-TE14011 (2-14))
(40) succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF10: succinyl-TE14011 (2-14))
(41) glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF11: glutaryl-TE14011 (2-14))
(42) deaminoTMG-APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF12: deaminoTMG-APA-TE14011 (2-14))
(43) nelfinabiryl-succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF13: nelfinabiryl-succinyl-TE14011 (2-14))
(44) AZT-glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF14: AZT-glutaryl-TE14011 (2-14))
(45) R-CH ₂ -Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF15: R-CH ₂ NH-RTE14011 (2-14))
(46) H-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF17: TE14011 (2-14))
(47) TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF18: TMguanyl-TC14012)
(48) ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF19: ACA-TC14012)
(49) ACA-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TF20: ACA-T140)
(50) H-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TZ14011)
(51) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTZ14011)
(52) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTN14003)
(53) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTN14005)
(54) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(4F-benzoyl-TN14003)
(55) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHMe
(4F-benzoyl-TN14011-Me)
(56) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHEt
(4F-benzoyl-TN14011-Et)
(57) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHiPr
(4F-benzoyl-TN14011-iPr)
(58) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-tyramine
(4F-benzoyl-TN14011-tyramine)

The peptide of the present invention also includes a peptide containing an amino acid sequence substantially identical to the amino acid sequence of any of the peptides described above, or an amide, ester or salt thereof. As used herein, “substantially identical amino acid sequence” refers to peptide activity (eg, ligand-receptor binding inhibitory activity) or peptide anti-cancer activity (eg, migration inhibitory activity, invasion inhibitory activity, and anti-metastatic activity). Etc.) or anti-rheumatic action (eg, migration inhibitory action) and the like are qualitatively homogeneous [therefore, to some extent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, More preferably, there may be a difference of about 0.5 to 2 times)]. Therefore, as long as the above characteristics are maintained, one or more amino acids may have a mutation in the amino acid sequence represented by any of the above formulas (Ia) to (Ij) and (1) to (58). .
That is, in the present invention, a significant (significant) change in the physiological and chemical characteristics of the original (unmutated) peptide (= characteristically different, or even if it is homogeneous, the quantity is significantly different. Peptides (mutant peptides) obtained as a result of mutations in amino acid sequences such as substitutions, deletions or insertions (additions) that do not cause such changes) The amino acid sequence of the mutant peptide is considered substantially identical to the amino acid sequence of the original (unmutated) peptide.
In general, mutations such as amino acid substitutions, deletions or insertions (additions) in a peptide sequence often do not result in large (significant) changes in the physiological and chemical properties of the peptide, This is a well-known fact. Examples of such substitution include those in which a certain amino acid is substituted with another amino acid having similar properties (characteristics), and in general, when substitution is performed between amino acids having strong similarity in characteristics It is considered that the change in the properties of the substitution on the original peptide before substitution is small. Amino acids are based on the similarity of their characteristics as one standard, for example: (i) Nonpolar (hydrophobic) amino acids (eg alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, etc.) ); (Ii) polar (neutral) amino acids (eg glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc.); (iii) positively charged (basic) amino acids (eg arginine, lysine, histidine) (Iv) are classified into negatively charged (acidic) amino acids (eg, aspartic acid, glutamic acid, etc.), so that amino acid substitutions within each class are conservative with respect to peptide properties (ie, “ Substitutions that result in “substantially identical” amino acid sequences.

In other words, “substantially identical amino acid sequences”
(I) One or more, preferably 1 to 10 and more preferably 1 to 5 in the amino acid sequence represented by any one of the above formulas (Ia) to (Ij) and (1) to (58) An amino acid sequence in which not more than one amino acid is substituted with another amino acid,
(Ii) 1 or more and 7 or less, preferably 1 or more and 5 or less, more preferably 1 in the amino acid sequence represented by any of the above formulas (Ia) to (Ij) and (1) to (58) An amino acid sequence in which not less than 3 amino acids are deleted,
(Iii) 1 or more and 15 or less, preferably 1 or more and 10 or less, more preferably 1 in the amino acid sequence represented by any of the above formulas (Ia) to (Ij) and (1) to (58) Or (iv) a constituent amino acid in a peptide having the amino acid sequence of (i), (ii) or (iii) above (in particular its side chain) Peptides containing modifications, or amides, esters or salts thereof may be included.
The peptide of the present invention is a stable peptide against heat or protease by intentionally or accidentally performing substitution, deletion, insertion (addition), modification, etc. of (i) to (iv) above in its amino acid sequence. Alternatively, the peptide can be converted into a highly active peptide having an increased inhibitory activity. The peptides of the present invention also include these mutant peptides or amides, esters or salts thereof.

  Furthermore, as the peptide of the present invention, in addition to the peptide consisting of the amino acid sequence represented by any of the above formulas (Ia) to (Ij) and (1) to (58), the amino acid sequence and about 50 to 99. Containing an amino acid sequence having a homology of 9% (preferably 70 to 99.9%, more preferably 80 to 99.9%, still more preferably 90 to 99.9%), and the above formulas (Ia) to ( Ij) and a peptide having substantially the same quality of activity as the peptide consisting of the amino acid sequence shown in any one of (1) to (58), its amide, its ester or its salt. Examples of the activity include inhibitory activities possessed by the peptide, such as a ligand-receptor binding inhibitory activity and a signal transduction inhibitory activity. The “substantially the same” inhibitory activity indicates that the properties such as the ligand binding inhibitory activity for the receptor are homogeneous. Accordingly, a strength that is not remarkably strong may be observed in the ligand binding inhibitory activity against the receptor, and a difference in molecular weight is not a problem.

  Examples of the amide, ester or salt of the peptide having the amino acid sequence represented by any of the above formulas (1) to (58) are the same as those exemplified for the peptide represented by the above general formula (Ia). . Preferably, it is desirable that the peptide having the amino acid sequence represented by any one of the above formulas (1) to (58) has an amidated carboxyl group at the C-terminal amino acid residue.

The peptides of the present invention including the peptides containing the amino acid sequences represented by the above formulas (1) to (58) can be produced according to peptide synthesis methods known per se. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the peptide of interest can be produced by condensing a partial peptide or amino acid that can constitute a peptide and the remaining portion, and removing the protective group when the product has a protective group. Examples of known condensation methods and protecting group removal include the methods described in the following 1) to 5).
1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
5) Supervised by Haruaki Yajima, Development of follow-up medicines Vol.14 Peptide synthesis

Specific peptide synthesis methods include, for example, the following methods.
Usually, commercially available resins for polypeptide synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid in which the α-amino group and the side chain functional group are appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target polypeptide. At the end of the reaction, the polypeptide is cut out from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the desired polypeptide or amide form thereof. For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for polypeptide synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the polypeptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for polypeptide bond formation reaction, and is usually selected appropriately from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like. The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like. The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like. As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

  Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol or 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine was removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan was the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means. As another method for obtaining an amide form of a polypeptide, for example, first, the α-carboxyl group of the carboxy-terminal amino acid is protected by amidation, and then the peptide chain is extended to the desired chain length on the amino group side. A polypeptide in which only the protecting group of the α-amino group at the N-terminus of the peptide chain is removed and a polypeptide from which only the protecting group of the carboxyl group at the C-terminus is removed are produced, and both the polypeptides are mixed as described above. Condense in. The details of the condensation reaction are the same as described above. After purifying the protected polypeptide obtained by condensation, all the protecting groups are removed by the above-described method to obtain the desired crude polypeptide. This crude polypeptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide of the desired polypeptide. In order to obtain a polypeptide ester, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired polypeptide ester is obtained in the same manner as the amide of the polypeptide. You can get a body.

  After the reaction, the peptide of the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. it can.

  Preferably, the novel peptide of the present invention having the amino acid sequence represented by the above formulas (1) to (58) can be produced by the method described in Examples below or a method analogous thereto. Moreover, the peptide of this invention can be manufactured by the method as described in the international publication 02/20561 pamphlet, or the method according to these.

  What is necessary is just to implement according to a conventional means, when using the peptide of this invention as a pharmaceutical or animal medicine. For example, tablets, capsules, elixirs, microcapsules and the like, as needed, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, mixing the peptides of the present invention with physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in unit dosage forms generally required for accepted pharmaceutical practice. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. .
Examples of aqueous solutions for injection include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, and suitable solubilizing agents. For example, you may use together with alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethyleneglycol), a nonionic surfactant (for example, polysorbate ^80TM , HCO-50), etc. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. The adjusted injection solution is usually aseptically filled into a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it is suitable for humans and mammals (eg, mouse, rat, guinea pig, rabbit, sheep, pig, cow, cat, dog, monkey, baboon, chimpanzee, etc.). Can be administered.
The dose of the peptide of the present invention varies depending on symptoms, but when administered orally, it is usually about 0.1 to 1000 mg, preferably about 1.0 to 500 mg, more preferably about once per 60 kg body weight. About 1.0 to 200 mg. When administered parenterally, the dose per 60 kg body weight varies depending on the administration subject, symptom, administration method, etc. For example, in the form of an injection, it is usually about 0.01 to 300 mg per dose. About 0.1 to 200 mg, more preferably about 0.1 to 100 mg may be administered by intravenous injection. In the case of other animals, an amount converted per 60 kg body weight can be administered.

The peptide of the present invention has an anticancer effect, that is, a cancer cell movement inhibitory effect and a cancer metastasis inhibitory effect. That is, since the peptide of the present invention has a cancer metastasis inhibitory effect, as will be apparent from Examples described later, it can be used as a preventive and therapeutic drug related to cancer, particularly cancer metastasis. Therefore, the peptide in the present invention is an oral cancer, pharyngeal cancer, lip cancer, tongue cancer, gingival cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer including colon cancer, liver cancer, Gallbladder cancer, pancreatic cancer, nasal cavity cancer, lung cancer, osteosarcoma, soft tissue cancer, skin cancer, melanoma, breast cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, penile cancer, bladder cancer, kidney cancer, brain tumor, thyroid gland It is useful as an agent for improving, preventing and treating cancer, lymphoma, leukemia and the like. Further, the peptide of the present invention has an anti-chronic rheumatoid effect, that is, a T cell movement inhibitory effect. That is, since the peptide of the present invention has a T cell movement inhibitory action, as will be apparent from the examples described later, it can be used as a prophylactic and therapeutic drug for rheumatoid arthritis. Therefore, the peptide in the present invention is useful as an agent for improving, preventing and treating chronic rheumatoid arthritis.
It is known that compounds having CXCR4 antagonistic activity have antiviral activity. Therefore, it will be apparent to those skilled in the art that the peptides of the present invention can also be used as preventive and therapeutic agents for viral infections (eg, AIDS, SARS, etc.).

For example, when the peptide of the present invention is used as an anticancer agent, other anticancer agents (for example, chemotherapeutic agents, immunotherapeutic agents, or agents that inhibit the action of cell growth factors and their receptors) (hereinafter, combined use) (Abbreviated as “drug”).
The peptide of the present invention exhibits an excellent anticancer effect even when used as a single agent, but further enhances its effect by using in combination with one or several of the above concomitant drugs (multi-drug combination). Can do.

Examples of the “chemotherapeutic agent” include alkylating agents, antimetabolites, anticancer antibiotics, plant-derived anticancer agents and the like.
Examples of the “alkylating agent” include nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambutyl, cyclophosphamide, ifosfamide, thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitoblonitol, Faran, dacarbazine, ranimustine, sodium estramustine phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, piprobroman, etoglucid, altretamine, ambmustine, dibrospdium hydrochloride, fotemustine, predonimustine, pumitepa, ribomustine, temosofredomestre , Trophosphamide, Dinostatin stymalamar, Carbocon, Adzelesin, Systemustin, Vizeresi , Platinum complexes (carboplatin, cisplatin, miboplatin, nedaplatin, such as oxaliplatin), and the like.

  Examples of the “antimetabolite” include, for example, mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, enocitabine, cytarabine, cytarabine okphosphat, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, Doxyfluridine, carmofur, garocitabine, emiteful, etc.), aminopterin, leucovorin calcium, tabloid, butosine, folinate calcium, levofolinate calcium, cladribine, emiteful, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, pyritroxime, idoxyuridine, mitoxifridin , Ambamustine, gemicitabine and the like.

  Examples of the “anticancer antibiotic” include anthracycline anticancer drugs (doxorubicin hydrochloride, daunorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, etc.), actinomycin D, actinomycin C, mitomycin C, chromomycin A3 Bleomycin hydrochloride, bleomycin sulfate, pepromycin sulfate, neocalcinostatin, misramycin, sarcomomycin, carcinophylline, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride, and the like.

Examples of the “plant-derived anticancer agent” include vinca alkaloid anticancer drugs (vinblastine sulfate, vincristine sulfate, vindesine sulfate, vinorelbine, etc.), taxane anticancer drugs (such as paclitaxel, docetaxel), etoposide, etoposide phosphate, teniposide, Examples include vinorelbine.
The “cell growth factor” in the “drug that inhibits the action of cell growth factor and its receptor” may be any substance that promotes cell growth, and usually has a molecular weight of 20,000. Examples of the following peptides include factors that exert an action at a low concentration by binding to a receptor. Specifically, (1) EGF (epidermal growth factor) or a substance having substantially the same activity as that [ (Eg, EGF, haregulin (HER2 ligand), etc.), (2) insulin or a substance having substantially the same activity (eg, insulin, IGF (insulin-like growth factor) -1, IGF-2, etc.), ( 3) FGF (fibroblast growth factor) or a substance having substantially the same activity (eg, acidic FGF, basic FGF, KGF (keratinocyte growth factor), FGF-10, etc.) (4) Other cell growth factors [eg, CSF (colony stimulating factor), EPO (erythropoietin), IL-2 (interleukin-2), NGF (nerve growth factor), PDGF (platelet-derived growth factor), TGFβ ( transforming growth factor β), HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor) and the like.

The “cell growth factor receptor” may be any receptor capable of binding to the above-mentioned cell growth factor, and specifically includes an EGF receptor, a haregulin receptor (HER2). Insulin receptor, IGF receptor, FGF receptor-1 or FGF receptor-2, HGF receptor (c-met), VEGF receptor, SCF receptor (c-kit) and the like.
Examples of the “drug that inhibits the action of cell growth factor” include Herceptin (HER2 antibody), GLEEVEC (c-met, c-kit, abl inhibitor), Iressa (EGF receptor inhibitor) and the like.

  In addition to the above drugs, topoisomerase I inhibitors (eg, irinotecan, topotecan etc.), topoisomerase II inhibitors (eg, sobuzoxane etc.), angiogenesis inhibitors and the like can also be used.

In addition, when the peptide of the present invention is used as a prophylactic and / or therapeutic agent for rheumatoid arthritis, it can be used in combination with a prophylactic and / or therapeutic agent for other joint diseases. Examples of the drugs used in combination include anti-inflammatory steroids (eg, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, etc.), nonsteroidal anti-inflammatory analgesics (eg, indomethacin, diclofenac, loxoprofen, ibuprofen, aspirin, piroxicam , Sulindac, etc.) or hyaluronic acid preparations (eg, sodium hyaluronate), COX-II inhibitors and the like.
The peptide of the present invention exhibits an excellent anti-chronic rheumatoid arthritis effect even when used as a single agent, but further enhances its effect when used in combination with one or several of the above concomitant drugs (multi-drug combination). Can be made.

  When the peptide of the present invention is used in combination with the concomitant drug, the administration timing of the peptide of the present invention and the concomitant drug is not limited, and the peptide of the present invention and the concomitant drug may be administered simultaneously to the administration subject. Alternatively, administration may be performed with a time difference. The dose of the concomitant drug may be determined according to the dose used clinically, and can be appropriately selected depending on the administration subject, administration route, disease, combination and the like.

  The administration mode of the peptide of the present invention and the concomitant drug is not particularly limited, as long as the polypeptide of the present invention or a salt thereof and the concomitant drug are combined at the time of administration. Examples of such administration forms include (1) administration of a single preparation obtained by simultaneously formulating the peptide of the present invention and a concomitant drug, and (2) separate preparation of the peptide of the present invention and the concomitant drug. Simultaneous administration of the two preparations obtained by the same administration by the same administration route, (3) with a time difference in the same administration route of the two preparations obtained by separately formulating the peptide of the present invention and the concomitant drug. (4) Simultaneous administration of two kinds of preparations obtained by separately formulating the peptide of the present invention and a concomitant drug by different administration routes, (5) Preparation of the peptide of the present invention and the concomitant drug separately Administration of the two types of preparations obtained by combining the two different administration routes at different time intervals (for example, administration in the order of the peptide of the present invention → concomitant drug, or administration in the reverse order). Hereinafter, these administration forms are collectively abbreviated as the combination agent of the present invention.

  The concomitant drug of the present invention has low toxicity. For example, the peptide of the present invention and / or the above concomitant drug is mixed with a pharmacologically acceptable carrier according to a method known per se, for example, a pharmaceutical composition such as a tablet (sugar-coated tablet). , Including film-coated tablets), powders, granules, capsules (including soft capsules), liquids, injections, suppositories, sustained-release agents, etc., orally or parenterally (eg, topical, rectal, intravenous) Administration, etc.). Injections can be administered intravenously, intramuscularly, subcutaneously, into organs, intranasally, intradermally, instilled, intracerebral, rectal, intravaginally and intraperitoneally, inside tumors, proximal to tumors, etc. Can be administered.

As the pharmacologically acceptable carrier that may be used in the production of the concomitant drug of the present invention, the same carriers as those used in the pharmaceutical composition of the present invention described above can be used.
The compounding ratio of the peptide of the present invention and the concomitant drug in the concomitant drug of the present invention can be appropriately selected depending on the administration subject, administration route, disease and the like.
The content of the concomitant drug in the concomitant drug of the present invention varies depending on the form of the preparation, but is usually about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight, more preferably based on the whole preparation. About 0.5 to 20% by weight.
The content of additives such as carriers in the combination agent of the present invention varies depending on the form of the preparation, but is usually about 1 to 99.99% by weight, preferably about 10 to 90% by weight, based on the whole preparation. .

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but these do not limit the scope of the present invention.

<Production Example 1: Production of polypeptide TC14003>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TC14003)
1. Synthesis of TC14003 protected polypeptide resin Fmoc-Cys (Trt) corresponding to position 13 after removing Fmoc group with 20% piperidine / DMF from Fcoc-Arg (Pbf) -Alko resin, the first arcoin introduced with 14th position arginine -OH (2.5 eq) was added and a condensation reaction was carried out in DMF by the DIPCDI-HOBt method. The degree of progress of the condensation reaction was examined by the ninhydrin test of Kaiser, E. et al. (Anal. Biochem., 34: 595 (1970)).

2. Introduction of amino acids at positions 12 to 1 In the same manner, Cit, Arg (Pbf), Tyr (t-Bu), Pro, DLys (Boc), Lys (Boc), Cit, Tyr (t-Bu) ), Cys (Trt), Nal, Arg (Pbf) and Arg (Pbf) residues were introduced into the resin to obtain a protecting group-protected polypeptide resin.

3. Deprotection group, separation and purification of polypeptide from resin Protected group-protected polypeptide resin removed Fmoc group by treatment with 20% piperidine / DMF and then 1M TMSBr-thioanisole / TFA (trifluoro (Acetic acid) system (m-cresol (100 eq), ethanedithiol (300 eq) in the presence) was reacted at 25 ° C. for 2 hours. The resin was separated from the reaction mixture by filtration, washed twice with TFA, the filtrate and washings combined, concentrated under reduced pressure, water-cooled dry ether was added to the residue, and the resulting precipitate was supernatant by centrifugation and decantation. Separated from. The obtained residue was washed with cold ether, dissolved in 1N acetic acid, and diluted with distilled water.

4). Cyclization by air oxidation The diluted aqueous solution of the above-mentioned polypeptide was adjusted to pH 7.5 with concentrated ammonia water, and oxidization was performed by air oxidation by aeration. This aqueous solution was purified by large-scale preparative HPLC (Cosmozil 5C18 AR-II column: acetonitrile-water) and gel chromatography (Sephadex G-15, eluent: 0.1N AcOH) to obtain a single peak polypeptide. Lyophilized. Purity was confirmed by HPLC.

<Production Example 2: Production of TC14005>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TC14005)
TC14005 was produced in the same manner as in Production Example 1. However, DCit was used in place of DLys (Boc) at position 8 and Arg (Pbf) was used instead of Cit at position 6 at the introduction of the 12th to 1st amino acids.

<Production Example 3: Production of TC14011>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TC14011)
TC14011 was produced in the same manner as in Production Example 1. However, DCit was used in place of DLys (Boc) at position 8 at the introduction of amino acids at positions 12 to 1.

<Production Example 4: Production of TC14013>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(TC14013)
TC14013 was produced in the same manner as in Production Example 1. However, Cit was used in place of Arg (Pbf) at position 11 at the introduction of the 12th to 1st amino acids.

<Production Example 5: Production of TC14015>
H-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TC14015)
TC14015 was produced in the same manner as in Production Example 1. However, Cit was used in place of Arg (Pbf) at position 1 when the amino acids at positions 12 to 1 were introduced.

<Production Example 6: Production of TC14017>
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TC14017)
TC14017 was produced in the same manner as in Production Example 1. However, when introducing amino acids 12 to 1, DCit is used instead of DLys (Boc) at position 8, Arg (Pbf) is used instead of Cit at position 6, and Cit is used instead of Arg (Pbf) at position 1. It was.

<Production Example 7: Production of TC14019>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(TC14019)
TC14019 was produced in the same manner as in Production Example 1. However, when introducing amino acids 12 to 1, Cit is used instead of Arg (Pbf) at position 11, DCit is used instead of DLys (Boc) at position 8, Arg (Pbf) is used instead of Cit at position 6. It was.

<Production Example 8: Production of TC14021>
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(TC14021)
TC14021 was produced in the same manner as in Production Example 1. However, when introducing amino acids 12 to 1, Cit is used instead of Arg (Pbf) at position 11, Arg (Pbf) instead of Cit at position 6, and Cit is used instead of Arg (Pbf) at position 1. It was.

<Production Example 9: Production of TC14012>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TC14012)
1. Synthesis of TC14012 protected polypeptide resin
After removing the Fmoc group from Fmoc-Rink amide resin with 20% piperidine / DMF, Fmoc-Arg (Pbf) -OH (2.5 eq) corresponding to position 14 was added, and a condensation reaction was carried out in DMF by the DIPCDI-HOBt method. . The degree of progress of the condensation reaction was examined by the ninhydrin test of Kaiser, E. et al. (Anal. Biochem., 34: 595 (1970)).

2. Introduction of amino acids at positions 12 to 1 In the same manner, Cys (Trt), Cit, Arg (Pbf), Tyr (t-Bu), Pro, DCit, Lys (Boc), Cit, Tyr (t -Bu), Cys (Trt), Nal, Arg (Pbf), Arg (Pbf) residues were introduced into Rink amide resin to obtain a protecting resin with a protecting group protected.

3. Deprotection group, separation and purification of polypeptide from resin Protected group-protected polypeptide resin removed Fmoc group by treatment with 20% piperidine / DMF and then 1M TMSBr-thioanisole / TFA (trifluoro (Acetic acid) system (m-cresol (100 eq), ethanedithiol (300 eq) in the presence) was reacted at 25 ° C. for 3 hours. The resin was separated from the reaction mixture by filtration, washed twice with TFA, the filtrate and washings combined, concentrated under reduced pressure, water-cooled dry ether was added to the residue, and the resulting precipitate was supernatant by centrifugation and decantation. Separated from. The obtained residue was washed with cold ether, dissolved in 1N acetic acid, and diluted with distilled water.

4). Cyclization by air oxidation The diluted aqueous solution of the above-mentioned polypeptide was adjusted to pH 7.5 with concentrated ammonia water, and oxidization was performed by air oxidation by aeration. This aqueous solution was purified by large-scale preparative HPLC (Cosmozil 5C18 AR-II column: acetonitrile-water) and gel chromatography (Sephadex G-15, eluent: 0.1N AcOH) to obtain a single peak polypeptide. Lyophilized. Purity was confirmed by HPLC.

<Production Example 10: Production of TC14014>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(TC14014)
TC14014 was produced in the same manner as in Production Example 9. However, Cit was used in place of Arg (Pbf) at position 11 and DLys (Boc) was used in place of DCit at position 8 when the amino acids at positions 12 to 1 were introduced.

<Production Example 11: Production of TC14016>
H-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TC14016)
TC14016 was produced in the same manner as in Production Example 9. However, DLys (Boc) was used in place of DCit at position 8 and Cit was used in place of Arg (Pbf) at position 1 in the introduction of the 12th to 1st amino acids.

<Production Example 12: Production of TC14018>
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TC14018)
TC14018 was produced in the same manner as in Production Example 9. However, Arg (Pbf) was used in place of Cit at position 6 and Cit was used in place of Arg (Pbf) at position 1 at the introduction of the 12th to 1st amino acids.

<Production Example 13: Production of TC14020>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(TC14020)
AcTC14020 was produced in the same manner as in Production Example 9. However, Cit was used instead of Arg (Pbf) at position 11 and Arg (Pbf) was used instead of Cit at position 6 when the amino acids at positions 12 to 1 were introduced.

<Production Example 14: Production of TC14022>
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(TC14022)
TC14022 was produced in the same manner as in Production Example 9. However, at the introduction of amino acids at positions 12 to 1, Cit instead of Arg (Pbf) at position 11, DLys (Boc) instead of DCit at position 8, Arg (Pbf), 1 instead of Cit at position 6 Cit was used in place of Arg (Pbf).

<Production Example 15: Production of TA14001, TA14005 to TA14009, TC14001, and TC14004>
H-Ala-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TA14001)
H-Arg-Arg-Nal-Cys-Tyr-Ala-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TA14005)
H-Arg-Arg-Nal-Cys-Tyr-Arg-Ala-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TA14006)
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DAla-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TA14007)
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Ala-Tyr-Arg-Cit-Cys-Arg-OH
(TA14008)
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Ala-Arg-Cit-Cys-Arg-OH
(TA14009)
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TC14001)
H-Arg-Arg-Nal-Cys-Tyr-Arg-Cit-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TC14004)
By changing the amino acid to be introduced by the same method as in Production Example 1 or 9, TA14001, TA14005 to TA14009, TC14001 and TC14004 shown above can be produced.

<Production Example 16: Production of AcTC14003, AcTC14005, AcTC14011 to AcTC14022>
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14003)
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14005)
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14011)
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(AcTC14013)
Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14015)
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(AcTC14017)
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(AcTC14019)
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
(AcTC14021)
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTC14012)
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(AcTC14014)
Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTC14016)
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTC14018)
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(AcTC14020)
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH ₂
(AcTC14022)
Acetylated forms of TC14003, TC14005, and TC14011 to TC14022 were produced by the same method as in Production Examples 1-14. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, acetylates by acetic anhydride (100 eq) -s lysine (100 eq) / DMF treatment, and then 1M against the resin. TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 2 hours (when C-terminal is carboxylic acid) or 3 hours ( The reaction was carried out (when the C-terminal is an amide).

<Production Example 17: Production of polypeptide TE14005>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14005)
1. Synthesis of TE14005 protected polypeptide resin After removal of Fmoc group with 20% piperidine / DMF from 270 mg (0.2 mmol) of Fmoc-Arg (Pbf) -Alko resin (0.74 mmol / g) Fmoc-Cys (Trt) -OH (2.5 eq) corresponding to position 12 was added, and a condensation reaction was performed in DMF by the DIPCDI-HOBt method. The degree of progress of the condensation reaction was examined by the ninhydrin test of Kaiser, E. et al. (Anal. Biochem., 34: 595 (1970)).

2. Introduction of amino acids at positions 12 to 1 In the same manner, Cit, Arg (Pbf), Tyr (t-Bu), Pro, DGlu (0-t-Bu), Lys (Boc), Arg (Pbf ), Tyr (t-Bu), Cys (Trt), Nal, Arg (Pbf), Arg (Pbf) residues were introduced into the resin to obtain a protecting group-protected polypeptide resin.

3. Deprotection Group, Separation and Purification of Polypeptide from Resin Protected group-protected polypeptide resin removes the Fmoc group by treatment with 20% piperidine / DMF, then 1M TMSBr-thioanisole / TFA (200 mg to 200 mg resin) The reaction was carried out with 10 mL of trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 2 hours. The resin was removed from the reaction mixture by filtration, washed twice with 1 mL of TFA, the filtrate and washings combined, concentrated under reduced pressure, 30 mL of water-cooled dry ether was added to the residue, and the resulting precipitate was centrifuged. Separated from the supernatant by decantation. The obtained residue was washed with cold ether, dissolved in 50 mL of 1N acetic acid, and diluted to 250 mL with distilled water.

4). Cyclization by air oxidation The diluted aqueous solution of the above-mentioned polypeptide was adjusted to pH 7.5 with concentrated ammonia water, and oxidization was performed by air oxidation by aeration. The aqueous solution was purified by large-scale preparative HPLC (Cosmozil 5C18 AR-II column: acetonitrile-water) and gel chromatography (Sephadex G-15, eluent: 0.1N AcOH) to obtain a single peak polypeptide. Lyophilized. Purity was confirmed by HPLC.
Yield 24.1 mg (7 AcOH salt) (21.3%)
[α] _D ^23.6 =-5.36 (c 1.12, H ₂ O)
Ion spray mass spectrum (IS-MS): C ₈₉ H ₁₃₆ N ₃₂ 0 ₂₀ S ₂
Calculated value: 2038.38 Actual value: 2038
(Triple Stage Quadrupole Mass Spectrometer API-IIIE (Sciex))

<Production Example 18: Production of polypeptide TE14001>
H-DGlu-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14001)
TE14001 was produced in the same manner as in Production Example 17. However, DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 and DGlu (Ot-Bu) was used in place of Arg (Pbf) at position 1 at the introduction of the 12th to 1st amino acids.

<Production Example 19: Production of polypeptide TE14002>
H-Arg-Glu-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14002)
TE14002 was produced in the same manner as in Production Example 17. However, DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 and Glu (Ot-Bu) was used in place of Arg (Pbf) at position 2 at the introduction of the 12th to 1st amino acids.

<Production Example 20: Production of polypeptide TE14003>
H-Arg-Arg-Nal-Cys-Tyr-Glu-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14003)
TE14003 was produced in the same manner as in Production Example 17. However, DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 and Glu (Ot-Bu) was used in place of Arg (Pbf) at position 6 at the introduction of the 12th to 1st amino acids.

<Production Example 21: Production of polypeptide TE14004>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Glu-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TE14004)
TE14004 was produced in the same manner as in Production Example 17. However, DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 and Glu (Ot-Bu) was used in place of Lys (Boc) at position 7 at the introduction of the 12th to 1st amino acids.

<Production Example 22: Production of polypeptide TE14006>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Glu-Cit-Cys-Arg-OH
(TE14006)
TE14006 was produced in the same manner as in Production Example 17. However, DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 and Glu (Ot-Bu) was used in place of Arg (Pbf) at position 11 at the introduction of the 12th to 1st amino acids.

<Production Example 23: Production of polypeptide TE14007>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Glu-OH
(TE14007)
TE14007 was produced in the same manner as in Production Example 17. However, in place of the first 14-position arginine-introduced arco resin Fmoc-Arg (Pbf) -Alko resin, the first 14-position glutinic acid-introduced arco resin Fmoc-Glu (Ot-Bu) -Alko resin was used. DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 when the amino acid at position 1 to position 1 was introduced.

<Production Example 24: Production of polypeptide TE14011>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14011)
1. Synthesis of TE14011 protected polypeptide resin
After removing the Fmoc group from Fmoc-Rink amide resin with 20% piperidine / DMF, Fmoc-Arg (Pbf) -OH (2.5 eq) corresponding to position 14 was added, and a condensation reaction was carried out in DMF by the DIPCDI-HOBt method. . The degree of progress of the condensation reaction was examined by the ninhydrin test of Kaiser, E. et al. (Anal. Biochem., 34: 595 (1970)).

2. Introduction of amino acids at positions 12 to 1 In the same manner, Cys (Trt), Cit, Arg (Pbf), Tyr (t-Bu), Pro, DGlu (Ot-Bu), Lys (Boc), Cit, Tyr (t-Bu), Cys (Trt), Nal, Arg (Pbf) and Arg (Pbf) residues were introduced into Rink amide resin to obtain a protecting group-protected polypeptide resin.

3. Deprotection group, separation and purification of polypeptide from resin Protected group-protected polypeptide resin removed Fmoc group by treatment with 20% piperidine / DMF and then 1M TMSBr-thioanisole / TFA (trifluoro (Acetic acid) system (m-cresol (100 eq), ethanedithiol (300 eq) in the presence) was reacted at 25 ° C. for 3 hours. The resin was separated from the reaction mixture by filtration, washed twice with TFA, the filtrate and washings combined, concentrated under reduced pressure, water-cooled dry ether was added to the residue, and the resulting precipitate was supernatant by centrifugation and decantation. Separated from. The obtained residue was washed with cold ether, dissolved in 1N acetic acid, and diluted with distilled water.

4). Cyclization by air oxidation The diluted aqueous solution of the above-mentioned polypeptide was adjusted to pH 7.5 with concentrated ammonia water, and oxidization was performed by air oxidation by aeration. This aqueous solution was purified by large-scale preparative HPLC (Cosmozil 5C18 AR-II column: acetonitrile-water) and gel chromatography (Sephadex G-15, eluent: 0.1N AcOH) to obtain a single peak polypeptide. Lyophilized. Purity was confirmed by HPLC.

<Production Example 25: Production of polypeptide TE14012>
H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14012)
TE14012 was produced in the same manner as in Production Example 24. However, DCit was used in place of DGlu (Ot-Bu) at position 8 and DGlu (Ot-Bu) was used instead of Cit at position 6 at the introduction of the 12th to 1st amino acids.

<Production Example 26: Production of polypeptide TE14013>
H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14013)
TE14013 was produced in the same manner as in Production Example 24. However, DGlu (Ot-Bu) was used in place of Cit at position 6 at the introduction of amino acids at positions 12 to 1.

<Production Example 27: Production of polypeptide TE14014>
H-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TE14014)
TE14014 was produced in the same manner as in Production Example 24. However, DGlu (Ot-Bu) was used in place of Arg (Pbf) at position 1 at the introduction of the 12th to 1st amino acids.

<Production Example 28: Production of polypeptide TE14015>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH ₂
(TE14015)
TE14015 was produced in the same manner as in Production Example 24. However, DGlu (Ot-Bu) was used in place of Tyr (t-Bu) at the 10th position at the introduction of the 12th to 1st amino acids.

<Production Example 29: Production of polypeptide TE14016>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH ₂
(TE14016)
TE14016 was produced in the same manner as in Production Example 24. However, DGlu (Ot-Bu) was used in place of Cit at position 12 at the introduction of the 12th to 1st amino acids.

<Production Example 30: Production of polypeptides AcTE14014 to AcTE14016>
Ac-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTE14014)
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH ₂
(AcTE14015)
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH ₂
(AcTE14016)
TE14014 to TE14016 acetylated products were produced in the same manner as in Production Examples 27 to 29. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, acetylates by acetic anhydride (100 eq) -s lysine (100 eq) / DMF treatment, and then 1M against the resin. The reaction was carried out in a TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 31: Production of polypeptide TF1>
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF1: AcTE14011)
TF1 was produced in the same manner as in Production Example 24. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, acetylates by acetic anhydride (100 eq) -s lysine (100 eq) / DMF treatment, and then 1M against the resin. The reaction was carried out in a TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 32: Production of polypeptide TF2>
guanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF2: guanyl-TE14011)
TF2 was produced in the same manner as in Production Example 24. However, the protecting group-protected polypeptide resin removes the Fmoc group by treatment with 20% piperidine / DMF, and guanyl by treatment with 1H-pyrazole-1-carboxamidine (5 eq)-, N-diisopropylethylamine (10 eq) / DMF. Then, the resin was reacted with 1M TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 33: Production of polypeptide TF3>
TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF3: TMguanyl-TE14011)
TF3 was produced in the same manner as in Production Example 24. However, the protecting group-protected polypeptide resin removes the Fmoc group by treatment with 20% piperidine / DMF to give 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetra Tetramethylguanylation by fluoroborate (5 eq) / DMF treatment, followed by 1M TMSBr-thioanisole / TFA (trifluoroacetic acid) system (m-cresol (100 eq), ethanedithiol (300 eq) in the presence of the resin ) For 3 hours at 25 ° C.

<Production Example 34: Production of polypeptide TF4>
TMguanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF4: TMguanyl-TE14011 (2-14))
TF4 was produced in the same manner as in Production Example 24. However, the 1st arginine was not condensed.

<Production Example 35: Production of polypeptide TF5>
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF5: 4F-benzoyl-TE14011)
TF5 was produced in the same manner as in Production Example 24. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, condenses 4-fluorobenzoic acid (2.5 eq) by the DIPCDI-HOBt method, and then adds 1M TMSBr- The reaction was carried out at 25 ° C. for 3 hours in a thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)).

<Production Example 36: Production of polypeptide TF6>
2F-benzoyl-Arg-Arg-Nrg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ (TF6: 2F-benzoyl-TE14011)
TF6 was produced in the same manner as in Production Example 24. However, the protecting group-protected polypeptide resin is prepared by removing Fmoc group by 20% piperidine / DMF treatment, condensing 2-fluorobenzoic acid (2.5 eq) by DIPCDI-HOBt method, and then adding 1M TMSBr- The reaction was carried out at 25 ° C. for 3 hours in a thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)).

<Production Example 37: Production of polypeptide TF7>
APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF7: APA-TE14011 (2-14))
TF7 was produced in the same manner as in Production Example 24. However, 5-aminopentanoic acid (Fmoc protector) was introduced in place of Arg (Pbf) at position 1 when the amino acids at positions 12 to 1 were introduced.

<Production Example 38: Production of polypeptide TF8>
desamino-R-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF8: desamino-R-TE14011 (2-14))
TF8 was produced in the same manner as in Production Example 24. However, at the introduction of the 12th to 1st amino acids, 5-aminopentanoic acid (Fmoc protector) was introduced instead of Arg (Pbf) at the 1st position, and the protecting group-protected polypeptide resin was 20 The Fmoc group was removed by treatment with% piperidine / DMF, guanylated by treatment with 1H-pyrazole-1-carboxamidine (5 eq)-, N-diisopropylethylamine (10 eq) / DMF, then 1M TMSBr-thioanisole against the resin / TFA (trifluoroacetic acid) system (m-cresol (100 eq), in the presence of ethanedithiol (300 eq)) was reacted at 25 ° C. for 3 hours.

<Production Example 39: Production of polypeptide TF9>
guanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF9: guanyl-TE14011 (2-14))
TF9 was produced using the same method as in Production Example 32. However, the 1st arginine was not condensed.

<Production Example 40: Production of polypeptide TF10>
succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF10: succinyl-TE14011 (2-14))
TF10 was produced in the same manner as in Production Example 24. However, the 1st arginine was not condensed. In addition, the protecting group-protected polypeptide resin is prepared by removing Fmoc group by 20% piperidine / DMF treatment, hemisuccinylation by succinic anhydride (5 eq) / pyridine treatment, and then adding 1M TMSBr-thioanisole / TFA to the resin. The reaction was carried out in the (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 41: Production of polypeptide TF11>
glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF11: glutaryl-TE14011 (2-14))
TF11 was produced in the same manner as in Production Example 40. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, hemisuccinylation by glutaric anhydride (5 eq) / pyridine treatment, and then adds 1M TMSBr-thioanisole / TFA to the resin. The reaction was carried out in the (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 42: Production of polypeptide TF12>
deaminoTMG-APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF12: deaminoTMG-APA-TE14011 (2-14))
TF12 was produced in the same manner as in Production Example 24. However, at the introduction of the 12th to 1st amino acids, 5-aminopentanoic acid (Fmoc protector) was introduced instead of Arg (Pbf) at the 1st position, and the protecting group-protected polypeptide resin was 20 The Fmoc group was removed by treatment with% piperidine / DMF, and tetramethyl by 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (5 eq) / DMF. After guanylation, the resin was then reacted with 1M TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 43: Production of polypeptide TF15>
R-CH ₂ -Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF15: R-CH ₂ NH-RTE14011)
TF15 was produced in the same manner as in Production Example 24. However, instead of condensing Fmoc-Arg (Pbf) -OH at the 1-position at the introduction of amino acids at positions 12 to 1, Fmoc-Arg (Pbf) -H (aldehyde) was condensed by reductive amination ( NaB (CN) H ₃ (3 eq), AcOH (1 eq) / DMF).

<Production Example 44: Production of polypeptide TF17>
H-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ (TF17)
(TF17: TE14011 (2-14))
TF17 was produced in the same manner as in Production Example 24. However, the 1st arginine was not condensed.

<Production Example 45: Production of polypeptide TF18>
TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF18: TMguanyl-TC14012)
TF18 was produced in the same manner as in Production Example 9. However, the protecting group-protected polypeptide resin removes the Fmoc group by treatment with 20% piperidine / DMF to give 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetra Tetramethylguanylation by fluoroborate (5 eq) / DMF treatment, followed by 1M TMSBr-thioanisole / TFA (trifluoroacetic acid) system (m-cresol (100 eq), ethanedithiol (300 eq) in the presence of the resin ) For 3 hours at 25 ° C.

<Production Example 46: Production of polypeptide TF19>
ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TF19: ACA-TC14012)
TF19 was produced in the same manner as in Production Example 9. However, 6-aminohexanoic acid (Fmoc protector) was introduced after Arg (Pbf) at position 1 when the amino acids at positions 12 to 1 were introduced.

<Production Example 47: Production of polypeptide TF20>
ACA-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
(TF20: ACA-T140)
TF20 was produced in the same manner as in Production Example 17. However, at the introduction of amino acids 12 to 1, DLys (Boc) instead of DGlu (Ot-Bu) at position 8, 6-aminohexanoic acid instead of Arg (Pbf) at position 1 (Fmoc protector) Was used.

<Production Example 48: Production of polypeptide TZ14011>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TZ14011)
TZ14011 was produced in the same manner as in Production Example 24. However, DLys (Boc) was used in place of DGlu (Ot-Bu) at position 8 and Arg (Pbf) was used in place of Lys (Boc) at position 7 when introducing amino acids at positions 12 to 1.

<Production Example 49: Production of polypeptide AcTZ14011>
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTZ14011)
AcTZ14011 was produced in the same manner as in Production Example 48. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, acetylates by acetic anhydride (100 eq) -s lysine (100 eq) / DMF treatment, and then 1M against the resin. The reaction was carried out in a TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 50: Production of polypeptide TN14003>
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TN14003)
TN14003 was produced in the same manner as in Production Example 9. However, DLys (Boc) was used in place of DCit at the 8-position at the introduction of the 12th to 1st amino acids.

<Production Example 51: Production of polypeptide TN14005>
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(TN14005)
TN14005 was produced in the same manner as in Production Example 9. However, Arg (Pbf) was used in place of Cit at position 6 at the introduction of the 12th to 1st amino acids.

<Production Example 52: Production of polypeptide AcTN14003>
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTN14003)
AcTN14003 was produced in the same manner as in Production Example 50. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, acetylates by acetic anhydride (100 eq) -s lysine (100 eq) / DMF treatment, and then 1M against the resin. The reaction was carried out in a TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 53: Production of polypeptide AcTN14005>
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(AcTN14005)
AcTN14005 was produced in the same manner as in Production Example 51. However, the protecting group-protected polypeptide resin removes the Fmoc group by 20% piperidine / DMF treatment, acetylates by acetic anhydride (100 eq) -s lysine (100 eq) / DMF treatment, and then 1M against the resin. The reaction was carried out in a TMSBr-thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours.

<Production Example 54: Production of polypeptide 4F-benzoyl-TN14003>
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂
(4F-benzoyl-TN14003)
1. Synthesis of 4F-benzoyl-TN14003 protected polypeptide resin
Fmoc-Rink amide resin (0.34 mmol / g) From 2.94 g (1 mmol), the Fmoc group was removed with 20% piperidine / DMF, then Fmoc-Arg (Pbf) -OH (2.5 eq) corresponding to position 14 was added and DMF was added. In the middle, the condensation reaction was carried out by the DIPCDI-HOBt method. The degree of progress of the condensation reaction was examined by the ninhydrin test of Kaiser, E. et al. (Anal. Biochem., 34: 595 (1970)).

2. Introduction of amino acids at positions 13 to 1 In the same manner, Cys (Trt), Cit, Arg (Pbf), Tyr (t-Bu), Pro, DLys (Boc), Lys (Boc), Cit, Tyr (t-Bu), Cys (Trt), Nal, Arg (Pbf), Arg (Pbf) residues are introduced into Rink amide resin, and 4-fluorobenzoic acid (2.5 eq) is added to the last N-terminus with DIPCDI -HOBt Condensation by the method yielded a protecting group-protected polypeptide resin.

3. Deprotection group, separation and purification of polypeptide from resin 1M TMSBr-thioanisole / TFA (trifluoroacetic acid) system (m-cresol (100 eq), ethanedithiol (300 eq) against polypeptide resin (1 mmol) ) In the presence) 270 mL was reacted at 25 ° C. for 3 hours. The resin was filtered off from the reaction mixture, washed twice with 5 mL of TFA, and the combined filtrate and washings were concentrated under reduced pressure. To the residue was added 300 mL of water-cooled dry ether, and the resulting precipitate was decanted. Separated from the supernatant. The obtained residue was washed with cold ether, dissolved in 500 mL of 1N acetic acid, and diluted to 2.5 L with distilled water.

4). Cyclization by air oxidation The diluted aqueous solution of the above-mentioned polypeptide was adjusted to pH 7.5 with concentrated ammonia water, and oxidization was performed by air oxidation by aeration. This aqueous solution was purified by large-scale preparative HPLC (Cosmozil 5C18 AR-II column: acetonitrile-water) to obtain a single peak polypeptide, which was lyophilized. Purity was confirmed by HPLC.
Yield 551.5 mg (6 TFA salt) (19.4%)
[α] _D ^28.6 =-10.25 (c 0.39, H ₂ O)
Ion spray mass spectrum (IS-MS): C ₉₇ H ₁₄₄ FN ₃₃ 0 ₁₉ S ₂
Calculated value: 2159.52 Actual value: 2161
(Triple Stage Quadrupole Mass Spectrometer API-IIIE (Sciex))

<Production Example 55: Production of polypeptide 4F-benzoyl-TE14011-Me>
4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHMe
(4F-benzoyl-TE14011-Me)
1. Synthesis of 4F-benzoyl-TE14011-Me protected polypeptide resin
Fmoc-Arg (Pbf) -OH (4 eq) corresponding to position 14 is added to 4-sulfamylbutyryl AM NovaGel resin and condensed in CHCl ₃ at -0 ° C by PyBOP (3 eq) -DIPEA (6 eq) method Reaction was performed (this condensation reaction was performed twice). After removing the Fmoc group from this resin with 20% piperidine / DMF, Fmoc-Cys (Trt) -OH (2.5 eq) corresponding to position 13 was added, and a condensation reaction was performed in DMF by the DIPCDI-HOBt method. The degree of progress of the condensation reaction was examined by the ninhydrin test of Kaiser, E. et al. (Anal. Biochem., 34: 595 (1970)). Hereinafter, in the same manner for the introduction of amino acids at positions 12 to 1, Cit, Arg (Pbf), Tyr (t-Bu), Pro, DGlu (Ot-Bu), Lys (Boc), Cit, Tyr (t- Bu), Cys (Trt), Nal, Arg (Pbf), Arg (Pbf) residues are introduced into sulfamylbutyryl resin, and 4-fluorobenzoic acid (2.5 eq) is condensed at the N-terminus by DIPCDI-HOBt method for protection A group-protected polypeptide resin was obtained.

2. C-terminal alkylamidation, deprotection group, separation and purification of polypeptide from resin Protected group-protected polypeptide resin is treated with ICH ₂ CN (40 eq), DIPEA (10 eq) / NMP treatment (48 hours) for cyanomethyl Then, methylamine (excess) in THF / DMF was allowed to act on the resin to separate the C-terminal methylamidated protecting group-protected polypeptide from the resin. Next, the protecting group-protected polypeptide was reacted in a 1 M thioanisole / TFA (trifluoroacetic acid) system (in the presence of m-cresol (100 eq), ethanedithiol (300 eq)) at 25 ° C. for 3 hours. The reaction solution was concentrated under reduced pressure, water-cooled dry ether was added to the residue, and the resulting precipitate was separated from the supernatant by centrifugal sedimentation and decantation. The obtained residue was washed with cold ether, dissolved in 1N acetic acid, and diluted with distilled water.

3. Cyclization by air oxidation The diluted aqueous solution of the above-mentioned polypeptide was adjusted to pH 7.5 with concentrated ammonia water, and oxidization was performed by air oxidation by aeration. This aqueous solution was purified by large-scale preparative HPLC (Cosmozil 5C18 AR-II column: acetonitrile-water) and gel chromatography (Sephadex G-15, eluent: 0.1N AcOH) to obtain a single peak polypeptide. Lyophilized. Purity was confirmed by HPLC.

<Production Example 56: Production of polypeptide 4F-benzoyl-TE14011-Et>
4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHEt
(4F-benzoyl-TE14011-Et)
4F-benzoyl-TE14011-Et was produced in the same manner as in Production Example 55. However, ethylamine was used in place of methylamine.

<Production Example 57: Production of polypeptide: 4F-benzoyl-TE14011-iPr>
4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHiPr
(4F-benzoyl-TE14011-iPr)
4F-benzoyl-TE14011-iPr was produced in the same manner as in Production Example 55. However, isopropylamine was used instead of methylamine.

<Production Example 58: Production of polypeptide 4F-benzoyl-TE14011-tyramine>
4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-tyramine
(4F-benzoyl-TE14011-tyramine)
4F-benzoyl-TE14011-tyramine was produced in the same manner as in Production Example 55. However, tyramine (p-hydroxyphenylethylamine) was used instead of methylamine.

<Test Example 1: Inhibitory activity of polypeptide of the present invention for binding of CXCL12 to CXCR4 receptor>
Test compounds diluted in buffer with 50 μL of Jurkat human T cell leukemia cells prepared to ⁶ × 10 ⁶ cells / mL in buffer (Dulbecco's PBS solution (pH 7.0) containing 0.5% BSA, 20 mM HEPES) on the assay plate (Table) 1; each compound was synthesized in the form of an acetate salt) and 200 μM ¹²⁵ I-CXCL12 solution was dispensed in an amount of 25 μL, and reacted at room temperature for 1 hour to carry out a binding reaction. After the reaction, the reaction solution was suction filtered through a 96-well GF / C filter plate, and the radioactivity of each well was measured by top count. The inhibitory activity of each test compound was determined by setting the radioactivity when no test compound was added to 100% and the radioactivity when adding 100 nM non-radiolabeled CXCL12 as 0%. The results are shown in Table 1 below.

  From the results of Table 1, it was shown that this compound has a strong binding inhibitory activity.

<Test Example 2: Inhibitory activity of TE-14005 on breast cancer cell migration induced by CXCL12>
A transwell filter (polycarbonate filter, 8 μm diameter, Costar) was treated in a 10 μg / mL fibronectin solution at 37 ° C. for 6 hours and then air-dried. CXCL12 (R & D Systems) 100 nM and buffer A (0.1% bovine serum albumin, 12 mM HEPES) DMEM (GibcoBRL) containing 600 μL / well were added to the lower chamber of the transwell. Substances and human breast cancer MDA-MB-231 cells (purchased from American Tissue Culture Collection) were added 100 μL / well of buffer A containing 2 × 10 ⁶ cells / mL, and cultured for 15 hours in a 37 ° C., 5% CO ₂ incubator. The upper surface of the filter was wiped off, the cells were removed, the cells on the lower surface of the filter were fixed and stained with a 25% methanol solution containing 0.5% crystal violet (Wako Pure Chemical Industries), washed with distilled water, and then air-dried. Cut off, 0.1 M sodium citrate / 50 The ethanol solution was added to dissolve the crystal violet, and the absorbance at 550 nm was measured, and the results are shown in Fig. 1. The negative control shows the migration in the absence of CXCL12 and MDA-MB-231 by adding CXCL12. The cell migration was enhanced (positive control), and the CXCL12-induced MDA-MB-231 cell migration was inhibited by an antagonist, TE14005, up to 10 nM.

<Test Example 3: Inhibitory activity of TC14012 and TN14003 for binding of CXCL12 to CXCR4 receptor>
Test compounds diluted in buffer with 50 μL of Jurkat human T cell leukemia cells prepared to ⁶ × 10 ⁶ cells / mL in buffer (Dulbecco's PBS solution (pH 7.0) containing 0.5% BSA, 20 mM HEPES) on the assay plate (Table) 2; each compound was synthesized in the form of acetate) and 200 μM ¹²⁵ I-CXCL12 solution was dispensed in an amount of 25 μL, and reacted at room temperature for 1 hour to carry out a binding reaction. After the reaction, the reaction solution was suction filtered through a 96-well GF / C filter plate, and the radioactivity of each well was measured by top count. The inhibitory activity of each test compound was determined by setting the radioactivity when no test compound was added to 100% and the radioactivity when adding 100 nM non-radiolabeled CXCL12 as 0%. The results are shown in Table 2 below.

  From the results of Table 2, it was shown that this compound has a strong binding inhibitory activity.

<Test Example 4: Inhibitory activity of TC-14012 on breast cancer cell migration induced by CXCL12>
A transwell filter (polycarbonate filter, 8 μm diameter, Costar) was treated in a 10 μg / mL fibronectin solution at 37 ° C. for 6 hours and then air-dried. CXCL12 (R & D Systems) 100 nM and buffer A (0.1% bovine serum albumin, 12 mM HEPES) DMEM (GibcoBRL) containing 600 μL / well were added to the lower chamber of the transwell. Substances and human breast cancer MDA-MB-231 cells (purchased from American Tissue Culture Collection) were added 100 μL / well of buffer A containing 2 × 10 ⁶ cells / mL, and cultured for 15 hours in a 37 ° C., 5% CO ₂ incubator. The upper surface of the filter was wiped off, the cells were removed, the cells on the lower surface of the filter were fixed and stained with a 25% methanol solution containing 0.5% crystal violet (Wako Pure Chemical Industries), washed with distilled water, and then air-dried. Cut off, 0.1 M sodium citrate / 50 The ethanol solution was added to dissolve crystal violet, and the absorbance at 550 nm was measured, and the results are shown in Fig. 2. The negative control shows the migration when CXCL12 is not added, and the positive control shows the migration when CXCL12 is added. By adding CXCL12, the migration of MDA-MB-231 cells was enhanced, and the migration of MDA-MB-231 cells induced by CXCL12 was inhibited by antagonist, TC-14012 up to 10 nM.

<Test Example 5: Inhibitory activity of 4Fbenzoyl-TN-14003 on T cell-derived leukemia cell migration induced by CXCL12>
600 μL of DMEM (GibcoBRL) containing 30 nM CXCL12 (R & D Systems) 30 nM and test substance buffer A (0.1% bovine serum albumin, 12 mM HEPES) in the lower chamber of the transwell (polycarbonate filter, 8 μm diameter, Costar) The test substance and human T cell-derived leukemia cell SUP-T1 cells (purchased from American Tissue Culture Collection), buffer A containing 2 × 10 ⁶ cells / mL, 100 μL / well were added to the upper chamber at 37 ° C. After culturing in a 5% CO ₂ incubator for 4 hours, the number of cells that migrated to the lower chamber was measured using a Coulter counter, and the results are shown in Fig. 3. The negative control shows the migration when CXCL12 is not added. Control is migration when CXCL12 is added By adding CXCL12, the SUP-T1 cell migration was enhanced, and the SUP-T1 cell migration induced by CXCL12 was inhibited to 10 nM by the antagonist, 4Fbenzoyl-TN-14003. 4Fbenzoyl-TN-14003 is thought to be useful as an inhibitor of rheumatoid arthritis because it inhibits T cell motility at low concentrations.

<Test Example 6: Inhibitory activity of 4F-benzoyl-TN-14003 on breast cancer cell migration induced by CXCL12>
A transwell filter (polycarbonate filter, 8 μm diameter, Costar) was treated in a 10 μg / mL fibronectin solution at 37 ° C. overnight and then air-dried. To the lower chamber of the transwell was added 600 μL / well of DMEM (GibcoBRL) containing buffer A (0.1% bovine serum albumin, 12 mM HEPES) containing 100 nM CXCL12 (R & D Systems) and 4F-benzoyl-TN-14003. 4 Fbenzoyl-TN-14003 and human breast cancer MDA-MB-231 cells (purchased from American Tissue Culture Collection) were added to the chamber at 100 μL / well of buffer A containing 2 × 10 ⁶ cells / mL at 37 ° C. in a 5% CO ₂ incubator. After culturing for 15 hours, the upper surface of the filter is wiped off, the cells are removed, the cells on the lower surface of the filter are fixed and stained with a 25% methanol solution containing 0.5% crystal violet (Wako Pure Chemical Industries), and washed with distilled water. Air dried. The filter part was cut off, 0.1 M sodium citrate / 50% ethanol solution was added to dissolve crystal violet, and the absorbance at 550 nm was measured, and the results are shown in Fig. 4. The negative control is the migration without CXCL12 addition. The positive control shows the migration when CXCL12 was added, and the migration of MDA-MB-231 cells was enhanced by the addition of CXCL12.The migration of MDA-MB-231 cells induced by CXCL12 The sex antagonist, 4Fbenzoyl-TN-14003, inhibited up to 100 nM.

<Test Example 7: Anti-metastatic activity of 4Fbenzoyl-TN-14003>
10 ⁶ MDA-MB-231 human breast cancer cells were transplanted from the tail vein into 5-week-old female CB-17 SCID mice (Claire Japan). 4Fbenzoyl-TN-14003 was adjusted to 80 mg / mL with physiological saline and enclosed in a sustained release osmotic pump (Alzette pump, Alza, 2 weeks sustained release). / kg / day), the mouse was subcutaneously mounted on the back of the mouse the day before transplantation. Furthermore, 14 days after transplantation, an Alzette pump containing the same dose of drug was additionally attached. The control group was additionally equipped with an Alzette pump infused with physiological saline. 28 days after transplantation, the mice were dissected and about 2 mL of 0.2% Evans blue solution was injected from the trachea, and the lungs were stained. The lungs were removed, immersed in Bouin's solution, stained and fixed. It was judged whether or not there was a clear anti-metastatic activity by visually observing the metastatic lesion (portion stained yellow). The results are shown in FIG. In the lung (A) of the control group, a yellow-stained portion was uniformly recognized and metastasis to the lung was observed. On the other hand, in the 4Fbenzoyl-TN-14003 administration group (B), cases with a small proportion of yellow staining were observed. In comparison, it was observed that metastasis was suppressed in the 4Fbenzoyl-TN-14003 administration group.

<Test Example 8: Inhibitory activity of 4Fbenzoyl-TN14003 on the migration of human T cell line (Jurkat) and mouse splenocytes induced by CXCL12>
1. Effects of human T cell lines on the migration response
RPMI-1640 and fetal calf serum (FCS) purchased from BioWhittaker, penicillin-streptomycin solution, RPMI-1640 (without phenol red) and HEPES from Invitrogen, BSA from Sigma, human SDF-1α (CXCL12) from Genzyme did. The human T lymphocyte cell line Jurkat was purchased from ATCC and cultured in RPMI-1640 10% FCS. 4F-benzoyl-TN14003 was dissolved in PBS and used for the experiment.
Migration reaction was performed using 24-well Transwell (Costar, membrane, pore size 5 μm). SDF-1α (final concentration 1 ng / mL) 600 μL was added to the Transwell lower layer, 5 × 10 ⁵ cells (200 μL) were added to the insert, and reacted at 37 ° C. for 4 hours. Cells were preincubated with drug for 30 minutes at 37 ° C. The migration reaction was performed in RPMI-1640 medium containing 20 mmol / L HEPES and 0.5% BSA. Cells that migrated to the lower layer were collected, and the number of cells was counted using a Coulter Counter. The inhibition rate (%) for the migration reaction by each concentration of drug was determined from the following equation, and the IC ₅₀ value was calculated from the inhibition rate.

As a result, as shown in FIG. 6, Jurkat cells showed strong cell migration reactivity against SDF-1α. 4F-benzoyl-TN14003 inhibited this reaction in a concentration-dependent manner, and its IC ₅₀ value was 0.65 nmol / L.

2. Effects on mouse spleen cell migration
Spleens were collected from BALB / c mice (male, Nihon Charles River) to prepare single cell suspensions, and erythrocytes were disrupted to prepare spleen cells.
SDF-1α (Peprotech, final concentration 100 ng / mL) was added to the lower layer of Transwell (pore size 5 μm), and 1 × 10 ⁶ / well cells (100 μL) were added to the insert and reacted at 37 ° C. for 2.5 hours. Cells were preincubated with drug for 30 minutes at 37 ° C. The migration reaction was performed in RPMI-1640 medium containing 20 mmol / L HEPES and 0.5% BSA. The number of cells that migrated to the lower layer was counted using a Coulter Counter. In the same manner as in the previous section, the inhibition rate of migration reaction by each concentration of drug and the IC ₅₀ value of the inhibitory action were calculated.
As a result, as shown in FIG. 7, 4F-benzoyl-TN14003 exhibited a concentration-dependent inhibitory effect on the migration response of mouse splenocytes to SDF-1α. Its IC ₅₀ value was 0.54 nmol / L, indicating an inhibitory effect comparable to that of human cells. From this, it was confirmed that there is almost no species difference between human and mouse for this peptide.

<Test Example 9: Effect of 4Fbenzoyl-TN14003 on mouse delayed hypersensitivity reaction (DTH)>
Sheep preserved blood was purchased from the Japan Biomaterials Center. The sheep preserved blood was washed twice with physiological saline, suspended in physiological saline and used as sheep erythrocytes (SRBC). Since the OD541 nm value of oxyhemoglobin when hemolyzing SRBC suspension 1.0 × 10 ⁹ cells / mL with 14 times the amount of distilled water is approximately equivalent to 0.700, the SRBC density was adjusted accordingly.
BALB / c mice (male, 6 weeks old, Nippon Charles River) were sensitized by subcutaneous administration of 2 × 10 ⁷ cells / 50 μl SRBC to the left hind paw. Five days later, 10 ⁸ cells / 50 μl of SRBC was subcutaneously administered to the right hind footpad to induce a DTH reaction. The thickness of the right hind footpad was measured using a digital micrometer (Mitutoyo CD-15B) immediately before and 24 hours after antigen induction, and the increase in thickness of the footpad due to swelling (mm) was used as an indicator of DTH response.
4F-benzoyl-TN14003 was dissolved in PBS and continuously administered using an Alzet osmotic pump (Alza, 0.5 μL / hr for 7 days). The osmotic pump was implanted subcutaneously in the back under ether anesthesia the day before sensitization. As a control, a pump infused with PBS was similarly implanted. 4F-benzoyl-TN14003 was administered at doses of 4.8, 24 and 120 μg / day.
Data were expressed as mean ± standard error (n = 7). A Williams test was performed, and p ≦ 0.025 was considered significant.
As a result, as shown in FIG. 8, 4F-benzoyl-TN14003 (4.8, 24 and 120 μg / day) significantly inhibited toe edema in a dose-dependent manner, and the inhibition rates were 9, 31 and 51%, respectively. Met. This suggested that CXCR4 plays an important role in cellular immunity such as DTH reaction.

<Test Example 10: Treatment effect of 4Fbenzoyl-TN14003 for mouse collagen arthritis>
FK-506 was purified by a method known per se (Kino T. et al., J. Antibiot., 1987 40 (9): 1249-55). Methotrexate was purchased from Wako Pure Chemicals, indomethacin from Sigma, bovine type II collagen from the Collagen Technology Workshop, Freund's complete adjuvant (FCA) from Difco, and anti-mouse IgG2a antibody from Zymed.
Bovine type II collagen was dissolved in a 0.05 mol / L acetic acid solution to a concentration of 2 mg / mL, and an emulsion was prepared with an equal amount of FCA. DBA / 1JN mice (male, 6 weeks old, Japanese Charles River) were inoculated with 50 μL of emulsion in the ridge skin. Booster immunization was similarly performed 21 days later. Measurement of body weight and hind limb thickness and arthritis scoring were performed over 2 weeks from the booster immunization. The arthritis score was scored at 0-3 points on each limb and evaluated by the sum (up to 12 points) (0, normal; 1, mild or single finger swelling; 2, moderate swelling or multifinger swelling) ; 3, severe swelling). Limbs and serum were collected 2 weeks after boost.
After bovine type II collagen (10 μg / mL PBS solution) was coated on an immunoplate and blocked, 100 μL of 1000-fold diluted mouse serum was added and left at room temperature for 2 hours. After washing, anti-mouse IgG2a antibody (1000-fold diluted) was added. After washing, TMB was added, allowed to stand at room temperature for 30 minutes, an equal amount of H ₂ SO ₄ was added, and A450 nm was measured.
Indomethacin (1 mg / kg), methotrexate (3 mg / kg) and FK-506 (10 mg / kg) were suspended in 0.5% methylcellulose and administered orally daily for 2 weeks from the day of booster immunization in a volume of 0.1 mL / 10 g body weight. did. The same volume of 0.5% methylcellulose solution was orally administered to the control group. 4F-benzoyl-TN14003 was dissolved in PBS and continuously administered using an Alzet osmotic pump (Alza, 0.5 μL / hr for 2 weeks). The osmotic pump was implanted subcutaneously in the back of the mouse under ether anesthesia the day before the booster immunization. As a control, a pump infused with PBS was similarly implanted. Each drug was evaluated based on values after 2 weeks of booster immunization.
Data were expressed as mean ± standard error (n = 8-12). Comparison between the two groups was performed by Student's t test, and p ≦ 0.05 was considered significant. Multiple comparison was performed by Dunnett test, and p ≦ 0.05 was considered significant.
As a result, all of Indomethacin (1 mg / kg, po), methotrexate (3 mg / kg, po) and FK-506 (10 mg / kg, po) significantly suppressed hind limb swelling and A significant or clear inhibitory action was shown (FIG. 9).
4F-benzoyl-TN14003 (120 μg / day) significantly inhibited hind limb swelling, arthritis score and weight loss. In addition, the anti-type II collagen IgG2a antibody titer showed a tendency to be suppressed (FIG. 10). These inhibitory effects were equal to or greater than those of the existing drugs.

  Since the peptide compound of the present invention having CXCR4 antagonistic activity inhibits the binding between CXCR4 and CXCL12 / SDF-1α, cancer types expressing CXCR4 such as oral cancer, pharyngeal cancer, lip cancer, tongue Cancer, gingival cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer including colon cancer, liver cancer, gallbladder cancer, pancreatic cancer, nasal cavity cancer, lung cancer, osteosarcoma, soft tissue cancer, skin cancer, melanoma Can suppress the migration reaction of cancer cells such as breast cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, penile cancer, bladder cancer, kidney cancer, brain tumor, thyroid cancer, lymphoma, leukemia, etc. It is useful as a preventive and / or therapeutic agent. In addition, the peptide compound of the present invention can suppress the immune cell migration reaction induced by CXCL12 / SDF-1α, and is useful as a prophylactic and / or therapeutic agent for rheumatoid arthritis.

It is a figure which shows the inhibitory activity of TE-14005 with respect to the breast cancer cell migration property induced | guided | derived by CXCL12. The vertical axis shows cell migration (absorbance at OD 550 nm). From left to right when CXCL12 is not added (negative control), when CXCL12 is added (positive control), when CXCL12 and TE-14005 are added at 10 nM, when CXCL12 and TE-14005 are added at 100 nM, when CXCL12 and TE-14005 are added at 1 μM, CXCL12 and TE− 14005 The result when 10 μM was added is shown (mean value + standard deviation, n = 2). * Indicates the significance of each TE-14005 addition group with the positive control group (Williams test, p ≦ 0.025). It is a figure which shows the inhibitory activity of TC-14012 with respect to the breast cancer cell migration property induced | guided | derived by CXCL12. The vertical axis shows cell migration (absorbance at OD 550 nm). From left to right when CXCL12 is not added (negative control), when CXCL12 is added (positive control), when CXCL12 and TC-14012 are added at 1 nM, when CXCL12 and TC-14012 are added at 10 nM, when CXCL12 and TC-14012 are added at 100 nM, CXCL12 and TC- 14012 shows the results when 1 μM was added (mean value + standard deviation, n = 2). * Indicates the significance of each TC-14012 added group with the positive control group (Williams test, p ≦ 0.025). It is a figure which shows the inhibitory activity of 4Fbenzoyl-TN-14003 with respect to the migration of the T cell derived leukemia cell induced | guided | derived by CXCL12. The vertical axis represents cell migration (cell number). From left to right when CXCL12 is not added (negative control), when CXCL12 is added (positive control), when CXCL12 and 4Fbenzoyl-TN-14003 1 nM are added, when CXCL12 and 4Fbenzoyl-TN-14003 10 nM are added, CXCL12 and 4Fbenzoyl-TN-14003 The results when 1 μM of CXCL12 and 4Fbenzoyl-TN-14003 are added are shown (mean value + standard deviation, n = 2). * Indicates the significance of each 4Fbenzoyl-TN-14003 addition group with the positive control group (Williams test, p ≦ 0.025). It is a figure which shows the inhibitory activity of 4Fbenzoyl-TN-14003 with respect to the breast cancer cell migration property induced | guided | derived by CXCL12. The vertical axis shows cell migration (absorbance at OD 550 nm). From left to right when CXCL12 is not added (negative control), when CXCL12 is added (positive control), when CXCL12 and 4Fbenzoyl-TN-14003 are added at 10 nM, when CXCL12 and 4Fbenzoyl-TN-14003 are added at 100 nM, and when CXCL12 and 4Fbenzoyl-TN-14003 are added The results when CXCL12 and 4Fbenzoyl-TN-14003 10 μM are added are shown (mean value + standard deviation, n = 2). * Indicates the significance of each 4Fbenzoyl-TN-14003 addition group with the positive control group (Williams test, p ≦ 0.025). It is a lung-tissue dyeing | staining image which shows the lung metastasis inhibitory activity of 4Fbenzoyl-TN-14003 in the mouse | mouth transplanted with the human breast cancer cell. A is an image of a lung of a control group (raw food administration group), and B is an image of a lung of a 4Fbenzoyl-TN-14003 administration group. It is a figure which shows the inhibitory activity of 4Fbenzoyl-TN-14003 with respect to the migration of the Jurkat cell induced | guided | derived by SDF-1 (alpha) (CXCL12). The vertical axis shows cell migration (ratio of mobile cells to input). From the left, when SDF-1α is not added, when SDF-1α is added, when SDF-1α and 4Fbenzoyl-TN-14003 are added at 10 pM, when SDF-1α and 4Fbenzoyl-TN-14003 are added at 100 pM, SDF-1α and 4Fbenzoyl-TN-14003 The results of adding 1 nM, SDF-1α and 4Fbenzoyl-TN-14003, adding 10 nM, adding SDF-1α and 4Fbenzoyl-TN-14003, 100 nM are shown. It is a figure which shows the inhibitory activity of 4Fbenzoyl-TN-14003 with respect to the migration property of the mouse | mouth splenocytes induced | guided | derived by SDF-1 (alpha) (CXCL12). The vertical axis shows cell migration (ratio of mobile cells to input). From the left, when SDF-1α is not added, when SDF-1α is added, when SDF-1α and 4Fbenzoyl-TN-14003 are added at 10 pM, when SDF-1α and 4Fbenzoyl-TN-14003 are added at 100 pM, SDF-1α and 4Fbenzoyl-TN-14003 The results of adding 1 nM, SDF-1α and 4Fbenzoyl-TN-14003, adding 10 nM, adding SDF-1α and 4Fbenzoyl-TN-14003, 100 nM are shown. It is a figure which shows the inhibitory effect of 4Fbenzoyl-TN-14003 with respect to mouse | mouth DTH reaction by SRBC induction. The vertical axis represents the increase in footpad thickness due to swelling (average value ± standard error, n = 7). From the left, the results of the PBS administration (control) group, the 4F benzoyl-TN-14003 4.8 μg / day administration group, the 4F benzoyl-TN-14003 24 μg / day administration group, and the 4F benzoyl-TN-14003 120 μg / day administration group are shown. * Indicates P ≦ 0.025 (comparison with PBS administration group; Williams test). It is a figure which shows the effect | action of the existing drug with respect to mouse | mouth collagen arthritis. A is change in body weight [vertical axis: change in body weight (g) (mean ± standard error, n = 8), horizontal axis: days after booster immunization], B is change in disease incidence [vertical axis: disease incidence ( %) (N = 8), horizontal axis: days after booster immunization, C is arthritis score change [vertical axis: arthritis score (mean ± standard error, n = 8), horizontal axis: days after booster immunization ], D represents the variation in thickness (vertical axis: thickness (mm) (mean ± standard error, n = 8), horizontal axis: days after booster immunization) (□: normal mouse group, ■: Drug non-administration (control) group, Δ: indomethacin administration group, ▲: methotrexate administration group, ●: FK506 administration group). E is the effect of each drug on hindlimb swelling 2 weeks after booster immunity [vertical axis: hindlimb weight (mg) (mean ± standard error, n = 8)], F is anti-bovine 2 weeks after booster immunization The effect of each drug on the type II collagen IgG2a antibody titer [vertical axis: antibody titer (A450) (mean value ± standard error, n = 8)] is shown [normal mouse group from the left, no drug administration (control) group , An indomethacin (IND) administration group, a methotrexate (MTX) administration group, and an FK506 administration group]. ## is P ≦ 0.01 (comparison with normal mouse group; t test), * and ** are P ≦ 0.05 and P ≦ 0.01 (comparison with non-drug group, Dunnett test), respectively. Indicates. It is a figure which shows the effect | action of 4Fbenzoyl-TN-14003 with respect to mouse | mouth collagen arthritis. A is change in body weight [vertical axis: change in body weight (g) (mean value ± standard error), horizontal axis: days after booster immunization], B is change in disease incidence [vertical axis: disease incidence (%), horizontal Axis: days after booster immunization], C is arthritis score change [vertical axis: arthritis score (mean value ± standard error), horizontal axis: days after booster immunity], D is heel thickness change [vertical axis: 踵Thickness (mm) (mean value ± standard error), horizontal axis: days after booster immunization [□: normal mouse group (n = 8), ■: drug non-administered (control) group (n = 12) , Δ: 4Fbenzoyl-TN-14003 administration group (n = 11)]. E is the effect of 4Fbenzoyl-TN-14003 on hindlimb swelling 2 weeks after boosting [vertical axis: hindlimb weight (mg) (mean ± standard error)], F is anti-bovine II 2 weeks after boosting 4 shows the effect of 4Fbenzoyl-TN-14003 on the type I collagen IgG2a antibody titer [vertical axis: antibody titer (A450) (mean value ± standard error)] [normal mouse group (n = 8) from left, no drug administration ( Control) group (n = 12), 4Fbenzoyl-TN-14003 administration group (n = 11) is shown]. ## indicates P ≦ 0.01 (comparison with normal mouse group; t-test), and ** indicates P ≦ 0.01 (comparison with non-drug-administered group; t-test).

Claims (15)

A peptide represented by 4F-benzoyl-TN14003 (4F-benzoyl-Arg-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ );
Here, 4F-benzoyl on the left of the N-terminal amino acid is a 4-fluorobenzoyl group, Arg is an L-arginine residue, Nal is an L-3- (2-naphthyl) alanine residue, and Cys is L-cysteine. Tyr represents an L-tyrosine residue, Cit represents an L-citrulline residue, Lys represents an L-lysine residue, DLys represents a D-lysine residue, and Pro represents an L-proline residue. Two cysteine residues are linked by an intramolecular disulfide bond, indicating that the NH _{2 to} the right of the C-terminal amino acid is amidated at the amino group;
Or a salt thereof.   A pharmaceutical comprising any of the peptides according to claim 1 or a salt thereof. A CXCR4 antagonist comprising any of the peptides according to claim 1 or a salt thereof. The pharmaceutical product according to claim 2 for prevention and / or treatment of cancer or rheumatoid arthritis. The pharmaceutical product according to claim 4, which is used for breast cancer or pancreatic cancer. The pharmaceutical product according to claim 4, which is used for breast cancer, spleen cancer and leukemia expressing CXCR4 receptor. The pharmaceutical product according to claim 4, which is used for rheumatoid arthritis. The pharmaceutical product according to claim 4, which is used for inhibiting cancer metastasis.   Use of the peptide or its salt of Claim 1 for manufacturing the preventive and / or therapeutic agent of cancer or rheumatoid arthritis.   The use according to claim 9, wherein the preventive and / or therapeutic agent is used for the treatment of cancer except pancreatic cancer.   The use according to claim 9, wherein the prophylactic and / or therapeutic agent is used for breast cancer, spleen cancer, pancreatic cancer and leukemia expressing CXCR4 receptor.   The use according to claim 9, wherein the preventive and / or therapeutic agent is used for breast cancer, spleen cancer and leukemia expressing CXCR4 receptor.   Use according to claim 9, wherein the preventive and / or therapeutic agent is used for rheumatoid arthritis.   Use according to claim 9, wherein the prophylactic and / or therapeutic agent is used for the inhibition of cancer metastasis. 4F-benzoyl-TN14003 (4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys- for the manufacture of prophylactic and / or therapeutic agents for inhibiting delayed type hypersensitivity reactions (DTH) Peptide represented by Pro-Tyr-Arg-Cit-Cys-Arg-NH ₂ );
Here, 4F-benzoyl on the left of the N-terminal amino acid is a 4-fluorobenzoyl group, Arg is an L-arginine residue, Nal is an L-3- (2-naphthyl) alanine residue, and Cys is L-cysteine. Tyr represents an L-tyrosine residue, Cit represents an L-citrulline residue, Lys represents an L-lysine residue, DLys represents a D-lysine residue, and Pro represents an L-proline residue. Two cysteine residues are linked by an intramolecular disulfide bond, indicating that the NH _{2 to} the right of the C-terminal amino acid is amidated at the amino group;
Or use of its salts.

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