U.S. patent application number 11/467237 was filed with the patent office on 2007-10-04 for gastrin releasing peptide compounds.
This patent application is currently assigned to BRACCO IMAGING S.P.A.. Invention is credited to Enrico Cappelletti, Luciano Lattuada, Karen E. Linder, Edmund R. Marinelli, Palaniappa Nanjappan, Natarajan Raju, Rolf E. Swenson, Michael Tweedle.
Application Number | 20070231257 11/467237 |
Document ID | / |
Family ID | 32711539 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070231257 |
Kind Code |
A1 |
Cappelletti; Enrico ; et
al. |
October 4, 2007 |
Gastrin Releasing Peptide Compounds
Abstract
New and improved compounds for use in radiodiagnostic imaging or
radiotherapy having the formula M-N--O--P-G, wherein M is the metal
chelator (in the form complexed with a metal radionuclide or not),
N--O--P is the linker, and G is the GRP receptor targeting peptide.
Methods for imaging a patient and/or providing radiotherapy to a
patient using the compounds of the invention are also provided. A
method for preparing a diagnostic imaging agent from the compound
is further provided. A method for preparing a radiotherapeutic
agent is further provided.
Inventors: |
Cappelletti; Enrico; (Sergno
(MI), IT) ; Lattuada; Luciano; (Bussero (MI), IT)
; Linder; Karen E.; (Kingston, NJ) ; Marinelli;
Edmund R.; (Lawrenceville, NJ) ; Nanjappan;
Palaniappa; (Princeton, NJ) ; Raju; Natarajan;
(Kendall Park, NJ) ; Swenson; Rolf E.; (Princeton,
NJ) ; Tweedle; Michael; (Princeton, NJ) |
Correspondence
Address: |
KRAMER LEVIN NAFTALIS & FRANKEL LLP
INTELLECTUAL PROPERTY DEPARTMENT
1177 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Assignee: |
BRACCO IMAGING S.P.A.
Via Egidio Folli 50
Milan
IT
|
Family ID: |
32711539 |
Appl. No.: |
11/467237 |
Filed: |
August 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10341577 |
Jan 13, 2003 |
7226577 |
|
|
11467237 |
Aug 25, 2006 |
|
|
|
Current U.S.
Class: |
424/1.69 ;
530/309 |
Current CPC
Class: |
C07K 7/086 20130101;
A61P 43/00 20180101; A61P 1/18 20180101; A61P 35/00 20180101; A61K
51/088 20130101; A61P 13/08 20180101 |
Class at
Publication: |
424/001.69 ;
530/309 |
International
Class: |
A61K 51/08 20060101
A61K051/08; C07K 14/595 20060101 C07K014/595 |
Claims
1-68. (canceled)
69. A compound of the general formula: M-N--O--P-G wherein M is a
metal chelator, optionally complexed with a radionuclide; N is 0,
an alpha or non-alpha amino acid or other linking group; O is an
alpha or non-alpha amino acid; and P is 0, an alpha or non-alpha
amino acid or other linking group, and G is a GRP receptor
targeting peptide, wherein at least one of N, O or P is a non-alpha
amino acid.
70. The compound of claim 69, wherein G is an agonist.
71. The compound of claim 69, wherein the non-alpha amino acid is
selected from the group consisting of: 8-amino-3,6-dioxaoctanoic
acid; N-4-aminoethyl-N-1-piperazine-acetic acid; and polyethylene
glycol derivatives having the formula
NH.sub.2--(CH.sub.2CH.sub.2O)n-CH.sub.2CO.sub.2H or
NH.sub.2--(CH.sub.2CH.sub.2O)n-CH.sub.2CH.sub.2CO.sub.2H where n=2
to 100.
72. The compound of claim 69, wherein the metal chelator is
selected from the group consisting of DTPA, DOTA, DO3A, HP-DO3A,
EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM,
MECAM, CMDOTA and derivatives thereof.
73. The compound of claim 72, wherein M is selected from the group
consisting of EHPG and derivatives thereof.
74. The compound of claim 73, wherein M is selected from selected
from the group consisting of 5-Cl-EHPG, 5-Br-EHPG, 5-Me-EHPG,
5-t-Bu-EHPG, and 5-sec-Bu-EHPG.
75. The compound of claim 72, wherein M is selected from the group
consisting of benzodiethylenetriamine pentaacetic acid (benzo-DTPA)
and derivatives thereof.
76. The compound of claim 75, wherein M is selected from the group
consisting of dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA,
benzyl-DTPA, and dibenzyl DTPA.
77. The compound of claim 72, wherein M is selected from the group
consisting of HBED and derivatives thereof.
78. The compound of claim 72, wherein M is selected from the group
consisting of benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, benzo-TETA,
benzo-DOTMA, and benzo-TETMA.
79. The compound of claim 72, wherein M is selected from the group
consisting of derivatives of 1,3-propylenediaminetetraacetic acid
(PDTA) and triethylenetetraaminehexaacetic acid (TTHA); derivatives
of 1,5,10-N,N',N''-tris(2,3-dihydroxybenzoyl)-tricatecholate
(LICAM) and 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl)
aminomethylbenzene (MECAM).
80. The compound of claim 69, wherein M is selected from the group
consisting of: N,N-dimethylGly-Ser-Cys; N,N-dimethylGly-Thr-Cys;
N,N-diethylGly-Ser-Cys; and N,N-dibenzylGly-Ser-Cys.
81. The compound of claim 69, wherein M is selected from the group
consisting of: N,N-dimethylGly-Ser-Cys-Gly;
N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly; and
N,N-dibenzylGly-Ser-Cys-Gly.
82. The compound of claim 69, selected from the group consisting
of:
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Lys-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Arg-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Asp-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Ser-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Gly-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Glu-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Dala-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Lys-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Asp-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Ser-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Glu-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Dala-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO:
1; N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-2,3-diaminopropionic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-2,3-diaminopropionic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Asp-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Asp-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Ser-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Arg-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-2,3-diaminopropionic acid-Gly-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Lys-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys(Acm)-Gly-2,3-diaminopropionic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Asp-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Ser-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-2,3-diaminopropionic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Lys-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-1-piperazineacetic
acid-Asp-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-1-piperazineacetic
acid-Ser-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-1-piperazineacetic
acid-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-1-piperazineacetic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-1-piperazineacetic
acid-2,3-diaminopropionic acid BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-1-piperazineacetic
acid-Lys-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-4-Hydroxyproline-8-amino-3,6-dioxaoc-
tanoic acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO:
1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-4-aminoproline-8-amino-3,6-dioxaocta-
noic acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO:
1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Lys-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Arg-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Ser-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-Asp-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Asp-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Ser-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Arg-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-2,3-diaminopropionic acid-Gly-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-8-amino-3,6-dioxaoctanoic
acid-Lys-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; and
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-2,3-diaminopropionic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1.
83. The compound of claim 69, selected from the group consisting
of: N,N-dimethylglycine-Ser-Cys-Gly-Lys-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Arg-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Asp-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Ser-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Gly-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Glu-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Dala-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Lys-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Asp-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Ser-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Glu-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Dala-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO:
1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-2,3-diaminopropionic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-2,3-diaminopropionic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Asp-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Asp-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Ser-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Arg-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-2,3-diaminopropionic acid-Gly-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Lys-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-2,3-diaminopropionic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Asp-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Ser-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys(Acm)-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-2,3-diaminopropionic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-4-aminoethyl-N-1-piperazineacetic
acid-Lys-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-1-piperazineacetic
acid-Asp-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-1-piperazineacetic
acid-Ser-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-1-piperazineacetic
acid-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-1-piperazineacetic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-1-piperazineacetic
acid-2,3-diaminopropionic acid BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-N-1-piperazineacetic
acid-Lys-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-4-Hydroxyproline-8-amino-3,6-dioxaoctanoi-
c acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-4-aminoproline-8-amino-3,6-dioxaoctanoic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Lys-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Arg-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Ser-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-Asp-8-amino-3,6-dioxaoctanoic
acid-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Asp-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Ser-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Arg-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-2,3-diaminopropionic acid-Gly-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1;
N,N-dimethylglycine-Ser-Cys-Gly-8-amino-3,6-dioxaoctanoic
acid-Lys-Gly-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID
NO: 1; and N,N-dimethylglycine-Ser-Cys-Gly-2,3-diaminopropionic
acid-8-amino-3,6-dioxaoctanoic acid-Gly-BBN(7-14) wherein the
BBN(7-14) sequence is SEQ. ID NO: 1.
84. The compound of claim 69, selected from the group consisting
of: DO3A-monoamide-8-amino-3,6-dioxaoctanoic acid-diaminopropionic
acid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;
DO3A-monoamide-8-amino-3,6-dioxaoctanoic
acid-biphenylalanine-BBN(7-14) wherein the BBN(7-14) sequence is
SEQ. ID NO: 1; DO3A-monoamide-8-amino-3,6-dioxaoctanoic
acid-diphenylalanine-BBN(7-14) wherein the BBN(7-14) sequence is
SEQ. ID NO: 1; DO3A-monoamide-8-amino-3,6-dioxaoctanoic
acid-4-benzoylphenylalanine-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1; DO3A-monoamide-5-aminopentanoic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1; DO3A-monoamide-8-amino-3,6-dioxaoctanoic
acid-D-phenylalanine-BBN(7-14) wherein the BBN(7-14) sequence is
SEQ. ID NO: 1; and DO3A-monoamide-8-aminooctanoic
acid-8-amino-3,6-dioxaoctanoic acid-BBN(7-14) wherein the BBN(7-14)
sequence is SEQ. ID NO: 1.
85. A method of imaging comprising the steps of: administering to a
patient a diagnostic imaging agent comprising the compound of claim
69 complexed with a diagnostic radionuclide, and imaging said
patient.
86. A method of imaging comprising the steps of: administering to a
patient a diagnostic imaging agent comprising the compound of any
one of claims 81-84 complexed with a diagnostic radionuclide, and
imaging said patient.
87. A method for preparing a diagnostic imaging agent comprising
the step of adding to an injectable medium a substance comprising
the compound of claim 69.
88. A method of treating a patient in need of radiotherapy
comprising the step of administering to a patient a
radiotherapeutic agent comprising the compound of claim 69
complexed with a therapeutic radionuclide.
89. A method of treating a patient in need of radiotherapy
comprising the step of administering to a patient a
radiotherapeutic agent comprising the compound of any one of claims
81-84 complexed with a therapeutic radionuclide.
90. A method of preparing a radiotherapeutic agent comprising the
step of adding to an injectable medium a substance comprising the
compound of claim 69.
91. A method of preparing a radiotherapeutic agent comprising the
step of adding to an injectable medium a substance comprising the
compound of any one of claims 81-84.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel radionuclide-labeled gastrin
releasing peptide (GRP) compounds which are useful as diagnostic
imaging agents or radiotherapeutic agents. These GRP compounds
include the use of novel linkers between a metal chelator and the
targeting peptide, which provides for improved
pharmacokinetics.
BACKGROUND OF THE INVENTION
[0002] The use of radiopharmaceuticals (e.g., diagnostic imaging
agents, radiotherapeutic agents) to detect and treat cancer is well
known. In more recent years, the discovery of site-directed
radiopharmaceuticals for cancer detection and/or treatment has
gained popularity and continues to grow as the medical profession
better appreciates the specificity, efficacy and utility of such
compounds.
[0003] These newer radiopharmaceutical agents typically consist of
a targeting agent connected to a metal chelator, which can be
chelated to (e.g., complexed with) a diagnostic metal radionuclide
such as, for example, technetium or indium, or a therapeutic metal
radionuclide such as, for example, lutetium, yttrium, or rhenium.
The role of the metal chelator is to hold (i.e., chelate) the metal
radionuclide as the radiopharmaceutical agent is delivered to the
desired site. A metal chelator which does not bind strongly to the
metal radionuclide would render the radiopharmaceutical agent
ineffective for its desired use since the metal radionuclide would
therefore not reach its desired site. Thus, further research and
development led to the discovery of metal chelators, such as that
reported in U.S. Pat. No. 5,662,885 to Pollak et. al., hereby
incorporated by reference, which exhibited strong binding affinity
for metal radionuclides and the ability to conjugate with the
targeting agent. Subsequently, the concept of using a "spacer" to
create a physical separation between the metal chelator and the
targeting agent was further introduced, for example in U.S. Pat.
No. 5,976,495 to Pollak et. al., hereby incorporated by
reference.
[0004] The role of the targeting agent, by virtue of its affinity
for certain binding sites, is to direct the radiopharmaceutical
agent containing the metal radionuclide to the desired site for
detection or treatment. Typically, the targeting agent may include
a protein, a peptide, or other macromolecule which exhibits a
specific affinity for a given receptor. Other known targeting
agents include monoclonal antibodies (MAbs), antibody fragments
(F.sub.ab's and (F.sub.ab).sub.2's), and receptor-avid peptides.
Donald J. Buchsbaum, "Cancer Therapy with Radiolabeled Antibodies;
Pharmacokinetics of Antibodies and Their Radiolabels; Experimental
Radioimmunotherapy and Methods to Increase Therapeutic Efficacy,"
CRC Press, Boca Raton, Chapter 10, pp. 115-140, (1995); Fischman,
et al. "A Ticket to Ride: Peptide Radiopharmaceuticals," The
Journal of Nuclear Medicine, vol. 34, No. 12, (December 1993).
[0005] In recent years, it has been learned that some cancer cells
contain gastrin releasing peptide (GRP) receptors (GRP-R) of which
there are a number of subtypes. In particular, it has been shown
that several types of cancer cells have over-expressed or uniquely
expressed GRP receptors. For this reason, much research and study
have been done on GRP and GRP analogues which bind to the GRP
receptor family. One such analogue is bombesin (BBN), a 14 amino
acid peptide (i.e., tetradecapeptide) isolated from frog skin which
is an analogue of human GRP and which binds to GRP receptors with
high specificity and with an affinity similar to GRP.
[0006] Bombesin and GRP analogues may take the form of agonists or
antagonists. Binding of GRP or BBN agonists to the GRP receptor
increases the rate of cell division of these cancer cells and such
agonists are internalized by the cell, while binding of GRP or BBN
antagonists generally does not result in either internalization by
the cell or increased rates of cell division. Such antagonists are
designed to competitively inhibit endogenous GRP binding to GRP
receptors and reduce the rate of cancer cell proliferation. See,
e.g., Hoffken, K.; Peptides in Oncology II, Somatostatin Analogues
and Bombesin Antagonists (1993), pp. 87-112. For this reason, a
great deal of work has been, and is being pursued to develop BBN or
GRP analogues that are antagonists. E.g., Davis et al., Metabolic
Stability and Tumor Inhibition of Bombesin/GRP Receptor
Antagonists, Peptides, vol. 13, pp. 401-407, 1992.
[0007] In designing an effective radiopharmaceutical compound for
use as a diagnostic or therapeutic agent for cancer, it is
important that the drug have appropriate in vivo targeting and
pharmacokinetic properties. For example, it is preferable that the
radiolabeled peptide have high specific uptake by the cancer cells
(e.g., via GRP receptors). In addition, it is also preferred that
once the radionuclide localizes at a cancer site, it remains there
for a desired amount of time to deliver a highly localized
radiation dose to the site.
[0008] Moreover, developing radiolabeled peptides that are cleared
efficiently from normal tissues is also an important factor for
radiopharmaceutical agents. When biomolecules (e.g., MAb, F.sub.ab
or peptides) labeled with metallic radionuclides (via a chelate
conjugation), are administered to an animal such as a human, a
large percentage of the metallic radionuclide (in some chemical
form) can become "trapped" in either the kidney or liver parenchyma
(i.e., is not excreted into the urine or bile). Duncan et al.;
Indium-111-Diethylenetriaminepentaacetic Acid-Octreotide Is
Delivered in Vivo to Pancreatic, Tumor Cell, Renal, and Hepatocyte
Lysosomes, Cancer Research 57, pp. 659-671, (Feb. 15, 1997). For
the smaller radiolabeled biomolecules (i.e., peptides or F.sub.ab),
the major route of clearance of activity is through the kidneys
which can also retain high levels of the radioactive metal (i.e.,
normally >10-15% of the injected dose). Retention of metal
radionuclides in the kidney or liver is clearly undesirable.
Conversely, clearance of the radiopharmaceutical from the blood
stream too quickly by the kidney is also undesirable if longer
diagnostic imaging or high tumor uptake for radiotherapy is
needed.
[0009] Subsequent work, such as that in U.S. Pat. No. 6,200,546 and
US 2002/0054855 to Hoffman, et. al, hereby incorporated by
reference, has attempted to overcome this problem by forming a
compound having the general formula X-Y-B wherein X is a group
capable of complexing a metal, Y is a covalent bond on a spacer
group and B is a bombesin agonist binding moiety. Such compounds
were reported to have high binding affinities to GRP receptors, and
the radioactivity was retained inside of the cells for extended
time periods. In addition, in vivo studies in normal mice have
shown that retention of the radioactive metal in the kidneys was
lower than that known in the art, with the majority of the
radioactivity excreted into the urine.
[0010] New and improved radiopharmaceutical compounds which have
improved pharmacokinetics and improved kidney excretion (i.e.,
lower retention of the radioactive metal in the kidney) have now
been found for diagnostic imaging and therapeutic uses. For
diagnostic imaging, rapid renal excretion and low retained levels
of radioactivity are critical for improved images. For
radiotherapeutic use, slower blood clearance to allow for higher
tumor uptake and better tumor targeting with low kidney retention
are critical.
SUMMARY OF THE INVENTION
[0011] In an embodiment of the present invention, there is provided
new and improved compounds for use in radiodiagnostic imaging or
radiotherapy. The compounds include a chemical moiety capable of
complexing a medically useful metal ion or radionuclide (metal
chelator) attached to a GRP receptor targeting peptide by a linker
or spacer group.
[0012] In general, compounds of the present invention may have the
formula: M-N--O--P-G
[0013] wherein M is the metal chelator (in the form complexed with
a metal radionuclide or not), N--O--P is the linker, and G is the
GRP receptor targeting peptide.
[0014] The metal chelator M may be any of the metal chelators known
in the art for complexing with a medically useful metal ion or
radionuclide. Preferred chelators include DTPA, DOTA, DO3A,
HP-DO3A, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA,
LICAM, MECAM, or peptide chelators, such as, for example, those
discussed herein. The metal chelator may or may not be complexed
with a metal radionuclide, and may include an optional spacer such
as a single amino acid. Preferred metal radionuclides for
scintigraphy or radiotherapy include .sup.99mTc, .sup.51Cr,
.sup.67Ga, .sup.68Ga, .sup.47Sc, .sup.51C .sup.167Tm, .sup.141Ce,
.sup.168Yb, .sup.175Yb, .sup.140La, .sup.90Y, .sup.88Y, .sup.153S,
.sup.166Ho, .sup.165Dy, .sup.166Dy, .sup.62Cu, .sup.64Cu,
.sup.67Cu, .sup.97Ru, .sup.103Ru, .sup.186Re, .sup.188Re,
.sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi, .sup.214Bi,
.sup.105Rh, .sup.109Pd, .sup.117mSn, .sup.149Pm, .sup.161Tb,
.sup.177Lu, .sup.198Au and .sup.199Au. The choice of metal will be
determined based on the desired therapeutic or diagnostic
application. For example, for diagnostic purposes the preferred
radionuclides include .sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.99mTc,
and .sup.111In, with .sup.99mTc, and .sup.111In being particularly
preferred. For therapeutic purposes, the preferred radionuclides
include .sup.64Cu, .sup.90Y, .sup.105Rh .sup.111In, .sup.117mSn,
.sup.149Pm, .sup.153Sm, .sup.161Tb, .sup.166Dy, .sup.166Ho,
.sup.175Yb, .sup.177Lu, .sup.186/188Re, and .sup.199Au, with
.sup.177Lu and .sup.90Y being particularly preferred. A most
preferred chelator used in compounds of the invention is
1-substituted 4,7,10-tricarboxymethyl 1,4,7,10
tetraazacyclododecane triacetic acid (DO3A).
[0015] In one embodiment, the linker N--O--P contains at least one
non-alpha amino acid.
[0016] In another embodiment, the linker N--O--P contains at least
one substituted bile acid.
[0017] In yet another embodiment, the linker N--O--P contains at
least one non-alpha amino acid with a cyclic group.
[0018] The GRP receptor targeting peptide may be GRP, bombesin or
any derivatives or analogues thereof In a preferred embodiment, the
GRP receptor targeting peptide is a GRP or bombesin analogue which
acts as an agonist. In a particularly preferred embodiment, the GRP
receptor targeting peptide is a bombesin agonist binding moiety
disclosed in U.S. Pat. No. 6,200,546 and US 2002/0054855,
incorporated herein by reference.
[0019] There is also provided a novel method of imaging using the
compounds of the present invention.
[0020] There is further provided a novel method for preparing a
diagnostic imaging agent comprising the step of adding to an
injectable imaging medium a substance containing the compounds of
the present invention.
[0021] A novel method of radiotherapy using the compounds of the
invention is also provided, as is a novel method for preparing a
radiotherapeutic agent comprising the step of adding to an
injectable therapeutic medium a substance comprising a compound of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A is a graphical representation of a series of
chemical reactions for the synthesis of intermediate C
((3.beta.,5.beta.)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic
acid), from A (Methyl-(3.beta.,5.beta.)-3-aminocholan-24-ate) and B
((3.beta.,5.beta.)-3-aminocholan-24-oic acid), as described in
Example I;
[0023] FIG. 1B is a graphical representation of the sequential
reaction for the synthesis of
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]cholan-24-yl]-L-glutaminyl-L-tryp-
tophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L62), as described in Example I;
[0024] FIG. 2A is a graphical representation of the sequential
reaction for the synthesis of
N-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-
-glycyl-L-histidyl-L-leucyl-L-methioninamide (L70), as described in
Example II;
[0025] FIG. 2B is a general graphical representation of the
sequential reaction for the synthesis of
N-[4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ac-
etyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glyc-
yl-L-histidyl-L-leucyl-L-methioninamide (L73),
N-[3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-
-L-histidyl-L-leucyl-L-methioninamide (L115), and
N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-g-
lycyl-L-histidyl-L-leucyl-L-methioninamide (L116), as described in
Example II;
[0026] FIG. 2C is a chemical structure of the linker used in the
synthesis reaction of FIG. 2B for synthesis of
N-[4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ac-
etyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glyc-
yl-L-histidyl-L-leucyl-L-methioninamide (L73), as described in
Example II;
[0027] FIG. 2D is a chemical structure of the linker used in the
synthesis reaction of FIG. 2B for synthesis of
N-[3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-
-L-histidyl-L-leucyl-L-methioninamide (L115), as described in
Example II;
[0028] FIG. 2E is a chemical structure of the linker used in the
synthesis reaction of FIG. 2B for synthesis of
N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-g-
lycyl-L-histidyl-L-leucyl-L-methioninamide (L116), as described in
Example II;
[0029] FIG. 2F is a graphical representation of the sequential
reaction for the synthesis of
N-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]gl-
ycyl-4-piperidinecarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycy-
l-L-histidyl-L-leucyl-L-methioninamide (L74), as described in
Example II;
[0030] FIG. 3A is a graphical representation of a series of
chemical reactions for the synthesis of intermediate
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxoch-
olan-24-oic acid (C), as described in Example III;
[0031] FIG. 3B is a graphical representation of the sequential
reaction for the synthesis of
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-gluta-
minyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionin-
amide (L67), as described in Example III;
[0032] FIG. 3C is a chemical structure of
(3.beta.,5.beta.)-3-Amino-12-oxocholan-24-oic acid (B), as
described in Example III;
[0033] FIG. 3D is a chemical structure of
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxoch-
olan-24-oic acid (C), as described in Example III;
[0034] FIG. 3E is a chemical structure of
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-gluta-
minyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionin-
amide (L67), as described in Example III;
[0035] FIG. 4A is a graphical representation of a sequence of
reactions to obtain intermediates
(3.beta.,5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amin-
o-12-hydroxy cholan-24-oic acid (3a) and
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as described in
Example IV;
[0036] FIG. 4B is a graphical representation of the sequential
reaction for the synthesis of
N-[(3.beta.,5.beta.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxochola-
n-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-le-
ucyl-L-methioninamide (L63), as described in Example IV;
[0037] FIG. 4C is a graphical representation of the sequential
reaction for the synthesis of
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-
-1,4,7,10-tetraazacyclo
dodec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]--
L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-me-
thioninamide (L64), as described in Example IV;
[0038] FIG. 4D is a chemical structure of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid (2b), as described in Example IV;
[0039] FIG. 4E is a chemical structure of
(3.beta.,5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amin-
o-12-hydroxycholan-24-oic acid (3a), as described in Example
IV;
[0040] FIG. 4F is a chemical structure of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as described in
Example IV;
[0041] FIG. 4G is a chemical structure of
N-[(3.beta.,5.beta.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxochola-
n-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-le-
ucyl-L-methioninamide (L63), as described in Example IV;
[0042] FIG. 4H is a chemical structure of
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-
-1,4,7,10-tetraazacyclo
dodec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]--
L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-me-
thioninamide (L64), as described in Example IV;
[0043] FIG. 5A is a general graphical representation of the
sequential reaction for the synthesis of
4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]-
amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-h-
istidyl-L-leucyl-L-methioninamide (L71); and
Trans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]a-
cetyl]amino]methyl]cyclohexylcarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-
-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L72) as
described in Example V;
[0044] FIG. 5B is a chemical structure of the linker used in
compound L71 as shown in FIG. 5A and as described in Example V;
[0045] FIG. 5C is a chemical structure of the linker used in
compound L72 as shown in FIG. 5B and as described in Example V;
[0046] FIG. 5D is a chemical structure of Rink amide resin
functionalised with bombesin[7-14] (B), as described in Example
V;
[0047] FIG. 5E is a chemical structure of
Trans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cyclohexanecarbo-
xylic acid (D), as described in Example V;
[0048] FIG. 6A is a graphical representation of a sequence of
reactions for the synthesis of intermediate linker
2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid (E),
as described in Example VI;
[0049] FIG. 6B is a graphical representation of a sequence of
reactions for the synthesis of intermediate linker
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic
acid (H), as described in Example VI;
[0050] FIG. 6C is a graphical representation of the synthesis of
N-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,1
0-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-try-
ptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L75), as described in Example VI;
[0051] FIG. 6D is a graphical representation of the synthesis of
N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,1
0-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-nitrobenzoyl]-L-glutamin-
yl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninami-
de (L76), as described in Example VI;
[0052] FIG. 6E is a chemical structure of
2-[(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid (C),
as described in Example VI;
[0053] FIG. 6F is a chemical structure of 2-(aminomethyl)benzoic
acid (D), as described in Example VI;
[0054] FIG. 6G is a chemical structure of
2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid (E),
as described in Example VI;
[0055] FIG. 6H is a chemical structure of
4-(aminomethyl)-3-nitrobenzoic acid (G), as described in Example
VI;
[0056] FIG. 6I is a chemical structure of
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic
acid (H), as described in Example VI;
[0057] FIG. 6J is a chemical structure of
N-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-
-L-histidyl-L-leucyl-L-methioninamide (L75), as described in
Example VI;
[0058] FIG. 6K is a chemical structure of
N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valy-
l-glycyl-L-histidyl-L-leucyl-L-methioninamide (L76), as described
in Example VI;
[0059] FIG. 7A is a graphical representation of a sequence of
reactions for the synthesis of intermediate linker
[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic
acid (E), as described in Example VII;
[0060] FIG. 7B is a graphical representation of the synthesis of
N-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-triptophyl-L-alanyl-L-valy-
l-glycyl-L-histidyl-L-leucyl-L-methioninamide (L124), as described
in Example VII;
[0061] FIG. 7C is a chemical structure of (4-Cyanophenoxy)acetic
acid ethyl ester (B), as described in Example VII;
[0062] FIG. 7D is a chemical structure of (4-Cyanophenoxy)acetic
acid (C), as described in Example VII;
[0063] FIG. 7E is a chemical structure of
[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic
acid (E), as described in Example VII;
[0064] FIG. 7F is a chemical structure of
N-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valy-
l-glycyl-L-histidyl-L-leucyl-L-methioninamide (L124), as described
in Example VII;
[0065] FIG. 8A is a graphical representation of a sequence of
reactions for the synthesis of intermediate
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic
acid (E), as described in Example VIII;
[0066] FIG. 8B is a graphical representation of the synthesis of
N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-va-
lyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L125), as
described in Example VIII;
[0067] FIG. 8C is a chemical structure of
4-(Azidomethyl)-3-methoxybenzoic acid methyl ester, (B), as
described in Example VIII;
[0068] FIG. 8D is a chemical structure of
4-(Aminomethyl)-3-methoxybenzoic acid methyl ester, (C), as
described in Example VIII;
[0069] FIG. 8E is a chemical structure of
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic
acid, (E), as described in Example VIII;
[0070] FIG. 8F is a chemical structure of
N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acet-
yl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-va-
lyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L125), as
described in Example VIII;
[0071] FIG. 9A and FIG. 9B are graphical representations of the
binding and competition curves described in Example X;
[0072] FIG. 10A is a graphical representation of the results of
radiotherapy experiments described in Example XXI;
[0073] FIG. 10B is a graphical representation of the results of
other radiotherapy experiments described in Example XXI;
[0074] FIG. 11 is a chemical structure of
DO3A-monoamide-Gly-Lys-(3,6,9)-trioxaundecane-1,11-dicarboxylic
acid-3,7-dideoxy-3-aminocholic
acid)-L-arginyl-L-glutaminyl-L-triptophyl-L-alanyl-L-valyl-glycyl-L-histi-
dyl-L-leucyl-L-methioninamide (L65);
[0075] FIG. 12 is a chemical structure of
N-[2-S-[[[[[12.alpha.-Hydroxy-17.beta.-(1-methyl-3-carboxypropyl)etiochol-
an-3.beta.-carbamoylmethoxyethoxyethoxyacetyl]-amino-6-[4,7,10-tris(carbox-
ymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]hexano-
yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
-methioninamide (L66);
[0076] FIG. 13A is a chemical structure of
N-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-
-glycyl-L-histidyl-L-leucyl-L-methioninamide (L70);
[0077] FIG. 13B is a chemical structure
N-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-3--
carboxypropionyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-a-
lanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L114);
[0078] FIG. 13C is a chemical structure
N-[4-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-2-hyd-
roxy-3-propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl--
L-histidyl-L-leucyl-L-methioninamide (L144);
[0079] FIG. 13D is a chemical structure
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[[4,7,10-Tris(carboxymethyl-
)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxyethoxy]acetyl]amino]-
-7,12-dihydroxycholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-gl-
ycyl-L-histidyl-L-leucyl-L-methioninamide (L69); and
[0080] FIG. 13E is a chemical structure
N-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]acetyl]amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L--
valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L146).
[0081] FIG. 14 discloses chemical structures of intermediates which
may be used to prepare compounds L64 and L70 as described in
Example XXII.
[0082] FIG. 15 is a graphical representation of the preparation of
L64 using segment coupling as described in Example XXII.
DETAILED DESCRIPTION OF THE INVENTION
[0083] In the following description, various aspects of the present
invention will be further elaborated. For purposes of explanation,
specific configurations and details are set forth in order to
provide a thorough understanding of the present invention. However,
it will also be apparent to one skilled in the art that the present
invention may be practiced without the specific details.
Furthermore, well known features may be omitted or simplified in
order not to obscure the present invention.
[0084] In an embodiment of the present invention, there is provided
a new and improved compound for use in radiodiagnostic imaging or
radiotherapy. The compound includes a chemical moiety capable of
complexing a medically useful metal ion or radionuclide (metal
chelator) attached to a GRP receptor targeting peptide by a linker
or spacer group.
[0085] In general, compounds of the present invention may have the
formula: M-N--O--P-G wherein M is the metal chelator (in the form
complexed with a metal radionuclide or not), N--O--P is the linker,
and G is the GRP receptor targeting peptide. Each of the metal
chelator, linker, and GRP receptor targeting peptide is described
in the discussion that follow.
[0086] In another embodiment of the present invention, there is
provided a new and improved linker or spacer group which is capable
of linking a metal chelator to a GRP receptor targeting peptide. In
general, linkers of the present invention may have the formula:
N--O--P wherein each of N, O and P are defined throughout the
specification.
[0087] Compounds meeting the criteria defined herein were
discovered to have improved pharmacokinetic properties compared to
other radiolabeled GRP receptor targeting peptide conjugates known
in the art. For example, compounds containing the linkers of the
present invention were retained in the bloodstream longer, and thus
had a longer half life than prior known compounds. The longer half
life was medically beneficial because it permitted better tumor
targeting which is useful for diagnostic imaging, and especially
for therapeutic uses, where the cancerous cells and tumors receive
greater amounts of the radiolabeled peptides.
1. Metal Chelator
[0088] The term "metal chelator" refers to a molecule that forms a
complex with a metal atom, wherein said complex is stable under
physiological conditions. That is, the metal will remain complexed
to the chelator backbone in vivo. More particularly, a metal
chelator is a molecule that complexes to a radionuclide metal to
form a metal complex that is stable under physiological conditions
and which also has at least one reactive functional group for
conjugation with the linker N--O--P. The metal chelator M may be
any of the metal chelators known in the art for complexing a
medically useful metal ion or radionuclide. The metal chelator may
or may not be complexed with a metal radionuclide. Furthermore, the
metal chelator can include an optional spacer such as a single
amino acid (e.g., Gly) which does not complex with the metal, but
which creates a physical separation between the metal chelator and
the linker.
[0089] The metal chelators of the invention may include, for
example, linear, macrocyclic, terpyridine, and N.sub.3S,
N.sub.2S.sub.2, or N.sub.4 chelators (see also, U.S. Pat. No.
5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556, U.S.
Pat. No. 5,075,099, U.S. Pat. No. 5,886,142, the disclosures of
which are incorporated by reference herein in their entirety), and
other chelators known in the art including, but not limited to,
HYNIC, DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT)
chelators (see also U.S. Pat. No. 5,720,934). For example, N.sub.4
chelators are described in U.S. Pat. Nos. 6,143,274; 6,093,382;
5,608,110; 5,665,329; 5,656,254; and 5,688,487, the disclosures of
which are incorporated by reference herein in their entirety.
Certain N.sub.3S chelators are described in PCT/CA94/00395,
PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. Nos. 5,662,885;
5,976,495; and 5,780,006, the disclosures of which are incorporated
by reference herein in their entirety. The chelator may also
include derivatives of the chelating ligand
mercapto-acetyl-glycyl-glycyl-glycine (MAG3), which contains an
N.sub.3S, and N.sub.2S.sub.2 systems such as MAMA
(monoamidemonoaminedithiols), DADS (N.sub.2S diaminedithiols),
CODADS and the like. These ligand systems and a variety of others
are described in Liu and Edwards, Chem Rev. 1999, 99, 2235-2268 and
references therein, the disclosures of which are incorporated by
reference herein in their entirety.
[0090] The metal chelator may also include complexes containing
ligand atoms that are not donated to the metal in a tetradentate
array. These include the boronic acid adducts of technetium and
rhenium dioximes, such as those described in U.S. Pat. Nos.
5,183,653; 5,387,409; and 5,118,797, the disclosures of which are
incorporated by reference herein, in their entirety.
[0091] Examples of preferred chelators include, but are not limited
to, diethylenetriamine pentaacetic acid (DTPA),
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),
1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10
tetraazacyclododecane triacetic acid (DO3A),
ethylenediaminetetraacetic acid (EDTA), and
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).
Additional chelating ligands are
ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives
thereof, including 5-Cl-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG,
and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid
(benzo-DTPA) and derivatives thereof, including dibenzo-DTPA,
phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl-DTPA; bis-2
(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and
derivatives thereof, the class of macrocyclic compounds which
contain at least 3 carbon atoms, more preferably at least 6, and at
least two heteroatoms (O and/or N), which macrocyclic compounds can
consist of one ring, or two or three rings joined together at the
hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and
benzo-NOTA, where NOTA is 1,4,7-triazacyclononane
N,N',N''-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic
acid), and benzo-TETMA, where TETMA is
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic
acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA)
and triethylenetetraaminehexaacetic acid (TTHA); derivatives of
1,5,10-N,N',N''-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM)
and 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl) aminomethylbenzene
(MECAM). Examples of representative chelators and chelating groups
contemplated by the present invention are described in WO 98/18496,
WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619,
PCT/US98/01473, PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S.
Pat. No. 5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No.
6,143,274, each of which is hereby incorporated by reference in its
entirety.
[0092] Particularly preferred metal chelators include those of
Formula 1, 2 and 3 (for .sup.111In and radioactive lanthanides,
such as, for example .sup.177Lu, .sup.90Y, .sup.153Sm, and
.sup.166Ho) and those of Formula 4, 5 and 6 (for radioactive
.sup.99mTc, .sup.186Re, and .sup.188Re) set forth below. These and
other metal chelating groups are described in U.S. Pat. Nos.
6,093,382 and 5,608,110, which are incorporated by reference herein
in their entirety. Additionally, the chelating group of formula 3
is described in, for example, U.S. Pat. No. 6,143,274; the
chelating group of formula 5 is described in, for example, U.S.
Pat. Nos. 5,627,286 and 6,093,382, and the chelating group of
formula 6 is described in, for example, U.S. Pat. Nos. 5,662,885;
5,780,006; and 5,976,495, all of which are incorporated by
reference. Specific metal chelators of formula 6 include
N,N-dimethylGly-Ser-Cys; N,N-dimethylGly-Thr-Cys;
N,N-diethylGly-Ser-Cys; N,N-dibenzylGly-Ser-Cys; and other
variations thereof. For example, spacers which do not actually
complex with the metal radionuclide such as an extra single amino
acid Gly, may be attached to these metal chelators (e.g.,
N,N-dimethylGly-Ser-Cys-Gly; N,N-dimethylGly-Thr-Cys-Gly;
N,N-diethylGly-Ser-Cys-Gly; N,N-dibenzylGly-Ser-Cys-Gly). Other
useful metal chelators such as all of those disclosed in U.S. Pat.
No. 6,334,996, also incorporated by reference (e.g.,
Dimethylgly-L-t-Butylgly-L-Cys-Gly;
Dimethylgly-D-t-Butylgly-L-Cys-Gly; Dimethylgly-L-t-Butylgly-L-Cys,
etc.)
[0093] Furthermore, sulfur protecting groups such as Acm
(acetamidomethyl), trityl or other known alkyl, aryl, acyl,
alkanoyl, aryloyl, mercaptoacyl and organothiol groups may be
attached to the cysteine amino acid of these metal chelators.
[0094] Additionally, other useful metal chelators include: ##STR1##
##STR2##
[0095] In the above Formulas 1 and 2, R is alkyl, preferably
methyl. In the above Formula 5, X is either CH.sub.2 or O, Y is
either C.sub.1-C.sub.10 branched or unbranched alkyl; Y is aryl,
aryloxy, arylamino, arylaminoacyl; Y is arylalkyl--where the alkyl
group or groups attached to the aryl group are C.sub.1-C.sub.10
branched or unbranched alkyl groups, C.sub.1-C.sub.10 branched or
unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or
polyhydroxy-polyalkoxyalkyl groups, J is C(.dbd.O)--, OC(.dbd.O)--,
SO.sub.2--, NC(.dbd.O)--, NC(.dbd.S)--, N(Y), NC(.dbd.NCH.sub.3)--,
NC(.dbd.NH)--, N.dbd.N--, homopolyamides or heteropolyamines
derived from synthetic or naturally occurring amino acids; all
where n is 1-100. J may also be absent. Other variants of these
structures are described, for example, in U.S. Pat. No. 6,093,382.
In Formula 6, the group S--NHCOCH.sub.3 may be replaced with SH or
S-Z wherein Z is any of the known sulfur protecting groups such as
those described above. Formula 7 illustrates one embodiment of
t-butyl compounds useful as a metal chelator. The disclosures of
each of the foregoing patents, applications and references are
incorporated by reference herein, in their entirety.
[0096] In a preferred embodiment, the metal chelator includes
cyclic or acyclic polyaminocarboxylic acids such as DOTA
(1,4,7,10-tetraazacyclododecane -1,4,7,10-tetraacetic acid), DTPA
(diethylenetriaminepentaacetic acid), DTPA-bismethylamide,
DTPA-bismorpholineamide, DO3A
N-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl,
HP-DO3A, DO3A-monoamide and derivatives thereof.
[0097] Preferred metal radionuclides for scintigraphy or
radiotherapy include .sup.99Tc, .sup.51Cr, .sup.67Ga, .sup.68G,
.sup.47Sc, .sup.51Cr, .sup.167Tm, .sup.141Ce, .sup.111In,
.sup.168Yb, .sup.175Yb, .sup.140La, .sup.90Y, .sup.88Y, .sup.153Sm,
.sup.166Ho, .sup.165Dy, .sup.166Dy, .sup.62Cu, .sup.64Cu,
.sup.67Cu, .sup.97Ru, .sup.103Ru, .sup.186Re, .sup.188Re,
.sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi, .sup.214Bi,
.sup.105Rh, .sup.109Pd, .sup.117mSn, .sup.149Pm, .sup.161Tb,
.sup.177Lu, .sup.198Au and .sup.199Au and oxides or nitrides
thereof The choice of metal will be determined based on the desired
therapeutic or diagnostic application. For example, for diagnostic
purposes (e.g., to diagnose and monitor therapeutic progress in
primary tumors and metastases), the preferred radionuclides include
.sup.64Cu, .sup.67Ga, .sup.67Ga, .sup.99mTc, and .sup.111In, with
.sup.99mTc and .sup.111In being especially preferred. For
therapeutic purposes (e.g., to provide radiotherapy for primary
tumors and metastasis related to cancers of the prostate, breast,
lung, etc.), the preferred radionuclides include .sup.64Cu,
.sup.90Y, .sup.105Rh, .sup.111In, .sup.117mSn, .sup.149Pm,
.sup.153Sm, .sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.175Yb,
.sup.177Lu, .sup.186/188Re, and .sup.199Au, with .sup.177Lu and
.sup.90Y being particularly preferred. .sup.99mTc is particularly
useful and is a preferred for diagnostic radionuclide because of
its low cost, availability, imaging properties, and high specific
activity. The nuclear and radioactive properties of .sup.99mTc make
this isotope an ideal scintigraphic imaging agent. This isotope has
a single photon energy of 140 keV and a radioactive half-life of
about 6 hours, and is readily available from a .sup.99Mo-.sup.99mTc
generator. For example, the .sup.99mTc labeled peptide can be used
to diagnose and monitor therapeutic progress in primary tumors and
metastases. Peptides labeled with .sup.177Lu, .sup.90Y or other
therapeutic radionuclides can be used to provide radiotherapy for
primary tumors and metastasis related to cancers of the prostate,
breast, lung, etc.
2A. Linkers Containing at Least One Non-Alpha Amino Acid
[0098] In one embodiment of the invention, the linker N--O--P
contains at least one non-alpha amino acid. Thus, in this
embodiment of the linker N--O--P,
[0099] N is 0 (where 0 means it is absent), an alpha or non-alpha
amino acid or other linking group;
[0100] O is an alpha or non-alpha amino acid; and
[0101] P is 0, an alpha or non-alpha amino acid or other linking
group,
[0102] wherein at least one of N, O or P is a non-alpha amino acid.
Thus, in one example, N=Gly, O=a non-alpha amino acid, and P=0.
[0103] Alpha amino acids are well known in the art, and include
naturally occurring and synthetic amino acids.
[0104] Non-alpha amino acids also include those which are naturally
occurring or synthetic. Preferred non-alpha amino acids include:
[0105] 8-amino-3,6-dioxaoctanoic acid; [0106]
N-4-aminoethyl-N-1-acetic acid; and [0107] polyethylene glycol
derivatives having the formula
NH.sub.2--(CH.sub.2CH.sub.2O)n-CH.sub.2CO.sub.2H or
NH.sub.2--(CH.sub.2CH.sub.2O)n-CH.sub.2CH.sub.2CO.sub.2H where n=2
to 100.
[0108] Examples of compounds having the formula M-N--O--P-G which
contain linkers with at least one non-alpha amino acid are listed
in Table 1. TABLE-US-00001 TABLE 1 Compounds Containing Linkers
With At Least One Non-alpha Amino Acid HPLC HPLC Compound
method.sup.1 RT.sup.2 MS.sup.3 IC50.sup.5 M N O P G* L1 10-40% B
5.43 1616.6 5 N,N- Lys 8-amino-3,6- none BBN(7-14) dimethylglycine-
dioxaoctanoic Ser-Cys(Acm)-Gly acid L2 10-40% B 5.47 1644.7 3 N,N-
Arg 8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L3 10-40% B 5.97 1604.6 >50 N,N- Asp
8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L4 10-40% B 5.92 1575.5 4 N,N- Ser
8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L5 10-40% B 5.94 1545.5 9 N,N- Gly
8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L6 10-30% B 7.82 1639(M + Na) >50 N,N- Glu
8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L7 10-30% B 8.47 1581(M + Na) 7 N,N- Dala
8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L8 10-30% B 6.72 1639(M + Na) 4 N,N- 8- Lys
none BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L9 10-30% B 7.28 823.3(M + 2/2) 6 N,N- 8- Arg
none BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L10 10-30% B 7.94 1625.6(M + Na) >50 N,N- 8-
Asp none BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L11 10-30% B 7.59 1575.6 36 N,N- 8- Ser none
BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L12 10-30% B 7.65 1567.5(M + Na) >50 N,N- 8-
Gly none BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L13 10-30% B 7.86 1617.7 >50 N,N- 8- Glu none
BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L14 10-30% B 7.9 1581.7(M + Na) 11 N,N- 8- Dala
none BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L15 10-30% B 7.84 1656.8(M + Na) 11.5 N,N- 8-
8-amino-3,6- none BBN(7-14) dimethylglycine- amino- dioxaoctanoic
Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L16 10-30% B 6.65
1597.4(M + Na) 17 N,N- 8- 2,3- none BBN(7-14) dimethylglycine-
amino- diaminopropionic Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic
acid L17 10-30% B 7.6 1488.6 8 N,N- none 8-amino-3,6- none
BBN(7-14) dimethylglycine- dioxaoctanoic Ser-Cys(Acm)-Gly acid L18
10-30% B 7.03 1574.6 7.8 N,N- 2,3- 8-amino-3,6- none BBN(7-14)
dimethylglycine- diaminopropionic dioxaoctanoic Ser-Cys(Acm)-Gly
acid acid L19 10-35% B 5.13 1603.6 >50 N,N- Asp 8-amino-3,6- Gly
BBN(7-14) dimethylglycine- dioxaoctanoic Ser-Cys(Acm)-Gly acid L20
10-35% B 5.19 1603.6 37 N,N- 8- Asp Gly BBN(7-14) dimethylglycine-
amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L21 10-35% B 5.04
1575.7 46 N,N- 8- Ser Gly BBN(7-14) dimethylglycine- amino-
Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L22 10-35% B 4.37 1644.7
36 N,N- 8- Arg Gly BBN(7-14) dimethylglycine- amino-
Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L23 10-35% B 5.32 1633.7
>50 N,N- 8- 8-amino-3,6- Gly BBN(7-14) dimethylglycine- amino-
dioxaoctanoic Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L24
10-35% B 4.18 1574.6 38 N,N- 8- 2,3- Gly BBN(7-14) dimethylglycine-
amino- diaminopropionic Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic
acid L25 10-35% B 4.24 1616.6 26 N,N- 8- Lys Gly BBN(7-14)
dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid
L26 10-35% B 4.45 1574.6 30 N,N- 2,3- 8-amino-3,6- Gly BBN(7-14)
dimethylglycine- diaminopropionic dioxaoctanoic Ser-Cys(Acm)-Gly
acid acid L27 10-35% B 4.38 1627.3 >50 N,N- N-4- Asp none
BBN(7-14) dimethylglycine- aminoethyl- Ser-Cys(Acm)-Gly N- 1-
piperazineacetic acid L28 10-35% B 4.1 1600.3 25 N,N- N-4- Ser none
BBN(7-14) dimethylglycine- aminoethyl- Ser-Cys(Acm)-Gly N- 1-
piperazineacetic acid L29 10-35% B 3.71 1669.4 36 N,N- N-4- Arg
none BBN(7-14) dimethylglycine- aminoethyl- Ser-Cys(Acm)-Gly N- 1-
piperazineacetic acid L30 10-35% B 4.57 1657.2 36 N,N- N-4-
8-amino-3,6- none BBN(7-14) dimethylglycine- aminoethyl-
dioxaoctanoic Ser-Cys(Acm)-Gly N- acid 1- piperazineacetic acid L31
10-35% B 3.69 1598.3 >50 N,N- N-4- 2,3- none BBN(7-14)
dimethylglycine- aminoethyl- diaminopropionic Ser-Cys(Acm)-Gly N-
acid 1- piperazineacetic acid L32 10-35% B 3.51 1640.3 34 N,N- N-4-
Lys none BBN(7-14) dimethylglycine- aminoethyl- Ser-Cys(Acm)-Gly N-
1- piperazineacetic acid L33 10-35% B 4.29 1584.5 >50 N,N- N-1-
Asp none BBN(7-14) dimethylglycine- piperazineacetic
Ser-Cys(Acm)-Gly acid L34 10-35% B 4.07 1578.7(M + Na) 38 N,N- N-1-
Ser none BBN(7-14) dimethylglycine- piperazineacetic
Ser-Cys(Acm)-Gly acid L35 10-35% B 3.65 1625.6 26 N,N- N-1- Arg
none BBN(7-14) dimethylglycine- piperazineacetic Ser-Cys(Acm)-Gly
acid L36 10-35% B 4.43 1636.6 7 N,N- N-1- 8-amino-3,6- none
BBN(7-14) dimethylglycine- piperazineacetic dioxaoctanoic
Ser-Cys(Acm)-Gly acid acid L37 10-35% B 3.66 1555.7 23 N,N- N-1-
2,3- none BBN(7-14) dimethylglycine- piperazineacetic
diaminopropionic Ser-Cys(Acm)-Gly acid acid L38 10-35% B 3.44
1619.6 7 N,N- N-1- Lys none BBN(7-14) dimethylglycine-
piperazineacetic Ser-Cys(Acm)-Gly acid L42 30-50% B 5.65 1601.6 25
N,N- 4- 8-amino-3,6- none BBN(7-14) dimethylglycine- Hydroxyproline
dioxaoctanoic Ser-Cys(Acm)-Gly acid L48 30-50% B 4.47 1600.5 40
N,N- 4- 8-amino-3,6- none BBN(7-14) dimethylglycine- aminoproline
dioxaoctanoic Ser-Cys(Acm)-Gly acid L51 15-35% B 5.14 1673.7 49
N,N- Lys 8-amino-3,6- Gly BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L52 15-35% B 6.08 1701.6 14 N,N- Arg
8-amino-3,6- Gly BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L53 15-35% B 4.16 1632.6 10 N,N- Ser
8-amino-3,6- Gly BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L54 15-35% B 4.88 1661.6 >50 N,N- Asp
8-amino-3,6- Gly BBN(7-14) dimethylglycine- dioxaoctanoic
Ser-Cys(Acm)-Gly acid L55 15-35% B 4.83 1683.4(M + Na) 43 N,N- 8-
Asp Gly BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L56 15-35% B 4.65 1655.7(M + Na) 4 N,N- 8- Ser
Gly BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L57 15-35% B 4.9 1701.8 50 N,N- 8- Arg Gly
BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-
dioxaoctanoic acid L58 15-35% B 4.22 846.4(M + H/2) >50 N,N- 8-
8-amino-3,6- Gly BBN(7-14) dimethylglycine- amino- dioxaoctanoic
Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L59 15-35% B 4.03
1635.5 42 N,N- 8- 2,3- Gly BBN(7-14) dimethylglycine- amino-
diaminopropionic Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L60
15-35% B 4.11 1696.6(M + Na) 20 N,N- 8- Lys Gly BBN(7-14)
dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid
L61 15-35% B 4.32 1631.4 43 N,N- 2,3- 8-amino-3,6- Gly BBN(7-14)
dimethylglycine- diaminopropionic dioxaoctanoic Ser-Cys(Acm)-Gly
acid acid L78 20-40% B 6.13 1691.4(M + Na) 35 DO3A-monoamide 8-
Diaminopropionic none BBN(7-14) amino- acid 3,6- dioxaoctanoic acid
L79 20-40% B 7.72 1716.8(M + Na) 42 DO3A-monoamide 8-
Biphenylalanine none BBN(7-14) amino- 3,6- dioxaoctanoic acid L80
20-40% B 7.78 1695.9 >50 DO3A-monoamide 8- Diphenylalanine none
BBN(7-14) amino- 3,6- dioxaoctanoic acid L81 20-40% B 7.57 1513.6
37.5 DO3A-monoamide 8- 4- none BBN(7-14) amino-
Benzoylphenylalanine 3,6- dioxaoctanoic acid L92 15-30% B 5.63
1571.6 5 DO3A-monoamide 5- 8-amino-3,6- none BBN(7-14)
aminopentanoic dioxaoctanoic acid acid L94 20-36% B 4.19 1640.8(M +
Na) 6.2 DO3A-monoamide 8- D- none BBN(7-14)
amino- Phenylalanine 3,6- dioxaoctanoic acid L110 15-45% B 5.06
1612.7 36 DO3A-monoamide 8- 8-amino-3,6- none BBN(7-14)
aminooctanoic dioxaoctanoic acid acid *BBN(7-14) is [SEQ ID NO: 1]
.sup.1HPLC method refers to the 10 minute time for the HPLC
gradient. .sup.2HPLC RT refers to the retention time of the
compound in the HPLC. .sup.3MS refers to mass spectra where
molecular weight is calculated from mass/unit charge (m/e).
.sup.4IC.sub.50 refers to the concentration of compound to inhibit
50% binding of iodinated bombesin to a GRP receptor on cells.
2B. Linkers Containing at Least One Substituted Bile Acid
[0109] In another embodiment of the present invention, the linker
N--O--P contains at least one substituted bile acid. Thus, in this
embodiment of the linker N--O--P, [0110] N is 0 (where 0 means it
is absent), an alpha amino acid, a substituted bile acid or other
linking group; [0111] O is an alpha amino acid or a substituted
bile acid; and [0112] P is 0, an alpha amino acid, a substituted
bile acid or other linking group, [0113] wherein at least one of N,
O or P is a substituted acid.
[0114] Bile acids are found in bile (a secretion of the liver) and
are steroids having a 10 hydroxyl group and a five carbon atom side
chain terminating in a carboxyl group. In substituted bile acids,
at least one atom such as a hydrogen atom of the bile acid is
substituted with another atom, molecule or chemical group. For
example, substituted bile acids include those having a 3-amino,
24-carboxyl function optionally substituted at positions 7 and 12
with hydrogen, hydroxyl or keto functionality.
[0115] Other useful substituted bile acids in the present invention
include substituted cholic acids and derivatives thereof. Specific
substituted cholic acid derivatives include: [0116]
(3.beta.,5.beta.)-3-aminocholan-24-oic acid; [0117]
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid;
[0118]
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid; [0119]
Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholic
acid); [0120]
(3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxy-12-oxocholan-24-oic
acid; and [0121]
(3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxycholan-24-oic acid.
[0122] Examples of compounds having the formula M-N--O--P-G which
contain linkers with at least one substituted bile acid are listed
in Table 2. TABLE-US-00002 TABLE 2 Compounds Containing Linkers
With At Least One Substituted Bile Acid HPLC HPLC Compound
method.sup.1 RT.sup.2 MS.sup.3 IC50.sup.5 M N O P G* L62 20-80% B
3.79 1741.2 >50 DO3A- Gly (3.beta.,5.beta.)-3- none BBN(7-14)
monoamide aminocholan- 24-oic acid L63 20-80% B 3.47 1757.0 23
DO3A- Gly (3.beta.,5.beta.,12.alpha.)-3- none BBN(7-14) monoamide
amino-12- hydroxycholan- 24-oic acid L64 20-50% B 5.31 1773.7 8.5
DO3A- Gly (3.beta.,5.beta.,7.alpha.,12.alpha.)- none BBN(7-14)
monoamide 3-amino- 7,12- dihydroxycholan- 24-oic acid L65 20-80% B
3.57 2246.2 >50 DO3A- Gly Lys-(3,6,9- Arg BBN(7-14) monoamide
trioxaundecane- 1,11- dicarbonyl- 3,7- dideoxy-3- aminocholic acid)
L66 20-80% 3.79 2245.8 >50
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3- Lys(DO3A- Arg BBN(7-14)
amino-7,12- monoamide- dihydroxycholan-24-oic Gly) acid-3,6,9-
trioxaundecane-1,11- dicarbonyl L67 20-80% 3.25 1756.9 4.5 DO3A-
Gly (3.beta.,5.beta.,7.alpha.,12.alpha.)- none BBN(7-14) monoamide
3-amino-12- oxacholan-24- oic acid L69 20-80% 3.25 1861.27 not
DO3A- 1- (3.beta.,5.beta.,7.alpha.,12.alpha.)- none BBN(7-14)
tested monoamide amino- 3-amino- 3,6- 7,12- dioxaoctanoic
dihydroxycholan- acid 24-oic acid *BBN(7-14) is [SEQ ID NO: 1]
.sup.1HPLC method refers to the 10 minute time for the HPLC
gradient. .sup.2HPLC RT refers to the retention time of the
compound in the HPLC. .sup.3MS refers to mass spectra where
molecular weight is calculated from mass/unit charge (m/e).
.sup.4IC.sub.50 refers to the concentration of compound to inhibit
50% binding of iodinated bombesin to a GRP receptor on cells.
2C. Linkers Containing at Least One Non-Alpha Amino Acid With a
Cyclic Group
[0123] In yet another embodiment of the present invention, the
linker N--O--P contains at least one non-alpha amino acid with a
cyclic group. Thus, in this embodiment of the linker N--O--P,
[0124] N is 0 (where 0 means it is absent), an alpha amino acid, a
non-alpha amino acid with a cyclic group or other linking group;
[0125] O is an alpha amino acid or a non-alpha amino acid with a
cyclic group; and [0126] P is 0, an alpha amino acid, a non-alpha
amino acid with a cyclic group, or other linking group, [0127]
wherein at least one of N, O or P is a non-alpha amino acid with a
cyclic group.
[0128] Non-alpha amino acids with a cyclic group include
substituted phenyl, biphenyl, cyclohexyl or other amine and
carboxyl containing cyclic aliphatic or heterocyclic moieties.
[0129] Examples of such include: [0130] 4-aminobenzoic acid [0131]
4-aminomethyl benzoic acid [0132] trans-4-aminomethylcyclohexane
carboxylic acid [0133] 4-(2-aminoethoxy)benzoic acid [0134]
isonipecotic acid [0135] 2-aminomethylbenzoic acid [0136]
4-amino-3-nitrobenzoic acid [0137]
4-(3-carboxymethyl-2-keto-1-benzimidazolyl-piperidine [0138]
6-(piperazin-1-yl)-4-(3H)-quinazolinone-3-acetic acid [0139]
(2S,5S)-5-amino-1,2,4,5,6,7-hexahydro-azepino[3,21-hi]indole-4-one-2-carb-
oxylic acid [0140]
(4S,7R)-4-amino-6-aza-5-oxo-9-thiabicyclo[4.3.0]nonane-7-carboxylic
acid [0141]
3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one [0142]
N1-piperazineacetic acid [0143] N-4-aminoethyl-N-1-piperazineacetic
acid [0144] (3 S)-3-amino-1-carboxymethylcaprolactam [0145] (2S
,6S,9)-6-amino-2-carboxymethyl-3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione
[0146] Examples of compounds having the formula M-N--O--P-G which
contain linkers with at least one alpha amino acid with a cyclic
group are listed in Table 3. TABLE-US-00003 TABLE 3 Compounds
Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl
Or Heterocyclic) Carboxylates Com- HPLC HPLC pound method.sup.1
RT.sup.2 MS.sup.3 IC50.sup.5 M N O P G* L70 10-40% B 6.15 1502.6 5
DO3A- Gly 4-aminobenzoic none BBN(7-14) monoamide acid L71 1482.2(M
+ Na) 7 DO3A- none 4-aminomethyl none BBN(7-14) monoamide benzoic
acid L72 1504.0(M + K) 8 DO3A- none trans-4- none BBN(7-14)
monoamide aminomethylcyclohexyl carboxylic acid L73 5-35% 7.01
1489.8 5 DO3A- none 4-(2- none BBN(7-14) monoamide
aminoethoxy)benzoic acid L74 5-35% 6.49 1494.8 7 DO3A- Gly
isonipecotic none BBN(7-14) monoamide acid L75 5-35% 6.96 1458.0 23
DO3A- none 2- none BBN(7-14) monoamide aminomethylbenzoic acid L76
5-35% 7.20] 1502.7 4 DO3A- none 4-aminomethyl- none BBN(7-14)
monoamide 3-nitrobenzoic acid L77 20-40% B 6.17 1691.8(M + Na) 17.5
DO3A- 8- 1- none BBN(7-14) monoamide amino- Naphthylalanine 3,6-
dioxaoctanoic acid L82 20-40% B 6.18 1584.6 8 DO3A- none 4-(3- none
BBN(7-14) monoamide carboxymethyl- 2-keto-1- benzimidazolyl-
piperidine L83 20-40% B 5.66 1597.5 >50 DO3A- none
6-(piperazin-1- none BBN(7-14) monoamide yl)-4-(3H)- quinazolinone-
3-acetic acid L84 20-40% B 6.31 1555.5 >50 DO3A- none (2S,5S)-5-
none BBN(7-14) monoamide amino- 1,2,4,5,6,7- hexahydro-
azepino[3,21- hi]indole- 4-one-2- carboxylic acid L85 20-40% B 5.92
1525.5 >50 DO3A- none (4S,7R)-4- none BBN(7-14) monoamide
amino-6-aza-5- oxo-9- thiabicyclo[4.3. 0]nonane-7- carboxylic acid
L87 20-40% B 5.47 1593.8(M + Na) >50 DO3A- none 3- none
BBN(7-14) monoamide carboxymethyl- 1-phenyl-1,3,8- triazaspiro[4.5]
decan-4-one L88 20-40% B 3.84 1452.7 >50 DO3A- none N1- none
BBN(7-14) monoamide piperazineacetic acid L89 20-40% B 5.68
1518.5(M + Na) 23 DO3A- none N-4- none BBN(7-14) monoamide
aminoethyl-N- 1-piperazine- acetic acid L90 20-40% B 7.95 1495.4 50
DO3A- none (3S)-3-amino- none BBN(7-14) monoamide 1-
carboxymethylcaprolactam L91 20-40% B 3.97 1535.7 >50 DO3A- none
(2S,6S,9)-6- none BBN(7-14) monoamide amino-2- carboxymethyl- 3,8-
diazabicyclo- [4,3,0]-nonane- 1,4-dione L93 15-30% B 7.57 1564.7
5.8 DO3A- 5- trans-4- none BBN(7-14) monoamide aminopentanoic
aminomethylcyclohexane- acid 1- carboxylic acid L95 15-35% B 5.41
1604.6 14 DO3A- trans-4- D- none BBN(7-14) monoamide amino
Phenylalanine methyl cyclohexane- 1- carboxylic acid L96 20-36% B
4.75 1612.7 35 DO3A- 4- 8-amino-3,6- none BBN(7-14) monoamide amino
dioxaoctanoic methyl acid benzoic acid L97 15-35% B 5.86 1598.8 4.5
DO3A- 4- trans-4- none BBN(7-14) monoamide benzoyl-
aminomethylcyclohexane- (L)- 1- phenylalanine carboxylic acid L98
15-35% B 4.26 1622.7 16 DO3A- trans-4- Arg none BBN(7-14) monoamide
aminomethylcyclohexane- 1- carboxylic acid L99 15-35% B 4.1 1594.7
22 DO3A- trans-4- Lys none BBN(7-14) monoamide
aminomethylcyclohexane- 1- carboxylic acid L100 15-35% B 4.18
1613.6 10 DO3A- trans-4- Diphenylalanine none BBN(7-14) monoamide
aminomethylcyclohexane- 1- carboxylic acid L101 15-35% B 5.25
1536.7 25 DO3A- trans-4- 1- none BBN(7-14) monoamide
aminomethylcyclohexane- Naphthylalanine 1- carboxylic acid L102
15-35% B 5.28 1610.8 9.5 DO3A- trans-4- 8-amino-3,6- none BBN(7-14)
monoamide aminomethylcyclohexane- dioxaoctanoic 1- acid carboxylic
acid L103 15-35% B 4.75 1552.7 24 DO3A- trans-4- Ser none BBN(7-14)
monoamide aminomethylcyclohexane- 1- carboxylic acid L104 15-35% B
3.91 1551.7 32 DO3A- trans-4- 2,3- none BBN(7-14) monoamide
aminomethylcyclohexane- diaminopropionic 1- acid carboxylic acid
L105 20-45% B 7.68 1689.7 3.5 DO3A- trans-4- Biphenylalanine none
BBN(7-14) monoamide aminomethylcyclohexane- 1- carboxylic acid L106
20-45% B 6.97 1662.7 3.8 DO3A- trans-4- (2S,5S)-5- none BBN(7-14)
monoamide aminomethylcyclohexane- amino- 1- 1,2,4,5,6,7- carboxylic
hexahydro- acid azepino[3,21- hi]indole- 4-one-2- carboxylic acid
L107 15-35% B 5.79 1604.7 5 DO3A- trans-4- trans-4- none BBN(7-14)
monoamide aminomethylcyclohexane- aminomethylcyclohexane- 1- 1-
carboxylic carboxylic acid acid L108 15-45% B 6.38 1618.7 10 DO3A-
8- Phenylalanine none BBN(7-14) monoamide amino- 3,6- dioxaoctanoic
acid L109 15-45% B 6.85 1612.7 6 DO3A- trans-4- Phenylalanine none
BBN(7-14) monoamide aminomethylcyclohexane- 1- carboxylic acid L111
20-45% B 3.75 1628.6 8 DO3A- 8- trans-4- none BBN(7-14) monoamide
aminooctanoic aminomethylcyclohexane- acid 1- carboxylic acid L112
20-47% B 3.6 1536.5 4.5 DO3A- none 4'- none BBN(7-14) in 9 min
monoamide aminomethyl- biphenyl-1- carboxylic acid L113 20-47% B
3.88 1558.6(M + Na) 5 DO3A- none 3'- none BBN(7-14) in 9 min
monoamide aminomethyl- biphenyl-3- carboxylic acid L114 10-40% B
5.47 1582.8 4.5 CMDOTA Gly 4-aminobenzoic none BBN(7-14) acid L124
5-35% B 7.04 1489.9 8.0 DO3A- none 4- none BBN(7-14) monoamide
aminomethylphenoxyacetic acid L143 5-35% B 6.85 1516.8 NT** DO3A-
Gly 4- none BBN(7-14) monoamide aminophenylacetic acid L144 5-35% B
6.85 1462.7 NT HPDO3A none 4-phenoxy none BBN(7-14) L145 20-80% B
1.58 1459.8 5 DO3A- none 3- none BBN(7-14) monoamide
aminomethylbenzoic acid L146 20-80% B 1.53 1473.7 9 DO3A- none 4-
none BBN(7-14) monoamide aminomethylphenylacetic acid L147 20-80% B
1.68 1489.7 NT DO3A- none 4-aminomethyl- none BBN(7-14) monoamide
3- methoxybenzoic acid *BBN(7-14) is [SEQ ID NO: 1] **NT is defined
as "not tested." .sup.1HPLC method refers to the 10 minute time for
the HPLC gradient. .sup.2HPLC RT refers to the retention time of
the compound in the HPLC. .sup.3MS refers to mass spectra where
molecular weight is calculated from mass/unit charge (m/e).
.sup.4IC.sub.50 refers to the concentration of compound to inhibit
50% binding of iodinated bombesin to a GRP receptor on cells.
2D. Other Linking Groups
[0147] Other linking groups which may be used within the linker
N--O--P include a chemical group that serves to couple the GRP
receptor targeting peptide to the metal chelator while not
adversely affecting either the targeting function of the GRP
receptor targeting peptide or the metal complexing function of the
metal chelator. Suitable other linking groups include peptides
(i.e., amino acids linked together) alone, a non-peptide group
(e.g., hydrocarbon chain) or a combination of an amino acid
sequence and a non-peptide spacer.
[0148] In one embodiment, other linking groups for use within the
linker N--O--P include L-glutamine and hydrocarbon chain, or a
combination thereof.
[0149] In another embodiment, other linking groups for use within
the linker N--O--P include a pure peptide linking group consisting
of a series of amino acids (e.g., diglycine, triglycine,
gly-gly-glu, gly-ser-gly, etc.), in which the total number of atoms
between the N-terminal residue of the GRP receptor targeting
peptide and the metal chelator in the polymeric chain is .ltoreq.12
atoms.
[0150] In yet a further embodiment, other linking groups for use
within the linker N--O--P can also include a hydrocarbon chain
[i.e., R.sub.1--(CH.sub.2).sub.n--R.sub.2]wherein n is 0-10,
preferably n=3 to 9, R.sub.1 is a group (e.g., H.sub.2N--, HS--,
--COOH) that can be used as a site for covalently linking the
ligand backbone or the preformed metal chelator or metal complexing
backbone; and R.sub.2 is a group that is used for covalent coupling
to the N-terminal NH.sub.2-group of the GRP receptor targeting
peptide (e.g., R.sub.2 is an activated COOH group). Several
chemical methods for conjugating ligands (i.e., chelators) or
preferred metal chelates to biomolecules have been well described
in the literature [Wilbur, 1992; Parker, 1990; Hermanson, 1996;
Frizberg et al., 1995]. One or more of these methods could be used
to link either the uncomplexed ligand (chelator) or the radiometal
chelate to the linker or to link the linker to the GRP receptor
targeting peptides. These methods include the formation of acid
anhydrides, aldehydes, arylisothiocyanates, activated esters, or
N-hydroxysuccinimides [Wilbur, 1992; Parker, 1990; Hermanson, 1996;
Frizberg et al., 1995].
[0151] In a preferred embodiment, other linking groups for use
within the linker N--O--P may be formed from linker precursors
having electrophiles or nucleophiles as set forth below: [0152]
LP1: a linker precursor having on at least two locations of the
linker the same electrophile E1 or the same nucleophile Nu1; [0153]
LP2: a linker precursor having an electrophile E1 and on another
location of the linker a different electrophile E2; [0154] LP3: a
linker precursor having a nucleophile Nu1 and on another location
of the linker a different nucleophile Nu2; or [0155] LP4: a linker
precursor having one end functionalized with an electrophile E1 and
the other with a nucleophile Nu1.
[0156] The preferred nucleophiles Nu1/Nu2 include --OH, --NH, --NR,
--SH, --HN--NH.sub.2, --RN--NH.sub.2, and --RN--NHR', in which R'
and R are independently selected from the definitions for R given
above, but for R' is not H.
[0157] The preferred electrophiles E1/E2 include --COOH, --CH.dbd.O
(aldehyde), --CR.dbd.OR' (ketone), --RN--C.dbd.S, --RN--C.dbd.O,
--S--S-2-pyridyl, --SO.sub.2--Y, --CH.sub.2C(.dbd.O)Y, and
##STR3##
[0158] wherein Y can be selected from the following groups:
##STR4##
3. GRP Receptor Targeting Peptide
[0159] The GRP receptor targeting peptide (i.e., G in the formula
M-N--O--P-G) is any peptide, equivalent, derivative or analogue
thereof which has a binding affinity for the GRP receptor
family.
[0160] The GRP receptor targeting peptide may take the form of an
agonist or an antagonist. A GRP receptor targeting peptide agonist
is known to "activate" the cell following binding with high
affinity and may be internalized by the cell. Conversely, GRP
receptor targeting peptide antagonists are known to bind only to
the GRP receptor on the cell without being internalized by the cell
and without "activating" the cell. In a preferred embodiment, the
GRP receptor targeting peptide is an agonist.
[0161] In a more preferred embodiment of the present invention, the
GRP agonist is a bombesin (BBN) analogue and/or a derivative
thereof. The BBN derivative or analog thereof preferably contains
either the same primary structure of the BBN binding region (i.e.,
BBN(7-14) [SEQ ID NO: 1]) or similar primary structures, with
specific amino acid substitutions that will specifically bind to
GRP receptors with better or similar binding affinities as BBN
alone (i.e., Kd<25 nM). Suitable compounds include peptides,
peptidomimetics and analogues and derivatives thereof. The presence
of L-methionine (Met) at position BBN-14 will generally confer
agonistic properties while the absence of this residue at BBN-14
generally confers antagonistic properties [Hoffken, 1994].
[0162] It is well documented in the art that there are a few and
selective number of specific amino acid substitutions in the BBN
(8-14) binding region (e.g., D-Ala.sup.11 for L-Gly.sup.11 or
D-Trp.sup.8 for L-Trp.sup.8), which can be made without decreasing
binding affinity [Leban et al., 1994; Qin et al., 1994; Jensen et
al., 1993]. In addition, attachment of some amino acid chains or
other groups to the N-terminal amine group at position BBN-8 (i.e.,
the Trp.sup.8 residue) can dramatically decrease the binding
affinity of BBN analogues to GRP receptors [Davis et al., 1992;
Hoffken, 1994; Moody et al., 1996; Coy, et al., 1988; Cai et al.,
1994]. In a few cases, it is possible to append additional amino
acids or chemical moieties without decreasing binding affinity.
[0163] Analogues of BBN receptor targeting peptides include
molecules that target the GRP receptors with avidity that is
greater than or equal to BBN, as well as muteins, retropeptides and
retro-inverso-peptides of GRP or BBN. One of ordinary skill will
appreciate that these analogues may also contain modifications
which include substitutions, and/or deletions and/or additions of
one or several amino acids, insofar that these modifications do not
negatively alter the biological activity of the peptides described
therein. These substitutions may be carried out by replacing one or
more amino acids by their synonymous amino acids. Synonymous amino
acids within a group are defined as amino acids that have
sufficient physicochemical properties to allow substitution between
members of a group in order to preserve the biological function of
the molecule. Synonymous amino acids as used herein include
synthetic derivatives of these amino acids and may include those
listed in the following Table. In the chart and throughout this
application amino acids are abbreviated interchangeably either by
their three letter or single letter abbreviations, which are well
known to the skilled artisan. Thus, for example, T or Thr stands
for threonine, K or Lys stands for lysine, P or Pro stands for
proline and R or Arg stands for arginine. TABLE-US-00004 Amino
Acids Synonymous Groups Arg His, Lys, Glu, Gln Pro Ala, Thr, Gly,
N-methyl Ala, pipecolic acid, azetidine carboxylic acid Thr
3-hydroxy proline, 4-hydroxy proline, Ser, Ala, Gly, His, Gln Lys
Lys, ornithine, Arg, diaminopropionic acid, HArg, His
[0164] Deletions or insertions of amino acids may also be
introduced into the defined sequences provided they do not alter
the biological functions of said sequences. Preferentially such
insertions or deletions should be limited to 1, 2, 3, 4 or 5 amino
acids and should not remove or physically disturb or displace amino
acids which are critical to the functional conformation. Muteins of
the GRP receptor targeting peptides described herein may have a
sequence homologous to the sequence disclosed in the present
specification in which amino acid substitutions, deletions, or
insertions are present at one or more amino acid positions. Muteins
may have a biological activity that is at least 40%, preferably at
least 50%, more preferably 60-70%, most preferably 80-90% of the
peptides described herein. However, they may also have a biological
activity greater than the peptides specifically exemplified, and
thus do not necessarily have to be identical to the biological
function of the exemplified peptides. Analogues of GRP receptor
targeting peptides also include peptidomimetics or pseudopeptides
incorporating changes to the amide bonds of the peptide backbone,
including thioamides, methylene amines, and E-olefins. Also
peptides based on the structure of GRP, BBN or their peptide
analogues with amino acids replaced by N-substituted hydrazine
carbonyl compounds (also known as aza amino acids) are included in
the term analogues as used herein.
[0165] The GRP receptor targeting peptide can be prepared by
various methods depending upon the selected chelator. The peptide
can generally be most conveniently prepared by techniques generally
established and known in the art of peptide synthesis, such as the
solid-phase peptide synthesis (SPPS) approach. Solid-phase peptide
synthesis (SPPS) involves the stepwise addition of amino acid
residues to a growing peptide chain that is linked to an insoluble
support or matrix, such as polystyrene. The C-terminal residue of
the peptide is first anchored to a commercially available support
with its amino group protected with an N-protecting agent such as a
t-butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc)
group. The amino protecting group is removed with suitable
deprotecting agents such as TFA in the case of Boc or piperidine
for Fmoc and the next amino acid residue (in N-protected form) is
added with a coupling agent such as N,N'-dicyclohexylcarbodiimide
(DCC), or N,N'-diisopropylcarbodiimide (DIC) or
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU). Upon formation of a peptide bond, the
reagents are washed from the support. After addition of the final
residue, the peptide is cleaved from the support with a suitable
reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride
(HF).
[0166] The linker may then be coupled to form a conjugate by
reacting the free amino group of the Trp.sup.8 residue of the GRP
receptor targeting peptide with an appropriate functional group of
the linker. The entire construct of chelator, linker and targeting
moiety discussed above may also be assembled on resin and then
cleaved by agency of suitable reagents such as trifluoroacetic acid
or HF, as well.
4. Labeling and Administration of Compounds
[0167] Incorporation of the metal within the conjugate can be
achieved by various methods commonly known in the art of
coordination chemistry. When the metal is .sup.99mTc, a preferred
radionuclide for diagnostic imaging, the following general
procedure can be used to form a technetium complex. A
peptide-chelator conjugate solution is formed by initially
dissolving the conjugate in water, dilute acid, or in an aqueous
solution of an alcohol such as ethanol. The solution is then
optionally degassed to remove dissolved oxygen. When an --SH group
is present in the peptide, a thiol protecting group such as Acm
(acetamidomethyl), trityl or other thiol protecting group may
optionally be used to protect the thiol from oxidation. The thiol
protecting group(s) are removed with a suitable reagent, for
example with sodium hydroxide, and are then neutralized with an
organic acid such as acetic acid (pH 6.0-6.5). Alternatively, the
thiol protecting group can be removed in situ during technetium
chelation. In the labeling step, sodium pertechnetate obtained from
a molybdenum generator is added to a solution of the conjugate with
a sufficient amount of a reducing agent, such as stannous chloride,
to reduce technetium and is either allowed to stand at room
temperature or is heated. The labeled conjugate can be separated
from the contaminants .sup.99mTcO.sub.4.sup.- and colloidal
.sup.99mTcO.sub.2 chromatographically, for example with a C-18 Sep
Pak cartridge [Millipore Corporation, Waters Chromatography
Division, 34 Maple Street, Milford, Mass. 01757]or by HPLC using
methods known to those skilled in the art.
[0168] In an alternative method, the labeling can be accomplished
by a transchelation reaction. In this method, the technetium source
is a solution of technetium that is reduced and complexed with
labile ligands prior to reaction with the selected chelator, thus
facilitating ligand exchange with the selected chelator. Examples
of suitable ligands for transchelation includes tartrate, citrate,
gluconate, and heptagluconate. It will be appreciated that the
conjugate can be labeled using the techniques described above, or
alternatively, the chelator itself may be labeled and subsequently
coupled to the peptide to form the conjugate; a process referred to
as the "prelabeled chelate" method. Re and Tc are both in row VIIB
of the Periodic Table and they are chemical congeners. Thus, for
the most part, the complexation chemistry of these two metals with
ligand frameworks that exhibit high in vitro and in vivo
stabilities are the same [Eckelman, 1995]and similar chelators and
procedures can be used to label with Re. Many .sup.99mTc or
.sup.186/188Re complexes, which are employed to form stable
radiometal complexes with peptides and proteins, chelate these
metals in their +5 oxidation state [Lister-James et al., 1997].
This oxidation state makes it possible to selectively place
.sup.99mTc- or .sup.186/188Re into ligand frameworks already
conjugated to the biomolecule, constructed from a variety of
.sup.99mTc(V) and/or .sup.186/188Re(V) weak chelates (e.g,
.sup.99mTc-glucoheptonate, citrate, gluconate, etc.) [Eckelman,
1995; Lister-James et al., 1997; Pollak et al., 1996].
5. Diagnostic and Therapeutic Uses
[0169] When labeled with diagnostically and/or therapeutically
useful metals, compounds of the present invention can be used to
treat and/or detect cancers, including tumors, by procedures
established in the art of radiodiagnostics and radiotherapeutics.
[Bushbaum, 1995; Fischman et al., 1993; Schubiger et al., 1996;
Lowbertz et al., 1994; Krenning et al., 1994].
[0170] The compounds of the invention, which, as explained in more
detail in the Examples, show higher uptake in tumors in vivo than
compounds without the novel linkers disclosed herein, exhibit an
improved ability to target GRP receptor-expressing tumors and thus
to image or deliver radiotherapy to these tissues. Indeed, as shown
in the Examples, radiotherapy is more effective (and survival time
increased) using compounds of the invention.
[0171] The diagnostic application of these compounds can be as a
first line diagnostic screen for the presence of neoplastic cells
using scintigraphic imaging, as an agent for targeting neoplastic
tissue using hand-held radiation detection instrumentation in the
field of radioimmuno guided surgery (RIGS), as a means to obtain
dosimetry data prior to administration of the matched pair
radiotherapeutic compound, and as a means to assess GRP receptor
population as a function of treatment over time.
[0172] The therapeutic application of these compounds can be
defined either as an agent that will be used as a first line
therapy in the treatment of cancer, as combination therapy where
these radiolabeled agents could be utilized in conjunction with
adjuvant chemotherapy, and as the matched pair therapeutic agent.
The matched pair concept refers to a single unmetallated compound
which can serve as both a diagnostic and a therapeutic agent
depending on the radiometal that has been selected for binding to
the appropriate chelate. If the chelator cannot accommodate the
desired metals appropriate substitutions can be made to
accommodaste the different metal whilst maintaining the
pharmacology such that the behaviour of the diagnostic compound in
vivo can be used to predict the behaviour of the radiotherapeutic
compound.
[0173] A conjugate labeled with a radionuclide metal, such as
.sup.99mTc, can be administered to a mammal, including human
patients or subjects, by intravenous, subcutaneous or
intraperitoneal injection in a pharmaceutically acceptable carrier
and/or solution such as salt solutions like isotonic saline.
Radiolabeled scintigraphic imaging agents provided by the present
invention are provided having a suitable amount of radioactivity.
In forming .sup.99mTc radioactive complexes, it is generally
preferred to form radioactive complexes in solutions containing
radioactivity at concentrations of from about 0.01 millicurie (mCi)
to 100 mCi per mL. Generally, the unit dose to be administered has
a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1
mCi to 30 mCi. The solution to be injected at unit dosage is from
about 0.01 mL to about 10 mL. The amount of labeled conjugate
appropriate for administration is dependent upon the distribution
profile of the chosen conjugate in the sense that a rapidly cleared
conjugate may need to be administered in higher doses than one that
clears less rapidly. In vivo distribution and localization can be
tracked by standard scintigraphic techniques at an appropriate time
subsequent to administration; typically between thirty minutes and
180 minutes depending upon the rate of accumulation at the target
site with respect to the rate of clearance at non-target tissue.
For example, after injection of the diagnostic radionuclide-labeled
compounds of the invention into the patient, a gamma camera
calibrated for the gamma ray energy of the nuclide incorporated in
the imaging agent can be used to image areas of uptake of the agent
and quantify the amount of radioactivity present in the site.
Imaging of the site in vivo can take place in a few minutes.
However, imaging can take place, if desired, hours or even longer,
after the radiolabeled peptide is injected into a patient. In most
instances, a sufficient amount of the administered dose will
accumulate in the area to be imaged within about 0.1 hour to permit
the taking of scintiphotos.
[0174] The compounds of the present invention can be administered
to a patient alone or as part of a composition that contains other
components such as excipients, diluents, radical scavengers,
stabilizers, and carriers, all of which are well-known in the art.
The compounds can be administered to patients either intravenously
or intraperitoneally.
[0175] There are numerous advantages associated with the present
invention. The compounds made in accordance with the present
invention form stable, well-defined .sup.99mTc or .sup.186/188Re
labeled compounds. Similar compounds of the invention can also be
made by using appropriate chelator frameworks for the respective
radiometals, to form stable, well-defined products labeled with
.sup.153Sm, .sup.90Y, .sup.166Ho, .sup.105Rh, .sup.199Au,
.sup.149Pm, .sup.177Lu, .sup.111In or other radiometal. The
radiolabeled GRP receptor targeting peptides selectively bind to
neoplastic cells expressing GRP receptors, and if an agonist is
used, become internalized, and are retained in the tumor cells for
extended time periods. The radioactive material that does not reach
(i.e., does not bind) the cancer cells is preferentially excreted
efficiently into the urine with minimal retention of the radiometal
in the kidneys.
6. Radiotherapy
[0176] Radioisotope therapy involves the administration of a
radiolabeled compound in sufficient quantity to damage or destroy
the targeted tissue. After administration of the compound (by e.g.,
intravenous, subcutaneous, or intraperitonal injection), the
radiolabeled pharmaceutical localizes preferentially at the disease
site (in this instance, tumor tissue that expresses the GRP
receptor). Once localized, the radiolabeled compound then damages
or destroys the diseased tissue with the energy that is released
during the radioactive decay of the isotope that is
administered.
[0177] The design of a successful radiotherapeutic involves several
critical factors:
[0178] 1. selection of an appropriate targeting group to deliver
the radioactivity to the disease site;
[0179] 2. selection of an appropriate radionuclide that releases
sufficient energy to damage that disease site, without
substantially damaging adjacent normal tissues; and
[0180] 3. selection of an appropriate combination of the targeting
group and the radionuclide without adversely affecting the ability
of this conjugate to localize at the disease site. For radiometals,
this often involves a chelating group that coordinates tightly to
the radionuclide, combined with a linker that couples said chelate
to the targeting group, and that affects the overall
biodistribution of the compound to maximize uptake in target
tissues and minimize uptake in normal, non-target organs.
[0181] The present invention provides radiotherapeutic agents that
satisfy all three of the above criteria, through proper selection
of targeting group, radionuclide, metal chelate and linker.
[0182] Radiotherapeutic agents may contain a chelated 3+ metal ion
from the class of elements known as the lanthanides (elements of
atomic number 57-71) and their analogs (i.e. M.sup.3+ metals such
as yttrium and indium). Typical radioactive metals in this class
include the isotopes 90-Yttrium, 111-Indium, 149-Promethium,
153-Samarium, 166-Dysprosium, 166-Holmium, 175-Ytterbium, and
177-Lutetium. All of these metals (and others in the lanthanide
series) have very similar chemistries, in that they remain in the
+3 oxidation state, and prefer to chelate to ligands that bear hard
(oxygen/nitrogen) donor atoms, as typified by derivatives of the
well known chelate DTPA (diethylenetriaminepentaacetic acid) and
polyaza-polycarboxylate macrocycles such as DOTA
(1,4,7,10-tetrazacyclododecane-N,N',N'',N'''-tetraacetic acid and
its close analogs. The structures of these chelating ligands, in
their fully deprotonated form are shown below. TABLE-US-00005 DTPA
DOTA ##STR5## ##STR6##
[0183] These chelating ligands encapsulate the radiometal by
binding to it via multiple nitrogen and oxygen atoms, thus
preventing the release of free (unbound) radiometal into the body.
This is important, as in vivo dissociation of 3.sup.+ radiometals
from their chelate can result in uptake of the radiometal in the
liver, bone and spleen [Brechbiel M W, Gansow O A,
"Backbone-substituted DTPA ligands for .sup.90Y
radioimmunotherapy", Bioconj. Chem. 1991; 2: 187-194; Li, W P, Ma D
S, Higginbotham C, Hoffman T, Ketring A R, Cutler C S, Jurisson, S
S, "Development of an in vitro model for assessing the in vivo
stability of lanthanide chelates." Nucl. Med. Biol. 2001; 28(2):
145-154; Kasokat T, Urich K. Arzneim.-Forsch, "Quantification of
dechelation of gadopentetate dimeglumine in rats". 1992; 42(6):
869-76]. Unless one is specifically targeting these organs, such
non-specific uptake is highly undesirable, as it leads to
non-specific irradiation of non-target tissues, which can lead to
such problems as hematopoietic suppression due to irradiation of
bone marrow.
[0184] For radiotherapy applications any of the chelators for
therapeutic radionuclides disclosed herein may be used. However,
forms of the DOTA chelate [Tweedle M F, Gaughan G T, Hagan J T,
"1-Substituted-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane
and analogs." U.S. Pat. No. 4,885,363, Dec. 5, 1989]are
particularly preferred, as the DOTA chelate is expected to
de-chelate less in the body than DTPA or other linear chelates.
[0185] General methods for coupling DOTA-type macrocycles to
targeting groups through a linker (e.g. by activation of one of the
carboxylates of the DOTA to form an active ester, which is then
reacted with an amino group on the linker to form a stable amide
bond), are known to those skilled in the art. (See e.g. Tweedle et
al. U.S. Pat. No. 4,885,363). Coupling can also be performed on
DOTA-type macrocycles that are modified on the backbone of the
polyaza ring.
[0186] The selection of a proper nuclide for use in a particular
radiotherapeutic application depends on many factors,
including:
[0187] a. Physical half-life--This should be long enough to allow
synthesis and purification of the radiotherapeutic construct from
radiometal and conjugate, and delivery of said construct to the
site of injection, without significant radioactive decay prior to
injection. Preferably, the radionuclide should have a physical
half-life between about 0.5 and 8 days.
[0188] b. Energy of the emission(s) from the
radionuclide--Radionuclides that are particle emitters (such as
alpha emitters, beta emitters and Auger electron emitters) are
particularly useful, as they emit highly energetic particles that
deposit their energy over short distances, thereby producing highly
localized damage. Beta emitting radionuclides are particularly
preferred, as the energy from beta particle emissions from these
isotopes is deposited within 5 to about 150 cell diameters.
Radiotherapeutic agents prepared from these nuclides are capable of
killing diseased cells that are relatively close to their site of
localization, but cannot travel long distances to damage adjacent
normal tissue such as bone marrow.
[0189] c. Specific activity (i.e. radioactivity per mass of the
radionuclide)--Radionuclides that have high specific activity (e.g.
generator produced 90-Y, 111-In, 177-Lu) are particularly
preferred. The specific activity of a radionuclide is determined by
its method of production, the particular target that is used to
produce it, and the properties of the isotope in question.
[0190] Many of the lanthanides and lanthanoids include
radioisotopes that have nuclear properties that make them suitable
for use as radiotherapeutic agents, as they emit beta particles.
Some of these are listed in the table below. TABLE-US-00006
Approximate range of b- particle Half-Life Max b-energy Gamma
energy (cell Isotope (days) (MeV) (keV) diameters) .sup.149-Pm 2.21
1.1 286 60 .sup.153-Sm 1.93 0.69 103 30 .sup.166-Dy 3.40 0.40 82.5
15 .sup.166-Ho 1.12 1.8 80.6 117 .sup.175-Yb 4.19 0.47 396 17
.sup.177-Lu 6.71 0.50 208 20 .sup.90-Y 2.67 2.28 -- 150 .sup.111-In
2.810 Auger electron 173, 247 <5 .mu.m emitter Pm: Promethium,
Sm: Samarium, Dy: Dysprosium, Ho: Holmium, Yb: Ytterbium, Lu:
Lutetium, Y: Yttrium, In: Indium
[0191] Methods for the preparation of radiometals such as
beta-emitting lanthanide radioisotopes are known to those skilled
in the art, and have been described elsewhere [e.g. Cutler C S,
Smith C J, Ehrhardt G J.; Tyler T T, Jurisson S S, Deutsch E.
"Current and potential therapeutic uses of lanthanide
radioisotopes." Cancer Biother. Radiopharm. 2000; 15(6): 531-545].
Many of these isotopes can be produced in high yield for relatively
low cost, and many (e.g. .sup.90-Y, .sup.149-Pm, .sup.177-Lu) can
be produced at close to carrier-free specific activities (i.e. the
vast majority of atoms are radioactive). Since non-radioactive
atoms can compete with their radioactive analogs for binding to
receptors on the target tissue, the use of high specific activity
radioisotope is important, to allow delivery of as high a dose of
radioactivity to the target tissue as possible.
[0192] Radiotherapeutic derivatives of the invention containing
beta-emitting isotopes of rhenium (.sup.186-Re and .sup.188-Re) are
also particularly preferred.
7. Dosages and Additives
[0193] Proper dose schedules for the radiopharmaceutical compounds
of the present invention are known to those skilled in the art. The
compounds can be administered using many methods which include, but
are not limited to, a single or multiple IV or IP injections, using
a quantity of radioactivity that is sufficient to permit imaging
or, in the case of radiotherapy, to cause damage or ablation of the
targeted GRP-R bearing tissue, but not so much that substantive
damage is caused to non-target (normal tissue). The quantity and
dose required for scintigraphic imaging is discussed supra. The
quantity and dose required for radiotherapy is also different for
different constructs, depending on the energy and half-life of the
isotope used, the degree of uptake and clearance of the agent from
the body and the mass of the tumor. In general, doses can range
from a single dose of about 30-50 mCi to a cumulative dose of up to
about 3 Curies.
[0194] The radiopharmaceutical compositions of the invention can
include physiologically acceptable buffers, and can require
radiation stabilizers to prevent radiolytic damage to the compound
prior to injection. Radiation stabilizers are known to those
skilled in the art, and may include, for example, para-aminobenzoic
acid, ascorbic acid, gentistic acid and the like.
[0195] A single, or multi-vial kit that contains all of the
components needed to prepare the radiopharmaceuticals of this
invention, other than the radionuclide, is an integral part of this
invention.
[0196] A single-vial kit preferably contains a
chelator/linker/targeting peptide conjugate of the formula
M-N--O--P-G, a source of stannous salt (if reduction is required,
e.g., when using technetium), or other pharmaceutically acceptable
reducing agent, and is appropriately buffered with pharmaceutically
acceptable acid or base to adjust the pH to a value of about 3 to
about 9. The quantity and type of reducing agent used will depend
highly on the nature of the exchange complex to be formed. The
proper conditions are well known to those that are skilled in the
art. It is preferred that the kit contents be in lyophilized form.
Such a single vial kit may optionally contain labile or exchange
ligands such as glucoheptonate, gluconate, mannitol, malate, citric
or tartaric acid and can also contain reaction modifiers such as
diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine
tetraacetic acid (EDTA), or .alpha., .beta., or
.gamma.-cyclodextrin that serve to improve the radiochemical purity
and stability of the final product. The kit may also contain
stabilizers, bulking agents such as mannitol, that are designed to
aid in the freeze-drying process, and other additives known to
those skilled in the art.
[0197] A multi-vial kit preferably contains the same general
components but employs more than one vial in reconstituting the
radiopharmaceutical. For example, one vial may contain all of the
ingredients that are required to form a labile Tc(V) complex on
addition of pertechnetate (e.g. the stannous source or other
reducing agent). Pertechnetate is added to this vial, and after
waiting an appropriate period of time, the contents of this vial
are added to a second vial that contains the chelator and targeting
peptide, as well as buffers appropriate to adjust the pH to its
optimal value. After a reaction time of about 5 to 60 minutes, the
complexes of the present invention are formed. It is advantageous
that the contents of both vials of this multi-vial kit be
lyophilized. As above, reaction modifiers, exchange ligands,
stabilizers, bulking agents, etc. may be present in either or both
vials.
General Preparation of Compounds
[0198] The compounds of the present invention can be prepared by
various methods depending upon the selected chelator. The peptide
portion of the compound can be most conveniently prepared by
techniques generally established and known in the art of peptide
synthesis, such as the solid-phase peptide synthesis (SPPS)
approach. Because it is amenable to solid phase synthesis,
employing alternating FMOC protection and deprotection is the
preferred method of making short peptides. Recombinant DNA
technology is preferred for producing proteins and long fragments
thereof.
[0199] Solid-phase peptide synthesis (SPPS) involves the stepwise
addition of amino acid residues to a growing peptide chain that is
linked to an insoluble support or matrix, such as polystyrene. The
C-terminal residue of the peptide is first anchored to a
commercially available support with its amino group protected with
an N-protecting agent such as a t-butyloxycarbonyl group (Boc) or a
fluorenylmethoxycarbonyl (Fmoc) group. The amino protecting group
is removed with suitable deprotecting agents such as TFA in the
case of Boc or piperidine for Fmoc and the next amino acid residue
(in N-protected form) is added with a coupling agent such as
diisopropylcarbodiimide (DIC). Upon formation of a peptide bond,
the reagents are washed from the support. After addition of the
final residue, the peptide is cleaved from the support with a
suitable reagent such as trifluoroacetic acid (TFA) or hydrogen
fluoride (HF).
Alternative Preparation of the Compounds Via Segment Coupling
[0200] The compounds of the invention may also be prepared by the
process known in the art as segment coupling or fragment
condensation (Barlos, K. and Gatos, D.; 2002 "Convergent Peptide
Synthesis" in Fmoc Solid Phase Synthesis--A Practical Approach;
Eds. Chan, W. C. and White, P. D.; Oxford University Press, New
York; Chap. 9, pp 215-228). In this method segments of the peptide
usually in side-chain protected form, are prepared separately by
either solution phase synthesis or solid phase synthesis or a
combination of the two methods. The choice of segments is crucial
and is made using a division strategy that can provide a manageable
number of segments whose C-terminal residues and N-terminal
residues are projected to provide the cleanest coupling in peptide
synthesis. The C-terminal residues of the best segments are either
devoid of chiral alpha carbons (glycine or other moieties achiral
at the carbon .alpha. to the carboxyl group to be activated in the
coupling step) or are compromised of amino acids whose propensity
to racemization during activation and coupling is lowest of the
possible choices. The choice of N-terminal amino acid for each
segment is based on the ease of coupling of an activated acyl
intermediate to the amino group. Once the division strategy is
selected the method of coupling of each of the segments is chosen
based on the synthetic accessibility of the required intermediates
and the relative ease of manipulation and purification of the
resulting products (if needed). The segments are then coupled
together, both in solution, or one on solid phase and the other in
solution to prepare the final structure in fully or partially
protected form.
[0201] The protected target compound is then subjected to removal
of protecting groups, purified and isolated to give the final
desired compound. Advantages of the segment coupling approach are
that each segment can be purified separately, allowing the removal
of side products such as deletion sequences resulting from
incomplete couplings or those derived from reactions such as
side-chain amide dehydration during coupling steps, or internal
cyclization of side-chains (such as that of Gln) to the alpha amino
group during deprotection of Fmoc groups. Such side products would
all be present in the final product of a conventional resin-based
`straight through` peptide chain assembly whereas removal of these
materials can be performed, if needed, at many stages in a segment
coupling strategy. Another important advantage of the segment
coupling strategy is that different solvents, reagents and
conditions can be applied to optimize the synthesis of each of the
segments to high purity and yield resulting in improved purity and
yield of the final product. Other advantages realized are decreased
consumption of reagents and lower costs.
EXAMPLES
[0202] The following examples are provided as examples of different
methods which can be used to prepare various compounds of the
present invention. Within each example, there are compounds
identified in single bold capital letter (e.g., A, B, C), which
correlate to the same labeled corresponding compounds in the
drawings identified.
General Experimental
[0203] A. Definitions of Abbreviations Used
[0204] The following common abbreviations are used throughout this
specification: [0205] 1,1-dimethylethoxycarbonyl (Boc or Boc);
[0206] 9-fluorenylmethyloxycarbonyl (Fmoc); [0207]
1-hydroxybenozotriazole (HOBt); [0208] N,N'-diisopropylcarbodiimide
(DIC); [0209] N-methylpyrrolidinone (NMP); [0210] acetic anhydride
(Ac.sub.2O); [0211]
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (iv-Dde);
[0212] trifluoroacetic acid (TFA); [0213] Reagent B
(TFA:H.sub.2O:phenol:triisopropylsilane, 88:5:5:2); [0214]
diisopropylethylamine (DIEA); [0215]
O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU); [0216]
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorphosphate (HATU); [0217] N-hydroxysuccinimide (NHS);
[0218] solid phase peptide synthesis (SPPS); [0219]
dimethylsulfoxide (DMSO); [0220] dichloromethane (DCM); [0221]
dimethylformamide (DMF); [0222] dimethylacetamide (DMA); [0223]
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);
[0224] Triisopropylsilane (TIPS);
[0225] 11,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid
(DOTA)
(1R)-1-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]e-
thane-1,2-dicarboxylic acid (CMDOTA); [0226] fetal bovine serum
(FBS); [0227] human serum albumin (HSA); [0228] human prostate
cancer cell line (PC3); [0229] radiochemical purity (RCP); and
[0230] high performance liquid chromatography (HPLC).
[0231] B. Materials
[0232] The Fmoc-protected amino acids used were purchased from
Nova-Biochem (San Diego, Calif., USA), Advanced Chem Tech
(Louisville, Ky., USA), Chem-Impex International (Wood Dale Ill.,
USA), and Multiple Peptide Systems (San Diego, Calif., USA). Other
chemicals, reagents and adsorbents required for the syntheses were
procured from Aldrich Chemical Co. (Milwaukee, Wis., USA) and VWR
Scientific Products (Bridgeport, N.J., USA). Solvents for peptide
synthesis were obtained from Pharmco Co. (Brookfield Conn., USA).
Columns for HPLC analysis and purification were obtained from
Waters Co. (Milford, Mass., USA). Experimental details are given
below for those that were not commercially available.
[0233] C. Instrumentation for Peptide Synthesis
[0234] Peptides were prepared using an Advanced ChemTech 496
.OMEGA. MOS synthesizer, an Advanced ChemTech 357 FBS synthesizer
and/or by manual peptide synthesis. However the protocols for
iterative deprotection and chain extension employed were the same
for all.
[0235] D. Instrumentation Employed for Analysis and
Purification
[0236] Analytical HPLC was performed using a Shimadzu-LC-10A dual
pump gradient analytical LC system employing Shimadzu-ClassVP
software version 4.1 for system control, data acquisition, and post
run processing. Mass spectra were acquired on a Hewlett-Packard
Series 1100 MSD mass spectrometer interfaced with a Hewlett-Packard
Series 1100 dual pump gradient HPLC system fitted with an Agilent
Technologies 1100 series autosampler fitted for either direct flow
injection or injection onto a Waters Associates XTerra MS C18
column (4.6 mm.times.50 mm, 5.mu. particle, 120 .ANG. pore). The
instrument was driven by a HP Kayak workstation using `MSD Anyone`
software for sample submission and HP Chemstation software for
instrument control and data acquisition. In most cases the samples
were introduced via direct injection using a 5 .mu.L injection of
sample solution at a concentration of 1 mg/mL and analyzed using
positive ion electrospray to obtain m/e and m/z (multiply charged)
ions for confirmation of structure. .sup.1H-NMR spectra were
obtained on a Varian Innova spectrometer at 500 MHz. .sup.13C-NMR
spectra were obtained on the same instrument at 125.73 MHz.
Generally the residual .sup.1H absorption, or in the case of
.sup.13C-NMR, the .sup.13C absorption of the solvent employed, was
used as an internal reference; in other cases tetramethylsilane
(.delta.=0.00 ppm) was employed. Resonance values are given in
.delta. units. Micro-analysis data was obtained from Quantitative
Technologies Inc. Whitehouse N.J. Preparative HPLC was performed on
a Shimadzu-LC-8A dual pump gradient preparative HPLC system
employing Shimadzu-ClassVP software version 4.3 for system control,
data acquisition, fraction collection and post run processing
[0237] E. General Procedure for Peptide Synthesis:
[0238] Rink Amide-Novagel HL resin (0.6 mmol/g) was used as the
solid support.
[0239] F. Coupling Procedure:
[0240] In a typical experiment, the first amino acid was loaded
onto 0.1 g of the resin (0.06 mmol). The appropriate Fmoc-amino
acid in NMP (0.25M solution; 0.960 mL was added to the resin
followed by N-hydroxybenzotriazole (0.5M in NMP; 0.48 mL)) and the
reaction block (in the case of automated peptide synthesis) or
individual reaction vessel (in the case of manual peptide
synthesis) was shaken for about 2 min. To the above mixture,
diisopropylcarbodiimide (0.5M in NMP; 0.48 mL) was added and the
reaction mixture was shaken for 4 h at ambient temperature. Then
the reaction block or the individual reaction vessel was purged of
reactants by application of a positive pressure of dry
nitrogen.
[0241] G. Washing Procedure:
[0242] Each well of the reaction block was filled with 1.2 mL of
NMP and the block was shaken for 5 min. The solution was drained
under positive pressure of nitrogen. This procedure was repeated
three times. The same procedure was used, with an appropriate
volume of NMP, in the case of manual synthesis using individual
vessels.
[0243] H. Removal of Fmoc Group:
[0244] The resin containing the Fmoc-protected amino acid was
treated with 1.5 mL of 20% piperidine in DMF (v/v) and the reaction
block or individual manual synthesis vessel was shaken for 15 min.
The solution was drained from the resin. This procedure was
repeated once and the resin was washed employing the washing
procedure described above.
[0245] I. Final Coupling of Ligand (DOTA and CMDOTA):
[0246] The N-terminal amino group of the resin bound peptide linker
construct was deblocked and the resin was washed. A 0.25M solution
of the desired ligand and HBTU in NMP was made, and was treated
with a two-fold equivalency of DIEA. The resulting solution of
activated ligand was added to the the resin (1.972 mL; 0.48 mmol)
and the reaction mixture was shaken at ambient temperature for
24-30 h. The solution was drained and the resin was washed. The
final wash of the resin was conducted with 1.5 mL dichloromethane
(3.times.).
[0247] J. Deprotection and Purification of the Final Peptide:
[0248] A solution of reagent B (2 mL;
88:5:5:2--TFA:Phenol:Water:TIPS) was added to the resin and the
reaction block or individual vessel was shaken for 4.5 h at ambient
temperature. The resulting solution containing the deprotected
peptide was drained into a vial. This procedure was repeated two
more times with 1 mL of reagent B. The combined filtrate was
concentrated under reduced pressure using a Genevac HT-12 series II
centrifugal concentrator. The residue in each vial was then
triturated with 2 mL of Et.sub.2O and the supernatant was decanted.
This procedure was repeated twice to provide the peptides as
colorless solids. The crude peptides were dissolved in
water/acetonitrile and purified using either a Waters XTerra MS C18
preparative HPLC column (50 mm.times.19 mm, 5 micron particle size,
120 .ANG. pore size) or a Waters-YMC C18 ODS column (250
mm.times.30 mm i.d., 10 micron particle size. 120 .ANG. pore size).
The fractions with the products were collected and analyzed by
HPLC. The fractions with >95% purity were pooled and the
peptides isolated by lyophilization.
[0249] Conditions for Preparative HPLC (Waters XTerra Column):
[0250] Elution rate: 50 mL/min [0251] Detection: UV, .lamda.=220 nm
[0252] Eluent A: 0.1% aq. TFA; Solvent B: Acetonitrile (0.1% TFA).
Conditions for HPLC Analysis: [0253] Column: Waters XTerra (Waters
Co.; 4.6.times.50 mm; MS C18; 5 micron particle, 120 .ANG. pore).
[0254] Elution rate: 3 mL/min; Detection: UV, .lamda.=220 nm.
[0255] Eluent A:0.1% aq. TFA; Solvent B: Acetonitrile (0.1%
TFA).
EXAMPLE I
FIGS. 1A-B
Synthesis of L62
[0256] Summary: As shown in FIGS. 1A-B, L62 was prepared using the
following steps: Hydrolysis of
(3.beta.,5.beta.)-3-aminocholan-24-oic acid methyl ester A with
NaOH gave the corresponding acid B, which was then reacted with
Fmoc-Cl to give intermediate C. Rink amide resin functionalised
with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2
(BBN[7-14][SEQ ID NO: 1]) was sequentially reacted with C,
Fmoc-glycine and DOTA tri-t-butyl ester. After cleavage and
deprotection with reagent B the crude was purified by preparative
HPLC to give L62. Overall yield: 2.5%. More details are provided
below:
[0257] A. Preparation of intermediates B and C: [0258] 1. Synthesis
of (3.beta.,5.beta.)-3-Aminocholan-24-oic acid (B) [0259] A 1 M
solution of NaOH (16.6 mL; 16.6 mmol) was added dropwise to a
solution of (3.beta.,5.beta.)-3-aminocholan-24-oic acid methyl
ester (5.0 g; 12.8 mmol) in MeOH (65 mL) at 45.degree. C. After 3 h
stirring at 45.degree. C., the mixture was concentrated to 25 mL
and H.sub.2O (40 mL) and 1 M HCl (22 mL) were added. The
precipitated solid was filtered, washed with H.sub.2O (2.times.50
mL) and vacuum dried to give B as a white solid (5.0 g; 13.3 mmol).
Yield 80%. [0260] 2. Synthesis of
(3.beta.,5.beta.)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid
(C) [0261] A solution of 9-fluorenylmethoxycarbonyl chloride (0.76
g; 2.93 mmol) in 1,4-dioxane (9 mL) was added dropwise to a
suspension of (3.beta.,5.beta.)-3-aminocholan-24-oic acid B (1.0 g;
2.66 mmol) in 10% aq. Na.sub.2CO.sub.3 (16 mL) and 1,4-dioxane (9
mL) stirred at 0.degree. C. After 6 h stirring at room temperature
H.sub.2O (90 mL) was added, the aqueous phase washed with Et.sub.2O
(2.times.90 mL) and then 2 M HCl (15 mL) was added (final pH: 1.5).
The aqueous phase was extracted with EtOAc (2.times.100 mL), the
organic phase dried over Na.sub.2SO.sub.4 and evaporated. The crude
was purified by flash chromatography to give C as a white solid
(1.2 g; 2.0 mmol). Yield 69%.
[0262] B. Synthesis of L62
(N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraaza-
cyclododec-1-yl]acetyl]amino]acetyl]amino]-cholan-24-yl]-L-glutaminyl-L-tr-
yptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide):
[0263] Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase
peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10
min, the solution was emptied and fresh 50% morpholine in DMA (7
mL) was added. The suspension was shaken for 20 min then the
solution was emptied and the resin washed with DMA (5.times.7 mL).
(3.beta.,5.beta.)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid
C (0.72 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2
mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and
DMA (7 mL) were added to the resin, the mixture shaken for 24 h at
room temperature, and the solution was emptied and the resin washed
with DMA (5.times.7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied,
fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken
for another 20 min. The solution was emptied and the resin washed
with DMA (5.times.7 mL). N-.alpha.-Fmoc-glycine (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol) and DMA (7 mL)
were added to the resin. The mixture was shaken for 3 h at room
temperature, the solution was emptied and the resin washed with DMA
(5.times.7 mL). The resin was then shaken with 50% morpholine in
DMA (7 mL) for 10 min, the solution was emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for
another 20 min. The solution was emptied and the resin washed with
DMA (5.times.7 mL) followed by addition of
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) to the resin. The mixture was shaken for
24 h at room temperature, the solution was emptied and the resin
washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2 (5.times.7 mL) and
vacuum dried. The resin was shaken in a flask with reagent B (25
mL) for 4.5 h. The resin was filtered and the solution was
evaporated under reduced pressure to afford an oily crude which was
triturated with Et.sub.2O (20 mL) gave a precipitate. The
precipitate was collected by centrifugation and washed with
Et.sub.2O (3.times.20 mL), then analysed by HPLC and purified by
preparative HPLC. The fractions containing the product were
lyophilised to give L62 (6.6 mg; 3.8.times.10.sup.-3 mmol) as a
white solid. Yield 4.5%.
EXAMPLE II
FIGS. 2A-F
Synthesis of L70, L73, L74, L115 and L116
[0264] Summary: The products were obtained by coupling of the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14][SEQ
ID NO:1]) (with appropriate side chain protection) on the Rink
amide resin with different linkers, followed by functionalization
with DOTA tri-t-butyl ester. After cleavage and deprotection with
reagent B the final products were purified by preparative HPLC.
Overall yields 3-9%.
[0265] A. Synthesis of L70: [0266] Resin A (0.5 g; 0.3 mmol) was
shaken in a solid phase peptide synthesis vessel with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin
washed with DMA (5.times.7 mL). Fmoc-4-aminobenzoic acid (0.43 g;
1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA
(7 mL) were added to the resin, the mixture shaken for 3 h at room
temperature, the solution was emptied and the resin washed with DMA
(5.times.7 mL). The resin was then shaken with 50% morpholine in
DMA (7 mL) for 10 min, the solution was emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture was shaken for
20 min. The solution was emptied and the resin washed with DMA
(5.times.7 mL). Fmoc-glycine (0.36 g; 1.2 mmol),
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate (HATU) (0.46 g; 1.2 mmol) and DIEA (0.40 mL;
2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution
was added to the resin, the mixture shaken for 2 h at room
temperature, the solution was emptied and the resin washed with DMA
(5.times.7 mL). The resin was then shaken with 50% morpholine in
DMA (7 mL) for 10 min, the solution was emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for 20
min. The solution was emptied and the resin washed with DMA
(5.times.7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic
acid tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40
mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture
was shaken for 24 h at room temperature, the solution was emptied
and the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
filtrate solution was evaporated under reduced pressure to afford
an oily crude that was triturated with Et.sub.2O (5 mL). The
precipitate was collected by centrifugation and washed with
Et.sub.2O (5.times.5 mL), then analysed by HPLC and purified by
preparative HPLC. The fractions containing the product were
lyophilised to give L70 as a white fluffy solid (6.8 mg; 0.005
mmol). Yield 3%.
[0267] B. Synthesis of L73, L115 and L116: [0268] Resin A (0.5 g;
0.3 mmol) was shaken in a solid phase peptide synthesis vessel with
50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension
was stirred for 20 min then the solution was emptied and the resin
washed with DMA (5.times.7 mL). Fmoc-linker (1.2 mmol),
N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2 mmol),
N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture was shaken for 3 h at room
temperature, the solution was emptied and the resin was washed with
DMA (5.times.7 mL). The resin was shaken with 50% morpholine in DMA
(7 mL) for 10 min, the solution was emptied, fresh 50% morpholine
in DMA (7 mL) was added and the mixture was shaken for 20 min. The
solution was emptied and the resin washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that was triturated with Et.sub.2O (5 mL). The precipitate
was collected by centrifugation and washed with Et.sub.2O
(5.times.5 mL), then analysed by HPLC and purified by preparative
HPLC. The fractions containing the product were lyophilised.
[0269] D. Synthesis of L74: [0270] Resin A (0.5 g; 0.3 mmol) was
shaken in a solid phase peptide synthesis vessel with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin was
washed with DMA (5.times.7 mL). Fmoc-isonipecotic acid (0.42 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture was shaken for 3 h at room
temperature, the solution was emptied and the resin was washed with
DMA (5.times.7 mL). The resin was shaken with 50% morpholine in DMA
(7 mL) for 10 min, the solution was emptied, fresh 50% morpholine
in DMA (7 mL) was added and the mixture was shaken for 20 min. The
solution was emptied and the resin was washed with DMA (5.times.7
mL). Fmoc-glycine (0.36 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC
(0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the
mixture was shaken for 3 h at room temperature, the solution was
emptied and the resin washed with DMA (5.times.7 mL). The resin was
then shaken with 50% morpholine in DMA (7 ML) for 10 min, the
solution was emptied, fresh 50% morpholine in DMA (7 mL) was added
and the mixture shaken for 20 minutes. The solution was emptied and
the resin was washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin was washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that was triturated with Et.sub.2O (5 mL). The precipitate
was collected by centrifugation and washed with Et.sub.2O
(5.times.5 mL), then analysed by HPLC and purified by HPLC. The
fractions containing the product were lyophilised to give L74 as a
white fluffy solid (18.0 mg; 0.012 mmol). Yield 8%.
EXAMPLE III
FIGS. 3A-E
Synthesis of L67
[0271] Summary: Hydrolysis of
(3.beta.,5.beta.)-3-amino-12-oxocholan-24-oic acid methyl ester A
with NaOH gave the corresponding acid B, which was then reacted
with Fmoc-Glycine to give intermediate C. Rink amide resin
functionalised with the octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14][SEQ ID NO:1])
was sequentially reacted with C, and DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the crude was purified by
preparative HPLC to give L67. Overall yield: 5.2%.
[0272] A. Synthesis (3.beta.,5.beta.)-3-Amino-12-oxocholan-24-oic
acid, (B) [0273] A 1 M solution of NaOH (6.6 mL; 6.6 mmol) was
added dropwise to a solution of
(3.beta.,5.beta.)-3-amino-12-oxocholan-24-oic acid methyl ester A
(2.1 g; 5.1 mmol) in MeOH (15 mL) at 45.degree. C. After 3 h
stirring at 45.degree. C., the mixture was concentrated to 25 mL
then H.sub.2O (25 mL) and 1 M HCl (8 mL) were added. The
precipitated solid was filtered, washed with H.sub.2O (2.times.30
mL) and vacuum dried to give B as a white solid (1.7 g; 4.4 mmol).
Yield 88%.
[0274] B. Synthesis of
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxoch-
olan-24-oic acid (C). [0275] Tributylamine (0.7 mL; 3.1 mmol) was
added dropwise to a solution of N-.alpha.-Fmoc-glycine (0.9 g; 3.1
mmol) in THF (25 mL) stirred at 0.degree. C. Isobutyl chloroformate
(0.4 mL; 3.1 mmol) was subsequently added and, after 10 min, a
suspension of tributylamine (0.6 mL; 2.6 mmol) and
(3.beta.,5.beta.)-3-amino-12-oxocholan-24-oic acid B (1.0 g; 2.6
mmol) in DMF (30 mL) was added dropwise, over 1 h, into the cooled
solution. The mixture was allowed to warm up and after 6 h the
solution was concentrated to 40 mL, then H.sub.2O (50 mL) and 1 N
HCl (10 mL) were added (final pH: 1.5). The precipitated solid was
filtered, washed with H.sub.2O (2.times.50 mL), vacuum dried and
purified by flash chromatography to give C as a white solid (1.1 g;
1.7 mmol). Yield 66%.
[0276] C. Synthesis of L67
(N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraaza-
cyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glut-
aminyl-L-trytophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionin-
amide). [0277] Resin D (0.5 g; 0.3 mmol) was shaken in a solid
phase peptide synthesis vessel with 50% morpholine in DMA (7 mL)
for 10 min, the solution was emptied and fresh 50% morpholine in
DMA (7 mL) was added. The suspension was stirred for 20 min then
the solution was emptied and the resin was washed with DMA
(5.times.7 mL).
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino]-12-oxoc-
holan-24-oic acid C (0.80 g; 1.2 mmol), N-hydroxybenzotriazole
(HOBt) (0.18 g; 1.2 mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19
mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture
was shaken for 24 h at room temperature, the solution was emptied
and the resin was washed with DMA (5.times.7 mL). The resin was
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution
was emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture was shaken for 20 min. The solution was emptied and the
resin was washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin was washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4.5 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that was triturated with Et.sub.2O (20 mL).
EXAMPLE IV
FIGS. 4A-H
Synthesis of L63 and L64
[0278] Summary: Hydrolysis of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid methyl ester 1b with NaOH gave the intermediate 2b, which was
then reacted with Fmoc-glycine to give 3b. Rink amide resin
functionalised with the octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14][SEQ ID NO:1])
was reacted with 3b and then with DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the crude was purified by
preparative HPLC to give L64. The same procedure was repeated
starting from intermediate 2a, already available, to give L63.
Overall yields: 9 and 4%, respectively.
[0279] A. Synthesis of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-Amino-7,12-dihydroxycholan-24-oic
acid, (2b) [0280] A 1 M solution of NaOH (130 mL; 0.13 mol) was
added dropwise to a solution of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid methyl ester 1b (42.1 g; 0.10 mol) in MeOH (300 mL) heated at
45.degree. C. After 3 h stirring at 45.degree. C., the mixture was
concentrated to 150 mL and H.sub.2O (350 mL) was added. After
extraction with CH.sub.2Cl.sub.2 (2.times.100 mL) the aqueous
solution was concentrated to 200 mL and 1 M HCl (150 mL) was added.
The precipitated solid was filtered, washed with H.sub.2O
(2.times.100 mL) and vacuum dried to give 2b as a white solid (34.8
g; 0.08 mol). Yield 80%.
[0281] B. Synthesis of
(3.beta.,5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amin-
o-12-hydroxycholan-24-oic acid, (3a) [0282] Tributylamine (4.8 mL;
20.2 mmol) was added dropwise to a solution of
N-.alpha.-Fmoc-glycine (6.0 g; 20.2 mmol) in THF (120 mL) stirred
at 0.degree. C. Isobutyl chloroformate (2.6 mL; 20.2 mmol) was
subsequently added and, after 10 min, a suspension of tributylamine
(3.9 mL; 16.8 mmol) and
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid 2a
(6.6 g; 16.8 mmol) in DMF (120 mL) was added dropwise, over 1 h,
into the cooled solution. The mixture was allowed to warm up and
after 6 h the solution was concentrated to 150 mL, then H.sub.2O
(250 mL) and 1 N HCl (40 mL) were added (final pH: 1.5). The
precipitated solid was filtered, washed with H.sub.2O (2.times.100
mL), vacuum dried and purified by flash chromatography to give 3a
as a white solid (3.5 g; 5.2 mmol). Yield 31%.
[0283] C. Synthesis of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) [0284]
Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a solution
of N-.alpha.-Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL) stirred
at 0.degree. C. Isobutyl chloroformate (1.7 mL; 13.5 mmol) was
subsequently added and, after 10 min, a suspension of tributylamine
(2.6 mL; 11.2 mmol) and
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxychol-
an-24-oic acid 3a (4.5 g; 11.2 mmol) in DMF (80 mL) was added
dropwise, over 1 h, into the cooled solution. The mixture was
allowed to warm up and after 6 h the solution was concentrated to
120 mL, then H.sub.2O (180 mL) and 1 N HCl (30 mL) were added
(final pH: 1.5). The precipitated solid was filtered, washed with
H.sub.2O (2.times.100 mL), vacuum dried and purified by flash
chromatography to give 3a as a white solid (1.9 g; 2.8 mmol). Yield
25%. In an alternative method,
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) can be prepared
as follows: [0285] N-Hydroxysuccinimide (1.70 g, 14.77 mmol) and
DIC (1.87 g, 14.77 mmol) were added sequentially to a stirred
solution of Fmoc-Gly-OH (4.0 g, 13.45 mmol) in dichloromethane (15
mL); the resulting mixture was stirred at room temperature for 4 h.
The N,N'-diisopropylurea formed was removed by filtration and the
solid was washed with ether (20 mL). The volatiles were removed and
the solid Fmoc-Gly-succinimidyl ester formed was washed with ether
(3.times.20 mL). Fmoc-Gly-succinimidyl ester was then redissolved
in dry DMF (15 mL) and 3-aminodeoxycholic acid (5.21 g, 12.78 mmol)
was added to the clear solution. The reaction mixture was stirred
at room temperature for 4 h, water (200 mL) was added and the
precipitated solid was filtered, washed with water, dried and
purified by silica gel chromatography (TLC (silica): (R.sub.f:
0.50, silica gel, CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1) (eluant:
CH.sub.2Cl.sub.2/CH.sub.3OH (9:1) to give Fmoc-Gly-3-aminocholic
acid as a colorless solid. Yield: 7.46 g (85%).
[0286] D. Synthesis of L63
(N-[(3.beta.,5.beta.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,1-
0-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxochol-
an-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-l-
eucyl-L-methioninamide) [0287] Resin A (0.5 g; 0.3 mmol) was shaken
in a solid phase peptide synthesis vessel with 50% morpholine in
DMA (7 mL) for 10 min, the solution was emptied and fresh 50%
morpholine in DMA (7 mL) was added. The suspension was stirred for
20 min then the solution was emptied and the resin washed with DMA
(5.times.7 mL).
(3.beta.,5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amin-
o-12-hydroxycholan-24-oic acid 3a (0.82 g; 1.2 mmol),
N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2 mmol),
N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture was shaken for 24 h at
room temperature, the solution was emptied and the resin was washed
with DMA (5.times.7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied,
fresh 50% morpholine in DMA (7 mL) was added and the mixture was
shaken for 20 min. The solution was emptied and the resin washed
with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that after treatment with Et.sub.2O (5 mL) gave a
precipitate. The precipitate was collected by centrifugation and
washed with Et.sub.2O (5.times.5 mL), then analysed and purified by
HPLC. The fractions containing the product were lyophilised to give
L63 as a white fluffy solid (12.8 mg; 0.0073 mmol). Yield 9%.
[0288] E. Synthesis of L64
(N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl-
)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydro-
xy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
-histidyl-L-leucyl-L-methioninamide) [0289] Resin A (0.5 g; 0.3
mmol) was shaken in a solid phase peptide synthesis vessel with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min, the solution was emptied and the resin was
washed with DMA (5.times.7 mL).
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid 3b (0.81 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0. 19 mL; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture was shaken for 24 h at room
temperature, the solution was emptied and the resin was washed with
DMA (5.times.7 mL). The resin was shaken with 50% morpholine in DMA
(7 mL) for 10 min, the solution was emptied, fresh 50% morpholine
in DMA (7 mL) was added and the mixture was shaken for 20 min. The
solution was emptied and the resin was washed with DMA (5.times.7
mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,1 0-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that was triturated with Et.sub.2O (5 mL). The precipitate
was collected by centrifugation and washed with Et.sub.2O
(5.times.5 mL). Then it was dissolved in H.sub.2O (20 mL), and
Na.sub.2CO.sub.3 (0.10 g; 0.70 mmol) was added; the resulting
mixture was stirred 4 h at room temperature. This solution was
purified by HPLC, the fractions containing the product lyophilised
to give L64 as a white fluffy solid (3.6 mg; 0.0021 mmol). Yield
4%.
EXAMPLE V
FIGS. 5A-E
Synthesis of L71 and L72
[0290] Summary: The products were obtained in two steps. The first
step was the solid phase synthesis of the octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14][SEQ ID NO:1])
(with appropriate side chain protecting groups) on the Rink amide
resin. The second step was the coupling with different linkers
followed by functionalization with DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the final products were
purified by preparative HPLC. Overall yields 3-9%.
[0291] A. Rink amide resin functionalised with bombesin[7-14] (B)
[0292] In a solid phase peptide synthesis vessel (see enclosure No.
1) Fmoc-aminoacid (24 mmol), N-hydroxybenzotriazole (HOBt) (3.67 g;
24 mmol), and N,N'-diisopropylcarbodiimide (DIC) (3.75 mL; 24 mmol)
were added sequentially to a suspension of Rink amide NovaGel.TM.
resin (10 g; 6.0 mmol) A in DMF (45 mL). The mixture was shaken for
3 h at room temperature using a bench top shaker, then the solution
was emptied and the resin was washed with DMF (5.times.45 mL). The
resin was shaken with 25% piperidine in DMF (45 mL) for 4 min, the
solution was emptied and fresh 25% piperidine in DMF (45 mL) was
added. The suspension was shaken for 10 min, then the solution was
emptied and the resin was washed with DMF (5.times.45 mL). [0293]
This procedure was applied sequentially for the following amino
acids: N-.alpha.-Fmoc-L-methionine, N-.alpha.-Fmoc-L-leucine,
N-.alpha.-Fmoc-N-im-trityl-L-histidine, N-.alpha.-Fmoc-glycine,
N-.alpha.-Fmoc-L-valine, N-.alpha.-Fmoc-L-alanine,
N-.alpha.-Fmoc-N-in-Boc-L-tryptophan. [0294] In the last coupling
reaction N-.alpha.-Fmoc-N-.gamma.-trityl-L-glutamine (14.6 g; 24
mmol), HOBt (3.67 g; 24 mmol), and DIC (3.75 mL; 24 mmol) were
added to the resin in DMF (45 mL). The mixture was shaken for 3 h
at room temperature, the solution was emptied and the resin was
washed with DMF (5.times.45 mL), CH.sub.2Cl.sub.2 (5.times.45 mL)
and vacuum dried.
[0295] B. Bombesin [7-14] functionalisation and cleavage procedure
The resin B (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied and fresh 50% morpholine in DMA (7 mL) was
added. The suspension was stirred for 20 min then the solution was
emptied and the resin was washed with DMA (5.times.7 mL). The
Fmoc-linker (1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2
mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 3 h at room temperature, the solution was emptied and
the resin washed with DMA (5.times.7 mL). The resin was then shaken
with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture was shaken for 20 min. The solution was emptied and the
resin was washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl C (0.79 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40
mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture
was shaken for 24 h at room temperature. The solution was emptied
and the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
filtrate was evaporated under reduced pressure to afford an oily
crude that was triturated with ether (5 mL). The precipitate was
collected by centrifugation and washed with ether (5.times.5 mL),
then analyzed by analytical HPLC and purified by preparative HPLC.
The fractions containing the product were lyophilized.
[0296] C. Products [0297] 1. L71
(4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl-
]amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycil-L--
histidyl-L-leucyl-L-methioninamide) [0298] The product was obtained
as a white fluffy solid (7.3 mg; 0.005 mmol). Yield 7.5%. [0299] 2.
L72
(Trans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-
acetyl]amino]methyl]cyclohexylcarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl--
L-valyl-glycil-L-histidyl-L-leucyl-L-methioninamide) [0300] The
product was obtained as a white fluffy solid (7.0 mg; 0.005 mmol).
Yield 4.8%.
[0301] D.
Trans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cycloh-
exanecarboxylic acid, (D) [0302] A solution of
N-(9-fluorenylmethoxycarbonyloxy)succinimide (4.4 g; 14.0 mmol) in
1,4-dioxane (40 mL) was added dropwise to a solution of
trans-4-(aminomethyl)cyclohexanecarboxylic acid (2.0 g; 12.7 mmol)
in 10% Na.sub.2CO.sub.3 (30 mL) cooled to 0.degree. C. The mixture
was then allowed to warm to ambient temperature and after 1 h
stirring at room temperature was treated with 1 N HCl (32 mL) until
the final pH was 2. The resulting solution was extracted with
n-BuOH (100 mL); the volatiles were removed and the crude residue
was purified by flash chromatography to give D as a white solid
(1.6 g; 4.2 mmol). Yield 33%.
EXAMPLE VI
FIGS. 6A-K
Synthesis of L75 and L76
[0303] Summary: The two products were obtained by coupling of the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14][SEQ ID
NO:1]) on the Rink amide resin with the two linkers E and H,
followed by functionalization with DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the final products were
purified by preparative HPLC. Overall yields: 8.5% (L75) and 5.6%
(L76).
[0304] A. 2-[(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic
acid, (C) [0305] The product was synthesized following the
procedure reported in the literature (Bornstein, J; Drummon, P. E.;
Bedell, S. F. Org Synth. Coll. Vol. IV 1963, 810-812).
[0306] B. 2-(Aminomethyl)benzoic acid, (D) [0307] A 40% solution of
methylamine (6.14 mL; 7.1 mmol) was added to
2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid C (2
g; 7.1 mmol) and then EtOH (30 mL) was added. After 5 minutes
stirring at room temperature the reaction mixture was heated at
50.degree. C. After 2.5 h, the mixture was cooled and the solvent
was evaporated under reduced pressure. The crude product was
suspended in 50 mL of absolute ethanol and the suspension was
stirred at room temperature for 1 h. The solid was filtered and
washed with EtOH to afford 2-(aminomethyl)benzoic acid D (0.87 g;
5.8 mmol). Yield 81%.
[0308] C. 2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic
acid, (E) [0309] The product was synthesized following the
procedure reported in the literature (Sun, J-H.; Deneker, W. F.
Synth. Commun. 1998, 28, 4525-4530).
[0310] D. 4-(Aminomethyl)-3-nitrobenzoic acid, (G) [0311]
4-(Bromomethyl)-3-nitrobenzoic acid (3.2 g; 12.3 mmol) was
dissolved in 8% NH.sub.3 in EtOH (300 mL) and the resulting
solution was stirred at room temperature. After 22 h the solution
was evaporated and the residue suspended in H.sub.2O (70 mL). The
suspension was stirred for 15 min and filtered. The collected solid
was suspended in H.sub.2O (40 mL) and dissolved by the addition of
few drops of 25% aq. NH.sub.4OH (pH 12), then the pH of the
solution was adjusted to 6 by addition of 6 N HCl. The precipitated
solid was filtered, and washed sequentially with MeOH (3.times.5
mL), and Et.sub.2O (10 mL) and was vacuum dried (1.3 kPa;
P.sub.2O.sub.5) to give 4-(aminomethyl)-3-nitrobenzoic acid as a
pale brown solid (1.65 g; 8.4 mmol). Yield 68%.
[0312] E.
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzo- ic
acid, (H)
[0313] 4-(Aminomethyl)-3-nitrobenzoic acid G (0.8 g; 4 mmol) was
dissolved in 10% aq. Na.sub.2CO.sub.3 (25 mL) and 1,4-dioxane (10
mL) and the solution was cooled to 0.degree. C. A solution of
9-fluorenylmethyl chloroformate (Fmoc-C1) (1.06 g; 4 mmol) in
1,4-dioxane (10 mL) was added dropwise for 20 min. After 2 h at
0-5.degree. C. and 1 h at 10.degree. C. the reaction mixture was
filtered and the solution was acidified to pH 5 by addition of 1 N
HCl. The precipitate was filtered, washed with H.sub.2O (2.times.2
mL) dried under vacuum (1.3 kPa; P.sub.2O.sub.5) to give
4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic
acid as a white solid (1.6 g; 3.7 mmol). Yield 92%.
[0314] F. L75
(N-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycy-
l-L-histidyl-L-leucyl-L-methioninamide) [0315] Resin I (0.5 g; 0.3
mmol) was shaken in a solid phase peptide synthesis vessel with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin
washed with DMA (5.times.7 mL).
2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid, E
(0.45 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2
mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and
DMA (7 mL) were added to the resin, the mixture shaken for 24 h at
room temperature, the solution was emptied and the resin was washed
with DMA (5.times.7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied,
fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken
for 20 min. The solution was emptied and the resin washed with DMA
(5.times.7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic
acid tris(1,1-dimethylethyl) ester adduct with NaCl (DOTA
tri-t-butyl ester) (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC
(0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were
added to the resin. The mixture was shaken for 24 h at room
temperature, the solution was emptied and the resin was washed with
DMA (5.times.7 mL), CH.sub.2Cl.sub.2 (5.times.7 mL) and vacuum
dried. The resin was shaken in a flask with reagent B (25 mL) for
4.5 h. The resin was filtered and the filtrate was evaporated under
reduced pressure to afford an oily crude that after treatment with
Et.sub.2O (20 mL) gave a precipitate. The resulting precipitate was
collected by centrifugation and was washed with Et.sub.2O
(3.times.20 mL) to give L75 (190 mg; 0.13 mmol) as a white solid.
Yield 44%.
[0316] G. L76
(N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-val-
yl-glycyl-L-histidyl-L-leucyl-L-methioninamide) [0317] Resin I (0.5
g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied and fresh 50% morpholine in DMA (7 mL) was added. The
suspension was stirred for 20 min then the solution was emptied and
the resin was washed with DMA (5.times.7 mL).
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic
acid, H (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL;
1.2 mmol) and DMA (7 mL) were added to the resin, the mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin was washed with DMA (5.times.7 mL). The resin was then
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution
was emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture was shaken for 20 min. The solution was emptied and the
resin was washed with DMA (5.times.7 mL). DOTA tri-t-butyl ester
(0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2
mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the
resin. The mixture was shaken for 24 h at room temperature, the
solution was emptied and the resin was washed with DMA (5.times.7
mL), CH.sub.2Cl.sub.2 (5.times.7 mL) and vacuum dried. The resin
was shaken in a flask with reagent B (25 mL) for 4.5 h. The resin
was filtered and the solution was evaporated under reduced pressure
to afford an oily crude that was triturated with Et.sub.2O (20 mL).
The precipitate was collected by centrifugation and was washed with
Et.sub.2O (3.times.20 mL) to give a solid (141 mg) which was
analysed by HPLC. A 37 mg portion of the crude was purified by
preparative HPLC. The fractions containing the product were
lyophilised to give L76 (10.8 mg; 7.2.times.10.sup.-3 mmol) as a
white solid. Yield 9%.
EXAMPLE VII
FIGS. 7A-F
Synthesis of L124
[0318] Summary: 4-Cyanophenol A was reacted with ethyl bromoacetate
and K.sub.2CO.sub.3 in acetone to give the intermediated B, which
was hydrolysed with NaOH to the corresponding acid C. Successive
hydrogenation of C with H.sub.2 and PtO.sub.2 at 355 kPa in
EtOH/CHCl.sub.3 gave the corresponding aminoacid D, which was
directly protected with FmocOSu to give E. Rink amide resin
functionalised with the octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14][SEQ ID NO:1])
was reacted with E and then with DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the crude was purified by
preparative HPLC to give L124. Overall yield: 1.3%
[0319] A. Synthesis of (4-Cyanophenoxy)acetic acid ethyl ester, (B)
[0320] The product was synthesized following the procedure reported
in the literature (Archimbault, P.; LeClerc, G.; Strosberg, A. D.;
Pietri-Rouxel, F. PCT Int. Appl. WO 980005, 1998).
[0321] B. Synthesis of (4-Cyanophenoxy)acetic acid, (C [0322] A 1 N
solution of NaOH (7.6 mL; 7.6 mmol) was added dropwise to a
solution of (4-cyanophenoxy)acetic acid ethyl ester B (1.55 g; 7.6
mmol) in MeOH (15 mL). After 1 h the solution was acidified with 1
N HCl (7.6 mL; 7.6 mmol) and evaporated. The residue was taken up
with water (20 mL) and extracted with CHCl.sub.3 (2.times.30 mL).
The organic phases were evaporated and the crude was purified by
flash chromatography to give (4-cyanophenoxy)acetic acid C (0.97 g;
5.5 mmol) as a white solid. Yield 72%.
[0323] C. Synthesis of
[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic
acid, (E [0324] PtO.sub.2 (150 mg) was added to a solution of
(4-cyanophenoxy)acetic acid C (1.05 g; 5.9 mmol) in EtOH (147 mL)
and CHCl.sub.3 (3 mL). The suspension was stirred 30 h under a
hydrogen atmosphere (355 kPa; 20.degree. C.). The mixture was
filtered through a Celite.RTM. pad and the solution evaporated
under vacuum. The residue was purified by flash chromatography to
give acid D (0.7 g) which was dissolved in H.sub.2O (10 mL), MeCN
(2 mL) and Et.sub.3N (0.6 mL) at 0.degree. C., then a solution of
N-(9-fluorenylmethoxycarbonyloxy)succinimide (1.3 g; 3.9 mmol) in
MeCN (22 mL) was added dropwise. After stirring 16 h at room
temperature the reaction mixture was filtered and the volatiles
were removed under vacuum. The residue was treated with 1 N HCl (10
mL) and the precipitated solid was filtered and purified by flash
chromatography to give
[4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic
acid E (0.56 g; 1.4 mmol) as a white solid. Overall yield 24%.
[0325] E. Synthesis of L124
(N-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ac-
etyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-trytophyl-L-alanyl-L-valy-
l-glycyl-L-histidyl-L-leucyl-L-methioninamide) [0326] Resin F (480
mg; 0.29 mmol) was shaken in a solid phase peptide synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied and fresh 50 % morpholine in DMA (7 mL) was added. The
suspension was stirred for 20 min, the solution was emptied and the
resin was washed with DMA (5.times.7 mL).
[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic
acid E (480 mg; 1.19 mmol), N-hydroxybenzotriazole (HOBt) (182 mg;
1.19 mmol), N,N'-diisopropylcarbodiimide (DIC) (185 .mu.L; 1.19
mmol) and DMA (7 mL) were added to the resin, the mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin was washed with DMA (5.times.7 mL). The resin was then
shaken with 50% morpholine in DMA (6 mL) for 10 min, the solution
was emptied, fresh 50% morpholine in DMA (6 mL) was added and the
mixture was shaken for 20 min. The solution was emptied and the
resin was washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (750 mg; 1.19 mmol),
HOBt (182 mg; 1.19 mmol), DIEA (404 .mu.L; 2.36 mmol), DIC (185
.mu.L; 1.19 mmol) and DMA (6 mL) were added to the resin. The
mixture was shaken for 24 h at room temperature, the solution was
emptied, the resin was washed with DMA (2.times.7 mL),
CH.sub.2Cl.sub.2 (5.times.7 mL) and vacuum dried. The resin was
shaken in a flask with Reagent B (25 mL) for 4 h. The resin was
filtered and the filtrate was evaporated under reduced pressure to
afford an oily crude that was triturated with Et.sub.2O (5 mL). The
precipitate was collected by centrifugation and washed with
Et.sub.2O (5.times.5 mL) to give a solid (148 mg) which was
analysed by HPLC. A 65 mg portion of the crude was purified by
preparative HPLC. The fractions containing the product were
lyophilised to give L127 as a white solid (15 mg; 0.01 mmol). Yield
7.9%.
EXAMPLE VIII
FIGS. 8A-F
Synthesis of L125
[0327] Summary: 4-(Bromomethyl)-3-methoxybenzoic acid methyl ester
A was reacted with NaN.sub.3 in DMF to give the intermediate azide
B, which was then reduced with Ph.sub.3P and H.sub.2O to amine C.
Hydrolysis of C with NaOH gave acid D, which was directly protected
with FmocOSu to give E. Rink amide resin functionalised with the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14][SEQ
ID NO: 1]) was reacted with E and then with DOTA tri-t-butyl ester.
After cleavage and deprotection with reagent B the crude was
purified by preparative HPLC to give L125. Overall yield: 0.2%.
[0328] A. Synthesis of 4-(Azidomethyl)-3-methoxybenzoic acid methyl
ester, (B) [0329] A solution of 4-(bromomethyl)-3-methoxybenzoic
acid methyl ester (8 g; 31 mmol) and NaN.sub.3(2 g; 31 mmol) in DMF
(90 mL) was stirred overnight at room temperature. The volatiles
were removed under vacuum and the crude product was dissolved in
EtOAc (50 mL). The solution was washed with water (2.times.50 mL)
and dried. The volatiles were evaporated to provide
4-(azidomethyl)-3-methoxybenzoic acid methyl ester (6.68 g; 30
mmol). Yield 97%.
[0330] B. 4-(Aminomethyl)-3-methoxybenzoic acid methyl ester, (C)
[0331] Triphenylphosphine (6.06 g; 23 mmol) was added to a solution
of (4-azidomethyl)-3-methoxybenzoic acid methyl ester B (5 g; 22
mmol) in THF (50 mL): hydrogen evolution and formation of a white
solid was observed. The mixture was stirred under nitrogen at room
temperature. After 24 h more triphenylphosphine (0.6 g; 2.3 mmol)
was added. After 24 h the azide was consumed and H.sub.2O (10 mL)
was added. After 4 h the white solid disappeared. The mixture was
heated at 45.degree. C. for 3 h and was stirred overnight at room
temperature. The solution was evaporated to dryness and the crude
was purified by flash chromatography to give
4-(aminomethyl)-3-methoxybenzoic acid methyl ester C (1.2 g; 6.1
mmol). Yield 28%.
[0332] C.
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxyben-
zoic acid, (E) [0333] A 1 N solution of NaOH (6.15 mL; 6.14 mmol)
was added dropwise to a solution of
4-(aminomethyl)-3-methoxybenzoic acid methyl ester C (1.2 g; 6.14
mmol) in MeOH (25 mL) heated at 40.degree. C. After stirring 8 h at
45.degree. C. the solution was stirred over night at room
temperature. A 1 N solution of NaOH (0.6 mL; 0.6 mmol) was added
and the mixture heated at 40.degree. C. for 4 h. The solution was
concentrated, acidified with 1 N HCl (8 mL; 8 mmol), extracted with
EtOAc (2.times.10 mL) then the aqueous layer was concentrated to 15
mL. This solution (pH 4.5) was cooled at 0.degree. C. and Et.sub.3N
(936 .mu.L; 6.75 mmol) was added (pH 11). A solution of
N-(9-fluorenylmethoxycarbonyloxy)succinimide (3.04 g; 9 mmol) in
MeCN (30 mL) was added dropwise (final pH 9) and a white solid
precipitated. After stirring 1 h at room temperature the solid was
filtered, suspended in 1N HCl (15 mL) and the suspension was
stirred for 30 min. The solid was filtered to provide
4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic
acid E as a white solid (275 mg; 0.7 mmol). [0334] The filtrate was
evaporated under vacuum and the resulting white residue was
suspended in 1N HCl (20 mL) and stirred for 30 minutes. The solid
was filtered and purified by flash chromatography to give more acid
E (198 mg; 0.5 mmol). Overall yield 20%.
[0335] D. L125
(N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]ace-
tyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-v-
alyl-glycyl-L-histidyl-L-leucyl-L-methioninamide) [0336] Resin F
(410 mg; 0.24 mmol) was shaken in a solid phase peptide synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution
was emptied and fresh 50% morpholine in DMA (7 mL) was added. The
suspension was stirred for 20 min then the solution was emptied and
the resin was washed with DMA (5.times.7 mL).
4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic
acid E (398 mg; 0.98 mmol), N-hydroxybenzotriazole (HOBt) (151 mg;
0.98 mmol), N,N'-diisopropylcarbodiimide (DIC) (154 .mu.L; 0.98
mmol) and DMA (6 mL) were added to the resin; the mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin was washed with DMA (5.times.7 mL). The resin was then
shaken with 50% morpholine in DMA (6 mL) for 10 min, the solution
was emptied, fresh 50% morpholine in DMA (6 mL) was added and the
mixture was shaken for 20 min. The solution was emptied and the
resin washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (618 mg; 0.98 mmol),
HOBt (151 mg; 0.98 mmol), DIC (154 .mu.L; 0.98 mmol), DIEA (333
.mu.L; 1.96 mmol) and DMA (6 mL) were added to the resin. The
mixture was shaken for 24 h at room temperature, the solution was
emptied and the resin was washed with DMA (5.times.7 mL),
CH.sub.2Cl.sub.2 (5.times.7 mL) and vacuum dried. The resin was
shaken in a flask with reagent B (25 mL) for 4 h. The resin was
filtered and the solution was evaporated under reduced pressure to
afford an oily crude that was triturated with Et.sub.2O (5 mL). The
resulting precipitate was collected by centrifugation, was washed
with Et.sub.2O (5.times.5 mL), was analysed by HPLC and purified by
preparative HPLC. The fractions containing the product were
lyophilised to give L125 as a white solid (15.8 mg; 0.011 mmol).
Yield 4.4%.
EXAMPLE IX
Synthesis of Additional GRP Compounds
[0337] A. General procedure for the preparation of
4,4'-Aminomethylbiphenylcarboxylic acid (B2) and
3,3'-aminomethylbiphenylcarboxylic acid (B3):
[0338] 1. Methyl-hydroxymethylbiphenylcarboxylates: [0339]
Commercially available (Aldrich Chemical Co.)
4-hydroxymethylphenylboric acid or 3-hydroxymethylphenylboric acid
(1.0 g, 6.58 mmol) was stirred with isopropanol (10 mL) and 2M
sodium carbonate (16 mL) until the solution became homogeneous. The
solution was degassed by passing nitrogen through the solution and
then treated with solid methyl-3-bromobenzoate, or
methyl-4-bromobenzoate (1.35 g, 6.3 mmol) followed by the Pd (0)
catalyst {[(C.sub.6H.sub.5).sub.3P].sub.4Pd; 0.023 g, 0.003 mmol}.
The reaction mixture was kept at reflux under nitrogen until the
starting bromobenzoate was consumed as determined by TLC analysis
(2-3 h). The reaction mixture was then diluted with 250 mL of water
and extracted with ethyl acetate (3.times.50 mL). The organic
layers were combined and washed with saturated sodium bicarbonate
solution (2.times.50 mL) and dried (Na.sub.2SO.sub.4). The solvent
was removed under reduced pressure and the residue was
chromatographed over flash silica gel (100 g). Elution with 40%
ethyl acetate in hexanes yielded the product either as a solid or
oil.
[0340] Yield:
[0341] B2-0.45g (31%); m.p. -170-171.degree. C.
[0342] B3-0.69 g (62%); oil.
[0343] .sup.1H NMR (CDCl.sub.3) .delta. B2-3.94 (s,3H,
--COOCH.sub.3), 4.73 (s, 2H, --CH.sub.2-Ph), 7.475 (d, 2H, J=5 Hz),
7.6 (d, 2H, J=10 Hz), 7.65 (d, 2H, J=5 Hz) and 8.09 (d, 2H, J=10
Hz).
[0344] M.S.--m/e--243.0 [M+H]
[0345] B3-3.94 (s, 3H, --COOCH.sub.3), 4.76 (s, 2H, --CH.sub.2-Ph),
7.50 (m, 4H), 7.62 (s, 1H), 7.77 (s, 1 H), 8.00 (s, 1H) and 8.27
(s, 1H).
[0346] M.S.-m/e-243.2 [M+H]
[0347] 2. Azidomethylbiphenyl carboxylates: [0348] The above
biphenyl alcohols (2.0 mmol) in dry dichloromethane (10 mL) were
cooled in ice and treated with diphenylphosphoryl azide (2.2 mol)
and DBU (2.0 mmol) and stirred under nitrogen for 24 h. The
reaction mixture was diluted with water and extracted with ethyl
acetate (2.times.25 mL).The organic layers were combined and washed
successfully with 0.5 M citric acid solution (2.times.25 mL), water
(2.times.25 mL) and dried (Na.sub.2SO.sub.4). The solution was
filtered and evaporated under reduced pressure to yield the crude
product. The 4,4'-isomer was crystallized from hexane/ether and the
3,3'-isomer was triturated with isopropyl ether to remove all the
impurities; the product was homogeneous as determined on TLC
analysis and further purification was not required.
[0349] Yield:
[0350] Methyl-4-azidomethyl-4-biphenylcaroxylate--0.245 g (46%);
m.p.--106-108.degree. C.
[0351] Methyl-4-azidomethyl-4-biphenylcaroxylate--0.36 g (59%,
oil)
[0352] .sup.1H NMR (CDCl.sub.3) .delta.-4,4'-isomer--3.95 (s, 3H,
--COOCH.sub.3), 4.41 (s, 2H, --CH.sub.2N.sub.3). 7.42 (d, 2H, J=5
Hz), 7.66 (m, 4H) and 8.11 (d, 2H, J=5 Hz)
[0353] 3,3'-Isomer -3.94 (s, 3H, --COOCH.sub.3), 4.41 (s, 2H,
--CH.sub.2N.sub.3), 7.26-7.6 (m, 5H), 7.76 (d, 1H, J=10 Hz), 8.02
(d, 1H, J=5 Hz) and 8.27 (s, 1H).
[0354] 3. Hydrolysis of the methyl esters of biphenylcarboxylates:
[0355] About 4 mmol of the methyl esters were treated with 20 mL of
2M lithium hydroxide solution and stirred until the solution was
homogeneous (20-24 h). The aqueous layer was extracted with
2.times.50 mL of ether and the organic layer was discarded. The
aqueous layer was then acidified with 0.5 M citric acid and the
precipitated solid was filtered and dried. No other purification
was necessary and the acids were taken to the next step.
[0356] Yield:
[0357] 4,4'-isomer--0.87 g of methyl ester yielded 0.754 g of the
acid (86.6%); m.p.--205-210.degree. C. 3,3'-isomer--0.48 g of the
methyl ester furnished 0.34 g of the acid (63.6%);
m.p.--102-105.degree. C.
[0358] .sup.1H NMR (DMSO-d.sub.6) .delta.: 4,4'-isomer--4.52 (s,
2H, --CH.sub.2N.sub.3), 7.50 (d, 2H, J=5 Hz), 7.9 (m, 4H), and 8.03
(d, 2H, J=10 Hz)
[0359] 3,3'-isomer--4.54 (s, 2H, --CH.sub.2N.sub.3), 7.4 (d, 1H,
J=10 Hz), 7.5-7.7 (m, 4H), 7.92 (ABq, 2H) and 8.19 (s, 1H).
[0360] 4. Reduction of the azides to the amine: [0361] This was
carried out on the solid phase and the amine was never isolated.
The azidocarboxylic acid was loaded on the resin using the standard
peptide coupling protocols. After washing, the resin containing the
azide was shaken with 20 equivalents of triphenylphosphine in
THF/water (95:5) for 24 h. The solution was drained under a
positive pressure of nitrogen and then washed with the standard
washing procedure. The resulting amine was employed in the next
coupling.
[0362] 5.
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[{(9H-Flouren-9ylmethoxy-
)amino]acetyl}amino-7,12-dihydroxycholan-24-oic acid: [0363]
Tributylamine (3.2 mL); 13.5 mmol) was added dropwise to a solution
of Fmoc-glycine (4.0 g, 13.5 mmol) in THF (80 mL) stirred at
0.degree. C. Isobutylchloroformate (1.7 mL; 13.5 mmol) was
subsequently added and, after 10 min, a suspension of tributylamine
(2.6 mL; 11.2 mmol) and (3.beta.,
5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid (4.5 g; 11.2 mmol) in DMF (80 mL) was added dropwise, over 1
h, into the cooled solution. The mixture was allowed to warm up to
ambient temperature and after 6 h, the solution was concentrated to
120 mL, then water (180 mL) and 1N HCl (30 mL) were added (final pH
1.5). The precipitated solid was filtered, washed with water
(2.times.100 mL), vacuum dried and purified by flash
chromatography. Elution with chloroform/methanol (8:2) yielded the
product as a colorless solid.
[0364] Yield: 1.9 g (25%). TLC: R.sub.f 0.30
(CHCl.sub.3/MeOH/NH.sub.4OH--6:3:1).
In Vitro and In Vivo Testing of Compounds
Example X
In Vitro Binding Assay for GRP Receptors in PC3 Cell Lines--FIGS.
9A-B
[0365] To identify potential lead compounds, an in vitro assay that
identifies compounds with high affinity for GRP-R was used. Since
the PC3 cell line, derived from human prostate cancer, is known to
exhibit high expression of GRP-R on the cell surface, a radio
ligand binding assay in a 96-well plate format was developed and
validated to measure the binding of .sup.125I-BBN to GRP-R positive
PC3 cells and the ability of the compounds of the invention to
inhibit this binding. This assay was used to measure the IC.sub.50
for RP527 ligand, L134 ligand (controls) and compounds of the
invention which inhibit the binding of .sup.125I-BBN to GRP-R.
(RP527=N,N-dimethylglycine-Ser-Cys(Acm)-Gly-5-aminopentanoic
acid-BBN (7-14) [SEQ. ID. NO: 1], which has MS=1442.6 and
IC50-0.84). Van de Wiele C, Dumont F et al., Technetium-99m RP527,
a GRP analogue for visualization of GRP receptor-expressing
malignancies: a feasibility study. Eur. J. Nucl. Med., (2000) 27;
1694-1699.; L134=DO3A-monoamide-8-amino-octanoic acid-BBN (7-14)
[SEQ. ID. NO: 1], which has MS=1467.0.
[0366] The Radioligand Binding Plate Assay was validated for BBN
and BBN analogues (including commercially available BBN and L1) and
also using .sup.99mTc RP527 as the radioligand.
A. Materials and Method:
[0367] 1. Cell Culture: [0368] PC3 (human prostate cancer cell
line) were obtained from the American Type Culture Collection and
cultured in RPMI 1640 in tissue culture flasks (Corning). This
growth medium was supplemented with 10% heat inactivated FBS
(Hyclone, SH30070.03), 10 mM HEPES (GibcoBRL, 15630-080), and
antibiotic/antimycotic (GibcoBRL, 15240-062) for a final
concentration of penicillin-streptomycin (100 units/mL), and
fungizone (0.25 .mu.g/mL). All cultures were maintained in a
humidified atmosphere containing 5% CO.sub.2/95% air at 37.degree.
C., and passaged routinely using 0.05% trypsin/EDTA (GibcoBRL
25300-054) where indicated. Cells for experiments were plated at a
concentration of 2.0.times.10.sup.4 /well either in 96-well white
/clear bottom microtiter plates (Falcon Optilux-I) or 96 well
black/clear collagen I cellware plates (Beckton Dickinson Biocoat).
Plates were used for binding studies on day 1 or 2
post-plating.
[0369] 2. Binding Buffer: [0370] RPMI 1640 supplemented with 20 mM
HEPES, 0.1% BSA (w/v), 0.5 mM PMSF (AEBSF), bacitracin (50 mg/500
ml), pH 7.4. [0371] .sup.125I-BBN (carrier free, 2200 Ci/mmole) was
obtained from Perkin-Elmer.
[0372] B. Competition Assay With .sup.125I-BBN for GRP-R in PC3
Cells: [0373] A 96-well plate assay was used to determine the
IC.sub.50 of various compounds of the invention to inhibit binding
of .sup.125I-BBN to human GRP-R. The following general procedure
was followed: [0374] All compounds tested were dissolved in binding
buffer and appropriate dilutions were also done in binding buffer.
PC3 cells (human prostate cancer cell line) for assay were plated
at a concentration of 2.0.times.10.sup.4/well either in 96-well
white /clear bottomed microtiter plates (Falcon Optilux-I) or 96
well black/clear collagen I cellware plates (Beckton Dickinson
Biocoat). Plates were used for binding studies on day 1 or 2
post-plating. The plates were checked for confluency (>90%
confluent) prior to assay. For the assay, RP527 or L134 ligand,
(controls), or compounds of the invention at concentrations ranging
from 1.25.times.10.sup.-9M to 5.times.10.sup.-9 M, was co-incubated
with .sup.125I-BBN (25,000 cpm/well). These studies were conducted
with an assay volume of 75 .mu.l per well. Triplicate wells were
used for each data point. After the addition of the appropriate
solutions, plates were incubated for 1 h at 4.degree. C. to prevent
internalization of the ligand-receptor complex. Incubation was
ended by the addition of 200 .mu.l of ice-cold incubation buffer.
Plates were washed 5 times and blotted dry. Radioactivity was
detected using either the LKB CompuGamma counter or a microplate
scintillation counter.
[0375] Competition binding curves for RP527 (control) and L70, a
compound of the invention can be found in FIGS. 9A-B These data
show that the IC50 of the RP527 control is 2.5 nM and that of L70,
a compound of this invention is 5 nM. The IC50 of the L134 control
was 5 nM. IC50 values for those compounds of the invention tested
can be found in tables 1-3 and show that they are comparable to
that of the controls and thus would be expected to have sufficient
affinity for the receptor to allow uptake by receptor bearing cells
in vivo.
[0376] C. Internalization & Efflux Assay:
[0377] These studies were conducted in a 96-well plate. After
washing to remove serum proteins, PC3 cells were incubated with
.sup.125I-BBN, .sup.177Lu-L134 or radiolabelled compounds of this
invention for 40 min, at 37.degree. C. Incubations were stopped by
the addition of 200 .mu.l of ice-cold binding buffer. Plates were
washed twice with binding buffer. To remove surface-bound
radioligand, the cells were incubated with 0.2M acetic acid (in
saline), pH 2.8 for 2 min. Plates were centrifuged and the acid
wash media were collected to determine the amount of radioactivity
which was not internalized. The cells were collected to determine
the amount of internalized .sup.125I-BBN, and all samples were
analyzed in the gamma counter. Data for the internalization assay
was normalized by comparing counts obtained at the various time
points with the counts obtained at the final time point (T40 min).
For the efflux studies, after loading the PC3 cells with
.sup.125I-BBN or radiolabelled compounds of the invention for 40
min at 37.degree. C., the unbound material was washed off, and the
% of internalization was determined as above. The cells were then
resuspended in binding buffer at 37.degree. C. for up to 3 h At
0.5, 1, 2, or 3 h. the amount remaining internalized relative to
the initial loading level was determined as above and used to
calculate the percent efflux recorded in Table 4. TABLE-US-00007
TABLE 4 Internalisation and efflux ofI-BBN and the Lu-177 complexes
of L134 (control) and compounds of this invention I-BBN L134 L63
L64 L70 Internalisation (40 minutes) 59 89 64 69 70 Efflux (2 h) 35
28 0 20 12
These data show that the compounds of this invention are
internalized and retained by the PC3 cells to a similar extent to
the controls.
EXAMPLE XI
Preparation of Tc-Labeled GRP Compounds
[0378] Peptide solutions of compounds of the invention identified
in Table 5 were prepared at a concentration of 1 mg/mL in 0.1%
aqueous TFA. A stannous chloride solution was prepared by
dissolving SnCl.sub.2.2H.sub.2O (20 mg/mL) in 1 N HCl. Stannous
gluconate solutions containing 20 .mu.g of SnCl.sub.2.2H.sub.2O/100
.mu.L were prepared by adding an aliquot of the SnCl.sub.2 solution
(10 .mu.L) to a sodium gluconate solution prepared by dissolving 13
mg of sodium gluconate in water. A hydroxypropyl gamma cyclodextrin
[HP-.gamma.-CD]solution was prepared by dissolving 50 mg of
HP-.gamma.-CD in 1 mL of water.
[0379] The .sup.99mTc labeled compounds identified below were
prepared by mixing 20 .mu.L of solution of the unlabeled compounds
(20 .mu.g), 50 .mu.L of HP-.gamma.-CD solution, 100 .mu.L of
Sn-gluconate solution and 20 to 50 .mu.L of .sup.99mTc
pertechnetate (5 to 8 mCi, Syncor). The final volume was around 200
.mu.L and final pH was 4.5-5. The reaction mixture was heated at
100.degree. C. for 15 to 20 min. and then analyzed by reversed
phase HPLC to determine radiochemical purity (RCP). The desired
product peaks were isolated by HPLC, collected into a stabilizing
buffer containing 5 mg/mL ascorbic acid, 16 mg/mL HP-.gamma.-CD and
50 mM phosphate buffer, pH 4.5, and concentrated using a speed
vacuum to remove acetonitrile. The HPLC system used for analysis
and purification was as follows: C18 Vydac column, 4.6.times.250
mm, aqueous phase: 0.1% TFA in water, organic phase: 0.085% TFA in
acetonitrile. Flow rate: 1 mL/min. Isocratic elution at 20% -25%
acetonitrile/0.085% TFA was used, depending on the nature of
individual peptide.
[0380] Labeling results are summarized in Table 5. TABLE-US-00008
TABLE 5 HPLC RCP.sup.4 (%) retention Initial immediately time
RCP.sup.3 following Compound.sup.1 Sequence.sup.2 (min) (%)
purification L2 -RJQWAVGHLM 5.47 89.9 95.6 L4 -SJQWAVGHLM 5.92 65
97 L8 -JKQWAVGHLM 6.72 86 94 L1 -KJQWAVGHLM 5.43 88.2 92.6 L9
-JRQWAVGHLM 7.28 91.7 96.2 L7 -aJQWAVGHLM 8.47 88.6 95.9 n.d. = not
detected .sup.1All compounds were conjugated with an
N,N'-dimethylglycine-Ser-Cys-Gly metal chelator. The Acm protected
form of the ligand was used. Hence, the ligand used to prepare the
99mTc complex of L2 was N,N'-dimethylglycine-Ser-Cys
(Acm)-Gly-RJQWAVGHLM. The Acm group was removed during chelation to
Tc. .sup.2In the Sequence, "J" refers to 8-amino-3,6-dioxaoctanoic
acid and "a" refers to D-alanine. .sup.3Initial RCP measurement
taken immediately after heating and prior to HPL purification.
.sup.4RCP determined following HPLC isolation and acetonitrile
removal via speed vacuum.
EXAMPLE XII
Preparation of .sup.177Lu-L64 for Cell Binding and Biodistribution
Studies
[0381] This compound was synthesized by incubating 10 .mu.g L64
ligand (10 .mu.L of a 1 mg/mL solution in water), 100 .mu.L
ammonium acetate buffer (0.2M, pH 5.2) and .about.1-2 mCi of
.sup.177LuCl.sub.3 in 0.05N HCl (MURR) at 90.degree. C. for 15 min.
Free .sup.177Lu was scavenged by adding 20 .mu.L of a 1%
Na.sub.2EDTA.2H.sub.2O (Aldrich) solution in water. The resulting
radiochemical purity (RCP) was .about.95%. The radiolabeled product
was separated from unlabeled ligand and other impurities by HPLC,
using a YMC Basic C8 column [4.6.times.150 mm], a column
temperature of 30.degree. C. and a flow rate of 1 mL/min, with a
gradient of 68% A/32% B to 66% A/34% B over 30 min., where A is
citrate buffer (0.02M, pH 3.0), and B is 80% CH.sub.3CN/20%
CH.sub.3OH. The isolated compound had an RCP of 100% and an HPLC
retention time of 23.4 minutes.
[0382] Samples for biodistribution and cell binding studies were
prepared by collecting the desired HPLC peak into 1000 .mu.L of
citrate buffer (0.05 M, pH 5.3, containing 1% ascorbic acid, and
0.1% HSA). The organic eluent in the collected eluate was removed
by centrifugal concentration for 30 min. For cell binding studies,
the purified sample was diluted with cell-binding media to a
concentration of 1.5 .mu.Ci/mL within 30 minutes of the in vitro
study. For biodistribution studies, the sample was diluted with
citrate buffer (0.05 M, pH 5.3, containing 1% sodium ascorbic acid
and 0.1% HSA) to a final concentration of 50 .mu.Ci/mL within 30
minutes of the in vivo study.
EXAMPLE XIII
Preparation of .sup.177Lu-L64 for Radiotherapy Studies
[0383] This compound was synthesized by incubating 70 .mu.g L64
ligand (70 .mu.L of a 1 mg/mL solution in water), 200 .mu.L
ammonium acetate buffer (0.2M, pH 5.2) and .about.30-40 mCi of
.sup.177LuCl.sub.3 in 0.05N HCl (MURR) at 85.degree. C. for 10 min.
After cooling to room temperature, free .sup.177Lu was scavenged by
adding 20 .mu.L of a 2% Na.sub.2EDTA.2H.sub.2O (Aldrich) solution
in water. The resulting radiochemical purity (RCP) was .about.95%.
The radiolabeled product was separated from unlabeled ligand and
other impurities by HPLC, using a 30OVHP Anion Exchange column
(7.5.times.50 mm) (Vydac) that was sequentially eluted at a flow
rate of 1 mL/min with water, 50% acetonitrile/water and then 1 g/L
aqueous ammonium acetate solution. The desired compound was eluted
from the column with 50% CH.sub.3CN and mixed with 1 mL of citrate
buffer (0.05 M, pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and
0.9% (v:v) benzyl alcohol. The organic part of the isolated
fraction was removed by spin vacuum for 40 min, and the
concentrated solution (.about.20-25 mCi) was adjusted within 30
minutes of the in vivo study to a concentration of 7.5 mCi/mL using
citrate buffer (0.05 M, pH 5.3) containing 5% ascorbic acid, 0.2%
HSA, and 0.9% (v:v) benzyl alcohol. The resulting compound had an
RCP of >95%.
EXAMPLE XIV
Preparation of .sup.111In-L64
[0384] This compound was synthesized by incubating 10 .mu.g L64
ligand (5 .mu.L of a 2 mg/mL solution in 0.01 N HCl), 60 .mu.L
ethanol, 1.12 mCi of .sup.111InCl.sub.3 in 0.05N HCl (80 .mu.L) and
155 .mu.L sodium acetate buffer (0.5M, pH 4.5) at 85.degree. C for
30 min. Free .sup.111In was scavenged by adding 20 .mu.L of a 1%
Na.sub.2EDTA.2H.sub.2O (Aldrich) solution in water. The resulting
radiochemical purity (RCP) was 87%. The radiolabeled product was
separated from unlabeled ligand and other impurities by HPLC, using
a Vydac C18 column, [4.6.times.250 mm], a column temperature of
50.degree. C. and a flow rate of 1.5 mL/min. with a gradient of 75%
A/25% B to 65% A/35% B over 20 min where A is 0.1% TFA in water, B
is 0.085% TFA in acetonitrile. With this system, the retention time
for .sup.111In-L64 is 15.7 min. The isolated compound had an RCP of
96.7%.
EXAMPLE XV
Preparation of .sup.177Lu-L134 (Control)
[0385] A stock solution of peptide was prepared by dissolving L134
ligand (prepared as described in US Application Publication No.
2002/0054855 and WO 02/87637, both incorporated by reference) in
0.01 N HCl to a concentration of 1 mg/mL .sup.177Lu-L134 was
prepared by mixing the following reagents in the order shown.
TABLE-US-00009 0.2 M NH.sub.4OAc, pH 6.8 100 .mu.l Peptide stock, 1
mg/mL, in 0.01 N HCl 5 .mu.l .sup.177LuCl.sub.3 (MURR) in 0.05M HCl
1.2 .mu.l (1.4 mCi)
The reaction mixture was incubated at 85.degree. C. for 10 min.
After cooling down to room temperature in a water bath, 20 .mu.l of
a 1% EDTA solution and 20 pl of EtOH were added. The compound was
analyzed by HPLC using a C18 column (VYDAC Cat # 218TP54) that was
eluted at flow rate of 1 mL/min with a gradient of 21 to 25% B over
20 min, where A is 0.1% TFA/H.sub.2O and B is 0.1% TFA/CH.sub.3CN).
.sup.177Lu-L 134 was formed in 97.1% yield (RCP) and had a
retention time of .about.16.1 min on this system.
EXAMPLE XVI
Preparation of .sup.177Lu-L63
[0386] This compound was prepared as described for .sup.177Lu-L134.
The compound was analyzed by HPLC using a C18 column (VYDAC Cat #
218TP54) that was eluted at flow rate of 1 mL/min with a gradient
of 30-34% B over 20 min (where solvent is A. 0.1% TFA/H.sub.2O and
B is 0.1% TFA/CH.sub.3CN). The .sup.177Lu-L63 that formed had an
RCP of 97.8% and a retention time of 14.2 min on this system.
EXAMPLE XVII
Preparation of .sup.177Lu-L70 for Cell Binding and Biodistribution
Studies
[0387] This compound was prepared following the procedures
described above, but substituting L70 (the ligand of Example II).
Purification was performed using a YMC Basic C8 column
(4.6.times.150 mm), a column temperature of 30.degree. C. and a
flow rate of 1 mL/min. with a gradient of 80% A/20% B to 75% A/25%
B over 40 min., where A is citrate buffer (0.02M, pH 4.5), and B is
80% CH.sub.3CN/20% CH.sub.3OH. The isolated compound had an RCP of
.about.100% and an HPLC retention time of 25.4 min.
EXAMPLE XVIII
Preparation of .sup.177Lu-L70 for Radiotherapy Studies
[0388] This compound was prepared as described above for L64.
EXAMPLE XIX
Preparation of .sup.111In-L70 for Cell Binding and Biodistribution
Studies
[0389] This compound was synthesized by incubating 10 .mu.g L70
ligand (10 .mu.L of a 1 mg/mL solution in 0.01 N HCl), 180 .mu.L
ammonium acetate buffer (0.2M, pH 5.3), 1.1 mCi of
.sup.111InCl.sub.3 in 0.05N HCl (61 .mu.L, Mallinckrodt) and 50 pL
of saline at 85.degree. C. for 30 min. Free .sup.111In was
scavenged by adding 20 .mu.L of a 1% Na.sub.2EDTA.2H.sub.2O
(Aldrich) solution in water. The resulting radiochemical purity
(RCP) was 86%. The radiolabeled product was separated from
unlabeled ligand and other impurities by HPLC, using a Waters
XTerra C18 cartridge linked to a Vydac strong anion exchange column
[7.5.times.50 mm], a column temperature of 30.degree. C. and a flow
rate of 1 mL/min. with the gradient listed in the Table below,
where A is 0.1 mM NaOH in water, pH 10.0, B is 1 g/L ammonium
acetate in water, pH 6.7 and C is acetonitrile. With this system,
the retention time for .sup.111In-L70 is 15 min while the retention
time for L70 ligand is 27 to 28 min. The isolated compound had an
RCP of 96%.
[0390] Samples for biodistribution and cell binding studies were
prepared by collecting the desired HPLC peak into 500 .mu.L of
citrate buffer (0.05 M, pH 5.3, containing 5% ascorbic acid, 1
mg/mL L-methionine and 0.2% HSA). The organic part of the
collection was removed by spin vacuum for 30 min. For cell binding
studies, the purified, concentrated sample was used within 30
minutes of the in vitro study. For biodistribution studies, the
sample was diluted with citrate buffer (0.05 M, pH 5.3, containing
5% sodium ascorbic acid and 0.2% HSA) to a final concentration of
10 .mu.Ci/mL within 30 minutes of the in vivo study. TABLE-US-00010
Time, min A B C 0-10 100% 10-11 100-50% 0-50% 11-21 50% 50% 21-22
50-0% 0-50% 50% 22-32 50% 50%
EXAMPLE XX
In Vivo Pharmacokinetic Studies
[0391] A. Tracer Dose Biodistribution:
[0392] Low dose pharmacokinetic studies (e.g., biodistribution
studies) were performed using the below-identified compounds of the
invention in xenografted, PC3 tumor-bearing nude mice
([Ncr]-Foxn1<nu>). In all studies, mice were administered 100
.mu.L of .sup.177Lu-labelled test compound at 200 .mu.Ci/kg, i.v.,
with a residence time of 1 and 24 h per group (n=3-4). Tissues were
analyzed in an LKB 1282 CompuGamma counter with appropriate
standards. TABLE-US-00011 TABLE 6 Pharmacokinetic comparison at 1
and 24 h in PC3 tumor-bearing nude mice (200 .mu.Ci/kg; values as %
ID/g) of Lu-177 labelled compounds of this invention compared to
control L134 control L63 L64 L70 Tissue 1 hr 24 hr 1 hr 24 hr 1 hr
24 hr 1 hr 24 hr Blood 0.44 0.03 7.54 0.05 1.87 0.02 0.33 0.03
Liver 0.38 0.04 12.15 0.20 2.89 0.21 0.77 0.10 Kidneys 7.65 1.03
7.22 0.84 10.95 1.45 6.01 2.31 Tumor 3.66 1.52 9.49 2.27 9.83 3.60
6.42 3.50 Pancreas 28.60 1.01 54.04 1.62 77.78 6.56 42.34 40.24
Whereas the distribution of radioactivity in the blood, liver and
kidneys after injection of L64 and L70 is similar to that of the
control compound, L134, the uptake in the tumor is much higher at 1
and 24 h for both L64 and L70. L63 also shows high tumour uptake
although with increased blood and liver values at early times.
Uptake in the mouse pancreas, a normal organ known to have GRP
receptors is much higher for L64, L70 and L63 than for L134.
EXAMPLE XXI
Radiotherapy Studies
A. Short Term Efficacy Studies:
[0393] Radiotherapy studies were performed using the PC3
tumor-bearing nude mouse model. Lu-177 labelled compounds of the
invention L64, L70, L63 and the treatment control compound L134
were compared to an untreated control group. (n=12 for each
treatment group and n=36 for the untreated control group). For the
first study, mice were administered 100 .mu.L of
.sup.177Lu-labelled compound of the invention at 30 mCi/kg, i.v, or
s.c. under sterile conditions. The subjects were housed in a
barrier environment for up to 30 days. Body weight and tumor size
(by caliper measurement) were collected on each subject 3 times per
week for the duration of the study. Criteria for early termination
included: death; loss of total body weight (TBW) equal to or
greater than 20%; tumor size equal to or greater than 2 cm.sup.3.
Results are displayed in FIG. 10A. These results show that animals
treated with L70, L64 or L63 have increased survival over the
control animals given no treatment and over those animals given the
same dose of L134. [0394] A repeat study was performed with L64 and
L70 using the same dose as before but using more animals per
compound (n=46) and following them for longer. The results of the
repeat study are displayed in FIG. 10B. Relative to the same
controls as before (n=36), both L64 and L70 treatment give
significantly increased survival (p<0.0001) with L70 being
better than L64 (p0.079).
EXAMPLE XXII
Alternative Preparation of L64 and L70 Using Segment Coupling
[0395] Compounds L64 and L70 can be prepared employing the
collection of intermediates generally represented by A-D (FIG. 14),
which themselves are prepared by standard methods known in the art
of solid and solution phase peptide synthesis (Synthetic
Peptides--A User's Guide 1992, Grant, G., Ed. WH. Freeman Co., NY,
Chap 3 and Chap 4 pp 77-258; Chan, W. C. and White, P. D. Basic
Procedures in Fmoc Solid Phase Peptide Synthesis--A Practical
Approach 2002, Chan, W. C. and White, P. D. Eds Oxford University
Press, New York, Chap. 3 pp 41-76; Barlos, K and Gatos, G.
Convergent Peptide Synthesis in Fmoc Solid Phase Peptide
Synthesis--A Practical Approach 2002, Chan, W. C. and White, P. D.
Eds Oxford University Press, New York, Chap. 9 pp 216-228.) which
are incorporated herein by reference.
[0396] These methods include Aloc, Boc, Fmoc or
benzyloxycarbonyl-based peptide synthesis strategies or judiciously
chosen combinations of those methods on solid phase or in solution.
The intermediates to be employed for a given step are chosen based
on the selection of appropriate protecting groups for each position
in the molecule, which may be selected from the list of groups
shown in FIG. 1. Those of ordinary skill in the art will also
understand that intermediates, compatible with peptide synthesis
methodology, comprised of alternative protecting groups can also be
employed and that the listed options for protecting groups shown
above serves as illustrative and not inclusive, and that such
alternatives are well known in the art.
[0397] This is amply illustrated in FIG. 15 which outlines the
approach. Substitution of the intermediate C2 in place of C1 shown
in the synthesis of L64, provides L70 when the same synthetic
strategies are applied.
Sequence CWU 1
1
1 1 8 PRT Artificial Description of Artificial Sequence This
peptide is the receptor binding site of bombesin and is also known
as BBN[7-14] 1 Gln Trp Ala Val Gly His Leu Met 1 5
* * * * *