CA2549318A1 - Improved gastrin releasing peptide compounds - Google Patents

Abstract

New and improved compounds for use in diagnostic imaging or therapy having the formula M-N-O-P-G, wherein M is an optical label or a 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 or phototherapy to a patient using the compounds of the invention are also provided. Methods and kits for preparing a diagnostic imaging agent from the compound is further provided. Methods and kits for preparing a radiotherapeutic agent are further provided.

Description

TITLE OF THE INVENTION
IMPROVED GASTRIN RELEASING PEPTIDE COMPOUNDS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001 ] This application claims benefit of U.S. Application No. 10/828,925 filed April 20, 2004, which is a continuation-in-part application of International Application PCT/US03/041328, filed December 24, 2003, which claims priority to U.S.
Application No.
10/341,577 filed January 13, 2003. All of these applications are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION

[0002] This invention relates to novel gastrin releasing peptide (GRP) compounds which are useful as diagnostic imaging agents or radiotherapeutic agents.
These GRP
compounds are labeled with radionuclides or labels detectable by in vivo light imaging and include the use of novel linkers between the label and the targeting peptide, which provides for improved pharmacokinetics.
BACKGROUND OF THE INVENTION

[0003] 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.

[0004] 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, fox example in U.S. Pat.
5,976,495 to Pollak et. al., hereby incorporated by reference.
[0005] The role of the targeting agent, by virtue of its affinity for certain binding sites, is to direct the diagnostic agent, such as a 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 specifac affinity for a given receptor. Other known targeting agents include monoclonal antibodies (MAbs), antibody fragments (Fab's and (Fab)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, (Dec. 1993). These references are hereby incorporated by reference in their entirety.

[0006] 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.

[0007] 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. S7-112. 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.

[0008] In designing an effective 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 fox a radiopharmaceutical, 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.

[0009] Moreover, developing radiolabeled peptides that are cleared efficiently from normal tissues is also an important factor for radiophannaceutical agents.
When biomolecules (e.g., MAb, Fab 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 Fab), 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.

[0010] Subsequent work, such as that in U.S. Pat. 6,200,546 and US
200210054855 to Hoffinan, et. al, hereby incorporated by reference in their entirety, have 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.

[0011 J New and improved radiopharmaceutical and other diagnostic 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
[0012] In an embodiment of the present invention, there is provided new and improved compounds for use in diagnostic 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. In another embodiment, these compounds include an optical label (e.g. a photolabel or other label detectable by light imaging, optoacoustical imaging or photoluminescence) attached to a GRP
receptor targeting peptide by a linker or spacer group.
[0013] 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), or the optical label, 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, D03A, HP-D03A, 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 99"'Tc, SICr, 67Ga, 68Ga, 47Sc, SICr 167Tm 141Ce 111In lbs~,-b 175 140La 90y 88Y 153sm 166H~ 165Dy 166Dy 62Cua 64Cu, > > > > > > > > > > >
67Cu 97Ru lo3Ru ls6Re lssRe Zo3Pb 211Bi 212Bi 213Bi 214Bi 225AC 105 lo9Pd 117~nSn > > > > > > > > > > > > > >
149Pm, 161Tb, 177Lu, lg$Au and 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 64Cu, 67Ga, 68Ga, 99mTc, and 111In, with 99mTc, and '''In being particularly preferred. For therapeutic purposes, the preferred radionuclides include 64Cu, 9°~,' IOSRh, 111In' 117msn' 149Pm' 153Sm' 161.Lb' 166Dy' 166H~' 175' 177L.u' 186/188Re, and 199Au, with 177Lu and 9°Y being particularly preferred. A most preferred chelator used in compounds ofthe invention is 1-substituted 4,7,10-tricarboxymethyl 1,4,7,10 5 tetraazacyclododecane triacetic acid (D03A).
[0015] The optical Label M may be any of various optical labels known in the art.
Preferred labels include, without limitation, optical dyes, including organic chromophores or fluorophores, such as cyanine dyes light absorbing compounds, light reflecting and scattering compounds, and bioluminescent molecules.
[0016] In one embodiment, the linker N-O-P contains at least one non-alpha amino acid.
[0017] In another embodiment, the linker N-O-P contains at least one substituted bile acid.
[0018] In yet another embodiment, the linker N-O-P contains at least one non-alpha amino acid With a cyclic group.
[0019] 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. 6,200,546 and US 2002/005455, incorporated herein by reference.
[0020] There is also provided a novel method of imaging using the compounds of the present invention.
[0021 ] A single or mufti-vial kit that contains all of the components needed to prepare the diagnostic or therapeutic agents of the invention is provided in an exemplary embodiment of the present invention.
[0022] 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.
[0023] 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 W vention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is a graphical representation of a series of chemical reactions for the synthesis of intermediate C ((3J3, 5/~)-3-(9H Fluoren-9-ylmethoxy)aminocholan-24-oic acid), from A (Methyl-(3J3, Sf3)-3-aminocholan-24-ate) and B ((3f3, SJ3)-3-aminocholan-24-oic acid), as described in Example I.
[0025] FIG. 1B is a graphical representation of the sequential reaction for the synthesis ofN [(313,513)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino] acetyl]amino] cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L62), as described in Example I.
[0026] FIG. 2A is a graphical representation of the sequential reaction for the synthesis ofN [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]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.
[0027] FIG. 2B is a general graphical representation of the sequential reaction for the synthesis ofN [4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-y1] acetyl] amino] ethoxy]benzoyl] -L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L73), N [3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alany1-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]
acetyl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L116), as described in Example II.
[0028] FIG. 2C is a chemical structure of the linker used in the synthesis reaction of FIG. 2B for synthesis ofN [4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl ] amino] ethoxy]benzoyl ]-L-glutaminyl-L-tryptophyl-L-al anyl-L-val yl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L73), as described in Example II.
[0029] FIG. 2D is a chemical structure of the linker used in the synthesis reaction of FIG. 2B for synthesis ofN [3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-y1) acetyl) amino)methyl)benzoylJ-L-glutaminyl-L-tryptophyl-L-al any!-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L115), as described in Example II.
[0030) FIG. 2E is a chemical structure of the linker used in the synthesis reaction of FIG. 2B for synthesis ofN [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJacetyl)amino)methyl)phenylacetyl)-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L116), as described in Example II.
[0031 ) FIG. 2F is a graphical representation of the sequential reaction for the synthesis ofN [[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetyl)glycyl-4-piperidinecarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L74), as described in Example II.
[0032) FIG. 3A is a graphical representation of a series of chemical reactions for the synthesis of intermediate (313,513)-3-[[(9H Fluoren-9-ylmethoxy)amino)acetyl)amino-12-oxocholan-24-oic acid (C), as described in Example III.
[0033] FIG. 3B is a graphical representation of the sequential reaction for the synthesis ofN [(313,5J3)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetylJaminoJacetyl]aminoJ-12,24-dioxocholan-24-ylJ-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L67), as described in Example III.
[0034) FIG. 3C is a chemical structure of (313,513)-3-Amino-12-oxocholan-24-oic acid (B), as described in Example III.
[0035) FIG. 3D is a chemical structure of (313,513)-3-[[(9H Fluoren-9-ylmethoxy)amino)acetyl)amino-12-oxocholan-24-oic acid (C), as described in Example III.
[0036) FIG 3E is a chemical structure ofN-[(313,5f3)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJacetylJamino)acetyl)amino)-12,24-dioxocholan-24-ylJ-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L67), as described in Example III.
[0037) FIG. 4A is a graphical representation of a sequence of reactions to obtain intermediates (313,513,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino)acetyl)amino-12-hydroxycholan-24-oic acid (3a) and (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl)amino-7,12-dihydroxycholan-24-oic acid (3b), as described in Example IV.

[0038] FIG. 4B is a graphical representation of the sequential reaction for the synthesis ofN [(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl] amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L63), as described in Example IV.
[0039] FIG. 4C is a graphical representation of the sequential reaction for the synthesis ofN [(313,513,7a,12a)-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-methioninamidc (L64), as described in Example IV.
[0040] FIG. 4D is a chemical structure of (313,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid (2b), as described in Example IV.
[0041] FIG. 4E is a chemical structure of (313,513,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic acid (3a), as described in Example IV;
[0042] FIG. 4F is a chemical structure of (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as described in Example IV.
[0043] FIG. 4G is a chemical structure ofN [(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L63), as described in Example IV.
[0044] FIG. 4H is a chemical structure ofN ((313,513,7a,12a)-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-methioninamide (L64), as described in Example IV.
[0045] FIG. 5A is a general graphical representation of the sequential reaction for the synthesis of4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-y1] acetyl] amino]methyl]b enzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L
histidyl-L-leucyl-L-methioninamide (L71); and 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-glycyl-L-histidyl-L-leucyl-L-methioninamide (L72) as described in Example V, wherein the linker is from Fig. 5B and Fig. 5C, respectively.
[0046] FIG. 5B is a chemical structure of the linker used in compound L71 as shown in Fig. 5A and as described in Example V.
[0047] FIG. SC is a chemical structure of the linker used in compound L72 as shown in Fig. 5A and as described in Example V.
[0048] FIG. SD is a chemical structure of Rink amide resin functionalised with bombesin[7-14] (B), as described in Example V.
[0049) FIG. SE is a chemical structure of Tf~an,r-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylic acid (D), as described in Example V;
[0050] 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.
[0051] 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.
[0052] FIG. 6C is a graphical representation of the synthesis ofN [2-[[[[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-histidyl-L-Ieucyl-L-methioninamide (L75), as described in Example VI.
[0053] FIG. 6D is a graphical representation of the synthesis of N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl) amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L76), as described in Example VI.
[0054] 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.
[0055] FIG. 7B is a graphical representation of the synthesis of N [[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-y1] acetyl] aminoJmethylJphenoxy] acetylJ-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L124), as described in Example VII.
[0056] FIG. 7C is a chemical structure ofN [[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec- I -yl] acetyl] amino]methylJphenoxy] acetyl]-L-glutaminyl-L-5 tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L124), as described in Example VII.
[0057] 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.
10 [0058] FIG. 8B is a graphical representation of the synthesis ofN [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-I -yl] acetyl] amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L125), as described in Example VIII.
[0059] FIG. SC is a chemical structure ofN [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJ acetyl] amino]methyl]-3-methoxybenzoylJ-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-Ieucyl-L-methioninamide, (L125), as described in Example VIII.
[0060] FIG. 9A is a graphical representation of a reaction for the synthesis of 3-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetylJaminobenzoic acid, (B), as described in Example IX.
[0061 ] FIG. 9B is a graphical representation of a reaction for the synthesis of 6-[[[(9H Fluoren-9-ylmethoxy)carbonylJaminoJacetyl]aminonaphthoic acid (C), as described in Example IX.
[0062] FIG. 9C is a graphical representation of a reaction for the synthesis of 4-[[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetylJmethylamino]benzoic acid3 (D), as described in Example IX.
[0063] FIG. 9D is a graphical representation of a reaction for the synthesis of N [4-[[[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-y1] acetyl] amino] acetyl] amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L146); N [3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJ acetyl] amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L233); N [6-[ [[ [ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]
amino] acetyl]
amino]naphthoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L234), andN [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl] methylamino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L235), as described in Example IX.
[0064] FIG. 10A is a graphical representation of a reaction for the synthesis of 7-[[Bis( I , l -dimethyl ethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid 4,10-bis(I,l-dimethylethyl) ester H, as described in Example X.
[0065] FIG. lOB is a graphical representation of a reaction for the synthesis ofN [4-[[[[[4,10-Bis(carboxymethyl)-7-(dihydroxyphosphinyl)methyl-1,4,7,10-tetraazacyclododec-I -yl] acetyl] amino]acetyl] amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucil-L-methioninamide, (L237), as described in Example X.
[0066] FIG.11A is a graphical representation of a reaction for the synthesis of N,N
Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L238), as described in Example XI.
[0067] FIG. 11B is a graphical representation of a reaction for the synthesis ofN,lV
Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-(313,513,7a,12a)-3-amino-7,12-dihydroxy-24-oxocholan-24-yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L239), as described in Example XI.
[0068] FIG. 12A is a graphical representation of a reaction for the synthesis of 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoic acid (A), as described in Example XII.
[0069) FIG. 12B is a graphical representation of a reaction for the synthesis of 4-[[
[(91I Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid, (D), as described in Example XII.
[0070] FIG. 12C is a graphical representation of a reaction for the synthesis of 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoic acid (E), as described in Example XII.

[007I] FIG. 12D is a chemical structure ofN [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-y1] acetyl]glycyl]amino]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-I-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L240) as described in Example XII.
[0072] FIG. 12E is a chemical structure of compound N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,lOtetraazacyclododec-1-yl] acetyl]glycyl]amino]3-chlorobenzoyl]L-glutaminyl-L-tryptophyl-I-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L241) as described in Example XII.
[0073] FIG. 12F is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,lOtetraazacyclododec-1-yl] acetyl]glycyl]amino]3-methylbenzoyl]L-glutaminyl-L-tryptophyl-I-alanyl-L-valyl-glycyl-L-histidyl-leucyl-L-methioninamide (L242), as described in Example XII.
[0074] FIG. 13A is a graphical representation of a reaction for the synthesis of 4-[N,N'-Bis[2-[(9 H fluoren-9-ylmethoxy)carbonyl]aminoethyl]amino]-4-oxobutanoic acid, (D), as described in Example XIII.
[0075] FIG. 13B is a graphical representation of a reaction for the synthesis ofN [4-[[4-[Bis [2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacycIododec-1-yl]acetyl]amino]ethyl]amino-1,4-dioxobutyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L244), as described in Example XIII.
[0076] FIG. 13C is a chemical structure of compound L244, as described in Example XIII.
[0077] FIG. 14A and FIG. 14B are graphical representations of the binding and competition curves described in Example XLIII.
[0078] FIG. 15A is a graphical representation of the results of radiotherapy experiments described in Example LV.
[0079] FIG. 15B is a graphical representation of the results of other radiotherapy experiments described in Example LV.
[0080] FIG. 16 is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10 tetraazacyclododec-1-yl] acetyl]glycyl]amino]-L-Lysinyl-(3,6,9)-trioxaundecane-1,1 I-dicarboxylic acid-3,7-dideoxy-3-aminocholic acid)-L-arginyl-L-glutaminyl-L-triptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L65).
[0081] FIG. 17 is a chemical structure ofN [2-S-[[[[(12a-Hydroxy-17a-(1-methyl-carboxypropyl)etiocholan-313-carbamoylmethoxyethoxyethoxyacetyl]-amino -6-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino)acetyl]amino] hexanoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L66).
[0082] FIG.18A is a chemical structure ofN [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl]amino)benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L70).
[0083] FIG.18B 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-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L114).
[0084] FIG.18C is a chemical structureN [4-[[4,7,10-Tris(carboxymethyl)-1,4,7,10 tetraazacyclododec-1-yl)-2-hydroxy-3-propoxy]benzoyl)-L-glutaminyl-L-tryptophyl-L
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L144).
[0085) FIG.18D is a chemical structure N [(313,513, 7a, l2a)-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-glycyl-L-histidyl-L-leucyl-L-methioninamide (L69).
[0086] FIG. 18E is a chemical structure ofN [4-([[[(4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl) amino)phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L146).
[0087] FIG. 19 dicloses chemical structures of intermediates which may be used to prepare compounds L64 and L70 as described in Example LVI.
[0088] FIG. 2O is a graphical representation of the preparation of L64 using segment coupling as described in Example LVI.
[0089) FIG. 21 is a graphical representation of the preparation of (1R)-1-(Bis f 2-[bis(carboxymethyl)amino]ethyl}amino)propane-3-carboxylic acid-1-carboxyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-Ieucyl-L-methioninamide (L201).
[0090] FIG. 22A is a graphical representation of chemical structure of chemical intermediates used to prepare L202.
[0091 ] FIG. 22B is a graphical representation of the preparation of N-[(3J3,513,12a)-3-[[[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-4-hydrazinobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L202).
[0092] FIG. 23A is a graphical representation of chemical structure of chemical intermediates used to prepare L203.
[0093] FIG. 23B is a graphical representation of the preparation of N-[(3J3,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L203).
[0094] FIG. 24 is a graphical representation of the preparation of N-[(313,513,12a)-3-[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]
amino]-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L204).
[0095] FIG. 25 is a graphical representation of the preparation of N-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L205).
[0096] FIG. 26A is a graphical representation of chemical structures of chemical intermediates used to prepare L206.
[0097] FIG. 26B is a graphical representation of the preparation ofN-[(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetylJamino]acetyl]amino]- [4'-Amino-2'-methyl biphenyl-4-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L206).
[0098] FIG. 27A is a graphical representation of chemical structures of chemical intermediates used to prepare L207.

[0099] FIG. 27B is a graphical representation of the preparation of N-[(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1- ' yl]acetyl]amino]acetyl]amino]- [3'-amino-biphenyl-3-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L207).
5 [00100] FIG. 28 is a graphical representation of the preparation of N-[(313,513,12a)-3-[[[ [[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]- [1,2-diaminoethyl-terephthalyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L208).
[00101] FIG. 29A is a graphical representation of chemical structures of chemical 10 intermediates used to prepare L209.
[001021 FIG. 29B is a graphical representation of the preparation of L209.
[00103] FIG. 30A is a graphical representation of chemical structures of chemical intermediates used to prepare L210.
[00104] FIG. 30B is a chemical structure of L210.

15 [00105] FIG. 31 is a chemical structure ofN-[(313,S13,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L211.
[00106] FIG. 32 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L212.
[00107] FIG. 33 is a chemical structure ofN-[(3J3,S13,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionine carboxylate L213.
[001 O8] FIG. 34 is a chemical structure of N-[(3l3,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]-glycyl-4-aminobenzoyl-D-phenyl al aryl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L214.

[00109] FIG. 35 is a chemical structure ofN-[(3J3,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec- I -yl] acetyl] amino]-glycyl-aminobenzoyl-L-glutaminyl-L-arginyl-L-leucyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L215.
[00110] FIG. 36 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-glutaminyl-arginyl-L-tyrosinyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L216.
[00111] FIG. 37 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L217.
[00112] FIG. 38 is a chemical structure of L218.
[00113] FIG. 39 is a chemical structure ofN-[(3J3,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-aminopentyl, L219.
[00114] FIG. 40 is a chemical structure of N-[(3J3,5l3,12a)-3-[[[4,7,10-Tris(carboxym ethyl)-1,4, 7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-serinyl-L-valyl-D-alanyl-L-histidyl-L-leucyl-L-methioninamide, L220.
[00115] FIG. 41 is a chemical structure ofN-[(313,SJ3,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]-glycyl-4-aminobenzoyl-D-phenyl al anyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-val yl-glycyl-L-histidyl-L-leucyl-L-leucinamide, L221.
[00116] FIG. 42 is a chemical structure of N-[(313,5l3,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]-glycyl-4-aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide, L222.
[00117] FIG. 43 is a chemical structure ofN-[(3J3,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide, L223.
[00118] FIG. 44 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,I0-Tris(carboxymethyl)-I ,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-glycyl-L-histidyl-L-phenylalanyl-L-leucinamide, L224.
[00119] FIG. 45 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminob enzoyl-L-1 eucyl-L-tryptophyl-L-al anyl-L-valinyl-glycyl-L-s erinyl-L-phenyl al anyl-L-methioninamide, L225.
[00120] FIG. 46 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, L226.
[00121] FIG. 47 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-serinyl-L-phenylalanyl-L-methioninamide L227.
[00122] FIG. 48 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-phenylalanyl-L-methioninamide, L228.
[00123] FIG. 49A is a graphical representation of a reaction for the synthesis of (313,513 7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oic acid (B) as described in Example LVII.
[00124] FIG. 49B is a graphical representation of a reaction for the synthesis of N-[313,513 7a,12a)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-hi stidyl-L-1 eucyl-L-methi oninamide, (L69), as described in Example LVII.

[00125] FIG. 50 is a graphical representation of a reaction for the synthesis of N-[4-[2-Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]propoxy]benzoyl]-L-glutaminyl-L-tryptophyI-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L144), as described in Example LVIII.
[00126 FTG. 51 is a chemical structure of L300.
[00127] FIG. 52 is a TOCSY spectrum of Lu-L70 in DMSO-d6 at 25°C.
[0012&] FIG. 53 is a COSY spectrum of Lu-L70 in DMSO-d6 at 25°C.
[00129] FIG. 54 is a NOESY spectrum of Lu-L70 in DMSO-d6 at 25°C.
[00130] FIG. 55 is a gHSQC spectrum of Lu-L70 in DMSO-d6 at 25°C.
[00131] FIG. 56 is a gHMBC spectrum of Lu-L70 in DMSO-d6 at 25 °C.
[00132] FIG. 57 is a gHSQCTOCSY spectrum of Lu-L?0 in DMSO-d6 at 25°C.
[00133] FIG. 58 is a Regular 1H-NMR (bottom) and selective homo-decoupling of the water peak at 3.5 ppm of Lu-L70 in DMSO-d6 at 15 °C.
[00134] FIG. 59 is a TOCSY Spectrum of ~~SLu-D03A-monoamide-Aoc-QWAVGHLM-NH2 in DMSO-d& at 25 °C.
[00135] FIG. 60 is a chemical structure of L70.
[00136] FIG. 61 is a chemical structure of ~~SLu-D03A-monoamide-Aoc-QWAVGHLM-NH2.
[00137] FIG. 62 is a chemical structure of ~~SLu-L70 with a bound water molecule.
~ [00138] FIG. 63 is a chemical structure of L301.
Abbreviations Used In the Application Aoc- ~-aminooctanoic acid A a3- 3-amino ro ionic acid Abu4- 4-aminobutanoic acid Adca3- (313,513 7a,,12a,)-3-amino-7,12-dihydroxycholan-24-oic acid or 3-Amino-3-deox cholic acid Ahl2ca- 3J3,5J3,12a -3-amino-12-h drox cholan-24-oic acid Akca- (3f3,513,7a,12a)-3-amino-12-oxacholan-24-oic acid Cha- L-C clohex lalanine Nall- ~ L-1-Naphthylalanine Bi - L-Bi hen lalanine Mo3abz4- 3-Methoxy-4-aminobenzoic acid or 4-aminomethyl-3-methox benzoic acid B a4- 4-benzo 1 hen lalanine C13 abz4- 3-Chloro-4-aminobenzoic acid M3abz4- 3-meth 1-4-aminobenzoic acid Ho3abz4- 3-h drox -4-aminobenzoic acid Hybz4- 4-h drazinobenzoic acid Nmabz4- 4-meth laminobenzoic acid Mo3amb4- 3-methox -4-aminobenzoic acid Amb4- 4-aminomethylbenzoic acid Aeb4- 4-(2-aminoethox benzoic acid Dae- 1,2-diaminoeth I

T a- Tere hthalic acid A4m~bi hc4- 4'-Amino-2'-meth 1 bi hen 1-4-carbox lic acid A3bi hc3- 3-amino-3'-bi hen lcarbox lic acid Amc4- trans-4-aminometh lc clohexane carbox Iic acid Ae a4- N-4-aminoethyl-N-1- i erazine-acetic acid In - Isoni ecotic acid Pial- N-1- i erazineacetic acid Ckb - 4- 3-Carbox eth 1-2-keto-1-benzimidazo 1 - i eridine Abz3 3-Aminobenzoic acid Abz4 4-Aminobenzoic acid J 8-amino-3,6-dioxaoctanoic acid AvaS 5-Aminovaleric acid f (D -Phe D -T

Ala2 (also Bala) Beta-alanine DETAILED DESCRIPTION OF THE INVENTION
[00139] 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.
[00140] In an embodiment of the present invention, there are provided new and improved compounds for use in diagnostic imaging or radiotherapy. The compounds include an optical label or 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.
[00141 ] In general, compounds of the present invention may have the formula:
M-N-O-P-G
5 wherein M is the metal chelator (in the form complexed with a metal radionuclide or not), or an optical label, N-O-P is the linker, and G is the GRP receptor targeting peptide. Each of the metal chelator, optical label, linker, and GRP receptor targeting peptide is described in the discussion that follow.
[00I42] In another embodiment of the present invention, there is provided a new and 10 improved linker or spacer group which is capable of linking an optical label or 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.
15 [00143] Compounds meeting the criteria defined herein were discovered to have improved pharmacokinetic properties compared to other 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 20 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.
Additionally, compounds of the present invention had improved tissue receptor specificity compared to prior art compounds.
1 A. Metal Chelator [00144] 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, for example, 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.
[00145] The metal chelators of the invention may include, for example, linear, macrocyclic, terpyridine, and N3S, N2Sz, or N4 chelators (see also, U.S.
5,367,080, U.S.
5,364,613, U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142, the disclosures ofwhich are incorporated by reference 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. 5,720,934). For example, Nd chelators are described in U.S. Patent 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 in their entirety. Certain N3S
chelators are described in PCT/CA94100395, PCT/CA94/00479, PCTlCA95/00249 and in U.S. Patent Nos.
5,662,885; 5,976,495; and 5,780,006, the disclosures of which are incorporated by reference in their entirety. The chelator may also include derivatives of the chelating ligand mercapto-acetyl-glycyl-glycyl-glycine (MAG3), which contains an N3S, and N2S2 systems such as MAMA (monoamidemonoaminedithiols), DADS (NZS 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 ofwhich are incorporated by reference in their entirety.
[00146] 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. Patent Nos.
5,183,653;
5,387,409; and 5,118,797, the disclosures of which are incorporated by reference in their entirety.
[00147] Examples ofpreferred 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 (D03A), ethylenediaminetetraacetic acid (EDTA), 4-carbonylmethyl-10-phosponomethyl-1,4,7,10-Tetraazacyclododecane-1,7-diacetic acid (Cm4pml0d2a); 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-(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, PCTlLJS98/20182, and U.S. 4,899,755, U.S.
5,474,756, U.S. 5,846,519 and U.S. 6,143,274, each of which is hereby incorporated by reference in its entirety.
[00148] Particularly preferred metal chelators include those of Formula l, 2 and 3 (for ~~lIn and radioactive lanthanides, such as, for example ~~~Lu, 901', ~s3Sm, and ~66Ho) and those of Formula 4, 5 and 6 (for radioactive 99"'Tc, ~ $6Re, and 188Re) set forth below. These and other metal chelating groups are described in U.S. Patent Nos. 6,093,382 and 5,608,110, which are incorporated by reference in their entirety. Additionally, the chelating group of formula 3 is described in, for example, U.S. Patent No. 6,143,274; the chelating group of formula 5 is described in, for example, U.S. Patent Nos. 5,627,286 and 6,093,382, and the chelating group of formula 6 is described in, for example, U.S. Patent 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-dibenzylGIy-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.) [00149] Furthermore, sulfur protecting groups such as Acm (acetamidornethyl), 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.
[00150] Additionally, other useful metal chelators include:
R R
H OOC---~ ~'~ ~--- COOH
N N
C
,N N
HOOC~ ~ COOH
~R CO
(1) R R
HOOC----~ n ~--COOH
N N
C
N N
HOOC~ U COOH
~R NH
(2) HOOC--~ n ~--COOH
N N
~---COOH
(3) O
HN ~ ~-O
n NH HN NH HN
~N N~ ~ ~N N~
HO OH O HO OH
(4a) (4b) YiJ ~n X~ X
NH HN
NH HN
iJ ~~ /
N Y ~~ I I
HO OH HO OH
(5a) (5b) OH
O O
O N N~ °
~\
_ a /N S/

(6) O
O NH HN
HS
(7) [00151] In the above Formulas I and 2, R is alkyl, preferably methyl. In the above 5 Formulas Sa and Sb, X is either CH2 or O; Y is C1-Clo branched or unbranched alkyl; aryl, aryloxy, arylamino, arylaminoacyl; arylalkyl - where the alkyl group or groups attached to the aryl group are C1-Clo branched or unbranched alkyl groups, C1-Clo branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups; J is optional, but if present is C(=O)-, OC(=O)-, SOZ-, NC(=O)-, 10 NC(=S)-, N(Y), NC(=NCH3)-, NC(=NH)-, N=N-, homopolyamides or heteropolyamines derived from synthetic or naturally occurring amino acids; all where n is 1-100. Other variants of these structures are described, for example, in U.S. Patent No.
6,093,382. In Formula 6, the group S-NHCOCH3 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 15 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 in their entirety.
[00152] 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-20 bismorpholineamide, Cm4pm10d2a (1,4-carbonylmethyl-10-phosponomethyl-1,4,7,10-Tetraazacyclododecane-1,7-diacetic acid), D03A N [[4,7,10-Tris(carboxyrnethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl, HP-DO3A, D03A-monoamide and derivatives thereof.
[00153] Preferred metal radionuclides for scintigraphy or radiotherapy include 9~"'Tc, SICr, 67Ga 68~a 47SC SlCr 167Tm 141Ce 111In 16s~ 175 140La 90Y 88Y 153Sm 166HO 165D
> > > > > > > > > > > > > > y~

25 166D 62~u 64Cu 67~u 97Ru 103Ru 186Re 188Re 203Pb 211$i 212$i 213$i 214Bi 105 109Pd y> > > > > > > > > > > > > > >
l lTnSn, 149Pm, 161Tb, 177Lu, l9sAu and 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 64Cu, 6~Ga, 68Ga, 99mTc, and "'In, with 99mTc and "'In 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 64Cu, 9oY~ ~os~~ mln, I'~mSn, ~49Pm, ~s3sm~ ~6~.Lb~ is6Dy~ ~66Ho~ ms~,b~ I~~Lu, ~s6nssRe, and ~99Au, with 3?~Lu and 9°Y being particularly preferred. 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 99"'Tc 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 99Mo 99mTc generator.
Fox example, the 99"'Tc labeled peptide can be used to diagnose and monitor therapeutic progress in primary tumors and metastases. Peptides labeled with ~~~Lu, 9°Y or other therapeutic radionuclides can be used to provide radiotherapy for primary tumors and metastasis related to cancers of the prostate, breast, lung, etc.
1B. Optical Labels [00154] In an exemplary embodiment, the compounds of the invention may be conjugated with photolabels, such as optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having absorption or emission maxima in the range of 400-1540 nm. The compounds of the invention may alternatively be derivatized with a bioluminescent molecule. The preferred range of absorption maxima for photolabels is between 600 and 1000 nm to minimize interference with the signal from hemoglobin.
Preferably, photoabsorption labels have large molar absorptivities, e.g. > !Os crri 1M-!, while fluorescent optical dyes will have high quantum yields. Examples of optical dyes include, but are not limited to those described in WO 98/18497, WO 98/18496, WO
98/18495, WO
98118498, WO 98/53857, WO 96!17628, WO 97/18841, WO 96/23524, WO 98/47538, and references cited therein. For example, the photolabels may be covalently linked directly to compounds of the invention, such as, for example, compounds comprised of GRP
receptor targeting peptides and linkers of the invention. Several dyes that absorb and emit light in the visible and near-infrared region of electromagnetic spectrum are currently being used for various biomedical applications due to their biocompatibility, high molar absorptivity, and/or high fluorescence quantum yields. The high sensitivity of the optical modality in conjunction with dyes as contrast agents parallels that of nuclear medicine, and permits visualization of organs and tissues without the undesirable effect of ionizing radiation.
Cyanine dyes with intense absorption and emission in the near-infrared (NIR) region are particularly useful because biological tissues are optically transparent in this region. For example, indocyanine green, which absorbs and emits in the NIR region has been used for monitoring cardiac output, hepatic functions, and liver blood flow and its functionalized derivatives have been used to conjugate biomolecules for diagnostic purposes (R. B. Mujumdar, L. A.
Ernst, S. R.
Mujumdar, et al., Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters.
Bioconjugate Chemistry, 1993, 4(2), 105-11 l; Linda G. Lee and Sam L. Woo. "N-Heteroaromatic ion and iminium ion substituted cyanine dyes for use as fluorescent labels", U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al. "Light imaging contrast agents", WO
98/48846; Jonathan Turner, et al. "Optical diagnostic agents for the diagnosis of neurodegenerative diseases by means of near infra-red radiation", WO 98/22146;
Kai Licha, et al. "In-vivo diagnostic process by near infrared radiation", WO 96/17628;
Robert A. Snow, et al., Compounds, WO 98/48838. Various imaging techniques and reagents are described in U.S. Patents 6,663,847, 6,656,451, 6,641,798, 6,485,704, 6,423,547, 6,395,257, 6,280,703, 6,277,841, 6,264,920, 6,264,919, 6,228,344, 6,217,848, 6,190,641, 6,183,726, 6,180,087, 6,180,086, 6,180,085, 6,013,243, and published U.S. Patent Applications 2003185756, 20031656432, 2003158127, 2003152577, 2003143159, 2003105300, 2003105299, 2003072763, 2003036538, 2003031627, 2003017164, 2002169107, 2002164287, and 2002156117. All of the above references are incorporated by reference in their entirety.
2A. Linkers Containing At Least One Non-alpha Amino Acid [00155] 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, N is 0 (where 0 means it is absent), 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, 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.
[00156] Alpha amino acids are well known in the art, and include naturally occurring and synthetic amino acids.

[00157 Non-alpha amino acids are also known in the art and include those which are naturally occurnng or synthetic. Preferred non-alpha amino acids include:
8-amino-3,6-dioxaoctanoic acid;
N-4-aminoethyI-N-1-acetic acid; and polyethylene glycol derivatives having the formula NHZ-(CHzCHzO)n-CHZCOzH or NHZ-(CHZCHZO)n-CH2CHZCOZH where n = 2 to 100.
[00158) 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. These compounds may be prepared using the methods disclosed herin, particularly in the Examples, as well as by similar methods known to one skilled in the art.

Table -Compounds Containing Linkers With At Least One Non-alpha Amino Acid Compo HPLC HPLC

and method'RTZ MS3 IC505M N O P G*

Ll 10-40%B5.431616.65 N,N- Lys 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser-~ dioxaoctanoic C s Acm acid -Gl L2 10-40%B5.471644.73 N,N- Arg 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s(Acm acid -GI

L3 10-40%B5.971604.6>50 N,N- Asp 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s Acm acid -GI

LA 10-40%B5.921575.54 N,N- Ser 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s(Acm acid -GI

L5 10-40%B5.941545.59 N,N- Gly 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s Acm acid -Gl L6 10-30%B7.821639(M>50 N,N- Glu 8-amino-3,6-none BBN(7-14) +Na) dimethylglycine-Ser- dioxaoctanoic C s Acm acid -GI

L7 10-30%B8.471581 7 N,N- Dala 8-amino-3,6-none BBN(7-14) (M+Na) dimethylglycine-Ser- dioxaoctanoic C s Acm acid -Gl L8 10-30%B6.721639 4 N,N- 8-amino-3,6-Lys none BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic C s(Acm acid -GI

L9 10-30%B7.28823.36 N,N- 8-amino-3,6-Arg none BBN(7-14) ~ (M+2/2) dimethylglycine-Ser-dioxaoctanoic C s Acm acid -GI

L10 10-30%B7.941625.6>50 N,N- 8-amino-3,6-Asp none BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic Cys(Acm acid -Gly 7('able -Compounds Containing Linkers With At Least One Non-alpha Amino Acid CompoHPLC HPLC

and method'RTZ MS3 IC505M N O P G*

L1 10-30%B7.591575.636 N,N- 8-amino-3,6-Ser none BBN(7-14) I

dimethylglycine-Ser-dioxaoctanoic C s Acm -GI acid L12 10-30%B7.651567.5>50 N,N- 8-amino-3,6-Gly none BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic C s(Acm -GI acid L13 10-30%B7.861617.7>50 N,N- 8-amino-3,6-Glu none BBN(7-14) dimethylglycine-Ser-dioxaoctanoic Cys(Acm -Gly acid L14 10-30%B7.9 1581.711 N,N- 8-amino-3,6-Dala none BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic C s Acm -GI acid L15 10-30%B7.841656.811.5N,N- 8-amino-3,6-8-amino-3,6-none BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoicdioxaoctanoic C s Acm -GI acid acid L16 10-30%B6.651597.417 N,N- 8-amino-3,6-2,3- none BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoicdiaminopropio C s Acm -GI acid nic acid L17 10-30%B7.6 1488.68 N,N- none 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s Acm -GI acid L18 10-30%B7.031574.67.8 N,N- 2,3- 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser-diaminopropionidioxaoctanoic C s Acm -GI c acid acid L19 10-35%B5.131603.6>50 N,N- Asp 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s Acm -GI acid L20 10-35%B5.191603.637 N,N- 8-amino-3,6-Asp Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoic C s(Acm -Gl acid L21 10-35%B5.041575.746 N,N- 8-amino-3,6-Ser Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoic C s Acm -Gl acid L22 10-35%B4.371644.736 N,N- 8-amino-3,6-Arg Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoic C s(Acm -Gl acid L23 10-35%B532 1633.7>50 N,N- 8-amino-3,6-8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoicdioxaoctanoic C s(Acm -GI acid acid L24 IO-35%B4.181574.638 N,N- 8-amino-3,6-2,3- Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoicdiaminopropio Cys(Acm -Gly acid nic acid L25 10-35%B4.241616.626 N,N- 8-amino-3,6-Lys Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoic C s Acm -Gl acid L26 10-35%B4.451574.630 N,N- 2,3- 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser-diaminopropionidioxaoctanoic C s(Acm -Gl c acid acid L27 10-35%B4.381627.3>50 N,N- N-4-aminoethyl-Asp none BBN(7-14) dimethylglycine-Ser-N-1-Cys(Acm)-Gly piperazineacetic acid Table -Compounds Containing Linkers With At Least One Non-alpha Amino Acid CompoHPLC HPLC

and method'RTZ MS3 IC505M N O P G*

L28 10-35%B4.1 1600.325 N,N- N-4-aminoethyl-Ser none BBN(7-14) d imethylglycine-Ser-N-1-Cys(Acm)-Glypiperazineacetic acid L29 10-35%B3.711669.436 N,N- N-4-aminoethyl-Arg none BBN(7-14) d imethylglycine-Ser-N-1-Cys(Acm)-Glypiperazineacetic acid L30 10-35%B4.571657.236 N,N- N-4-aminoethyl-8-amino-3,6-none BBN(7-14) dimethylglycine-Ser-N-1- dioxaoctanoic Cys(Acm)-Glypiperazineaceticacid acid L31 10-35%B3.691598.3>50 N,N- N-4-aminoethyl-2,3- none BBN(7-14) dimethylglycine-Sex-N-1- diaminopropio Cys(Acm)-Glypiperazineaceticnic acid acid L32 10-35%B3.511640.334 N,N- N-4-aminoethyl-Lys none BBN(7-14) dimethylglycine-Ser-N-1-Cys(Acm)-Glypiperazineacetic acid L33 10-35%B4.291584.5>50 N,N- N-1- Asp none BBN(7-14) dimethylglycine-Ser-piperazineacetic Cys(Acm)-Glyacid L34 10-35%B4.071578.738 N,N- N-1- Ser none BBN(7-14) (M+Na) dimethylglycine-Ser-piperazineacetic C s(Acm -Gl acid L35 10-35%B3.651625.626 N,N- N-1- Arg none BBN(7-14) dimethylglycine-Ser-piperazineacetic C s(Acm -Gl acid L36 10-35%B4.431636.67 N,N- N-1- 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser-piperazineaceticdioxaoctanoic Cys(Acm -Glyacid acid L37 10-35%B3.661555.723 N,N- N-I- 2,3- none BBN(7-14) dimethylglycine-Ser-piperazineaceticdiaminopropio C s(Acm-Gl acid nic acid L38 10-35%B3.441619.67 N,N- N-1- Lys none BBN(7-14) dimethylglycine-Ser-piperazineacetic C s Acm -Gl acid L42 30-50%B5.651601.625 N,N- 4- 8-amino-3,6-none BBN(7-14) dimethylglycine-Ser-Hydroxyprolinedioxaoctanoic C s Acm -G1 acid L48 30-50%B4.471600.540 N,N- 4-aminoproline8-amino-3,6-none BBN(7-14) dimethylglycine-Ser- dioxaoctanoic CsAcm-Gl acid L51 15-35%B5.141673.749 N,N- Lys 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C sAcm-Gl acid L52 15-35%B6.081701.614 N,N- Arg 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s Acm -GI acid L53 15-35%B4.161632.610 N,N- Ser 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s(Acm -Gl acid 'f able -Compounds Containing Linkers With At Least One Non-alpha Amino Acid CompoHPLC HPLC

and method'RTZ MS3 IC505M N O P G*

L54 15-35%B4.881661.6>50 N,N- Asp 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser- dioxaoctanoic C s Acm -Gl acid L55 15-35%B4.831683.443 N,N- 8-amino-3,6-Asp Gly BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic C s(Acm)-GI acid L56 IS-35%B4.651655.74 N,N- 8-amino-3,6-Ser Gly BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic Cys(Acm -Gly acid L57 15-35%B4.9 1701.850 N,N- 8-amino-3,6-Arg Gly BBN(7-14) dimethylglycine-Ser-dioxaoctanoic C s Acm -GI acid L58 15-35%B4.22846.4 >50 N,N- 8-amino-3,6-8-amino-3,6-Gly BBN(7-14) (M+H/2) dimethylglycine-Ser-dioxaoctanoicdioxaoctanoic C s Acm -GI acid acid L59 15-35%B4.031635.542 N,N- 8-amino-3,6-2,3- Gly BBN(7-14) dimethylgIycine-Ser-dioxaoctanoicdiaminopropio C s Acm -GI acid nic acid L60 IS-35%B4.111696.620 N,N- 8-amino-3,6-Lys Gly BBN(7-14) (M+Na) dimethylglycine-Ser-dioxaoctanoic C s(Acm -GI acid L61 15-35%B4.321631.443 N,N- 2,3- 8-amino-3,6-Gly BBN(7-14) dimethylglycine-Ser-diaminopropionidioxaoctanoic C s Acm -Gl c acid acid L78 20-40%B6.131691.435 D03A-monoamide8-amino-3,6-Diaminopropinone BBN(7-14) (M+Na) dioxaoctanoiconic acid acid L79 20-40%B7.721716.842 D03A-monoamide8-amino-3,6-Biphenylalaninone BBN(7-14) (M+Na) dioxaoctanoicne acid L80 20-40%B7.781695.9>50 D03A-monoamide8-amino-3,6-Diphenylalaninone BBN(7-14) dioxaoctanoicne acid L81 20-40%B7.571513.637.5D03A-monoamide8-amino-3,6-4- none BBN(7-14) dioxaoctanoicBenzoylpheny acid lalanine L92 I S-30%B5.631571.65 D03A-monoamide5- 8-amino-3,6-none BBN(7-14) aminopentanoicdioxaoctanoic acid ~ acid L94 20-36%B4.191640.86.2 D03A-monoamide8-amino-3,6-D- none BBN(7-14) (M+Na) dioxaoctanoicPhenylalanine acid L110 15-45%B5.061612.736 DO3A-monoamide8-aminooctanoic8-amino-3,6-none BBN(7-14) acid dioxaoctanoic acid L209 20-40%B4.623072.5437 D03A-monoamideE(G8-amino-8- 8- BBN(7-14) over 3,6- aminooctanoicaminoo minutes dioxaoctanoicacid ctanoic acid-8-amino- acid 3,6-dioxaoctanoic acid QWAVGHLM-NHZ

Table B
-Compounds Containing Linkers With At Least One Non-alpha Amino Acid CompoHPLC HPLC

and method'RTZ MS3 IC505M N O P G*

L210 20-50%B6.183056.76l D03A-monoamideE(G-Aoa-Aoa-8- 8- BBN(7-14) I

over QWAVGHLM-aminooctanoicaminoo minutes NI-IZ) acid ctanoic acid *BBN(7-14) is [SEQ ID NO:1 ' HPLC method refers to the 10 minute time for the HPLC gradient.
z HPLC RT refers to the retention time of the compound in the HPLC.
3 MS refers to mass spectra where molecular weight is calculated from mass/unit charge 5 (m/e).
4 ICso 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 [00159] In another embodiment of the present invention, the linker N-O-P
contains at least 10 one substituted bile acid. Thus, in this embodiment of the linker N-O-P, N is 0 (where 0 means it is absent), an alpha amino acid, a substituted bile acid or other linking group;
O is an alpha amino acid or a substituted bile acid; and P is 0, an alpha amino acid, a substituted bile acid or other linking group, wherein at least one of N, O or P is a substituted acid.
[00160] Bile acids are found in bile (a secretion of the liver) and are steroids having a 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.
[00161 ] Other useful substituted bile acids in the present invention include substituted cholic acids and derivatives thereof. Specific substituted cholic acid derivatives include:
(313,513)-3-aminocholan-24-oic acid;

(313,513,12a)-3-amino-12-hydroxycholan-24-oic acid;
(313,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid;
Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3 aminocholic acid);
(313,513,7a)-3-amino-7-hydroxy-12-oxocholan-24-oic acid; and (313,513,7a)-3-amino-7-hydroxycholan-24-oic acid.
[00162 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. These compounds may be prepared using the methods disclosed herein, particularly in the Examples, as well as by similar methods known to one skilled in the art.

Table -Compounds Containing Linkers With At Least One Substituted Bile Acid CompoHPLC HPLC

and method'RTz MS3 IC505M N O P G*

L62 20- 3.79 1741.2>50 D03A-monoamideGly (313,513)-3-aminocholan-none BBN(7-14) 80%B 24-oic acid L63 20- 3.47 1757.023 D03A-monoamideGly (313,5J3,12a)-3-amino-12-none BBN(7-14) 80%B hydroxycholan-24-oic acid L64 20- 5.31 1773.78.5 D03A-monoamideGly (313,513,7a,12a)-3-amino-none BBN(7-14) 50%B 7,12-dihydroxycholan-24-oic acid L65 20- 3.57 2246.2>50 D03A-monoamideGly Lys-(3,6,9- Arg BBN(7-14) 80%B trioxaundecane-l , l l -dicarbonyl-3,7-dideoxy-3-aminocholic acid L66 20-80%3.79 2245.8>50 D03A-monoamideGly Lys-(313,513,7a,12a)-3-Arg BBN(7-14) amino-7,12-dihydroxycholan-24-oic acid-3,6,9-trioxaundecane-1,11-dicarbon 1 L67 20-80%3.25 1756.94.5 D03A-monoamideGly (313,513,?a,l2a)-3-amino-none BBN(7-14) 12-oxacholan-24-oic acid L69 20-80%3.25 1861.278 D03A-monoamide1-amino-(313,513,7a,12a)-3-amino-none BBN(7-14) 3,6- 7,12-dihydroxycholan-dioxaocta24-oic acid noic acid L280 - - - - D03A-monoamideGly 3f3,5l37a,12a)-3-amino-none Q-W-A-V-7,12-dihydroxycholan- a-H-L-M-24-oic acid NHz L281 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-f Q-W-A-V-7,12-dihydroxycholan- G-H-L-M-24-oic acid NHz Table -Compounds Containing Linkers With At Least One Substituted Bile Acid CompoHPLC HPLC

and method'RTZ MS3 IC505M N O P G*

L282 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-f Q-W-A-V-7,12-dihydroxycholan- G-H-L-L-24-oic acid NHZ

L283 - - - - D03A-monoamideGly 3J3,SJ3 7a,12a)-3-amino-f Q-W-A-V-7,12-dihydroxycholan- G-H-L-24-oic acid NH-pentyl L284 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-y QWAVBal 7,12-dihydroxycholan- a-HFNle-24-oic acid NHZ

L285 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-f Q-W-A-V-7,12-dihydroxycholan- Bala-H-F-24-oic acid Nle-NHZ

L286 - - - - D03A-monoamideGly 3J3,513 7a,12a)-3-amino-none QWAVGH

7,12-dihydroxycholan- FL-NHZ

24-oic acid L287 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-none QWAVGN

7,12-dihydroxycholan- MeHis-24-oic acid LM-NHZ

L288 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-none LWAVGS

7,12-dihydroxycholan- F-M-NHZ

24-oic acid L289 - - - - D03A-rnonoamideGly 313,513 7a,12a)-3-amino-none HWAVGH

7,12-dihydroxycholan- L-M-NHZ

24-oic acid L290 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-none LWATGH

7,12-dihydroxycholan- -F-M-NHZ

24-oic acid L291 - - - - D03A-monoamideGly 3J3,513 7a,12a)-3-amino-none QWAVGH

7,12-dihydroxycholan- -FMNHZ

24-oic acid L292 - - - - D03A-monoamideGly 3J3,513 7a,12a)-3-amino-QRLG
QWAVGH

7,12-dihydroxycholan-N LM-NHz 24-oic acid L293 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-QRYG
QWAVGH

7,12-dihydroxycholan-N LM-NHZ

24-oic acid L294 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-QKYG
QWAVGH

7,12-dihydroxycholan-N LM-NHZ

24-oic acid L295 - - - - Pglu-Q-Lys Gly 313,513 7a,12a)-3-amino-LG-N QWAVGH

(D03A- 7,12-dihydroxycholan- LM-NHZ

monoamide) 24-oic acid L303 D03A-monoamideGly 3-amino-3-deoxycholicnone QRLGNQ

- - - - acid WAVGHL

M-NHZ

L304 D03A-monoamideGly 3-amino-3-deoxycholicnone QRYGNQ

- - - - acid WAVGHL

M-NHZ

L305 D03A-monoamideGly 3-amino-3-deoxycholicnone QKYGNQ

- - - - acid WAVGHL

M_NHZ

'f able -Compounds Containing Linkers With At Least One Substituted Bile Acid Compo-I-IPLCIIPLC

and method'I2T2 MS3 IC505M N O P G

L306 D03A-monoamideGly 3-amino-3-deoxycholicnone See FIG.

- - - - acid 38 for structure of targeting a tide *BBN(7-14) is [SEQ ID NO:1]
HPLC method refers to the 10 minute time for the HPLC gradient.
~ HPLC RT refers to the retention time of the compound in the HPLC.
3 MS refers to mass spectra where molecular weight is calculated from masslunit charge 5 (m1e).
4 ICSO 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 [00163] In yet another embodiment of the present invention, the linker N-O-P
contains at 10 least one non-alpha amino acid with a cyclic group. Thus, in this embodiment of the linker N-O-P, 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;
O is an alpha amino acid or a non-alpha amino acid with a cyclic 15 group; and P is 0, an alpha amino acid, a non-alpha amino acid with a cyclic group, or other linking group, wherein at least one of N, O or P is a non-alpha amino acid with a cyclic group.
20 [00164] Non-alpha amino acids with a cyclic group include substituted phenyl, biphenyl, cyclohexyl or other amine and carboxyl containing cyclic aliphatic or heterocyclic moieties.
Examples of such include:
4-aminobenzoic acid (hereinafter referred to as "Abz4 in the specification") 3-aminobenzoic acid 4-aminomethyl benzoic acid 8-aminooctanoic acid trans-4-aminomethylcyclohexane carboxylic acid 4-(2-aminoethoxy)benzoic acid isonipecotic acid 2-aminomethylbenzoic acid 4-amino-3-nitrobenzoic acid 4-(3-carboxymethyl-2-keto-1-benzimidazolyl-piperidine 6-(piperazin-1-yl)-4-(3H)-quinazolinone-3-acetic acid (25,5 S)-5-amino-1,2,4,5,6,7-hexahydro-azepino[3,21-hi]indole-4-one-2-carboxylic acid (4S,7R)-4-amino-6-aza-5-oxo-9-thiabicyclo[4.3.0]nonane-7-carboxylic acid 3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one N1-piperazineacetic acid N-4-aminoethyl-N-1-piperazineacetic acid (3 S)-3-amino-1-carboxymethylcaprolactam (2S,6S,9)-6-amino-2-carboxymethyl-3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione 3-amino-3-deoxycholic acid 4-hydroxybenzoic acid 4-aminophenylacetic acid 3-hydroxy-4-aminobenzoic acid 3-methyl-4-aminobenzoic acid 3-chloro-4-aminobenzoic acid 3-methoxy-4-aminobenzoic acid 6-aminonaphthoic acid N,N'-Bis(2-aminoethyl)-succinamic acid [00165] 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.
These compounds may be prepared using the methods disclosed herein, particularly in the Examples, as well as by similar methods known to one skilled in the art.

Table -Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl Or Heterocyclic) Carboxylates Comp HPLC HPLC

ound method'RTZ MS3 ICSOSM N O p G*

L70 10-40%B6.15 1502.65 DO3A- Gly 4-aminobenzoicnone BBN(7-14) acid monoamide L71 20-50%14.14 59.68 7 D03A- none 4-aminomethylnone BBN(7-14) over (M+Na) monoamide benzoic acid minutes L72 20-50%13.64 65.73 8 D03A- none trans-4- none BBN(7-14) over (M+K) monoamide aminomethylcyclohe minutes x 1 carbox lic acid L73 5-35%7.01 1489.85 D03A- none 4-(2- none BBN(7-14) monoamide aminoethoxy)benzoic acid L74 S-35%6.49 1494.87 D03A- Gly isonipecoticnone BBN(7-14) acid monoamide L75 5-35%6.96 1458.023 D03A- none 2- none BBN(7-14) monoamide aminomethylbenzoic acid L76 5-35%7.20] 1502.74 DO3A- none 4-aminomethyl-3-none BBN(7-14) monoamide nitrobenzoic acid L77 20-40%B6.17 1691.817.5D03A- 8-amino-1-Naphthylalaninenone BBN(7-14) (M+Na) monoamide3,6-dioxaoctan oic acid L82 20-40%B6.18 1584.68 D03A- none 4-(3-carboxymethyl-none BBN(7-14) monoamide 2-keto-1-benzimidazolyl-i eridine L83 20-40%B5.66 1597.5>50 D03A- none 6-(piperazin-I-yl)-4-none BBN(7-14) monoamide (3H)-quinazolinone-3-acetic acid L84 20-40%B6.31 1555.5>50 D03A- none (25,55)-5-amino-none BBN(7-14) monoamide 1,2,4,5,6,7-hexahydro-azepino[3,21-hi]indole-4-one-2-carboxylic acid L85 20-40%B5.92 1525.5>50 D03A- none (4S,7R)-4-amino-6-none BBN(7-14) monoamide aza-5-oxo-9-thiabicyclo[4.3.0]non ane-7-carbox lic acid L86 20-40%B6.46 1598.6>50 D03A- none N,N-dimethylglycinenone BBN(7-14) monoamide L87 20-40%B5.47 1593.8>50 D03A- none 3-carboxymethyl-1-none BBN(7-14) (M+Na) monoamide phenyl-1,3,8-t riazaspiro[4.5]decan - 4-one L88 20-40%B3.84 1452.7>50 D03A- none NI-piperazineaceticnone BBN(7-14) monoamidea cid 't'able -Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl Or Heterocyclic) Carboxylates CompHPLC HPLC

oundmethod'RTZ MS3 IC505M N O P G*

L89 20-40%B5.68 1518.523 D03A- none N-4-aminoethyl-N-1-none BBN(7-14) (M+Na) monoamide piperazine-acetic acid L90 20-40%B7.95 1495.450 D03A- none (3S)-3-amino-1-none BBN(7-14) monoamide carboxymethylcaprol actam L91 20-40%B3.97 1535.7>50 D03A- none (2S,6S,9)-6-amino-2-none BBN(7-14) monoamide carboxymethyl-3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione L93 15-30%B7.57 1564.75.8 D03A- 5- trans-4- none BBN(7-14) monoamideaminopentaminomethylcyclohe anoic xane-I-carboxylic acid acid L95 15-35%B5.41 1604.614 D03A- trans-4-D-Phenylalaninenone BBN(7-14) monoamideaminometh ylcyclohex ane-1-carboxylic acid L96 20-36%B4.75 1612.735 DO3A- 4- 8-amino-3,6-none BBN(7-14) monoamideaminomethdioxaoctanoic acid ylbenzoic acid L97 15-35%B5.86 1598.84.5 D03A- 4-benzoyl-trans-4- none BBN(7-14) monoamide(L)- aminomethylcyclohe phenylalanxane-1-carboxylic ine acid L98 15-35%B4.26 1622.716 D03A- trans-4-Arg none BBN(7-14) monoarnideaminometh ylcyclohex ane-1-carboxylic acid L99 15-35%B4.1 1594.722 D03A- trans-4-Lys none BBN(7-14) monoamideaminometh ylcyclohex ane-1-carboxylic acid L10015-35%B4.18 1613.610 D03A- trans-4-Diphenylalaninenone BBN(7-14) monoamideaminometh ylcyclohex ane-1-carboxylic acid L10115-35fB5.25 1536.725 D03A- trans-4-1-Naphthylalaninenone BBN(7-14) monoamideaminometh ylcyclohex ane-1-carboxylic acid Table -Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl Or Heterocyclic) Carboxylates CompHPLC HPLC

oundmethod'RTZ MS's IC505M N O P G*

L10215-35%B5.28 1610.89.5 D03A- traps-4-8-amino-3,6-none BBN(7-14) monoamideaminomethdioxaoctanoic acid ylcyclohex ape-1-carboxylic acid L10315-35%B4.75 1552.724 D03A- traps-4-Ser none BBN(7-14) monoamideaminometh ylcyclohex ape-1-carboxylic acid L10415-35%B3.91 1551.732 D03A- traps-4-2,3-diaminopropionicnone BBN(7-14) monoamideaminomethacid ylcyclohex ape-I-carboxylic acid L10520-45%B7.68 1689.73.5 D03A- traps-4-Biphenylalaninenone BBN(7-14) monoamideaminometh ylcyclohex ape-1-carboxylic acid L10620-45%B6.97 1662.73.8 D03A- traps-4-(2S,5S)-5-amino-none BBN(7-14) monoamideaminometh1,2,4,5,6,7-ylcyclohexhexahydro-ane-I-azepino[3,21-carboxylichi]indole-acid 4-one-2-carboxylic acid L10715-35%B5.79 1604.75 DO3A- traps-4-traps-4- none BBN(7-14) monoamideaminomethaminomethylcyclohe ylcyclohexxane-1-carboxylic ape-1-acid carboxylic acid L10815-45%B6.38 1618.710 D03A- 8-amino-Phenylalaninenone BBN(7-14) monoamide3,6-dioxaoctan oic acid Ll 15-45%B6.85 1612.76 DO3A- traps-4-Phenylalaninenone BBN(7-14) monoamideaminometh ylcyclohex ape-1-carboxylic acid L1 20-45%B3.75 1628.68 D03A- 8- raps-4- none BBN(7-14) 11 t monoamideaminooctaaminomethylcyclohe noic xane-1-carboxylic acid acid Ll 20-47%B3.6 1536.54.5 D03A- none 4'-aminomethyl-none BBN(7-14) in monoamide biphenyl-1-min carbox lic acid Table -Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl Or Heterocyclic) Carboxylates CompHPLC HPLC

oundmethod'RTZ MS3 IC505M N O P G*

L11320-47%B3.88 1558.65 DO3A- none 3'-aminomethyl-none BBN(7-14) in (M+Na) monoamide biphenyl-3-min carbox lic acid L11410-40%B5.47 1582.84.5 CMDOTA Gl 4-aminobenzoicnone BBN(7-14 acid L1245-35%B7.04 1489.98.0 D03A- none 4- none BBN(7-14) monoamide aminomethylphenox acetic acid L1435-35%B6.85 1516.8I1 D03A- Gly 4-aminophenylaceticnone BBN(7-14) monoamide acid L1445-35%B6.85 1462.79 HPD03A none 4- henox none BBN(7-14 L14520-80%B1.58 1459.85 D03A- none 3- none BBN(7-14) monoamide aminomethylbenzoic acid L14620-80%B1.53 1473.79 D03A- none 4- none BBN(7-14) monoamide aminomethylphenyla cetic acid L14720-80%B1.68 1489.73.5 D03A- none 4-aminomethyl-3-none BBN(7-14) monoamide methox benzoic acid L20110-46%B5.77 1563.736 Boa*** none Gly 4- BBN(7-14) over aminoben minutes zoic acid L20210-46%B5.68 1517.7413 D03A- none Gly 4- BBN(7-14) over monoamide hydrazino minutes benzo L20310-46%B5.98 1444.699 D03A- none none 4- BBN(7-14) over monoamide aminoben minutes zoic acid L20410-46%B5.82 1502.7350 D03A- none 4-aminobenzoicGly BBN(7-14) acid over monoamide minutes L20510-46%B5.36 1503.7245 D03A- Gly 6-Aminonicotinicnone BBN(7-14) over monoamide acid minutes L20610-46%B7.08 1592.854.5 D03A- Gly 4'-Amino-2'-methylnone BBN(7-14) over monoamide biphenyl-4-minutes carbox lic acid L20710-46%B7.59 1578.832.5 D03A- Gly 3'-Aminobiphenyl-3-none BBN(7-14) over monoamide carboxylic 12 acid minutes L20810-46%B5.9 1516.757.5 D03A- Gly 1,2-diaminoethylTerephthaBBN(7-14) over monoamide lic 12 acid minutes L21110-46JB5.76 1560.774 D03A- Gly Gly 4- BBN(7-14) over monoamide aminoben minutes zoic acid L21210-46!B6.05 1503.71NT** D03A- none Gly 4- EWAVGH

over monoamide aminobenLM-NHZ

minutes . zoic acid L21310-46fB5.93 1503.71NT** D03A- Gly 4-aminobenzoicnone QWAVGH
acid over monoamide LM-OH

minutes Table -Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl Or Heterocyclic) Carboxylates Comp HPLC HPLC

ound method'RTZ MS3 IC505M N O P G*

L2I4 10-46%B7.36 1649.91NT** D03A- Gly 4-aminobenzoic(D)-PheBBN(7-14) acid over monoamide minutes L215 10-46%B5.08 2071.37NT** D03A- Gly 4-aminobenzoicnone QRLGNQ
acid over monoamide WAVGHL

minutes M-NHz L216 10-46%B4.94 2121.38NT** DO3A- Gly 4-aminobenzoicnone QRYGNQ
acid over monoamide WAVGHL

minutes L217 10-46%B4.38 2093.37NT** DO3A- Gly 4-aminobenzoicnone QKYGNQ
acid over monoamide WAVGHL

minutes ' M-NH2 L218 10-46%B6.13 2154.45NT** DO3A- Gly 4-aminobenzoicnone See acid FIG.

over monoamide 38 for minutes structure of targeting a tide L219 10-46%B8.61 1588.84NT** DO3A- Gly 4-aminobenzoic(D)-PheQWAVGH
acid over monoamide L-NH-minutes Pent L220 10-46%B5.96 1516.75NT** D03A- Gly 4-aminobenzoicnone QWSVaH
acid over monoamide LM-NHZ

minutes L221 10-46%B7.96 1631.87NT** D03A- Gly 4-aminobenzoic(D)-PheQWAVGH
acid over monoamide LL-NHZ

minutes L222 10-46%B6.61 1695.91NT** DO3A- Gly 4-aminobenzoic(D)-TyrQWAV-acid over monoamide Bala-HF-minutes Nle-NH

L223 10-46%B7.48 1679.91NT** D03A- Gly 4-aminobenzoicPhe QWAV-acid over monoamide Bala-HF-minutes Nle-NHz L224 10-46%B5.40 1419.57NT** D03A- Gly 4-aminobenzoicnone QWAGHF
acid over monoamide L,-NHz minutes L225 10-46%B8.27 1471.71NT** D03A- Gly 4-arninobenzoicnone LWAVGS
acid over monoamide FM-NH2 minutes L226 10-46%B5.12 1523.75NT** D03A- Gly 4-aminobenzoicnone HWAVGH
acid over monoamide LM-NH2 minutes L227 10-46%B6.61 1523.75NT** D03A- Gly 4-aminobenzoienone LWAVGS
acid over monoamide FM-NH2 minutes L228 10-46%B5.77 1511 NT** D03A- Gly 4-aminobenzoicnone QWAVGH
acid over monoamide FM-NHZ

minutes L233 5-35%B7.04 1502.714.8 D03A- Gly 3-aminobenzoicnone BBN(7-14) acid over monoamide min L234 20-80%1.95 1552.763 D03A- Gly 6-aminonaphthoicnone BBN(7-14) over monoamide acid minutes Table -Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl, Cycloalkyl Or Heterocyclic) Carboxylates Comp HPLC HPLC

ound method'RTZ MS3 IC505M N O P G~

L235 20-80%1.95 1515.72? D03A- Gly 4- none BBN(7-14) over monoamide methylaminobenzoic minutes acid L237 20-80%1.52 1538.685 Cm4pmlOd2aGly 4-aminobenzoicnone BBN(7-14) acid over minutes L238 5-35%B7.17 1462.701.5 N,N- Gly 4-aminobenzoicnone BBN(7-14) acid over dimethylglycin min e-Ser-C s(Acm -Gl L239 20-80%3.36 1733.164.5 N,N- Gly 3-amino-3- none BBN(7-14) over dimethylglycin deoxycholic 10 acid minutes e-Ser-C s(Acm -Gl L240 20-80%1.55 1532.734 D03A- Gly 3-methoxy-4-none BBN(7-14) over monoamide aminobenzoic 10 acid minutes L241 20-80!1.63 1535.684 D03A- Gly 3-chloro-4- none BBN(7-14) over monoamide aminobenzoic 10 acid minutes L242 20-80%1.55 1516.755 D03A- Gly 3-methyl-4- none BBN(7-14) over monoamide aminobenzoic 10 acid minutes L243 20-80%1.57 1518.7014 D03A- Gly 3-hydroxy-4-none BBN(7-14) over monoamide aminobenzoic 10 acid minutes L244 5-50% 4.61 1898.16>50 (D03A- N,N'- none none BBN(7-14) over monoamide)2Bis(2-minutes aminoethyl )_ succinamic acid L300 10-46%- - - D03A- Gly 4-aminobenzoicnone QWAVGH
acid over monoamide FL-NHz minutes L301 20-45%7.18 - - DO3A- none 4- L-I- BBN(7-14) over monoamide aminomethylbenzoicNaphthyla minutes acid lanine L302 - - - - D03A- Gly 4-aminobenzoicnone QWAVGN
acid monoamide MeHis-L-M-NHa *BBN(7-14) is [SEQ ID N0:1]
**NT is defined as "not tested."
~'**BOA is defined as (1R)-1-(Bis{2-[bis(carboxymethyl)amino]ethyl~amino)propane-1,3-dicarboxylic acid.
' HPLC method refers to the 10 minute time for the HPLC gradient.
Z HPLC RT refers to the retention time of the compound in the HPLC.
' MS refers to mass spectra where molecular weight is calculated from mass/unit charge (m/e).

4 ICSO refers to the concentration of compound to inhibit 50% binding of iodinated bombesin to a GRP receptor on cells.
[00166 A subset of compounds containing preferred linkers and various GRP
receptor targeting peptides are set forth in Table 4. These compounds may be prepared using the methods disclosed herein, particularly in the Examples, as well as by similar methods known to one skilled in the art.

Table -Compounds Containing Linkers of the Invention With Various GRP-R
Targeting Moities CompouHPLC HPLC

nd method'RTZ MS3 IC505 M N O P G*

L214 10-46%B7.36 1649.91NT** D03A- Gly 4-aminobenzoic(D)-BBN(7-14) acid over monoamide Phe minutes L215 10-46%B5.08 2071.37NT** D03A- GIy 4-aminobenzoicnoneQRLGNQWA
acid over monoamide VGHLM-NHZ

minutes L216 10-46%B4.94 2121.38NT** DO3A- Gly 4-aminobenzoicnoneQRYGNQWA
acid over monoamide VGHLM-NHZ

minutes L217 10-46%B4.38 2093.37NT** D03A- Gly 4-aminobenzoicnoneQKYGNQWA
acid over monoamide VGHLM-NH2 minutes L218 10-46%B6.13 2154.45NT** D03A- Gly 4-aminobenzoicnoneSee FIG.
acid 38 over monoamide for structure 12 o minutes targeting a tide L219 10-46%B8.61 1588.84NT** D03A- Gly 4-aminobenzoic(D)-QWAVGHL-acid over monoamide Phe NH-Pentyl minutes L220 10-46%B5.96 1516.75NT** D03A- Gly 4-aminobenzoicnoneQWAVaHLM

over monoamide acid -NH2 12 minutes L221 10-46%B7.96 1631.87NT** D03A- Gly 4-aminobenzoic(D)-QWAVGHLL
acid over monoamide Phe -NHZ

minutes L222 10-46%B6.61 1695.91NT** D03A- Gly 4-aminobenzoic(D)-QWAV-Bala-acid over monoamide Tyr HF-Nle-NHZ

minutes L223 10-46%B7.48 1679.91NT** D03A- Gly 4-aminobenzoicPhe QWAV-Bala-acid over monoamide HF-Nle-NHZ

minutes L224 10-46%B5.40 1419.57NT** D03A- Gly 4-aminobenzoicnoneQWAGHFL-acid over monoamide NHz minutes L225 10-46%B8.27 1471.71NT** D03A- Gly 4-aminobenzoicnoneLWAVGSFM
acid over monoamide -NHa minutes L226 10-46%B5.12 1523.75NT** D03A- Gly 4-aminobenzoicnoneHWAVGHL
acid over monoamide M-NHz minutes Tahle -Compounds Containing Linkers of the Invention With Various GRP-R
Targeting Moities CornpouHPLC HPLC

nd method'RTZ MS3 IC505 M N O P G

L227 10-46%B6.61 1523.75NT** D03A- Gly 4-aminobenzoicnoneLWATGHFM
acid over monoamide -NHz minutes L228 10-46%B5.77 1511 NT** D03A- Gly 4-aminobenzoicnoneQWAVGHFM
acid over monoamide -NHz minutes L280 D03A- Gly (313,513 7a,12a)-3-noneQWAVaHLM

-- -- -- -- monoamide amino-7,12- -NHZ

dihydroxycholan-24-oic acid L281 D03A- Gly (313,513 7a,12a)-3-f QWAVGH--- -- -- -- monoamide amino-7,12- LM-NHz dihydroxycholan-24-oic acid L282 D03A- Gly (313,573 ?a,l2a)-3-f QWAVGHLL

-- -- -- -- monoamide amino-7,12- -NHz dihydroxycholan-24-oic acid L283 D03A- Gly (313,513 ?a,l2a)-3-f QWAVGHLN

-- -- -- -- monoamide amino-7,12- H-pentyl dihydroxycholan-24-oic acid L284 D03A- Gly (313,5137a,12a)-3-y QWAVBaIaH

-- -- -- -- monoamide amino-7,12- F-Nle-NHz dihydroxycholan-24-oic acid L285 D03A- Gly (3f3,513 7a,12a)-3-f QWAVBala--- -- -- -- monoamide amino-?,12- HF-Nle-NHz dihydroxycholan-24-oic acid L286 D03A- Gly (313,513 7a,I2a)-3- QWAVGHFL

-- -- -- -- monoamide amino-7,12- none-NHz dihydroxycholan-24-oic acid L287 DO3A- Gly (3I3,573 7a,12a)-3- QWAVGNMe -- -- -- -- monoamide amino-7,12- noneHis-L-M-NHz dihydroxycholan-24-oic acid L~$g D03A- Gly (313,513 7a,12a)-3- LWAVGSFM

-- -_ -- -- monoamide amino-7,12- none-NHz dihydroxycholan-24-oic acid L289 DO3A- Gly (313,513 7a,12a)-3- HWAVGHL

-- -- -- -- monoamide amino-7,12- noneM-NHZ

dihydroxycholan-24-oic acid L290 D03A- Gly (373,513 7a,12a)-3- LWATGHFM

-- -- -- -- monoamide amino-?,12- none-NHZ

dihydroxycholan-24-oic acid L291 _ D03A- Gly (313,513 7a,12a)-3- QWAVGHFM

- - - - monoamide amino-7,12- none-NHz dihydroxycholan-24-oic acid 'fable ~
-Compounds Containing Linkers of the Invention With Various GRP-R
Targeting Moities CompouHPLC HPLC

nd method'RTZ MS3 IC505 M N O P G*

L292 - - - - D03A- Gly 313,5J3 7a,12a)-3-QRLGQWAVGHL

monoamide amino-7,12- N M-NHZ

dihydroxycholan-24-oic acid L293 - - - - D03A- Gly 313,513 7a,12a)-3-QRYGQWAVGHL

monoamide amino-7,12- N M-NHZ

dihydroxycholan-24-oic acid L294 - - - - DO3A- Gly 313,513 7a,12a)-3-QKY QWAVGHL

monoamide amino-7,12- GN M-NHZ

dihydroxycholan-24-oic acid L295 - - - - Pglu-Q-LysGly 313,513 7a,12a)-3-LG-NQWAVGHL

(D03A- amino-7,12- M-NHZ

monoamide) dihydroxycholan-24-oic acid L304 D03A- Gly 3-amino-3- noneQRYGNQWA

- - - - monoamide deox cholic VGHLM-NHZ
acid L305 D03A- Gly 3-amino-3- noneQKYGNQWA

- - - - monoamide deox cholic VGHLM-NH2 acid L306 D03A- Gly 3-amino-3- noneSee FIG.

monoamide deoxycholic for structure acid o targeting a tide 2D. Other Linking Groups [00167] Other linking goups 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 or optical label while not adversely affecting either the targeting function of the GRP receptor targeting peptide or the metal complexing function of the metal chelator or the detectability of the optical label. 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.
10 [00168] In one embodiment, other linking groups for use within the linker N-O-P include L-glutamine and hydrocarbon chains, or a combination thereof.
[00I 69] 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 or the optical label in the polymeric chain is s 12 atoms.
[00170) 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~-(CHZ)"-R2] wherein n is 0-10, preferably n = 3 to 9, R~ is a group (e.g., H2N-, HS-, -COOH) that can be used as a site for covalently linking the ligand backbone or the prefonned metal chelator or metal complexing backbone or optical label; and R2 is a group that is used for covalent coupling to the N-terminal NH2-group of the GRP receptor targeting peptide (e.g., R2 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 or optical label 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].
[00171) 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:
LP 1: a linker precursor having on at least two locations of the linker the same electrophile El or the same nucleophile Nul;
LP2: a linker precursor having an electrophile E1 and on another location of the linker a different electrophile E2;
LP3: a linker precursor having a nucleophile Nul and on another location of the linker a different nucleophile Nu2; or LP4: a linker precursor having one end functionalized with an electrophile El and the other with a nucleophile Nul.
[00172] The preferred nucleophiles Nul/Nu2 include-OH, -NH, -NR, -SH, -HN-NHa, -RN-NH2, and -RN-NHR', in which R' and R are independently selected from the definitions for R given above, but for R' is not H.
[00173] The preferred electrophiles El/E2 include -COOH, -CH=O (aldehyde), -CR=OR' (ketone), -RN-C=S, -RN-C=O,-S-S-2-pyridyl, -SOZ-Y, -CHaC(=O)Y , and O ~N. O~~N~ O
' ' wherein Y can be selected from the following groups:
N-N=N CI, Br, F
O
N
N
~O_N N\ ~ \ N N ~ \ N~N~ <N N' 'N N ~ \\ _ N N -~ ~N- N
I I
O .r"-0 ,,,~-O
F CI NO~
0 \ F ,~O \ CI J~O \. f"'wO
l F CI
O
O ~ .~, O O~I I \ ~ O \ S S
N~ /
N N / N-N
F
'~,S '~S~S I ~ NC / F ~ F
N / I
"~., S wN ~ ~-.S / F
F
~O
S N
GRP receptor tar etin -~~eptide [00174] 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.
[00175] 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.
[00176] 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:l]) 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., I~d<25nM). 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].
Some useful bombesin analogues are disclosed in U.S. Patent Pub. 2003/0224998, incorporated here in its entirety.
[00177] 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-Alal' for L-Gly"
or D-Trp$ for L-TrpB), 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 TrpB
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.
[00178] 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.
[00179] 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 l, 2, 3, 4 or S 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.
[00180] 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-butyloxycarbonyI 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), orN,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).
[00181] The linker may then be coupled to form a conjugate by reacting the free amino group of the Trp$ residue of the GRP receptor targeting peptide with an appropriate functional group ofthe 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.
[00182] Bombesin (7-14) is subject to proteolytic cleavage in vitro and in vivo, which shortens the half life of the peptide. It is well known in the literature that the amide bond of 10 the backbone of the polypeptide may be substituted and retain activity, while resisting proteolytic cleavage. For example, to reduce or eliminate undesired proteolysis, or other degradation pathways that diminish serum stability, resulting in reduced or abolished bioactivity, or to restrict or increase conformational flexibility, it is common to substitute amide bonds within the backbone of the peptides with functionality that mimics the existing 15 conformation or alters the conformation in the manner desired. Such modifications may produce increased binding affinity or improved pharmacokinetic behavior. It is understood that those knowledgeable in the art of peptide synthesis can make the following amide bond-changes for any amide bond connecting two amino acids (e.g., amide bonds in the targeting moiety, linker, chelator, etc.) with the expectation that the resulting peptides could have the 20 same or improved activity: insertion of alpha-N-methylamides or backbone thioamides, removal of the carbonyl to produce the cognate secondary amines, replacement of one amino acid with an aza-aminoacid to produce semicarbazone derivatives, and use of E-olefins and substituted E-olefins as amide bond surrogates. The hydrolysis can also be prevented by incorporation of a D-amino acid of one of the amino acids of the labile amide bond, or by 25 alpha-methyl aminoacid derivatives. Backbone amide bonds have also been replaced by heterocycles such as oxazoles, pyrrolidinones, imidazoles, as well as ketomethylenes and fluoroolefins.
[00183] Some specific compounds including such amide bond modifications are listed in Table 4a. The abbreviations used in Table 4a for the various amide bond modifications are 30 exemplified below:

~ H~ H O
W N N W Ni N
H p II
O
~ NH ~ NH
Q W
NMeQ W \

H O H
~N~ N~ ~N~ _''J~~.~',N
H S H
NH ~ NH
Q 'I~ [CSNH] W
Q ~ [CH2NH) W \ /

O ... H
WN / _ ~N N
H H O
NH ~ NH
Q'Y [CH=CH] W \ ~' a.-MeQ W \ /

Table -Preferred Amide Bond Modified Analogs _ Comp M_N-O-P BBN
Analogue ound _ monoamide-G-~'neW A V G H L M-NHZ

~Oi Abz4 L402 monoamide-G-'Y[CSW A V G H L M-NHZ

Abz4 NH

L403 monoamide-G-'Y[CHW A V G H L M-NHZ

Abz4 ZNH

L404 monoamide-G-'Y[CHW A V G H L M-NHZ

Abz4 =CH

Table -Preferred Amide Bond Modified Analo s Comp M-N-O-P BBN
Analogue ound monoamide-G-Me W A V G H L M-NHz ~06 Abz4 Q

monoamide-G-Q Nme-W A V G H L M-NHz L4D6 Abz4 monoamide-G-Q ~ A V G H L M-NHZ

L407 CSC]

Abz4 monoamide-G-Q A V G H L M-NHz L408 iyCCHZNH]

Abz4 - - -, monoamide-G-Q A V G H L M-NHz L4D9 (CH=CH]

Abz4 monoamide-G-Q a -MeW A V G H L M-NHz L410 Abz4 .-.

monoamide-G-Q W Nme-A V G H L M-NHz L411 Abz4 412 monoamide-G-Q W ,1,[CSC]V G H L M-NHz L Abz4 D03A- A_ monoamide-G-Q W V G H L M-NHz L413 ~,[CHZ~]

Abz4 monoamide-G-Q W Aib V G H L M-NHz L414 Abz4 mnoamide-G-Q W A V Sar H L M-NHz L415 Abz4 monoamide-G-Q W A V ~~CS~] H L M-NHz L416 Abz4 D03A- _ G

monoamide-G-Q W A V H L M-NHz 'Y[CH=CH]

L417 Abz4 monoamide-G-Q W A V Dala H L M-NHz L418 Abz4 monoamide-G-Q W A V G Nme-His L M-NHZ

L419 Abz4 D03A- g_ monoamide-G-Q W A V G ~,[CSC] L M-NHz L420 Abz4 D03A- H_ monoamide-G-Q W A V G ~,[CH L M-NHz ~]

L,421Abz4 2 monoamide-G-Q W A V ~ G ~ ~y[CH=CH]'L [ M
~z Lt122Abz4 _ Table -Preferred Amide Bond Modified Analo s Comp M_N-O-P BBN
Analogue ound L423 monoamide-G-Q W A V G a. -MeH L M-NHZ

Abz4 monoamide-G-Q W A V G H Lme- M-NHZ

L424 Abz4 L425 'nonoamide-G-Q W A V G H M NHZ

Abz4 MeL

L300 monoamide-G-Q W A V G H F-L NHZ

ABz4 4. Labeling And Administration Of Radiopharmaceutical Compounds [00184] Incorporation of the metal within the radiopharmaceutical conjugates can be achieved by various methods commonly known in the art of coordination chemistry. When the metal is 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), trityI or other thiol protecting group may optionally be used to protect the thiol from oxidation. The thiol protecting groups) 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 I S 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 99mTc04 and colloidal 99mTc02 chromatographically, for example with a C-18 Sep Pak cartridge [Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, Massachusetts 01757] or by HPLC using methods known to those skilled in the art.
[00185] 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 99"'Tc or lg6»gBRe 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 99"'Tc- or ~86~~88Re into ligand frameworks already conjugated to the biomolecule, constructed from a variety of 99mTc(V) and/or 186~~88Re(V) weak chelates (e.g., 99"'Tc- glucoheptonate, citrate, gluconate, etc.) [Eckelman, 1995; Lister-James et al., 1997;
Pollak et al., 1996]. These references are hereby incorporated by reference in their entirety.
5. Diagnostic and Therapeutic Uses [00186) When labeled with diagnostically and/or therapeutically useful metals or optical labels, compounds of the present invention can be used to treat and/or detect any pathology involving overexpression of GRP receptors (or NMB receptors) by procedures established in the art of radiodiagnostics, radiotherapeutics and optical imaging. [See, e.g., Bushbaum, 1995; Fischman et al., 1993; Schubiger et al., 1996; Lowbertz et al., 1994;
I~renning et al., 1994; examples of optical dyes include, but are not limited to those described in WO
98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO
97/18841, WO 96/23524, WO 98/47538, and references cited therein, hereby incorporated by reference in their entirety.]
[00187] GRP-R expression is highly upregulated in a variety of human tumors.
See e.g., WO 99/62563. Thus, compounds of the invention may be widely useful in treating and diagnosing cancers, including prostate cancer (primary and metastatic), breast cancer (primary and metastatic), colon cancer, gastric cancer, pancreatic cancer, non small cell lung cancer, small cell lung cancer, gastrinomas, melanomas, glioblastomas, neuroblastomas, uterus leiomyosarcoma tumors, prostatic intraepithelial neoplasias [PIN], and ovarian cancer.
Additionally, compounds of the invention may be useful to distinguish between conditions in which GRP receptors are upregulated and those in which they are not (e.g.
chronic pancreatitis and ductal pancreatic carcinoma, respectively [00188] The compounds of the invention, which, as explained in more detail in the Examples, show greater specificity and higher uptake in tumors in vivo than compounds 5 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.
[00189] The diagnostic application of these compounds can be as a first line diagnostic 10 screen for the presence of neoplastic cells using scintigraphic, optical, sonoluminescence or photoacoustic 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.
15 [00190] The therapeutic application of these compounds can be defined as an agent that will be used as a first line therapy in the treatment of cancer, as combination therapy where these agents could be utilized in conjunction with adjuvant chemotherapy, and/or as a 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 20 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 accommodate the different metal while maintaining the pharmacology such that the behavior of the diagnostic compound in vivo can be used to predict the behavior of the radiotherapeutic compound.
When utilized in conjunction with adjuvant chemotherapy any suitable chemotherapeutic 25 may be used, including for example, antineoplastic agents, such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, a, L -PAM or phennylalanine mustard), mercaptopurine, mitotane. procarbazine hydrochloride, dactinomycin (actinomycin D), 30 daunorubcin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, arnsacrine (m-AMSA), asparaginase (L-asparaginase) EYwina aparaginase, etoposide -(VP-16), interferon a,-2a, interferon a-2b, teniposide (VM-26), vinblastine sulfate (VLB), and arabinosyl. In certain embodiments, the therapeutic may be monoclonal antibody, such as a monoclonal antibody capable of binding to melanoma antigen.
[00191] A conjugate labeled with a radionuclide metal, such as 99mTc, can be administered to a mammal, including human patients or subjects, by, for example, 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 99"'Tc 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 izz 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.
[00192] 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.
[00193] There are numerous advantages associated with the present invention.
The compounds made in accordance with the present invention form stable, well-defined 99mTc or ~ssnssRe 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 ' S3Sm, g°Y, ' 66I-Io, ~os~~ 199Au~ la9Pm, ' ~~Lu, "'In or other radiometals.
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. Optical Imaginn~, Sonoluminescence, Photoacoustic Imaging and Phototherapy [00194] In accordance with the present invention, a number of optical parameters may be employed to determine the location of a target with in vivo light imaging after injection of the subject with an optically-labeled compound of the invention. Optical parameters to be detected in the preparation of an image may include transmitted radiation, absorption, fluorescent or phosphorescent emission, light reflection, changes in absorbance amplitude or maxima, and elastically scattered radiation. For example, biological tissue is relatively translucent to light in the near infrared (NIR) wavelength range of 650-1000 nm. NIR
radiation can penetrate tissue up to several centimeters, permitting the use of compounds of the present invention to image target-containing tissue in vivo. The use of visible and near-infrared (NIR) light in clinical practice is growing rapidly. Compounds absorbing or emitting in the visible, NIR, or long-wavelength (UV-A, >350 nm) region of the electromagnetic spectrum are potentially useful for optical tomographic imaging, endoscopic visualization, and phototherapy.
[00195] A major advantage of biomedical optics lies in its therapeutic potential.
Phototherapy has been demonstrated to be a safe and effective procedure for the treatment of various surface lesions, both external and internal. Dyes are important to enhance signal detection and/or photosensitizing of tissues in optical imaging and phototherapy. Previous studies have shown that certain dyes can localize in tumors and serve as a powerful probe for the detection and treatment of small cancers (D. A. Bellnier et al., Murine pharmacokinetics and antitumor efficacy of the photodynamic sensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, J. Photochem. Photobiol., 1993, 20, pp. 55-61; G. A.
Wagnieres et al., In vivo fluorescence spectroscopy and imaging for oncological applications, Photochem.
Photobiol., 1998, 68, pp. 603-632; J. S. Reynolds et al., Imaging of spontaneous canine mammary tumors using fluorescent contrast agents, Photochem. Photobiol., 1999, 70, pp. 87-94). All of these listed references are hereby incorporated by reference in their entirety.
However, these dyes do not localize preferentially in malignant tissues.
[00196] In an exemplary embodiment, the compounds of the invention may be conjugated with photolabels, such as optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having absorption or emission maxima in the range of 400-1500 nm. The compounds of the invention may alternatively be derivatized with a bioluminescent molecule. The preferred range of absorption maxima for photolabels is between 600 and 1000 mn to minimize interference with the signal from hemoglobin.
Preferably, photoabsorption labels have large molar absorptivities, e.g., >
105 cm-~M-~, while fluorescent optical dyes will have high quantum yields. Examples of optical dyes include, but are not limited to those described in US 6,641,798, WO 98/18497, WO
98/18496, WO
98/18495, WO 98118498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO
98/47538, and references cited therein, all hereby incorporated by reference in their entirety.
For example, the photolabels may be covalently linked directly to compounds of the invention, such as, for example, compounds comprised of GRP receptor targeting peptides and linkers of the invention. Several dyes that absorb and emit light in the visible and near-infrared region of electromagnetic spectrum are currently being used for various biomedical applications due to their biocompatibility, high molar absorptivity, and/or high fluorescence quantum yields. The high sensitivity of the optical modality in conjunction with dyes as contrast agents parallels that of nuclear medicine, and permits visualization of organs and tissues without the undesirable effect of ionizing radiation. Cyanine dyes with intense absorption and emission in the near-infrared (NIR) region are particularly useful because biological tissues are optically transparent in this region (B. C. Wilson, Optical properties of tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). For example, indocyanine green, which absorbs and emits in the NIR region has been used for monitoring cardiac output, hepatic functions, and liver blood flow (Y-L. He, H. Tanigami, H.
Ueyama, T.
Mashimo, and I. Yoshiya, Measurement of blood volume using indocyanine green measured with pulse-spectrometry: Its reproducibility and reliability. Critical Care Medicine, 1998, 26(8), 1446-1451; J. Caesar, S. Shaldon, L. Chiandussi, et al., The use of Indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function.
Clin. Sci. 1961, 21, 43-57) and its functionalized derivatives have been used to conjugate biomolecules for diagnostic purposes (R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, et al., Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjugate Chemistry, l 993, 4(2), 105-111; Linda G. Lee and Sam L. Woo. "N-Heteroaromatic ion and iminium ion substituted cyanine dyes for use as fluorescent labels", U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al.
"Light imaging contrast agents", WO 98/48846; Jonathan Turner, et al. "Optical diagnostic agents for the diagnosis of neurodegenerative diseases by means of near infra-red radiation", WO 98/22146; Kai Licha, et al. "In-vivo diagnostic process by near infrared radiation", WO
96/17628; Robert A. Snow, et al., Compounds, WO 98/48838, US 6,641,798. All of these listed references are hereby incorporated by reference in their entirety.
[00197] After injection of the optically-labeled compound, the patient is scanned with one or more light sources (e.g., a laser) in the wavelength range appropriate for the photolabel employed in the agent. The light used may be monochromatic or polychromatic and continuous or pulsed. Transmitted, scattered, or reflected light is detected via a photodetector tuned to one or multiple wavelengths to determine the location of target-containing tissue (e.g., tissue containing GRP) in the subject. Changes in the optical parameter may be monitored over time to detect accumulation of the optically-labeled reagent at the target site (e.g., the tumor or other site with GRP receptors). Standard image processing and detecting devices may be used in conjunction with the optical imaging reagents of the present invention.
[00198] The optical imaging reagents described above may also be used for acousto-optical or sonoluminescent imaging performed with optically-labeled imaging agents (see U.S. 5,171,298, WO 98/57666, and references therein). In acousto-optical imaging, ultrasound radiation is applied to the subject and affects the optical parameters of the transmitted, emitted, or reflected light. In sonoluminescent imaging, the applied ultrasound actually generates the light detected. Suitable imaging methods using such techniques are described in WO 98/57666.
[00199] Various imaging techniques and reagents are described in U.S. Patents 6,663,847, 6,656,451, 6,641,798, 6,485,704, 6,423,547, 6,395,257, 6,280,703, 6,277,841, 6,264,920, 6,264,919, 6,228,344, 6,217,848, 6,190,641, 6,183,726, 6,180,087, 6,180,086, 6,180,085, 6,013,243, and published U.S. Patent Applications 2003185756, 20031656432, 2003158127, 2003152577, 2003143159, 2003105300, 2003105299, 2003072763, 2003036538, 2003031627, 2003017164, 2002169107, 2002164287, and 2002156117, all of which are hereby incorporated by reference.

7. Radiotherapy [00200] 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 5 pharmaceutical localizes preferentially at the disease site (in this instance, tumor tissue or other tissue that expresses the pertinent 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. As discussed herein, the invention also encompasses use of radiotherapy in combination with adjuvant chemotherapy 10 (or in combination with any other appropriate therapeutic agent).
[00201 ] The design of a successful radiotherapeutic involves several critical factors:
1. selection of an appropriate targeting group to deliver the radioactivity to the disease site;
2. selection of an appropriate radionuclide that releases sufficient energy 15 to damage that disease site, without substantially damaging adjacent normal tissues; and 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 20 affects the overall biodistribution of the compound to maximize uptake in target tissues and minimize uptake in normal, non-target organs.
[00202] 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.
25 [00203] 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.
M3+ 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, Holmium, 175-Ytterbium, and 1~~-Lutetium. All of these metals (and others in the lanthanide 30 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.
DTPA DOTA

O _.

O - -OOC----~
,O ,r-~
j ~ COO-O~ N N

~

N~
O N N
N ~
N~

- ~ ~ , -OOC-J
~-/ ~--COO-O
.
O O_ O

[00204] 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~ radiometals from their chelate can result in uptake of the radiometal in the liver, bone and spleen [Brechbiel MW, Gansow OA, "Backbone-substituted DTPA ligands for 9°Y radioimmunotherapy", Bioconj. Chem.
1991; 2:187-194; Li, WP, Ma DS, Higginbotham C, Hoffinan T, Ketring AR, Cutler CS, Jurisson, SS, "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.
[00205] For radiotherapy applications any of the chelators for therapeutic radionuclides disclosed herein may be used. However, forms of the DOTA chelate [Tweedle MF, Gaughan GT, Hagan JT, "1-Substituted-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane and analogs." US Patent 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. Compounds L64 and L70 (when labeled with an appropriate therapeutic radionuclide) are particularly preferred for radiotherapy.
[00206] 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. US Patent 4,885,363).
Coupling can also be performed on DOTA-type macrocycles that are modified on the backbone of the polyaza ring.
[00207) The selection of a proper nuclide for use in a particular radiotherapeutic application depends on many factors, including:
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.
b. Energy of the emissions) 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.
c. Specific activit~(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.
[00208] 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.
Approximate range of b-Half -Life Max b- energy Gamma energy particle Isoto a da s Me ke cell diameters i49-Pm 2.21 1.1 286 60 's3-Sm 1.93 0.69 103 30 Approximate range of b-Half -Life Max b- energy Gamma energy particle Isoto a da s MeV ke cell diameters '6~-Dy 3.40 0.40 82.5 15 166-HO 1.12 1.8 80.6 117 '~5-Yb 4.19 0.47 396 17 '~~-Lu 6.71 0.50 208 20 9o-Y 2.67 2.28 - -150 "'-In 2.810 Auger electron 173, 247 < S~m emitter Pm:Promethium, Sm:Samarium, Dy:Dysprosium, Ho:Holmium, Yb:Ytterbium, Lu:Lutetium, Y:Yttrium, In:Indium [00209] 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 CJ, Ehrhardt GJ.; Tyler TT, Jurisson SS, Deutsch E. "Current and potential therapeutic uses of lanthanide radioisotopes." Cancer Biother. Radiopharm.
2000; 15(6):
531-545]. Many ofthese isotopes can be produced in high yield for relatively low cost, and many (e.g. 9o-Y,'ø9-Pm,'~~_Lu) can be produced at close to Garner-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.
[00210] Radiotherapeutic derivatives of the invention containing beta-emitting isotopes of rhenium (' 86-Re and ' $8-Re) are also particularly preferred.
8. Dosages And Additives [00211] Proper dose schedules for the 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. For radiopharmaceuticals, one administers 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.
[00212] For optical imaging compounds, dosages sufficient to achieve the desired image enhancement are known to those skilled in the art and may vary widely depending on the dye or other compound used, the organ or tissue to be imaged, the imaging equipment used, etc.
[00213] The 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.
[00214] A single, or mufti-vial kit that contains all of the components needed to prepare the diagnostic or therapeutic agents of this invention is an integral part of this invention. In the case of radiopharmaceuticals, such kits will often include all necessary ingredients except the radionuclide.
[00215] For example, a single-vial kit for preparing a radiopharmaceutical of the invention 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 a, 13, or y-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.
[00216] A mufti-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 5 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 Preuaration Of Compounds [00217] The compounds of the present invention can be prepared by various methods 10 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 15 producing proteins and long fragments thereof.
[00218] 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-20 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, v the reagents are washed from the support. After addition of the final residue, the peptide is 25 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 [00219] 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 30 "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 oc 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.
[00220] The protected target compound is then subjected to removal of protecting groups, purified and isolated o 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
[00221] 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 A. Definitions of Additional Abbreviations Used [00222] The following common abbreviations are used throughout this specification:
l,l-dimethylethoxycarbonyl (Boc or Boc);
9-fluorenylmethyloxycarbonyl (Fmoc);
allyloxycarbonyl (Aloc);
1-hydroxybenozotriazole (HOBt or HOBT);
N,N'-diisopropylcarbodiimide (DIC);
N-methylpyrrolidinone (NMP);
acetic anhydride (Ac20);
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (iv-Dde);
trifluoroacetic acid (TFA);
Reagent B (TFA:H20:phenolariisopropylsilane, 88:5:5:2);
diisopropylethylamine (DIEA);
O-( 1 H-benzotri azole-I -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU);
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniurn hexafluorphosphate (HATU);
N-hydroxysuccinimide (NHS);
solid phase peptide synthesis (SPPS);
dimethylsulfoxide (DMSO);
dichloromethane (DCM);
dimethylformamide (DMF);
dimethylacetamide (DMA);

1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);
Triisopropylsilane (TIPS);
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA) ( 1 R)-1-[ 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]
ethane-1,2-dicarboxylic acid (CMDOTA);
fetal bovine serum (FBS);
human serum albumin (HSA);
human prostate cancer cell line (PC3);
isobutylchloroformate (IBCF);
tributyl amine (TBA);
radiochemical purity (RCP); and high performance liquid chromatography (HPLC).
B. Materials [00223] The Fmoc-protected amino acids used were purchased from Nova-Biochem (San Diego, CA, USA), Advanced Chem Tech (Louisville, KY., USA), Chem-Impex International (Wood Dale Ill., USA), and Multiple Peptide Systems (San Diego, CA., USA).
Other chemicals, reagents and adsorbents required for the syntheses were procured from Aldrich Chemical Co. (Milwaukee, WI, USA) and VWR Scientific Products (Bridgeport, NJ., USA). Solvents for peptide synthesis were obtained from Pharmco Co.
(Brookfield CT., USA). Columns for HPLC analysis and purification were obtained from Waters Co.
(Milford, MA., USA). Experimental details are given below for those that were not commercially available.
C. Instrumentation for Peptide Synthesis [00224] Peptides were prepared using an Advanced ChemTech 496 SZ 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.
D. Automated synthesis with the Symphony instrument (made by Rainin) [00225] The synthesis was run with Symphony Software (Version 3) supplied by Protein Technologies Inc. Novagel TGR resin, with a substitution of 0.25 mmol/g, was used, and each well contained 0.2 g of the resin (50 ~.mol). The amino acids were dissolved in NMP and the concentration was 0.25M. A 0.25M solution of HBTU and N-Methylmorpholine in DMF was prepared and used for the coupling. All the couplings were carried out for 2.0 h. The cleavage was done outside the machine by transferring the resin to another reaction vessel and using Reagent B as in the manual synthesis E. Instrumentation Employed for Analysis and Purification [00226] Analytical HPLC was performed using a Shimadzu-LC-l0A 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 x 50 mm, 5~ particle, 120 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 ~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. 1H-NMR spectra were obtained on a Varian Innova spectrometer at 500 MHz. 13C-NMR spectra were obtained on the same instrument at 125.73 MHz. Generally the residual 1H absorption, or in the case of 13C-NMR, the'3C absorption ofthe solvent employed, was used as an internal reference;
in other cases tetramethylsilane (8 = 0.00 ppm) was employed. Resonance values are given in 8 units.
Micro analysis data was obtained from Quantitative Technologies Inc., Whitehouse NJ.
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.
F. General Procedures for Peptide Synthesis [00227) Rink Amide-Novagel HL resin (0.6 mmol/g) was used as the solid support.
G. Coupling Procedure [00228] 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 5 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 4h 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.
H. Washing Procedure 10 [00229] 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.
I. Removal of Fmoc Protectin _ Group 15 [00230] The resin bearing 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.
J. Final coupling of ligand (DOTA and CMDOTA) 20 [00231] 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 25 and the resin was washed. The final wash of the resin was conducted with 1.5 mL
dichloromethane (3X).
K. Deprotection and purification of the final peptide [00232] 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 4.5h at ambient 30 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 Et20 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 x 19 mm, 5 micron particle size, 120 pore size) or a Waters-YMC C18 ODS column (250 mm x 30 mm i.d., 10 micron particle size. 120 ~ pore size). The product-containing fractions were collected and analyzed by HPLC. The fractions with >95% purity were pooled and the peptides isolated by lyophilization.
[00233] Conditions for Preparative HPLC (Waters XTerra Column):
Elution rate: 50 mL/min Detection: UV, ~, = 220 nm Eluent A: 0.1 % aq. TFA; Eluent B: Acetonitrile (0.1 % TFA).
Conditions for HPLC Analysis:
Column: Waters XTerra (Waters Co..; 4.6 x 50 mm; MS C18; 5 micron particle, 120 l~ pore).
Elution rate: 3 mL/min; Detection: UV, ~, = 220 nm.
Eluent A:O.1 % aq. TFA; Eluent B: Acetonitrile (0.1 % TFA).
Example I - Figures 1A-B
Synthesis of L62 [00234] Summary: As shown in Figures 1A-B, L62 was prepared using the following steps: Hydrolysis of (313,513)-3-aminocholan-24-oic acid methyl ester A with NaOH gave the corresponding acid B, which was then reacted with Fmoc-CI to give intermediate C. Rink amide resin functionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (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:
A. Rink amide resin functionalised with Bombesin[7-141, (A) [00235] 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 NovaGelTM 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 x 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 x 45 mL).
[00236] This procedure was applied sequentially for the following amino acids:
N a-Fmoc-L-methionine, N oc-Fmoc-L-leucine, N a,-Fmoc-Nm-trityl-L-histidine, N a.-Fmoc-glycine, N a-Fmoc-L-valine, N oc-Fmoc-L-alanine, N a.-Fmoc-N"'-Boc-L-tryptophan.
[00237] In the last coupling reaction N a-Fmoc-N y-trityl-L-glutamine (14.6 g;

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 x 45 mL}, CH2Cl2 (5 x 45 mL) and vacuum dried.
B. Preparation of intermediates B and C (FIG 1A):
Synthesis of (313,513)-3-Aminocholan-24-oic acid (B) [00238] A 1 M solution ofNaOH (16.6 mL; 16.6 mrnol) was added dropwise to a solution of (313,513)-3-aminocholan-24-oic acid methyl ester (5.0 g;
12.8 mmol) in MeOH (65 mL) at 45 °C. After 3 h stirring at 45 °C, the mixture was concentrated to 25 mL and H20 (40 mL) and 1 M HCl (22 mL) were added. The precipitated solid was filtered, washed with H20 (2 x 50 mL) and vacuum dried to give B as a white solid (5.0 g;
13.3 mmol). Yield 80%.
2. Synthesis of (313,513, -L(9H Fluoren-9-ylmethoxy,)aminocholan-24-oic acid C
[00239] 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 (313,513)-3-aminocholan-24-oic acid B (1.0 g; 2.66 mmol) in 10% aq.

Na2CO3 (16 mL) and 1,4-dioxane (9 mL) stirred at 0 °C. After 6 h stirring at room temperature H20 (90 mL) was added, the aqueous phase washed with Et20 (2 x 90 mL) and then 2 M HCI (15 mL) was added (final pH: 1.5). The aqueous phase was extracted with EtOAc (2 x 100 mL), the organic phase dried over Na2S04 and evaporated. The crude was purified by flash chromatography to give C as a white solid (1.2 g; 2.0 mmol). Yield 69%.
C. Synthesis ofL62 (N [(313,513)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino] acetyl]amino]-cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide) (FIG 1B):
[00240] 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 shaken for 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). (313,SJ3)-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 x 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 x 7 mL). N a-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 x 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 x 7 mL) followed by addition of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (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 x 7 mL), CH2C12 (5 x 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 Et2O (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et2O (3 x 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 x 10-3 mmol) as a white solid. Yield 4.5%.
Example II - Figures 2A-F
Synthesis of L70, L73, L74, L115 and L116 [00241] Summary: The products were obtained by coupling of the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14] [SEQ ID NO:l]) (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%.
A. Synthesis of L70 (FIG. 2A~:
[00242] 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 x 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 x 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 fox 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL). Fmoc-glycine (0.36 g; 1.2 mmol) 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 x 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) 5 was added and the mixture shaken for 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (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 10 was shaken for 24 h at room temperature, the solution was emptied and the resin washed with DMA (5 x 7 mL), CH2C12 (5 x 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 Et20 (5 mL). The precipitate was 15 collected by centrifugation and washed with Et2O (5 x 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%.
B. Synthesis of L73, L115 and L116 (FIGS. 2B - 2E):
20 [00243] 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 x 7 mL). Fmoc-linker-OH (1.2 mmol), HOBt (0.18 g; 1.2 mmol), 25 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 x 7 mL). The resin was shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50%
morphoIine in DMA (7 mL) was added and the mixture was shaken for 20 30 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (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 x 7 mL), CH2Cl2 (5 x 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 Et20 (5 mL). The precipitate was collected by centrifugation and washed with Et20 (5 x 5 mL), then analysed by HPLC and purified by preparative HPLC. The fractions containing the product were lyophilised.
C. Synthesis of L74 (FIG. 2F):
[00244 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 x 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 x 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 x 7 mL). Fmoc-glycine (0.36 g; 1.2 mmol), HOBt (0.18 g; 1.2 mrnol), 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 x 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 x 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 x 7 mL), CH2C12 (5 x 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 Et20 (5 mL). The precipitate was collected by centrifugation and washed with Et20 (5 x 5 mL), then analysed by HPLC and purified by HPPLC. 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 - Figures 3A-E
Synthesis of L67 [00245] Summary: Hydrolysis of (313,513)-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-NH2 (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%.
A. Synthesis (313,513)-3-Amino-12-oxocholan-24-oic acid (B) (FIG 3A) [00246] A 1 M solution of NaOH (6.6 mL; 6.6 mmol) was added dropwise to a solution of (313,513)-3-amino-12-oxocholan-24-oic acid methyl ester A (2.1 g;
5.1 mmol) in MeOH (15 mL) at 45 °C. After 3 h stirring at 45 °C, the mixture was concentrated to 25 mL then H20 (25 mL) and 1 M HCI (8 mL) were added. The precipitated solid was filtered, washed with H20 (2 x 30 mL) and vacuum dried to give B as a white solid (1.7 g; 4.4 mmol). Yield 88%.
B. Synthesis of (313,5131-3-[[(9H Fluoren-9-ylmethoxy, amino]'acetyl]'amino-12-oxocholan-24-oic acid ~C) (FIG 3A) [00247] Tributylamine (0.7 mL; 3.1 mmol) was added dropwise to a solution ofN
a-Fmoc-glycine (0.9 g; 3.1 mmol) in THF (25 mL) stirred at 0 °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 (3J3,513)-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 H20 (50 mL) and 1 N HC1 (10 mL) were added (final pH: 1.5). The precipitated solid was filtered, washed with H20 (2 x 50 mL), vacuum dried and purified by flash chromatography to give C as a white solid (1.l g; 1.7 mmol). Yield 66%.
C. Synthesis of L67 (N ((313 513)-3-[~[f f4 7 10-Tris(carbox methyl)-1 4 7 10-tetraazacyclododec-1-~)acetyllamino) acet~]'aminol-12 24-dioxocholan-24-yll-L-~lutaminyl-L-try .~~tophyl-L-alanyl-L-val~l-~lycyl-L-hi stidyl-L-leuc.
methioninamide) (FIG 3B and FIG 3E).
[00248) 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 x 7 mL). (313,513)-3-[[(9H Fluoren-9-ylmethoxy)amino)acetyl)amino)-12-oxocholan-24-oic acid C (0.80 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 x 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (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 x 7 mL), CH2Cl2 (5 x 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 Et20 (20 mL).
Example IV - Figures 4A-H
Synthesis of L63 and L64 [00249) Summary: Hydrolysis of (313,513,7a,12a)-3-amino-7,12-dihydroxychoIan-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-NH2 (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 Za, already available, to give L63. Overall yields: 9 and 4%, respectively.
A. Synthesis of (313,513,7a,12a)-3-Amino-7,12-dihydroxycholan-24-oic acid, (2b) (FIG. 4A1 [00250] A 1 M solution of NaOH (130 mL; 0.13 mol) was added dropwise to a solution of (3>3,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid methyl ester 1b (42.1 g; 0.10 mol) in MeOH (300 mL) heated at 45 °C. After 3 h stirring at 45°C, the mixture was concentrated to 150 mL and H20 (350 mL) was added. After extraction with CH2C12 (2 x 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 H20 (2 x 100 mL) and vacuum dried to give 2b as a white solid (34.8 g; 0.08 mol). Yield 80%.
B. Synthesis of (313,513,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-h droxycholan-24-oic acid, (3a~(FIG. 4A) [00251] Tributylamine (4.8 mL; 20.2 mmol) was added dropwise to a solution ofN-a-Fmoc-glycine (6.0 g; 20.2 mmol) in THF (120 mL) stirred at 0°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 (313,513,12a)-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 H20 (250 mL) and 1 N HCI (40 mL) were added (final pH: 1.5). The precipitated solid was filtered, washed with H20 (2 x 100 mL), vacuum dried and purified by flash chromatography to give 3a as a white solid (3.5 g; 5.2 mmol). Yield 31 %.

C. Synthesis of (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7 12-dihydroxycholan-24-oic acid (3b) (FIG
[00252) Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a solution of N-a-5 Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL) stirred at 0°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 (3J3,513,7a,12a)-3-amino-7,12-dihydroxycholan-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 I 0 allowed to warm up and after 6 h the solution was concentrated to 120 mL, then H20 (180 mL) and 1 N HCl (30 mL) were added (final pH: 1.5). The precipitated solid was filtered, washed with H20 (2 x 100 mL), vacuum dried and purified by flash chromatography to give 3a as a white solid (1.9 g; 2.8 mmol). Yield 25%.
15 [00253] In an alternative method, (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) can be prepared as follows:
[00254] 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 20 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 x 20 mL). Fmoc-Gly-succinimidyl ester was then redissolved in dry 25 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):
(Rf:
0.50, silica gel, CHZCI2/CH30H, 9:1) (eluant: CH2Cla/CH30H (9:1)) to give 30 (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino)acetyl]amino-7,12-dihydroxycholan-24-oic acid as a colorless solid. Yield: 7.46 g (85 %).
D. Synthesis ofL63 (N [(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10 tetraazacyclododec-1-yl] acetyl]amino]acetyl]amino]-12-hydroxy-24 oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide~FIG. 4B) [00255) Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with SO% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh SO% 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 x 7 mL). (313,5J3,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic acid 3a (0.82 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 x 7 mL).
The resin was then shaken with SO% 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 (S x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (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 x 7 mL), CH2C12 (5 x 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 Et20 (5 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et20 (5 x S 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%.
E. Synthesis ofL64 (N [(313,513,7a,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]acetyl]amino]-7,12-3 0 dihydroxy-24-oxo chol an-24-yl]-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl glycyl-L-histidyl-L-leucyl-L-methioninamide) (FIG 4C) [00256] 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 x 7 mL). (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]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 x 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (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 x 7 mL), CH2Cl2 (S x 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 Et20 (5 mL). The precipitate was collected by centrifugation and washed with Et20 (5 x 5 mL).
Then it was dissolved in H20 (20 mL), and Na2C03 (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 - Figures SA-E
Synthesis of L71 and L72 [00257] Summary: The products were obtained in two steps. The first step was the solid phase synthesis of the octapeptide GIn-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]
[SEQ ID NO:1]) (with appropriate side chain protecting groups) on the Rink amide resin discussed supra. 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%.

A. Bombesin [7-14]' functionalisation and cleavage procedure (FIGS SA and 5D) [0025] 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 x 7 mL). The Fmoc-linker-OH (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 x 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI 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 x 7 mL), CH2Cl2 (5 x 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 x 5 mL), then analyzed by analytical HPLC and purified by preparative HPLC. The fractions containing the product were lyophilized.
B. Products [00259] 1. L71 (4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-y1] acetyl] amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide) The product was obtained as a white fluffy solid (7.3 mg; 0.005 mmol). Yield 7.5%.
[00260] 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) The product was obtained as a white fluffy solid (7.0 mg; 0.005 mmol). Yield 4.8%.
S C. Ttrans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl-aminoJmethyl]''cyclohexanecarboxylic acid (D) (FIG.
SE
[00261] A solution ofN (9-fluorenylmethoxycarbonyloxy)succinimide (4.4 g; 14.0 mmol) in 1,4-dioxane (40 mL) was added dropwise to a solution of tr-ans-4-(aminomethyl)cyclohexanecarboxylic acid (2.0 g; 12.7 mmol) in 10%
Na2C03 (30 mL) cooled to 0 °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 - Figures 6A-F
Synthesis of L75 and L76 [00262] 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 N0:1]) (A) 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).
A. 2-[(1,3-Dihydro-1,3-dioxo-2H isoindol-2-~lmethy~'benzoic acid's (FIG.

[00263] 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).
B. 2-(Aminomethyl)benzoic acid, (D~(FIG. 6A~
[00264] A 40% solution ofmethylamine (6.14 mL; 7.lmmol) 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 °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 S 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 %.
C. 2-[[[9H Fluoren-9-ylmethoxy carbons amino]meth~lbenzoic acid (E) (FIG.
10 [00265] The product was synthesized following the procedure reported in the literature (Sun, J-H.; Deneker, W. F. Synth. Commun. 1998, 28, 4525-4530).
D. 4-(Aminomethyl)-3-nitrobenzoic acid, (G) (FIG. 6B) [00266] 4-(Bromomethyl)-3-nitrobenzoic acid (3.2 g; 12.3 mmol) was dissolved in 8%
NH3 in EtOH (300 mL) and the resulting solution was stirred at room 15 temperature. After 22 h the solution was evaporated and the residue suspended in H20 (70 mL). The suspension was stirred for 15 min and filtered. The collected solid was suspended in H20 (40 mL) and dissolved by the addition of few drops of 25% aq. NH40H (pH 12), then the pH of the solution was adjusted to 6 by addition of 6 N HCl. The precipitated solid was 20 filtered, and washed sequentially with MeOH (3 x 5 mL), and Et20 (10 mL) and was vacuum dried (1.3 kPa; P205) to give 4-(aminomethyl)-3-nitrobenzoic acid as a pale brown solid (1.65 g; 8.4 mmol). Yield 68%.
E. 4-[[[9H Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid, (H) (FIG. 6B) 25 [00267] 4-(Aminomethyl)-3-nitrobenzoic acid G (0.8 g; 4 mmol) was dissolved in 10% aq. Na2C03 (25 mL) and 1,4-dioxane (10 mL) and the solution was cooled to 0 °C. A solution of 9-fluorenylmethyl chloroformate (Fmoc-Cl) (1.06 g; 4 mmol) in 1,4-dioxane (10 mL) was added dropwise for 20 min.
After 2 h at 0-5 °C and 1 h at 10 °C the reaction mixture was filtered and the 30 solution was acidified to pH 5 by addition of 1 N HCI. The precipitate was filtered, washed with H20 (2 x 2 mL) dried under vacuum (1.3 kPa; P205) 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%.
F. L75 (N [2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino] methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-g-l~yl-L-histid.1-~ucyl-L-methioninamide) (FIG. 6C1 [00268] 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 x 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 x 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (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 x 7 mL), CH2C12 (5 x 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 Et20 (20 mL) gave a precipitate. The resulting precipitate was collected by centrifugation and was washed with Et20 (3 x 20 mL) to give L75 (190 mg; 0.13 mmol) as a white solid. Yield 44%.

G. L76 (N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino] methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-~l~yl-L-histidyl-L-leucyl-L-methioninamide) (FIG 6D~
[00269] 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 x 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 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for I 0 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 x 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 x 7 mL), CH2Cl2 (5 x 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 Et20 (20 mL). The precipitate was collected by centrifugation and was washed with Et20 (3 x 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 x 10-3 mmol) as a white solid. Yield 9%.
Example VII - Figures 7A-C
Synthesis of L124 [00270] Summary: 4-Cyanophenol A was reacted with ethyl bromoacetate and K2C03 in acetone to give the intermediate B, which was hydrolysed with NaOH to the corresponding acid C. Successive hydrogenation of C with H2 and Pt02 at 355 kPa in EtOH/CHC13 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-NH2 (BBN[7-14] [SEQ ID NO:l]) 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%
A. Synthesis of (4-C~anophenoxy)acetic acid ethyl ester, (B) (FIG. 7A) [00271 ] 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).
B. Synthesis of (4-CyanophenoxX)acetic acid, (C) (FIG. 7A) [00272] 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 HCI (7.6 mL; 7.6 mmol) and evaporated. The residue was taken up with water (20 mL) and extracted with CHC13 (2 x 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%.
C. Synthesis of [4-[[[9H Fluoren-9-ylmethoxX carbon~lamino]meth~lphenoxy]'acetic acid, (E (FIG. 7A) [00273] Pt02 (150 mg) was added to a solution of (4-cyanophenoxy)acetic acid C
(1.05 g; 5.9 mmol) in EtOH (147 mL) and CHCl3 (3 mL). The suspension was stirred 30 h under a hydrogen atmosphere (355 kPa; 20 °C). The mixture was filtered through a Celite~ 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 H20 (10 mL), MeCN (2 mL) and Et3N (0.6 mL) at 0 °C, then a solution ofN (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%.
D. Synthesis ofL124 (N [[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl] amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide) (FIG. 7B) [00274] Resin A (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 x 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 ~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 x 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (750 mg; 1.19 mmol), HOBt (182 mg; 1.19 mmol), DIEA (404 ~.L; 2.36 mmol), DIC (185 ~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 x 7 mL), CH2Cl2 (5 x 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 Et20 (5 mL). The precipitate was collected by centrifugation and washed with Et20 (5 x 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 L124 (FIG. 7C) as a white solid (15 mg; 0.01 mmol). Yield 7.9%.

Example VIII - Figures 8A-C
Synthesis of L125 [00275] Summary: 4-(Bromomethyl)-3-methoxybenzoic acid methyl ester A was reacted with NaN3 in DMF to give the intermediate azide B, which was then reduced with Ph3P and 5 H20 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-NH2 (BBN[7-14] [SEQ ID NO:l]) (A) 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%.
10 A. Synthesis of 4-(Azidometh~)-3-methoxybenzoic acid methyl ester., (B~FIG.

[00276] A solution of 4-(bromomethyl)-3-methoxybenzoic acid methyl ester (8 g;

mmol) and NaN3(2 g; 31 mmol) in DMF (90 mL) was stirred overnight at room temperature. The volatiles were removed under vacuum and the crude 15 product was dissolved in EtOAc (50 mL). The solution was washed with water (2 x 50 mL) and dried. The volatiles were evaporated to provide 4-(azidomethyl)-3-methoxybenzoic acid methyl ester (6.68 g; 30 mmol). Yield 97%.
B. 4-(Aminometh~)-3-methoxybenzoic acid methyl ester, (C) (FIG. 8A) 20 [00277] 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 25 consumed and H20 (10 mL) was added. After 4 h the white solid disappeared.
The mixture was heated at 45 °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%.

C. 4-[[[9H Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acid.~E) (FIG. 8A1 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 °C. After stirring 8 h at 45 °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°C for 4 h.
The solution was concentrated, acidified with 1 N HCl (8 mL; 8 mmol), extracted with EtOAc (2 x 10 mL) then the aqueous layer was concentrated to 15 mL.
This solution (pH 4.5) was cooled at 0°C and Et3N (936 ~.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).
[00278] 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%.
D. L125 (N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino] methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-~lycyl-L-histidyl-L-leucyl-L-methioninamide) (FIG. 8B) [00279] Resin A (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 x 7 mL). 4-[[[9H Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acid E (398 mg; 0.98 mmol), HOBt (151 mg; 0.98 mmol), DIC (154 pL; 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 x 7 mL). The resin was then shaken with 50% morpholine in DMA (6 mL) for 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (618 mg; 0.98 mmol), HOBt (151 mg; 0.98 mmol), DIC (154 ~L; 0.98 mmol), DIEA (333 ~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 x 7 mL), CH2Cl2 (5 x 7 mL) and 10 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 Et20 (5 mL). The resulting precipitate was collected by centrifugation, was washed with Et20 (5 x 5 mL), was analysed by HPLC and purified by preparative HPLC. The fractions containing the product were lyophilised to give L125 (FIG. 8C) as a white solid (15.8 mg; 0.011 mmol). Yield 4.4%.
Example IX - Figures 9A - 9D
Synthesis of L146, L233, L234, and L235 [00280] Summary: The products were obtained in several steps starting from the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2(BBN[7-14]) (A) on the Rink amide resin. After final cleavage and deprotection with Reagent B the crudes were purified by preparative HPLC to give L146, L233, L234 and L235. Overall yields: 10%, 11 %, 4.5%, 5.7% respectively.
A. 3-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminobenzoic acid, B.(FIG. 9A) [00281] A solution of 3-aminobenzoic acid (0.5 g; 3.8 mmol) and N
ethyldiisopropylamine (DIEA) (0.64 mL; 3.8 mmol) in THF (20 mL) was added dropwise to a solution of Fmoc-glycine chloride (1.2 g; 4.0 mmol) (3) in THF (10 mL) and CHZCI2 (10 mL). After 24 h stirring at room temperature 1 M HCl (50 mL) was added (final pH: 1.5). The precipitate was filtered, washed with HZO (2 x 100 mL), vacuum dried and crystallised from CHC13/CH30H (I:I) to give B as a white solid (0.7 g; 1.6 mmol). Yield 43%.

B. N [3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-~lycyl-L-histi~l-L-leucyl-L-methioninamide L233 (FIG.

[00282] 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 emptied and fresh 50% morphoIine in DMA (7 mL) was added.
[00283] The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 3-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminobenzoic acid, B (0.50 g;1.2 mmol), HOBt (0.I 8 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 6 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL).
DOTA tri-t-butyl ester adduct with NaCl2 (0.79 g; 1.2 mmol) (5), 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, emptied and the resin washed with DMA (5 x 7 mL), CH2Cl2 (5 x 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 after treatment with Et20 (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with EtaO (3 x 20 mL) to give a solid (152 mg) which was analysed by HPLC. An amount of crude (50 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L233 (17.0 mg; 11.3 x 10-3 mmol) as a white solid. Yield 11%.
C. N [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-3 0 1-yl] acetyl] amino] acetyl] amino]phenyl acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide. L146 (FIG. 9D) [00284] 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 filtered and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was filtered and the resin washed with DMA (5 x 7 mL). Fmoc-4-aminophenylacetic acid (0.45 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 6 h at room temperature, filtered and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution filtered, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was filtered and the resin washed with DMA (5 x 7 mL).
Fmoc-glycine (0.36 g; 1.2 mmol), 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, filtered and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution filtered, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was filtered and the resin washed with DMA (5 x 7 mL).
DOTA tri-t-butyl ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt (0.18 g;
I .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, filtered and the resin washed with DMA (5 x 7 mL), CH2Cl2 (5 x 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 after treatment with Et2O (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et20 (3 x 20 mL) to give a solid (203 mg) which was analysed by HPLC.
An amount of crude (50 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L146 (11.2 mg; 7.4 x 10-3 mmol) as a white solid. Yield 10%.
D. 6-[[[(9FI Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminonaphthoic acid, C (FIG. 9B) [00285] A solution of 6-aminonaphthoic acid (500 mg; 2.41 mmol); and DIEA (410 ~L 2.41 mmol) in THF (20 mL) was added dropwise to a solution of Fmoc glycine chloride (760 mg; 2.41 mmol) in CH2Clz/THF 1:1 (10 mL) and stirred at room temperature. After 24 h the solvent was evaporated under vacuum.
The residue was taken up with 0.5 N HCI (50 mL) and stirred for 1 h. The white solid precipitated was filtered and dried. The white solid was suspended in methanol (30 mL) and boiled for 5 min, then was filtered to give product C
5 (690 mg; 1.48 mmol). Yield 62%.
E. N [6-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl] amino]naphthoyl]-L-glutaminyl-L-tryptophyl- L-alanyl-L-val I-y~l~yl-L-histidyl-L-leucyl-L-methioninamide, L234 10 [00286] Resin A (500 mg; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50 % morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 6-[[[(9H Fluoren-9-15 ylmethoxy)carbonyl]amino]acetyl]aminonaphthoic acid C (560 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 ~.L; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 6 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (6 L) for 10 min, the solution emptied, 20 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 x 7 mL). DOTA tri-t-butyl ester adduct with NaCI (757 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 p.L; 1.2 mmol), and DIEA (537 ~.L; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken in a flask, 25 emptied and the resin washed with DMA (2 x 7 mL), CHZCI2 (5 x 7 mL) and vacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resin was filtrated and the solution was evaporated under reduced pressure to afford an oil crude that after treatment with Et20 (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with 30 Et20 (3 x 20 mL) to give a solid (144 mg) which was analysed by HPLC. An amount of crude (54 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L234 (8 mg; 5.1 x 10-3 mmol) as a white solid. Yield 4.5%.

F. 4-[[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]methylamino]benzoic acid, D
(FIG. 9C) [00287] A solution of 4-N methylaminonaphthoic acid (500 mg; 3.3 mmol) and DIEA
(562 ~L 3.3 mmol) in THF (20 mL) was added to a solution of Fmoc-glycine chloride (1.04 g; 3.3 mmol) in CH2C12/THF 1:1 (10 mL) and stirred at room temperature. After 24 h the solvent was evaporated under vacuum. The residue was taken up with 0.5 N HCl (30 mL) and was stirred for 3 h at 0 °C.
The white solid precipitated was filtered and dried. The crude was purified by flash chromatography to give Compound D (350 mg; 0.81 mmol). Yield 25%.
G. N [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl]
methylamino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, L235 (FIG.
9D) [00288] Resin A (500 mg; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50 % morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 4-[[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]-N methyl]amino-benzoic acid D (510 mg;
1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 ~L; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 6 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7mL) for 10 min, the solution 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 x 7 mL). DOTA tri-t-butyl ester adduct with NaCI (757 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 ~.L; 1.2 mmol), and DIEA (537 ~L; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken in a flask, emptied and the resin washed with DMA (2 x 7 mL), CH2CI2 (5 x 7 mL) and vacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resin was filtrated and the solution was evaporated under reduced pressure to afford an oil crude that after treatment with Et20 (20 mL) gave a precipitate.

[00289] The precipitate was collected by centrifugation and washed with Et20 (3 x 20 mL) to give a solid (126 mg) which was analysed by HPLC. An amount of crude (53 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L235 (11 mg; 7.2 x 10-3 mmol) as a white solid. Yield 5.7%.
EXAMPLE X - Figures l0A-B
Synthesis of L237 [00290] Summary: 1-Formyl-1,4,7,10-tetraazacyclododecane (A) was selectively protected with benzyl chloroformate at pH 3 to give B, which was alkylated with t-butyl bromoacetate and deformylated with hydroxylamine hydrochloride to give D.
Reaction with P(OtBu)3 and paraformaldehyde gave E, which was deprotected by hydrogenation and alkylated with benzyl bromoacetate to give G, which was finally hydrogenated to H. Rink amide resin functionalized with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NHZ
(BBN[7-14]) (A) was sequentially reacted with Fmoc-4-aminobenzoic acid, Fmoc-glycine and H. After cleavage and deprotection with Reagent B the crude was purified by preparative HPLC to give L237. Overall yield 0.21 %.
A. 7-Formyl-1,4,7,10-tetraazacyclododecane-1-carboxylic acid phenylmethyl ester dihydrochloride, B (FIG. 10A) [00291] 1-Formyl-1,4,7,10-tetraazacyclododecane A (14 g; 69.9 mmol) was dissolved in H20 (100 mL) and 12 N HGl (11 mL) was added until pH 3 then 1,4-dioxane (220 mL) was added. A solution of benzyl chloroformate (13.8 g; 77 mmol) in 1,4-dioxane (15 mL) was slowly added dropwise in 3.5 h, constantly maintaining the reaction mixture at pH 3 by continuous addition of 2 N NaOH
(68.4 mL) with a pHstat apparatus. At the end of the addition the reaction was stirred for 1 h then washed with n-hexane (4 x 100 mL) and'Pr20 (4 x 100 mL). The aqueous phase was brought to pH 13 by addition of 10 N NaOH
(6.1 mL) and extracted with CHC13 (4 x 100 mL). The organic phase was washed with brine (100 mL), dried (Na2S04), filtered and evaporated. The oily residue was dissolved in acetone (200 mL) and 6 N HCl (26 mL) was added. The solid precipitated was filtered, washed with acetone (2 x 50 mL) and dried under vacuum to give compound B (23.6 g; 58 mmol) as a white crystalline solid. Yield 83%.

B. 4-(Phenylmethoxy)carbonyl-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid bis(1,1-dimethylethyl) ester D (FIG 10A) [00292] A solution of B (14.4 g; 35.3 mmol) in H20 (450 mL) and 1 N NaOH (74 mL;
74 mmol) was stirred for 20 min then extracted with CHCl3 (4 x 200 mL).
The organic layer was evaporated to obtain an oily residue (12.3 g) which was dissolved in CH3CN (180 mL) and N ethyldiisopropylamine (DIEA) (15 mL;
88.25 mmol). A solution of t-butyl bromoacetate (16.8 g; 86.1 mmol) in CH3CN (15 mL) was added dropwise to the previous solution in 2.5 h. After 20 h at room temperature the solvent was evaporated and the oily residue was dissolved in CHCl3 (150 mL) and washed with H20 (5 x 100 mL). The organic layer was dried (Na2S04), filtered and evaporated to dryness to give C
as a yellow oil. Crude C (22 g) was dissolved in EtOH (250 mL), NH20H~HC1 (2.93g; 42.2 mmol) was added and the solution heated to reflux.
After 48 h the solvent was evaporated and the residue dissolved in CHZCh (250 mL), washed with HBO (3 x 250 mL) then with brine (3 x 250 mL). The organic layer was dried (Na2S04), filtered and evaporated. The oily residue (18.85 g) was purified by flash chromatography. The fractions containing the product were collected and evaporated to obtain a glassy white solid (17.62 g) which was dissolved in H20 (600 mL) and 1 N NaOH (90 mL; 90 mmol) and extracted with CHC13 (3 x 250 ml). The organic layer was dried (Na2S04) and evaporated to dryness to give D (16.6 g; 31 mmol) as an oil. Yield 88%.
C. 4-(Phenylmethoxy)carbonyl-10-[[bis( 1,1-dimethylethoxy)phosphinyl]methyl)-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid bis(1,1-dimethylethyl) ester. E (FIG. 10A) [00293] A mixture of Compound D (13.87 g; 26 mmol), P(OtBu)3 (7.6 g; 28.6 mmol) (10) and paraformaldeyde (0.9 g; 30 mmol) was heated at 60 °C. After 16 h more P(OtBu)3 (1 g; 3.76 mmol) and paraformaldeyde (0.1 g; 3.33 mmol) were added. The reaction was heated at 60 °C for another 20 h then at 80 °C
for 8 h under vacuum to eliminate the volatile impurities. The crude was purified by flash chromatography to give E (9.33 g; 8 mmol) as an oil. Yield 31 %.

D. 7-[ [Bis( 1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid 1-phenylmethyl 4,10-bis( 1,1-dimeth l~yl) ester G (FIG 10A) [00294] To the solution of E (6.5 g; 5.53 mmol) in GH3OH (160 mL) 5% Pd/C (1 g;
0.52 mmol) was added and the mixture was stirred under hydrogen atmosphere at room temperature. After 4 h (consumed HZ 165 mL;6.7 mmol) the mixture was filtered through a Millipore~ filter (FT 0.45 ~.m) and the solution evaporated under reduced pressure. The crude (5.9 g) was purified by flash chromatography to give F (4.2 g) as an oil. Benzyl bromoacetate (1.9 g;
8.3 mmol) dissolved in CH3CN (8 mL) was added dropwise in 1 h to a solution of F (4.2 g) in CH3CN (40 mL) and DIEA (1.5 mL; 8.72 mmol).
After 36 h at room temperature the solvent was evaporated and the residue (5.76 g) dissolved in CHC13 (100 mL), washed with H20 (2 x 100 mL) then with brine (2 x 70 mL). The organic layer was dried (Na2S04), filtered and evaporated. The crude (S.5 g) was purified twice by flash chromatography, the fractions were collected and evaporated to dryness to afford G (1.12 g;
1.48 mmol) as an oil. Yield 27%.
E. 7-[ [Bis( 1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid 4,10-bis(1,1-dimethylethyl) ester, H (FIG. 10A) [00295] 5% Pd/C (0.2 g; 0.087 mmol) was added to a solution of G (1.12 g; 1.48 mmol) in CH30H (27 mL) and the mixture was stirred under hydrogen atmosphere at room temperature. After 2 h (consumed H2 35 mL; 1.43 mmol) the mixture was ftltered through a Millipore~ filter (FT 0.45 Vim) and the solution evaporated to dryness to give H (0.94 g; I .41 mmol) as a pale yellow oil. Yield 97%.
F. N [4-[[[[[4,10-Bis(carboxymethyl)-7-(dihydroxyphosphinyl)methyl-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl] amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucil-L-methioninamide, L237 (FIG. 10B) [00296] Resin A (330 mg; 0.20 mmol) (17) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (5 mL) for 10 min, the solution emptied and fresh 50 % morpholine in DMA (5 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 5 mL). Fmoc-4-aminobenzoic acid (290 mg;
0.80 mmol), HOBt (120 mg; 0.80 mmol), DIC (130 ~L; 0.80 mmol) and DMA
(5 mL) were added to the resin, the mixture shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 5 mL). The resin was then shaken with 50% morpholine in DMA (5 mL) for I 0 min, the solution emptied, fresh 50% morpholine in DMA (5 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 5 mL). Fmoc-glycine (240 mg; 0.8 mmol), HATU
(310 mg; 0.8 mmol) and DIEA (260 ~.L; 1.6 mmol) were stirred for 15 min in DMA (5 mL) then the solution was added to the resin, the mixture shaken for 2 h at room temperature, emptied and the resin washed with DMA (5 x 5 mL).
The resin was then shaken with 50% morpholine in DMA (5 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (5 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 5 mL). H (532 mg; 0.80 mmol), HOBt (120 mg;
0.80 mmol), DIC (130 ~.L; 0.80 mmol), and DIEA (260 ~.L; 1.6 mmol) and DMA (5 mL) were added to the resin. The mixture was shaken in a flask for 40 h at room temperature, emptied and the resin washed with DMA (5 x 5 mL), CH2C12 (5 x 5 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 Et20 (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et20 (3 x 20 mL) to give a solid (90 mg) which was analysed by HPLC. An amount of crude (50 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L237 (6 mg; 3.9 x 10-3 mmol) as a white solid. Yield 3.5%.
EXAMPLE XI - Figures 11A-B
Synthesis of L238 and L239 [00297] Summary: The products were obtained in several steps starting from the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]) (A) on the Rink amide resin. After cleavage and deprotection with Reagent B the crude was purified by preparative HPLC to give L238 and L239. Overall yields: 14 and 9%, respectively.

A. N,N Dimethylglycyl-L-Beryl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, L238 (FIG.11A) [00298] 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 emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 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, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL).
Fmoc-glycine (0.36 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N
ethyldiisopropylamine (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, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N a-Fmoc-S-acetamidomethyl-L-cysteine (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 shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N a-Fmoc-O-t-butyl-L-serine (0.46 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, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min.
[00299] The solution was emptied and the resin washed with DMA (5 x 7 mL). N,N
Dimethylglycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N
ethyldiisopropylamine (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 was shaken for 2 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL), CH2Cl2 (5 x 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 after treatment with Et2O (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et20 (3 x 20 mL) to give a solid (169 mg) which was analysed by HPLC. An amount of crude (60 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L238 (22.0 mg; 0.015 mmol) as a white solid. Yield 14%.
B. N,N Dimethylglycyl-L-Beryl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-(313,513,7a, l 2a)-3-amino-7,12-dihydroxy-24-oxocholan-24-yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-Ieucyl-L-methioninamide. L239 (FIG. llBl [00300] 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 emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). (313,S13,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid B (0.82 g;
1.2 mmol) (7), 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 24 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N a-Fmoc-S-acetamidomethyl-L-cysteine (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 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N a-Fmoc-O-t-butyl-L-serine (0.46 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, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N,N Dimethylglycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution was added to the resin.
[00301] 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 emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid B (0.82 g;
1.2 mmol) HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mrnol) and DMA (7 mL) were added to the resin, the mixture shaken for 24 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N a-Fmoc-S-acetamidomethyl-L-cysteine (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 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N a-Fmoc-O-t-butyl-L-serine (0.46 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, emptied and the resin washed with DMA (5 x 7 rnL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). N,N Dimethylglycine (0.12 g; 1.2 mmol), HATU
(0.46 g; 1.2 mmol) and N ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution was added to the resin.
EXAMPLE XII - Figures 12A-F
Synthesis of L240, L241, L242 [00302] Summary: The products were obtained in several steps starting from the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]) (A) on the Rink amide resin. After cleavage and deprotection with Reagent B the crudes were purified by preparative HPLC to give L240, L241, and L242. Overall yields: 7.4, 3.2, 1.3%
respectively.
A. 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoic acid A (FIG. 12A) [00303] A solution of 4-amino-3-methoxybenzoic acid (1.0 g; 5.9 mmol); and N-ethyldiisopropylamine (1.02 mL 5.9 mmol) in THF (20 mL) was added dropwise to a solution of Fmoc-glycylchloride (1.88 g; 5.9 mmol) in CHaCl2 /THF 1:1 (20 rnL) and stirred at room temperature under N2. After 3 h the solvent was evaporated under vacuum. The residue was taken up with 0.5 N
HCl (50 mL), was stirred for 1 h at 0 °C then filtered and dried.
The white solid was suspended in MeOH (30 mL) and stirred for I h, then was filtered and suspended in a solution of CHC13/hexane 1:4 (75 mL). The suspension was filtered to give compound A as a with solid (1.02 g; 2.28 mmol). Yield 39%.

B. N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]
acetyl]glycylJaminoJ-3-methoxybenzoyl)-L-glutaminyl-L-tryptophyl-1-alanyl-L-valyl-~Iycvl-L-histidyl-L-leucyl L methioninamide L240 [00304] 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 emptied and fresh SO% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 4-[[[ (9H-Fluoren-9-ylmethoxy)carbonylJ amino] acetyl]
amino-3-methoxybenzoic acid, A (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 shaken for 5 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol}, N ethyldiisopropylamine (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, emptied and the resin washed with DMA (5 x 7 mL), CH2C12 (5 x 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 oil crude that after treatment with Et2O (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with EtzO (5 x 20 mL) to give a solid (152 mg) which was analysed by HPLC.
An amount of crude (52 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L240 (12.0 mg; 7.8 x 10-3 mmol) as a white solid. Yield 7.4%.
C. 4-amino-3-chlorobenzoic acid C (FIG 12B) [00305) 1 N NaOH (1 I mL; 11 mmol) was added to a solution ofmethyl 4-amino-3-chlorobenzoate (2 g; 10.8 mmol) in MeOH (20 mL) at 45 °C. The reaction mixture was stirred for 5 h at 45 °C and overnight at room temperature.
More 1N NaOH was added (5 mL; 5 mmol) and the reaction was stirred at 45 °C
for 2 h. After concentration of solvent was added 1N HCl (16 ml). The solid precipitate was filtered and dried to give 4-amino-3-chlorobenzoic acid, C, as a with solid (I,75 g; 10.2 mmol). Yield 94.6%.
D. 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid, D (FIG. 12B1 [00306] A solution of 4-amino-3-chlorobenzoic acid (1.5 g; 8.75 mmol) and N-ethyldiisopropylamine (1.46 mL 8.75 mmol) in THF (50 mL) was added dropwise to a solution of Fmoc-glycylchloride (2.76 g; 8.75 mmol) in CH2Cl2 /THF 1:1 (30 mL) and stirred at room temperature under N2. After 3 h the solvent was evaporated under vacuum. The residue was taken up with 0.5N
HCl (50 mL), filtered and dried.
The white solid was suspended in MeOH (30 mL) and stirred for 1 h, then was filtered and dried to give 4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid (2.95 g; 6.5 mmol). Yield 75%.
E. N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,lOtetraazacyclododec-1-yI]
acetyl] glycyl] amino] 3-chlorob enzoyl] L-glutaminyl-L-tryptophyl-1-alanyl-L-valy~l~yl-L-histidyl-L-leucyl-L-methioninamide L241 (FIG. 12E) [00307] 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 emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid, D (0.54 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 5 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL).
[00308] The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-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), N
ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken for 40 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL), CHZCl2 (5 x 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 oil crude that after treatment with Et20 (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et20 (5 x 20 mL) to give a solid (151 mg) which was analysed by HPLC. An amount of crude (56 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L241 (5.6 mg; 3.6 x 10-3 mmol) as a white solid. Yield 3.2%.
F. 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoic acid, E (FIG. 12C) [00309] A solution of 4-amino-3-methylbenzoic acid (0.81 g; 5.35 mmol) and N
ethyldiisopropylamine (0.9 mL 5.35 mmol) in THF (30 mL) was added dropwise to a solution of Fmoc-glycylchloride (1.69 g; 5.35 mmol) in CH2C12 /THF 1:1 (20 mL) and stirred at room temperature under N2. After 3 h the solvent was evaporated under vacuum. The residue was taken up with HCl 0.5 N (50 mL)and was stirred for 3 h at 0°C. then was filtered and dried. The white solid was suspended in MeOH (50 mL) and stirred for 1 h, then filtered and dried to give Compound E (1.69 g; 3.9 mmol). Yield 73%.
G. N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]
2 5 acetyl] glycyl] amino] 3-methylb enzoyl] L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L242 (FIG. 12F1 [00310] 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 emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 4-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-3-methylbenzoic acid, E (0.52 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 5 h at room temperature, emptied and the resin washed with DMA
(5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCl (0.76 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol), N
ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.
[00311] The mixture was shaken for 40 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL), CHZC12 (5 x 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 oil crude that after treatment with Et20 (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with EtZO (5 x 20 mL) to give a solid (134 mg) which was analysed by HPLC. An amount of crude (103 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L242 (4.5 mg; 2.9 x 10-3 mmol) as a white solid. Yield 1.3%.
EXAMPLE XIII - Figures 13A-C
Synthesis of L244 [00312] Summary: The product was obtained in several steps starting from the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]) on the Rink amide resin (A). The final coupling step with DOTA tri-t-butyl ester was done in solution phase after cleavage and deprotection with Reagent B of Linker-BBN [7-14]. The crude was purified by preparative HPLC to give L244. Overall yield: 0.4%.
A. N.N'-(Iminodi-2,1-ethanedi~)bis[2 2 2-trifluoroacetamide~~FIG

[00313] Trifluoroacetic acid ethyl ester (50 g; 0.35 mol) was dropped into a solution of diethylenetriamine (18 g; 0.175 mol) in THF (180 mL) at 0°C in 1 h.
After 20 h at room temperature, the mixture was evaporated to an oily residue (54 g). T
he oil was crystallized from Et20 (50 mL), filtered, washed with cooled Et2O

(2 x 30 mL) and dried to obtain A as a white solid (46 g; 0.156 mol). Yield 89%.
B. 4-[N,N'-Bis[2-(trifluoroacetyl)aminoethyl]amino]-4-oxobutanoic acid, B (FIG. 13A) [00314] Succinic anhydride (0.34 g; 3.4 mmol) was added in a solution of A (1 g; 3.4 mmol) in THF (5 mL) at room temperature. After 28 h the crude was concentrated to residue (1.59 g), washed with EtOAc (2 x 10 mL) and 1 N
HCl (2 x 15 mL). The organic layer was dried on Na2S04, filtered and evaporated to give an oily residue (1.3 g) that was purified by flash chromatography (5) to afford B as an oil (0.85 g; 2.15 mmol). Yield 63%.
C. 4-[N,N'-Bis[2-[(9-H fluoren-9-ylmethoxy)carbonyl]aminoethyl]amino]-4-oxobutanoic acid, D (FIG.
[00315] Succinic anhydride (2 g; 20 mmol) was added in a solution of A (5 g;
16.94 mmol) in THF (25 mL) at room temperature. After 28 h the crude was concentrated to residue (7 g), washed in ethyl acetate (100 mL) and in 1 N
HCI (2 x 50 mL). The organic layer was dried on Na2S04, filtered and evaporated to give crude B as an oily residue (6.53 g). 2 N NaOH (25 mL) was added to suspension of crude B (5 g) in EtOH (35 mL) obtaining a complete solution after 1 h at room temperature. After 20 h the solvent was evaporated to obtain C as an oil (8.48 g). A solution of 9-fluorenylmethyl chloroformate (6.54 g, 25.3 mmol) in 1,4-dioxane (30 mL), was dropped in the solution of C
in 10% aq. Na2C03 (30 mL) in 1 h at 0°C. After 20 h at r.t. a gelatinous suspension was obtained and filtered to give a white solid (3.5 g) and a yellow solution. The solution was evaporated and the remaining aqueous solution was diluted in H20 (150 mL) and extracted with EtOAc (70 mL). Fresh EtOAc (200 mL) was added to aqueous phase, obtaining a suspension which was cooled to 0°C and acidified to pH 2 with conc. HCI. The organic layer was washed with H20 (5 x 200 mL) until neutral pH, then dried to give a glassy solid (6.16 g). The compound was suspended in boiling n-Hexane (60 mL) for 1 h, filtered to give D as a white solid (5.53 g, 8.54 mmol). Overall yield 50%.

D. N [4-[[4-[Bis[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJacetylJamino]ethyl]amino-1,4-dioxobutyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-g-l~cyl-L-histidyl-L-leu~l-L-methioninamide. L244 FIG. 13B) [00316] 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 emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL). 4-[N,N'-Bis[2-[(9-H-fluoren-9-yl methoxy) carbonyl]
aminoethylJ amino]-4-oxo butanoic acid (777.3 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 ~,L; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 40 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution 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 (2 x 7 mL) and with CH2Cl2 (5 x 7 mL) then it 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 after treatment with Et20 (20 mL) gave a precipitate. The precipitate was collected by centrifugation and washed with Et20 (5 x 20 mL) to give F as a white solid (140 mg). DOTA tri-t-butyl ester (112 mg; 0.178 mmol) HATU (70 mg; 0.178 mmol) and DIEA
(60 ~.L; 0.356 mmol) were added to a solution of F (50 mg; 0.0445 mmol) in DMA (3 mL) and CH2Cl2 (2 mL) and stirred for 24 h at room temperature.
The crude was evaporated to reduced volume (1 rnL) and shaken with Reagent B (25 mL) for 4.5 h. After evaporation of the solvent, the residue was treated with EtaO (20 mL) to give a precipitate. The precipitate was collected by centrifugation and washed with Et20 (5 x 20 mL) to afford a beige solid (132 mg) that was analyzed by HPLC. An amount of crude (100 mg) was purified by preparative HPLC. The fractions containing the product were lyophilized to give L244 (FIG. 13C) (3.5 mg; 1.84 x 10-3 mmol) as a white solid. Yield 0.8 %.

General Experimentals for Examples XIV-Example XLII

A. Manual Cou~lyn~s [00317] 6.0 equivalents of the appropriately protected amino acid was treated with 6.0 equivalents each of HOBt and DIC and activated outside the reaction vessel.
This activated carboxylic acid in NMP was then transferred to the resin containing the amine and the reaction was carried out for 4-6 h and then the resin was drained and washed.
B. Special coupling of Fmoc-Gly-OH to 4-aminobenzoic acid and aminobiphenylcarboxylic acid amides' [00318) Fmoc-Gly-OH (10.0 equiv.) was treated with HATU (10.0 equiv.) and DIEA
(20.0 equiv.) in NMP (10 mL of NMP was used for one gram of the amino acid by weight) and the solution was stirred for 10-15 min at RT before transfernng to the vessel containing the amine loaded resin. The volume of the solution was made to 15.0 ml for every gram of the resin. The coupling was continued for 20h at RT and the resin was drained of all the reactants.
This procedure was repeated one more time and then washed with NMP
before moving on to the next step.
C. Preparation of D03A monoamide~
[00319) 8.0 equivalents of DOTA mono acid was dissolved in NMP and treated with 8.0 equivalents of HBTU and 16.0 equivalents of DIEA. This solution was stirred for 15 min at RT and then transferred to the amine on the resin and the coupling was continued for 24h at RT. The resin was then drained, washed and then the peptide was cleaved and purified.
D. Cleavage of the crude peptides from the resin and burification~
[00320] The resin was suspended in Reagent B (15.0 ml/g) and shaken for 4h at RT.
The resin was then drained and washed with 2 x 5 mL of Reagent B again and combined with the previous filtrate. The filtrate was then concentrated under reduced pressure to a pastelliquid at RT and triturated with 25.0 mL of anhydrous ether (for every gram of the resin used). The suspension was then centrifuged and the ether layer was decanted. This procedure was repeated two more times and the colorless precipitate after ether wash was purified by preparative HPLC.
Example XIV - Figure 21 Synthesis of L201 [00321 ] 0.5 g of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-M-Resin (0.4 mmol/g, O.Sg, 0.2 mmol) (Resin A) was used. The rest of the amino acid units were added as described in the general procedure to prepare ( 1 R)-I -(Bis {2-[bis(carboxymethyl)amino]ethyl}amino)propane-3-carboxylic acid-1-carboxyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L201), Yield: 17.0 mg (5.4%) Examine XV - Figures 22A and 22B
Synthesis of L202 A. 4-Fmoc-hydrazinobenzoic acid (Fig 2~A~
[00322] A suspension of 4-hydrazinobenzoic acid (5.0 g, 32.9 mmol) in water (100 ml) was treated with cesium carbonate (21.5 g, 66.0 mmol). Fmoc-CI (9.1 g, 35.0 mmol) in THF (25 mL) was added dropwise to the above solution with stirring over a period of 1h. The solution wasrstirred for 4h more after the addition and the reaction mixture was concentrated to about 75 mL and extracted with ether (2 x 100 mL). The ether layer was discarded and the aqueous layer was acidified with 2N HCl. The separated solid was filtered, washed with water (5 x 100 mL) and then recrystallized from acetonitrile to yield the product (compound B) as a colorless solid. Yield: 11.0 g (89%). 1H NMR (DMSO-db) d 4.5 (m, 1 H, Ar-CH -CH), 4.45 (m, 2H, Ar-CH ), 6.6 (bs, 1 H, Ar-H), 7.4 -7.9 (m, 9, Ar-H and Ar-CH ), 8.3 (s, 2H, Ar-H), 9.6 (s, 2H, Ar-H). M. S. -m/z 373.2 [M-H].
[00323] 0.5 g of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-M-Resin (0.4 mmol/g, O.Sg, 0.2 mmol) (Resin A) was used. The amino acid units were added as described in the general procedure, including Compound B to prepare N [(313,513,12a)-3-[ [[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-3 0 y1] acetyl] amino] acetyl] amino]-4-hydrazinob enzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninarnide (L202) (Fig. 22B), Yield: 25.0 mg (8.3%).
Example XVI - Figures 23A and 23B
Synthesis of L203 A. Preparation of 4-Boc-aminobenzyl benzoate Compound B FIG. 23A~
[00324] A suspension of 4-boc-aminobenzoic acid (0.95 g, 4.0 mmol) in dry acetonitrile (10.0 mL) was treated with powdered cesium carbonate (1.3 g, 4.0 mmol) and stirred vigorously under nitrogen. Benzyl bromide (0.75 g, 4.4 mmol) was added and the reaction mixture was refluxed for 20h under nitrogen. The reaction mixture was then poured into ice cold water (200 mL) and the solid separated was filtered and washed with water (5 x 50 mL). The crude material was then recrystallized from aqueous methanol to yield the product as a colorless solid (Compound B). Yield: 0.8 g (61 %). 'H NMR
(CDC13): d 1.5 (s, 9H, Tertiary methyls), 5.4 (s, 2H, Ar-CH ), 7.4 (m, 7H, Ar-H) and 8.0 (m, 2H, Ar-H). M. S. -m/z 326.1 [M+H].
B. 4-Aminobenzyl benzoate Compound C (FIG. 23A~
[00325] 4-Boc-aminobenzyl benzoate (0.8 g, 2.5 mmol) was dissolved in DCM (20 mL) containing TFA (25% by volume) and stirred for 2h at RT. The reaction mixture was poured into 100.0 g of crushed ice and neutralized with saturated sodium bicarbonate solution until the pH reached about 8.5. The organic layer was separated and the aqueous layer was extracted with DCM (3 x 20 mL) and all the organic layers were combined. The DCM layer was then washed with 1 x 50 mL of saturated sodium bicarbonate, water ( 2 x 50 mL) and dried (sodium sulfate). Removal of the solvent yielded a colorless solid (Compound C) that was taken to the next step without further purification. Yield: 0.51 g (91 %). 1H NMR (CDCI3): d 5.3 (s, 2H, Ar-CH ), 6.6 (d, 2H, Ar-H, j =1.0 Hz), 7.4 (m, SH, Ar-H, J= 1.0 Hz) and 7.9 (d, 2H, Ar-H, J= 1.0 Hz).
C. 4-(2-Chloroacetyl)aminobenzyl benzoate Compound D (FIG. 23A~
[00326] The amine (0.51 g, 2.2 mmol) was dissolved in dry dimethylacetamide ( 5.0 mL) and cooled in ice. Chloroacetyl chloride (0.28 g, 2.5 mmol) was added dropwise via a syringe and the solution was allowed to come to RT and stirred for 2h. An additional, 2.5 mmol of chloroacetyl chloride was added and stirring was continued for 2 h more. The reaction mixture was then poured into ice cold water (100 mL). The precipitated solid was filtered and washed with water and then recrystallized from hexane/ether to yield a colorless solid (Compound D). Yield: 0.38 g (56%). 'H NMR (CDCl3): d 4.25 (s, 2H, CH -CI), 5.4 (s, 2H, Ar-H), 7.4 (m, 5H, Ar-H), 7.6 (d, 2H, Ar-H) , 8.2 (d, 2H, Ar-H) and 8.4 (s, 1 H, -CONH).
[00327] text-Butyl2-{1,4,7,10-tetraaza-7,10-bis~[(tert-butyl)oxycarbonyl]methyl}-4-[(N-~4-[benzyloxycarbonyl]phenyl } carbamoyl] cyclododecyl } acetate, Compound E (FIG. 23A):
[00328] DO3A-tri-t-butyl ester.HCl (5.24 g, 9.5 mmol) was suspended in 30.0 mL
of dry acetonitrile and anhydrous potassium carbonate (2.76 g, 20 mmol) was added and stirred for 30 min. The chloroacetamide D (2.8 g, 9.2 mmol) in dry acetonitrile (20.0 mL) was then added dropwise to the above mixture for 10 min. The reaction mixture was then stirred overnight. The solution was filtered and then concentrated under reduced pressure to a paste. The paste was dissolved in about 200.0 mL of water and extracted with 5 x 50 mL of ethyl acetate. The combined organic layer was washed with water (2 x 100 mL) and dried (sodium sulfate). The solution was filtered and evaporated under reduced pressure to a paste and the paste was chromatographed over flash silica gel (600.0 g). Elution with 5% methanol in DCM eluted the product. All the fractions that were homogeneous on TLC were pooled and evaporated to yield a colorless gum. The gum was recrystallized from isopropylether and DCM to prepare Compound E. Yield: 4.1 g (55%). 1H
NMR (CDCl3): d 1.5 (s, 27H, methyls), 2.0 - 3.75 (m, 24H, NCH s), 5.25 (d, 2H, Ar-CHa), 7.3 (m, 5H, Ar-H), 7.8 (d, 2H, Ar-H) and 7.95(d, 2H, Ar-H). M.
S. - m/z 804.3 [M+H].
D. Reduction of the above acid E to prepare Compound F FIG. 23A):
[00329] The benzyl ester E from above (1.0 g, 1.24 mmol) was dissolved in methanol-water mixture (10.0 mL, 95:5) and palladium on carbon was added (10%, 0.2 g). The solution was then hydrogenated using a Parr apparatus at 50.0 psi for 8h. The solution was filtered off the catalyst and then concentrated under reduced pressure to yield a colorless fluffy solid F. It was not purified further and was taken to the next step immediately. MS: m/z 714.3 [M+NaJ.
E. Preparation of L203 (FIG. 23B) [00330) The above acid F was coupled to the amine on the resin [H-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-Resin) Resin A and F from above using standard coupling procedures described above. 0.5 g (0.2 mmol) of the resin yielded 31.5 mg of the final purified peptide (10.9%) N [(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJ acetyl) amino)-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L203) (FIG. 23B).
Example XVII - Figure 24 Synthesis of L204 [00331) Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) (Resin A) was used. Fmoc-Gly-OH was loaded first followed by F from the above procedure (FIG. 23A) employing standard coupling conditions. Yield: 24.5 mg (8.16%) of N
[(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJacetyl]amino)-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L204) (FIG. 24).
Example XVIII - Figure 25 Synthesis of L205 [00332) Fmoc-6-aminonicotinic acid' was prepared as described in the literature ("Synthesis of diacylhydrazine compiounds for therapeutic use." Hoelzemann, G.; Goodman, S. (Merck Patent G.m.b.H.., Germany). Ger.Offen. 2000, 16 pp. CODEN: GWXXBX DE
19831710 A1 20000120) and coupled with preloaded Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) Resin A, followed by the other amino groups as above to prepare N [(313,513,12a)-3-[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJ acetyl] amino)-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L205) Yield: 1.28 mg (0.4%).

Example XIX - Figures 26A and 26B
Synthesis of L206 A. 4'-Fmoc-amino-3'-methylbinhenyl-4-carboxylic acid B' [00333] The amino acid (0.41 g, 1.8 mmol) was dissolved in a solution of cesium carbonate (0.98 g, 3.0 mmol) in 10.0 mL of water. See "Rational Design of Diflunisal Analogues with Reduced Affinity for Human Serum Albumin "
Mao, H. et al J. Am. Chem. Soc., 2001, 123(43), 10429-10435. This solution was cooled in an ice bath and a solution of Fmoc-Cl (0.52 g, 2.0 mmol) in THF (10.0 mL) was added dropwise with vigorous stirring. After the addition, the reaction mixture was stirred at RT for 20h. The solution was then acidified with 2N HCI. The precipitated solid was filtered and washed with water (3 x 20 mL) and air dried. The crude solid was then recrystallized from acetonitrile to yield a colorless fluffy solid B (FIG. 26A). Yield: 0.66 g (75%). 'H NMR (DMSO-d6): d 2.2 (s, Ar-Me), 4.25 (t, 1H, Ar-CH, j = SHz), 4.5 (d, 2H, O-CH2, j = 5.0 Hz), 7.1 (bs, 1H, CONH), 7.4 - 8.0 (m, 8H, Ar-H) and 9.75 (bs, 1H, -COOH). M. S.: m/z 472.0 [M-H].
[00334] The acid B from above was coupled to Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2g, 0.08 mmol) resin A with the standard coupling conditions.
Additional groups were added as above to prepareN [(313,513,12a.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]- [4'-Amino-2'-methyl biphenyl-4-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L206). Yield: 30.5 mg (24%).
Example XX - Figures 27A-B
Synthesis of L207 [00335] 3'-Fmoc-amino-biphenyl-3-carboxylic acid was prepared from the corresponding amine using the procedure described above. See "Synthesis of 3'-methyl-4-'-nitrobiphenylcarboxylic acids by the reaction of 3-methyl-4-nitrobenzenenediazonium acetate with methyl benzoate", Boyland, E. and Gorrod, J., J. Chem. Soc., Abstracts (1962), 2209-11. 0.7G of the amine yielded 0.81 g of the Fmoc-derivative (58%) (Compound B, FIG.

27A). 'H NMR (DMSO-db): d 4.3 (t, 1H, Ar-CH), 4.5 (d, 2H, O-CH2), 7.25-8.25 (m, 16H, Ar-H) and 9.9 (s, 1 H, -COOH). M. S. - m/z 434 [M-H].
[00336] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2g, 0.08 mmol) resin A
was coupled to the above acid B and additional groups as above (FIG. 27B). 29.0 mg ofN
[(3J3,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]- [3'-amino-biphenyl-3-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L207) was prepared (23%).
Example XXI - Fi ug re 28 Synthesis of L208 [00337] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2g, 0.08 mmol) A was deblocked and coupled to terephthalic acid employing HATU as the coupling agent. The resulting acid on the resin was activated with DIC and NHS and then coupled to ethylenediamine. DOTA-mono acid was finally coupled to the amine on the resin.
N
[(313,513,12x,)-3-[[ [ [[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]- [1,2-diaminoethyl-terephthalyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L208) was prepared for a yield of 17.5 mg (14%) Example XXII - Figures 29A-B
Synthesis of L209 A. Boc-Glu(G-OBn)-G-OBn:
[00338] Boc-Glutamic acid (5.0 g, 20.2 mol) was dissolved in THF (50.0 mL) and cooled to 0°C in an ice bath. HATU (15.61 g, 41.0 mmol) was added followed by DIEA (6.5 g, 50.0 mmol). The reaction mixture was stirred at 0°C for min. Benzyl ester of glycine [8.45 g, 50 mmol, generated from neutralizing benzyl glycine hydrochloride with sodium carbonate and by extraction with DCM and solvent removal] was added in THF (25.0 mL). The reaction mixture was allowed to come to RT and stirred for 20h at RT. All the volatiles were removed under reduced pressure. The residue was treated with saturated sodium carbonate solution (100 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layers were combined and washed with 1N HCl (2 x 100 mL) and water (2 x 100 mL) and dried (sodium sulfate). The solution was filtered and solvent was removed under reduced pressure to yield a paste that was chromatographed over flash silica gel (500.0 g). Elution with 2%
methanol in DCM yielded the product as a colorless paste (Compound B, FIG.
29A). Yield: 8.5 g (74.5%). 1H NMR (CDCl3): d 1.4 (s, 9H, -CH3s), 2.0 - 2.5 (m, 4H,-CH-CHZ and CO-CH), 4.2 (m, SH, N-CH -CO), 5.15 (s, 4H, Ar-CH ), 5.45 (bs, 1 H, Boc-NH), 7.3 (m, 1 OH, Ar-H) and 7.6 (2bs, 2H, CONH).
M. S. - mlz 564.1 [M+H]. Analytical HPLC retention time - 8.29 min (>97%
pure, 20-65%B over 15 min).
B. H-GIu~G-OBn)-G-OBn:
[00339] The fully protected glutamic acid derivative (1.7 g, 3.2 mmol) B from above was dissolved in DCM/TFA (4:1, 20 mL) and stirred until the starting material disappeared on TLC (2 h). The reaction mixture was poured into ice cold saturated sodium bicarbonate solution (200 mL) and the organic layer was separated and the aqueous layer was extracted with 2 x 50 mL of DCM and combined with the organic layer. The DCM layer was washed with saturated sodium bicarbonate (2 x 100 mL), water (2 x 100 mL) and dried (sodium sulfate). The solution was filtered and evaporated under reduced pressure and the residue was dried under vacuum to yield a glass (Compound C, FIG. 29A) that was taken to the next step without further purification. Yield: 0.72 g (95%). M. S. - m/z 442.2 [M+H].
C. ~DOTA-tri-t-butyl -GIu-(G-OBn)-G-OBn:
[00340] The amine C from above (1.33 g, 3 mmol) in anhydrous DCM (10.0 mL) was added to an activated solution of DOTA-tri-t-butyl ester [2.27 g, 3.6 mmol was treated with HBTU, 1.36 g, 3.6 mmol and DIEA 1.04 g, 8 mmol and stirred for 30 min at RT in 25 mL of dry DCM] and stirred at RT for 20h].
The reaction mixture was diluted with 200 mL of DCM and washed with saturated sodium carbonate (2 x 150 mL) and dried (sodium sulfate). The solution was filtered and solvent was removed under reduced pressure to yield a brown paste. The crude product was chromatographed over flash silica gel (500.0 g). Elution with 2% methanol in DCM furnished the product as a colorless gum (Compound D, FIG. 29A). Yield: 1.7 g (56.8%). IH NMR
(CDCl3): d 1.3 and 1.4 (2s, 9H, three methyls each from the free base and the sodium adduct of DOTA), 2.0 - 3.5 (m, 20H, N-CH2s and -CH-CH and CO-CH2), 3.75 - 4.5 (m, 13H, N-CH -CO), 5.2 (m, 4H, Ar-CHZ) and 7.25 (m, 1 OH, Ar-H). M. S. m/z -1 Ol 8.3 [M+Na] and 996.5 [M+H] and 546.3 [M+Na+H]/2. HPLC - Retention Time: 11.24 min (>90% , 20-80% B over 30 min).
D. ~DOTA-tri-t-butt -GIu~G-OH)-G-OH:
[00341 ] The bis benzyl ester (0.2 g, 0.2 mmol) D from above was dissolved in methanol-water (20 mL, 9:1 ) and hydrogenated at 50 psi in the presence of 10% Pd/C catalyst (0.4 g, 50% by wt. water). After the starting material disappeared on HPLC and TLC (4h), the solution was filtered off the catalyst and the solvent was removed under reduced pressure and the residue was dried under high vacuum for about 20h (<0.1 mm) to yield the product as a colorless foam (Compound E, FIG. 29A). Yield: 0.12 g (73.5%). 'H NMR (DMSO-d6): d 1.3 and 1.4 (2s, 9H corresponding to methyls of free base and the sodium adduct of DOTA), 1.8 - 4.7 (m, 33H, NCH s, COCH and CH-CH2 and NH-CH-CO), 8.1, 8.2 and 8.4 (3bs, NHCO). M. S.: m/z - 816.3 [M+H]
and 838.3 [M+Na]. HPLC Retention Time: 3.52 min (20-80% B over 30 min, > 95% pure).
E. H-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met NHS:
[00342] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) A was deblocked and coupled twice sequentially to 8-amino-3,6-dioxaoctanoic acid to yield the above deprotected peptide (Compound F, FIG. 29B) after preparative HPLC purification. Yield: 91.0 mg (37%).
[00343] HPLC Retention Time: 8.98 min (>95% purity, 10-40% B in over 10 min).
M. S.: m/z - 1230.6 [M+H], 615.9 [M+2H]/2.
F. Solution phase coupling of the bis-acid E and the amine F from above: (FIG.

[00344] The bis-acid (13.5 mg, 0.0166 mmol) E was dissolved in 100~L of dry acetonitrile and treated with NHS (4.0 mg, 0.035 mmol) and DIC (5.05 mg, 0.04 mmol) and stirred for 24h at RT. To the above activated acid, the free amine F (51.0 mg, 0.41 mmol)[generated from the TFA salt by treatment with saturated sodium bicarbonate and freeze drying the solution to yield the amine as a fluffy solid] was added followed by 1 OO~L of NMP and the stirring was continued for 40h more at RT. The solution was diluted with anhydrous ether (10 mL) and the precipitate was collected by centrifugation and washed with 2 x 10 mL of anhydrous ether again. The crude solid was then purified by preparative HPLC to yield the product as a colorless fluffy solid L209 as in FIG. 29B with a yield of 7.5 mg (14.7%).
Example XXIII - Figures 30A-B
Synthesis of L210 A. H-8-aminooctanoyl-8-aminooctanoyl-Gln-Tm-Ala-Val-Gly-His-Leu-Met-NHS:
[00345] This was also prepared exactly the same way as in the case of Compound F
(FIG. 29B), but using 1-aminooctanoic acid and the amine (Compound B, FIG. 30A) was purified by preparative HPLC. Yield: 95.0 mg (38.9%).
HPLC Retention Time: 7.49 min (>95% purity; 10-40%B over 10.0 min). M.
S.: m/z -1222.7 [M+H], 611.8 [M+2H]/2.
[00346] (DOTA-tri-t-butyl)-Glu-(G-OH)-G-OH (0.0163 g, 0.02 mmol) was converted to its bis-NHS ester as in the case of L209 in 100~L of acetonitrile and treated with the free base, Compound B (60.0 mg, 0.05 mmol) in 1 OO~L of NMP and the reaction was continued for 40h and then worked up and purified as above to prepare L210 (FIG. 30B) for a yield of 11.0 mg (18%).
Examine XXIV - Fi ug re 31 Synthesis of L211 [00347] Prepared from 0.2 g Of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.08 mmol) using standard protocols. N [(313,513,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-glycyl-4-aminob enzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L211 was prepared in a yield of 4.7 mg (3.7%) (FIG. 31).

Example XXV - Figure 32 Synthesis of L212 [00348] Prepared from Rink Amide Novagel resin (0.47 mmol/g, 0.2 g, 0.094 mmol) by building the sequence on the resin by standard protocols. N [(313,5]3,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L212 was prepared for a yield of 25.0 mg (17.7%) (FIG. 32).
Example XXVI - Fi ure 33 Synthesis of L213 [00349] Prepared from Fmoc-Met-2-chlorotrityl chloride resin (NovaBioChem, 0.78 mmol/g, 0.26 g, 0.2 mmol) and the rest of the sequence were built using standard methodology. N [(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionine L213 was prepared for a yield of 49.05 mg (16.4%) (FIG.33).
Example XXVII - Figure 34 Synthesis of L214 [00350] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was used to prepare N [(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L214 using standard conditions.
8.5 mg of the product (6.4%) was obtained (FIG. 34).
Example XXVIII - Fi, ure 35 Synthesis of L215 [00351] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was used to prepare N [(313,S13,12a)-3-[[[4,7,10-Tris(carboxyrnethyl)-1,4,7,10-tetraazacyclododec-1 yl] acetyl] amino]-glycyl-4-aminob enzoyl-L-glutaminyl-L-arginyl-L-1 eucyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninarnide L215. 9.2 mg (5.5%) was obtained (FIG. 35).

Example ~I~ - Figure 36 Synthesis of L216 [00352] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was used to prepareN [(313,S13,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-I-yl)acetyl)amino)-glycyl-4-aminobenzoyl-L-glutaminyl-arginyl-L-tyrosinyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L216. 25.0 mg (14.7%) was obtained (FIG. 36).
Example XXX - Figure 37 Synthesis of L217 [00353) Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin A (0.2g, 0.08 mmol) was used to prepareN [(313,513,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl) acetyl) amino)-glycyl-4-aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L217. 58.0 mg (34.7%) was obtained (FIG. 37).
Example ~!:~I - Figure 38 Synthesis of L218 [00354) Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin A (0.2g, 0.08 mmol) was used.
Fmoc-Lys(ivDde) was employed for the introduction of lysine. After the linear sequence was completed, the protecting group of the lysine was removed using 10% hydrazine in DMF (2 x 10 mL; 10 min each and then washed). The rest of the amino acids were then introduced using procedures described in the "general" section to complete the required peptide sequence. L218 in FIG. 38 as obtained in a yield of 40.0 mg (23.2%).
Examule ~S;XYII- Figure 39 Synthesis of L219 [00355) 4-Sulfamylbutyryl AM Novagel resin was used (1.1 mrnol/g; 0.5 g; 0.55 mmol).
The first amino acid was loaded on to this resin at -20° C for 20h. The rest of the sequence was completed utilizing normal coupling procedures. After washing, the resin was alkylated with 20.0 eq. of iodoacetonitrile and 10.0 equivalents of DIEA for 20h. The resin Was then drained of the liquids and washed and then cleaved with 2.0 eq. of pentylamine in 5.0 mL of THF for 20h. The resin was then washed with 2 x 5.0 mL of THF and all the filtrates were combined. THF was then evaporated under reduced pressure and the residue was then deblocked with 10.0 mL of Reagent B and the peptide N [(313,SJ3,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-S histidyl-L-leucyl-aminopentyl, L219 was purified as previously described.
28.0 mg (2.8%) was obtained (FIG. 39).
Example XXXIII- Figure 40 Synthesis of L220 [00356] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(313,513,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-I-y1] acetyl] amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-D-alanyl-L-histidyl-L-leucyl-L-methioninamide, L220. 31.5 mg (41.4%) was obtained (FIG.
40).
Example ~:XXIV - Figure 41 Synthesis of L221 [00357] NovaSyn TGR (0.25 mmol/g; 0.1 S g, 0.05 mmol) resin A was used to prepare N
[(3 ~3,5[i,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-leucinamide, L221. 28.0 mg (34.3%) was obtained (FIG. 41).
Example XXXV - Figure 42 Synthesis of L222 [00358] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3 (3,5 (3,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide, L222. 34.0 mg (40.0%) was obtained (FIG. 42).

Example XXXVI - Fi ug~ re 43 Synthesis of L223 [00359] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3[i,5 J3,12a,)-3-[ [ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-ylJacetyl]amino]-glycyl-4-aminobenzoyl-L-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide, L223.
31.2 mg (37.1 %) was obtained (FIG. 43).
Example ~:XXVII - Figure 44 Synthesis of L224 [00360] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3[i,5 j3,12oc)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-glycyl-L-histidyl-L-phenylalanyl-L-leucinamide, L224. 30.0 mg (42.2%) was obtained (FIG. 44).
Example ~:XXVIII - Figure 45 Synthesis of L225 [00361] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3 (3,5(3,12a)-3-[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJ acetyl] aminoJ-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-al anyl-L-valinyl-glycyl-L-serinyl-L-phenylalanyl-L-methioninamide, L225. 15.0 mg (20.4%) was obtained (FIG. 45).
Examule XXXIX - Fi, ug re 46 Synthesis of L226 [00362] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3 /3,5(3,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]aminoJ-glycyl-4-aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, L226. 40.0 mg (52.9%) was obtained (FIG.
46).
Example XL - Figure 47 Synthesis of L227 [00363] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3 [3,5(3,12a.)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-threonyll-glycyl-L-histidyl-L-phenylalanyl-L-methioninamide L227. 28.0 mg (36.7%) was obtained (FIG.
47).
Example XLI - Figure 48 Synthesis of L228 [00364] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepare N
[(3 (395 (3,12oc)-3-[ [ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-phenylalanyl-L-methioninamide, L228. 26.0 mg (33.8%) was obtained (FIG.
48).
EXAMPLE XLII - Synthesis of Additional GRP Compounds A. General procedure for the preparation of 4,4'-Aminomethylbiphenylcarboxylic acid (B2) and 3,3'-aminometh~phenylcarboxylic acid (B3):
1. Meths d~ymethylbiphenylcarbox 1 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 f [(C6I-is)3P]4Pd; 0.0238, 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 x 50 mL). The organic layers were combined and washed with saturated sodium bicarbonate solution (2 x 50 mL) and dried (Na2SO4). 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.

Yield:
B2 -0.45g (31 %); m. p. - 170-171 ° C.
B3 - 0.69 g (62%); oil.
'H NMR (CDCl3) d B2- 3.94 (s,3H, -COOCH3), 4.73 (s, 2H, -CHZ-Ph), 7.475 (d, 2H, J=
SHz), 7.6 (d, 2H, J = 10 Hz), 7.65 (d, 2H, J = SHz) and 8.09 (d, 2H, J = 10 Hz).
Ii~I. S. - m/e - 243.0 [M+H]
B3 - 3.94 (s, 3H, -COOCH3), 4.76 (s, 2H, -CHZ-Ph), 7.50 (m, 4H), 7.62 (s, 1 H), 7.77 (s, 1 H), 8.00 (s, 1 H) and 8.27 (s, 1 H).
M. S. - m/e - 243.2 [M+H]
2. Azidomethylbiphenyl carboxylates:
[00365] 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 24h. The reaction mixture was diluted with water and extracted with ethyl acetate (2 x 25 mL). The organic layers were combined and washed successfully with 0.5 M citric acid solution (2 x 25 mL), water (2 x 25 mL) and dried (Na2S04). 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.
Yield:
Methyl-4-azidomethyl-4-biphenylcaroxylate- 0.245 g (46%); m. p. -106-108° C.
Methyl-4-azidomethyl-4-biphenylcaroxylate - 0.36 g (59%, oil) 1H NMR (CDCl3) d - 4,4'-isomer - 3.95 (s, 3H, -COOCH3), 4.41 (s, 2H, -CHZN3), 7.42 (d, 2H, J = 5 Hz), 7.66 (m, 4H) and 8.11 (d, 2H, J = 5 Hz) 3,3'-Isomer - 3.94 (s, 3H, -COOCH3), 4.41 (s, 2H, -CH2N3), 7.26-7.6 (m, SH), 7.76 (d, 1 H, J
=10 Hz), 8.02 (d, 1 H, J = 5 Hz) and 8.27 (s, 1 H).

3. Hydrolysis of the methyl esters of biphenylcarbox lates:
[00366] 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 x 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.
Yield:
4,4'-isomer - 0.87 g of methyl ester yielded 0.754 g of the acid (86.6%); m.
p. - 205-210° C
3,3'-isomer - 0.48 g of the methyl ester furnished 0.34 g of the acid (63.6%);
m. p. -102-105° C.
'H NMR (DMSO-d6) d : 4,4'-isomer- 4.52 (s, 2H, -CH2N3), 7.50 (d, 2H, J = 5 Hz), 7.9 (m, 4H), and 8.03 (d, 2H, J = 10 Hz) 3,3'-isomer- 4.54 (s, 2H, -CHaN3), 7.4 ( d, 1H, J = 10 Hz), 7.5-7.7 (m, 4H), 7.92 (ABq, 2H) and 8.19 (s, 1 H).
4. Reduction of the azides to the amine:
[00367] 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.
5. (3(3, 5(3, 7a., l2oc)-3-[ f (9H-Flouren-9ylmethoxy)amino] acetyl ~ amino-7,12-dih,~ycholan-24-oic [00368] 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° 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(3, 5(3, 7a, 12a)-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 HCI (30 mL) were added (final pH 1.5). The precipitated solid was filtered, washed with water (2 x 100 mL), vacuum dried and purified by flash chromatography. Elution with chloroform/methanol (8:2) yielded the product as a colorless solid.
Yield: 1.9 g (25%). TLC: Rf 0.30 (CHCl3/MeOH/NH40H - 6:3:1).

IN VITRO AND IN VIVO TESTING OF COMPOUNDS
Example XLIII: IsZ vitro Binding Assay for GRP Receptors in PC3 Cell Lines FIGS. 14 A-B
[00369] 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 I25I-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 ICSO for RP527 ligand, DO3A-monoamide-Aoc-QWAVGHLM-NH2 (controls) and compounds of the invention which inhibit the binding of l2sl-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.
27; 1694-1699 (2000). D03A-rnonoamide-Aoc-QWAVGHLM-NHa is also referred to as D03A-monoamide-8-amino-octanoic acid-BBN (7-14) (SEQ. ID. NO: 1], and has MS=1467Ø DO3A monoamide-aminooctanyl-BBN[7-14].
[00370] The Radioligand Binding Plate Assay was validated for BBN and BBN
analogues (including commercially available BBN and Ll) and also using 99mTc RP527 as the radioligand.
A. Materials and Methods:
1. Cell culture:
[00371 ] PC3 (human prostate cancer cell line) were obtained from the American Type Culture Collection and cultured in RPMI 1640 (ATCC) 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 ~g/mL). All cultures were maintained in a humidified atmosphere containing 5% COa/95%
air at 37°C, and passaged routinely using 0.05% trypsin/EDTA (GibcoBRL
25300-054) where indicated. Cells for experiments were plated at a concentration of 2.0x104 /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.
2. Binding buffer:
[00372] RPMI 1640 (ATCC) supplemented with 20mM HEPES, 0.1 % BSA (w/v), 0.5 mM PMSF (AEBSF), bacitracin (50 mg/S00 ml), pH 7.4. ~25I-BBN (carrier free, 2200 Ci/mmole) was obtained from Perkin-Elmer.
B. Competition assay with ~ZSI-BBN for GRP-R in PC3 cells:
[00373] A 96-well plate assay was used to determine the ICSn of various compounds of the invention to inhibit binding of ~25I-BBN to human GRP-R. The following general procedure was followed:
[00374] 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 2.0x 104 /well either in 96-1 S 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 D03A-monoamide-Aoc-QWAVGHLM-NH2 ligand, (controls), or compounds of the invention at concentrations ranging from 1.25 x 10-9 M to SxlO-9M, was co-incubated with jZSI-BBN (25,000 cpm/well). These studies were conducted with an assay volume of 75 ~1 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°C to prevent internalization of the ligand-receptor complex. Incubation was ended by the addition of 200 ~,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.
[00375] Competition binding curves for RP527 (control) and L70, a compound of the invention can be found in FIGS. 14A-B. These data show that the IC50 of the RP527 control is 2.SnM and that of L70, a compound of this invention is SnM. The IC50 of the monoamide-Aoc-QWAVGHLM-NH2 control was SnM. IC50 values for those compounds of the invention tested can be found in Tables 1-3, supra, 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.
C. Internalization & Efflux assay:
[00376] These studies were conducted in a 96-well plate. After washing to remove serum proteins, PC3 cells were incubated with lzsl-BBN, ~~~Lu-D03A-monoamide-Aoc-QWAVGHLM-NH2 or radiolabeled compounds of this invention for 40 min, at 37 °C. Incubations were stopped by the addition of 200 ~.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.~ 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 l2sI-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).
[00377) For the efflux studies, after loading the PC3 cells with l2sl-BBN or radiolabeled compounds of the invention for 40 min at 37°C, the unbound material was filtered, and the % of internalization was determined as above.
The cells were then resuspended in binding buffer at 37°C for up to 3h. 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 5.

Internalisation and efflux of ~25I-BBN and the Lu-177 complexes of D03A-monoamide-Aoc-QWAVGHLM-NHZ (control) and compounds of this invention I-BBN D03A-monoamide- L63 L64 L70 Aoc-QWAVGHLM-NHZ (control Internalisation (40 59 89 64 69 70 minutes) Efflux (2h) 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 XLIV - Preparation of Tc-labeled GRP compounds.
[00378] Peptide solutions of compounds of the invention identified in Table 6 were prepared at a concentration of 1 mg/mL in 0.1 % aqueous TFA. A stannous chloride solution was prepared by dissolving SnCl2~2H2O (20 mg/mL) in 1 N HCl. Stannous gluconate solutions containing 20 ~.g of SnCh~2HaO/100 ~L were prepared by adding an aliquot of the SnCl2 solution (10 ~L) to a sodium gluconate solution prepared by dissolving 13 mg of sodium gluconate in water. A hydroxypropyl gamma cyclodextrin [HP-y-CD]
solution was prepared by dissolving 50 mg of HP-y-CD in 1 mL of water.
[00379] The 99mTc labeled compounds identified below were prepared by mixing 20 ~L of solution of the unlabeled compounds (20 fig), 50 ~L of HP-y-CD solution, 100 ~.L of Sn-gluconate solution and 20 to 50 ~.L Of 9smTc pertechnetate (5 to 8 mCi, Syncor). The final volume was around 200 ~L and final pH was 4.5 - 5. The reaction mixture was heated at 100°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-y-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 x 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.
[00380] Labeling results are summarized in Table 6.

HPLC
retentionInitial RCP4 (%) time RCP3 immediately following om oundl e uence2 min % urification L2 -RJQWAVGHLM-NHZ 5.47 89.9 95.6 L4 -SJQWAVGHLM- NHZ 5.92 65 97 L8 -JKQWAVGHLM- NH2 6.72 86 94 Ll -KJQWAVGHLM- NH2 5.43 88.2 92.6 L9 -JRQWAVGHLM- NH2 7.28 91.7 96.2 L7 -aJQWAVGHLM- NH2 8.47 88.6 95.9 n.d. = not detected 1: All compounds were conjugated with an N,N'-dimethylglycyl-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'-dimethylglycyl-Ser-Cys (Acm)-Gly-RJQWAVGHLM-NHa.
The Acm group was removed during chelation to Tc.
2: In the Sequence, "J" refers to 8-amino-3,6-dioxaoctanoic acid and "a"
refers to D-alanine.
3: Initial RCP measurement taken immediately after heating and prior to HPL
purification.
4: RCP determined following HPLC isolation and acetonitrile removal via speed vacuum.
Example XLV - Preparation of i~~Lu-L64 for cell binding and biodistribution studies:
[00381] This compound was synthesized by incubating IO ~.g L64 ligand (10 ~,L
of a 1 mg/mL solution in water), 100 ~L ammonium acetate buffer (0.2M, pH 5.2) and ~1-2 mCi of ~~~LuCl3 in O.OSN HCl (MURK) at 90°C for 15 min. Free ~~~Lu was scavenged by adding 20 ~,L of a I % Na2EDTA~2H20 (Aldrich) solution in water. The resulting radiochemical purity (RCP) was N95%. The radiolabeled product was separated from unlabeled ligand and other impurities by HPLC, using a YMC Basic C8 column [4.6 x 150 mm], a column temperature of 30°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% CH3CN/20%
CH30H. The isolated compound had an RCP of 100% and an HPLC retention time of 23.4 minutes.
[00382] Samples for biodistribution and cell binding studies were prepared by collecting the desired HPLC peak into 1000 ~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 ~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 fnal concentration of 50 ~Ci/mL within 30 minutes of the in vivo study.
Example XLVI - Preuaration of I~~Lu-L64 for radiotherapy studies:
[00383] This compound was synthesized by incubating 70 qg L64 ligand (70 ~L of a 1 mg/mL solution in water), 200 ~.L ammonium acetate buffer (0.2M, pH 5.2) and ~30 - 40 mCi of l~~LuCl3 in O.OSN HCl (MURR) at 85°C for 10 min. After cooling to room temperature, free ~~~Lu was scavenged by adding 20 ~L of a 2% Na2EDTA~2H20 (Aldrich) solution in water. The resulting radiochemical purify (RCP) was ~95%. The radiolabeled product was separated from unlabeled ligand and other impurities by HPLC, using a 300VHP
Anion Exchange column (7.5 x 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% CH3CN and mixed with ~l 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 (~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%.
Examule XLVII - Preparation of lilln-L64:
[00384] This compound was synthesized by incubating 10 ~g L64 ligand (5 ~L of a 2 mg/mL solution in 0.01 N HCl), 60 ~L ethanol, 1.12 mCi of IIInCl3 in O.OSN HCl (80 ~,L) and 155 ~.L sodium acetate buffer (0.5M, pH 4.5) at 85°C for 30 min.
Free' IIIn was scavenged by adding 20 ~L of a 1 % Na2EDTA.2H20 (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 Cl 8 column, [4.6 x 250 mm], a column temperature of 50°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 "'In-L64 is 15.7 min. The isolated compound had an RCP of 96.7%.
Example XLVIII - Preparation of ~~~Lu-DO3A-monoamide-Aoc-OWAVGHLM-NH2 Control [00385] A stock solution of peptide was prepared by dissolving D03A-monoamide-Aoc-QWAVGHLM-NH2 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. ~~~Lu- D03A-monoamide-Aoc-QWAVGHLM-NH2 was prepared by mixing the following reagents in the order shown.
0.2 M NH40Ac, pH 6.8 100 ~L
Peptide stock, 1 mg/mL, in 0.01 N HCl 5 ~L
~~~LuCl3 (MURR) in 0.05M HCl 1.2 ~,L (1.4 mCi) [00386] The reaction mixture was incubated at 85 °C for 10 min. After cooling down to room temperature in a water bath, 20 ~L of a 1 % EDTA solution and 20 ~L 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/H20 and B is 0.1 %TFA/CH3CN). ' ~~Lu--D03A-monoamide-Aoc-QWAVGHLM-NH2 was formed in 97.1 % yield (RCP) and had a retention time of 16.1 min on this system.
Example XLIX - Preparation of l~~Lu-L63 [00387] This compound was prepared as described for l~~Lu- D03A-monoamide-Aoc-QWAVGHLM-NH2. 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/H20 and B is 0.1 %TFA/CH3CN). The'~~Lu-L63 that formed had an RCP of 97.8% and a retention time of 14.2 min on this system.
Example L - Preparation of ~~~Lu-L70 for cell binding and biodistribution studies:
[00388] 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 x 150 mm), a column temperature of 30°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% CH3CN/20% CH30H. The isolated compound had an RCP of 100%
and an HPLC retention time of 25.4 min.
Example LI - Preparation of ~~~Lu- L70 for radiotherapy studies:
[00389] This compound was prepared as described above for L64.
Example LII - Preparation of ~~lIn-L70 for cell binding and biodistribution studies:
[00390] This compound was synthesized by incubating 10 ~g L70 ligand (10 ~L of a 1 m~mL solution in 0.01 N HCI), 180 ~L ammonium acetate buffer (0.2M, pH 5.3), 1.1 mCi of "'InCl3 in O.OSN HCl (61 pL, Mallinckrodt) and 50 ~L of saline at 85°C for 30 min. Free "'In was scavenged by adding 20 ~.L of a 1 % Na2EDTA~2H20 (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 x 50 mm), a column temperature of 30°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 "'In-L70 is 15 min while the retention time for L70 ligand is 27 to 28 min. The isolated compound had an RCP of 96%.
[00391 ] Samples for biodistribution and cell binding studies were prepared by collecting the desired HPLC peak into 500 ~.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 ~Ci/mL within 30 minutes of the in vivo study.
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 LIII - IsZ vivo Pharmacokinetic Studies A. Tracer dose biodistribution:
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]-Foxnl <nu>). In all studies, mice were administered 100 ~L of ~~~Lu-labeled test compound at 200 ~Ci/kg, i.v., with a residence time of 1 and 24 h per goup (n=3-4). Tissues were analyzed in an LKB 1282 CompuGamma counter with appropriate standards.

[00392] Pharmacokinetic comparison at l and 24 h in PC3 tumor-bearing nude mice (200 ~.Cilkg; values as % ID/g) of ~~~Lu-177 labeled compounds of this invention compared to control monoamide-Aoc-QWAVGHL

M-NHZ

Tissue control L63 L64 L70 1 hr 24 1 hr 24 1 hr 24 1 24 hr hr hr hr 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 Pancreas28.60 1.01 54.04 1.62 77.78 6.56 42.3440.24 [00393] 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, DO3A-monoamide-Aoc-QWAVGHLM-NH2), 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 the control compound D03A-monoamide-Aoc-QWAVGHLM-NH2.

Example LIV-Receptor Subtype Specificity [00394] Currently, four mammalian members of the GRP receptor family are known: the GRP-prefernng receptor (GRP-R), neuromedin-B preferring receptor (NMB-R), the bombesin receptor subtype 3 (BB3-R) and the bombesin receptor subtype 4 (BB4-R). The receptor subtype specificity of ~~~Lu-L70 was investigated. The results indicate ~~~Lu-L70 binds specifically to GRP-R and NMB-R, and has little affinity for BB3-R.
[00395] The subtype specificity of the Lutetium complex of L70 (here, l~~Lu-L70) (prepared as described sutara) was determined by in vitro receptor autoradiography using the procedure described in Reubi et al., "Bombesin Receptor Subtypes in Human Cancers:
Detection with the Universal Radioligand IZSI-[D-Tyr6, beta-Ala, Phel3, N1e14]", CIin.Cancer Res. 8:1139-1146 (2002) and tissue samples that had been previously found to express only one subtype of GRP receptor, as well as non-neoplastic tissues including normal pancreas and colon, as well as chronic pancreatitis (shown below in Table 8a). Human ileal carcinoid tissue was used as a source for NMB-R, human prostate carcinoma for GRP-R and human bronchial carcinoid for BB3-R subtype receptors. For comparison, receptor autoradiography was also performed with other bombesin radioligands, such as ~ZSI-Tyr4-bombesin or a compound known as the Universal ligand, ~2sI-[DTyrb, (3A1a11, Phel3, Nlel4]-BBN(6-14), which binds to all three subsets of GRP-R, on adjacent tissue sections. For further discussion, see Fleischmann et al., "Bombesin Receptors in Distinct Tissue Compartments of Human Pancreatic Diseases," Lab. Invest. 80:1807-1817 (2000); Markwalder et al., "Gastin-Releasing Peptide Receptors in the Human Prostate: Relation to Neoplastic Transformation,"
Cancer Res. 59:1152-1159 (1999); Gugger et al., "GRP Receptors in Non-Neoplastic and Neoplastic Human Breast," Am. J. Pathol. 155:2067-2076 (1999).

Detection of bombesin receptor subtypes in various human tissues using different radioligands.
Receptor Receptor autoradiography autoradiography Tumor n using using ~~~Lu-L70 standard BN

radioligands*

Mammary Ca 8 8/8 0/8 0/8 8/8 0/8 0/8 Receptor Receptor autoradiography autoradiography Tumor n using using ~~~Lu-L70 standard BN

radioligands*

Prostate Ca 4 4/4 0/4 0/4 4/4 0l4 0/4 Renal Ca 6 5/6 0/6 O/6 4/6 0/6 0/6 heal carcinoid8 O/8 8/8 0/8 0/8 8/8 0/8 Bronchial 6 2/6 0/6 0/6 2/6 (weak)0/6 6/6 (weak) carcinoid Colon Ca - tumor 7 3/7 0/7 0/7 3/7 (weak)0/7 0/7 (weak) - smooth 7 7/7 0/7 0/7 7/7 0/7 0/7 muscle Pancreas Ca 4 0/4 0/4 0l4 0/4 0/4 0/4 Chronic 5 5/5 0/5 0/5 5/5 0/5 0/5 pancreatitis (acini) Human pancreas7 1/7 0/7 0/7 0/7 0/7 0/7 (weak) (acini) Mouse pancreas4 4/4 0/4 0/4 4/4 0l4 0/4 (acini) * i2sl-[DTyr6, ~iAlal~, Phe~3, NIe~4]-BBN(6-I4) and lasl-Tyr4-BBN.
[00396] A seen from Table 8a, all GRP-R-expressing tumors such as prostatic, mammary and renal cell carcinomas, identified as such with established radioligands, were also visualized in vitro with l~~Lu-L70. Due to a better sensitivity, selected tumors with low levels of GRP-R could be identified with l~~Lu-L70, but not with l2sl-Tyr4-BBN, as shown in Table 8a. All NMB-R-expressing tumors identified with established radioligands were also visualized with l~~Lu-L70. Conversely, none of the BB3 tumors were detected with i~~Lu-L70. One should not make any conclusion on the natural incidence of the receptor expression in the various types of tumors listed in Table 8a, as the tested cases were chosen as receptor-positive in the majority of cases, with only a few selected negative controls. The normal human pancreas is not labeled with l~~Lu-L70, whereas the mouse pancreas is strongly labeled under identical conditions. Although the normal pancreas is a very rapidly degradable tissue and one can never completely exclude degradation of protein, including receptors, factors suggesting that the human pancreas data are truly negative include the positive control of the mouse pancreas under similar condition and the strongly labeled BB3 found in the islets of the respective human pancreas, which represent a positive control for the quality of the investigated human pancreas. Furthermore, the detection of GRP-R in pancreatic tissues that are pathologically altered (chronic pancreatitis) indicate that GRP-R, when present, can be identified under the chosen experimental conditions in this tissue. In fact, ~~~Lu-L70 identifies these GRP-R in chronic pancreatitis with greater sensitivity than ~25I-Tyr4-BBN. While none of the pancreatic cancers had measurable amounts of GRP-R, a few colon carcinomas showed a low density of heterogeneously distributed GRP
receptors measured with l~~Lu-L70 (Table 8a). It should further be noticed that the smooth muscles of the colon express GRP-R and were detected in vitro with l~~Lu-L70 as well as with the established bombesin ligands.
TABLE r3B
[00397] Binding affinity of 1~$Lu-L70 to the 3 bombesin receptor subtypes expressed in human cancers. Data are expressed as ICso in nM (mean ~ SEM. n = number of experiments in parentheses).
Compound B. NMB-R C. GRP-R BB3 Universal ligand0.8 0.1 (3) 0.7 0.1 (3) l .l 0.1 (3) msLu-L70 0.9 0.1 (4) 0.8 0.1 (5) >1,000 (3) [00398] As shown in Table 8b, the cold labeled x~SLu-L70 had a very high affinity for human GRP and NMB receptors expressed in human tissues while it had only low affinity for BB3 receptors. These experiments used ~25I-[DTyr6, (3A1a11, Phe~3, Nle~4]-BBN(6-14) as radiotracer. Using the ~~~Lu-labeled L70 as radiotracer, the above mentioned data are hereby confirmed and extended. All GRP-R-expressing human cancers were very strongly labeled with ~~~Lu-L70. The same was true for all NMB-R-positive tumors. Conversely, tumors with BB3 were not visualized. The sensitivity of ~~~Lu-L70 seems better than that of Iasl-Tyr4-BBN or the'25I-labeled universal bombesin analog. Therefore, a few tumors expressing a low density of GRP-R can be readily identified with'~~Lu-L70, while they are not positive with'ZSI-Tyr4-BBN. The binding characteristics of ~~~Lu-L70 could also be confirmed in non-neoplastic tissues. While the mouse pancreas, as control, was shown to express a very high density of GRP-R, the normal human pancreatic acini were devoid of GRP-R.
However, in conditions of chronic pancreatitis GRP-R could be identified in acini, as reported previously in Fleisclunann et al., "Bombesin Receptors in Distinct Tissue Compartments of Human Pancreatic Diseases," Lab. Invest. 80:1807-1817 (2000) and tissue, again with better sensitivity by using l~~Lu-L70 than by using'25I-Tyr4-BBN.
Conversely, the BB3-expressing islets were not detected with l~~Lu-L70, while they were strongly labeled with the universal ligand, as reported previously in Fleischmann et al., "Bombesin Receptors in Distinct Tissue Compartments of Human Pancreatic Diseases," Lab. Invest.
80:1807-1817 (2000). While a minority of colon carcinomas had GRP-R, usually in very low density and heterogeneously distributed, the normal colonic smooth muscles expressed a high density of GRP-R.
[00399] The results in Tables 8a and 8b indicate that Lu labeled L70 derivatives are expected to bind well to human prostate carcinoma, which primarily expresses GRP-R. They also indicate that Lu labeled L70 derivatives are not expected to bind well to normal human pancreas (which primarily expresses the BB3-R receptor), or to cancers which primarily express the BB3-R receptor subtype.
Examine LV - Radiotherapy Studies A. Efficac~Studies:
[00400) Radiotherapy studies were performed using the PC3 tumor-bearing nude mouse model. In Short Term Efficacy Studies, '~~Lu labeled compounds of the invention L64, L70, L63 and the treatment control compound D03A-monoamide-Aoc-QWAVGHLM-NH2 were compared to an untreated control group. (n=12 for each treatment group for up to 30 days, and n=36 for the pooled untreated control group for up to 31 days). For all efficacy studies, mice were administered 100 p.L of'~~Lu-labeled 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 the duration of the study. 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 cm3. Results of the Short Term Efficacy Study are displayed in FIG.15A. 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 the D03A-monoamide-Aoc-QWAVGHLM-NH2 control.
[00401] Long Term Efficacy Studies were performed with L64 and L70 using the same dose as before but using more animals per compound (n=46) and following them for up to 120 days. The results of the Long Term Efficacy Study are displayed in FIG.15B. Relative to the same controls as before (n=36), both L64 and L70 treatment gave significantly increased survival (p<0.0001) with L70 being better than L64, although not statistically different from each other (p<0.067).
Example LVI
Alternative Preparation of L64 and L70 Using Segment Coupling [00402] Compounds L64 and L70 can be prepared employing the collection of intermediates generally represented by A-D (FIG.19), 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.
[00403] These methods include Aloc, Boc, Fmoc or benzyloxycarbonyl-based peptide synthesis strategies or judiciously chosen combinations ofthose 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.
[00404] This is amply illustrated in FIG. 20 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.
EXAMPLE LVII - Figures 49A and 49B
Synthesis of L69 [00405] Summary: Reaction of (313,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid A with Fmoc-Cl gave intermediate B. Rink amide resin functionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NHZ (BBN[7-14]) (A), was sequentially reacted with B, Fmoc-8-amino-3,6-dioxaoctanoic acid and DOTA tri-t-butyl ester. After cleavage and deprotection with Reagent B the crude was purified by preparative HPLC to give L230.
Overall yield: 4.2%.
A. (3J3,SJ3,7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oic acid, B (FIG. 49A) [00406] A solution of 9-fluorenylmethoxycarbonyl chloride (1.4 g; 5.4 mmol) in 1,4-dioxane (18 mL) was added dropwise to a suspension of (313,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid A (2.0 g; 4.9 mmol) (3) in 10% aq.
Na2C03 (30 mL) and 1,4-dioxane (18 mL) stirred at 0 °C. After 6 h stirring at room temperature H20 (100 mL) was added, the aqueous phase washed with Et20 (2 x 90 mL) and then 2 M HCl (15 mL) was added (final pH: 1.5). The precipitated solid was filtered, washed with H20 (3 x 100 mL), vacuum dried and then purified by flash chromatography to give B as a white solid (2.2 g;
3.5 mmol). Yield 71 %.
B. N-[313,513,7a,12a)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino] ethoxy]ethoxy] acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-g-l~yl-L-histidyl-L-leucyl-L-methioninamide, L69 (FIG. 49B1 [00407] 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 filtered and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was filtered and the resin washed with DMA (5 x 7 mL). (313,513,7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oic acid B (0.75 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, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution 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 x 7 mL). Fmoc-8-amino-3,6-dioxaoctanoic acid (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, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was filtered and the resin washed with DMA (5 x 7 mL) 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), N-ethyldiisopropylamine (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, filtered and the resin washed with DMA (5 x 7 mL), CH2Cl2 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) (2) for 4.5 h. The resin was filtered and the solution was evaporated under reduced pressure to afford an oily crude that after treatment with EtzO (20 mL) gave a precipitate.
The precipitate was collected by centrifugation and washed with Et20 (3 x 20 mL) to give a solid (248 mg) which was analysed by HPLC. An amount of crude (50 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L69 (6.5 mg; 3.5 x 10-3 mmol) (FIG.
49B) as a white solid. Yield 5.8%.
EXAMPLE LVIII - Figure 50 Synthesis of L144 [00408] Summary: Rink amide resin functionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NHZ (BBN[7-14]) (A) was reacted with 4-[2-hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-2-oxoethylJ-1,4,7,10-tetrazacyclododec-1-yl)propoxy]
benzoic acid.
After cleavage and deprotection with Reagent B (2) the crude was purified by preparative HPLC to give L144. Overall yield: 12%.
A. N [4-[2-Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec 1-yl]propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L
histidyl-L-leucyl-L-methioninamide, L144 (FIG. 50) [00409] Resin A (0.4 g; 0.24 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was filtered and the resin washed with DMA (5 x 7 mL). 4-[2-Hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]propoxy] benzoic acid B (0.5 g;
0.7 mmol), HOBt (0.11 g; 0.7 mmol), DIC (0.11 mL; 0.7 mmol)), N
ethyldiisopropylamine (0.24 mL; 1.4 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken for 24 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL), CHZC12 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) (2) for 4.5 h. The resin was filtered and the solution was evaporated under reduced pressure to afford an oily crude that after treatment with Et20 (20 mL) gave a precipitate.
The precipitate was collected by centrifugation and washed with Et20 (3 x 20 mL) to give a solid (240 mg) which was analysed by HPLC. An amount of crude (60 mg) was purified by preparative HPLC. The fractions containing the product were lyophilised to give L144 (10.5 mg; 7.2 x 10-3 mmol) as a white solid. Yield 12%.
EXAMPLE LIX
Preparation of L300 and ~~~Lu-L300 [00410] From 0.2 g of Rink amide Novagel resin (0.63 mmol/g, 0.126 mmol), L300 (0.033 g, 17%) was obtained after preparative column chromatography. The retention time was 6.66 minutes. The molecular formula is C~aH99N190~s. The calculated molecular weight is 151 x.71; 1519.6 observed. The sequence is D03A-Gly-Abz4-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH2. The structure of L300 is shown in Figure S1.

[00411 ] L300 (13.9 ~g in 13.9 ~L of 0.2M pH 4.8 sodium acetate buffer) was mixed with 150 ~L of 0.2M pH 4.8 sodium acetate buffer and 4 ~L of ~~~LuCl3 (1.136 mCi, Missouri Research Reactor). After 10 min at 100°C, the radiochemical purity (RCP) was 95%. The product was purified on a Vydac C18 peptide colunm (4.6 x 250 mm, 5 um pore size) eluted at a flow rate of 1 mL/min using an aqueous/organic gradient of 0.1 % TFA in water (A) and 0.085% TFA in acetonitrile (B). The following gradient was used: isocratic 22%
B for 30 min, to 60% B in 5 min, hold at 60% B for 5 min. The compound, which eluted at a retention time of 18.8 min., was collected into 1 mL of an 0.8% human serum albumin solution that was prepared by adding HSA to a 9:1 mixture of normal saline and Ascorbic Acid, Injection.
Acetonitrile was removed using a Speed Vacuum (Savant). After purification, the compound had an RCP of 100%.
EXAMPLE LX - Characterization of Linker Specificity in Relation to GRP
Receptor Subtypes [00412] Two cell lines, C6, an NMB-R expressing rodent glioblastoma cell line and PC3, a GRP-R expressing human prostate cancer cell line, were used in this assay.
The affnity of various unlabeled compounds for each receptor subtype (NMB-R and GRP-R) was determined indirectly by measuring its ability to compete with the binding of ~ZSI-NMB or izsl-BBN to its corresponding receptors in C6 and PC3 cells.
A. Materials and Methods:
1. Cell Culture:
[00413] C6 cells were obtained from ATCC (CCL-107) and cultured in F12K media (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate, 15% horse serum and 2.5% FBS. Cells for the assays were plated at a concentration of 9.6 x 104/ well in 48 well poly-lysine coated plates (Beckton Dickinson Biocoat). PC3 were obtained from ATCC (CRL-1435) and cultured in RPMI 1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate, 10 mM HEPES and 10% FBS. Both cultures were maintained in a humidified atmosphere containing 5% C02/95% air at 37°C.
PC3 cells for the assays were plated at a concentration of 2.0 x 104 cells/
well in 96-well white/clear bottom plates (Falcon Optilux-I). Plates were used for the assays on day 2 of the post-plating.

2. Binding buffer, and radio-li ands:
[00414] RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0 mMAEBSF (CAS # 3087-99-7) and 0.1 % Bacitracin (CAS # 1405-87-4), pH
7.4.
Custom made ~2sI-[Tyr°]NMB, > 2.0 Ci/pmole (Amersham Life Science) [~2sI-NMB] and commercially available ~ZSI-[Tyr4]BBN, >2.0 Cilpmole (Perkin Elmer Life Science) [l2sl-BBN] were used as radioligands.
B. In vitro Assay:
[00415] Using a 48-well plate assay system (for C6 study) competition experiments were performed using ~ZSI-NMB. All of the PC3 studies were performed as described in Example ~LIII using ~2sI-BBN. Selection of compounds for the assay was based on linker subtype. Results are shown in Table 9.

Number of selected compounds for the assay and their linkers biphenyl) [00416] The binding parameters obtained from the studies were analyzed using a one-site competition non-linear regression analysis with GraphPad Prism. The relative affinity of various compounds for NMB-R in C6 cells were compared with those obtained using commercially available [Tyr4]-BBN and [Tyr°]-NMB. To distinguish the GRP-R preferring compounds from NMB-R plus GRP-R prefernng compounds, ICs° values obtained for each compound was compared with those obtained from [Tyr°]-BBN with ~zsI-NMB
on C6 cells.
The cut off point between the two classes of compounds was taken as 1 OX the ICs° of [Tyr4]-BBN. Among the compounds tested, 8 compounds preferentially bind to GRP-R (as shown in Table 10) while 32 compounds bind to both GRP-R and NMB-R with similar affinity, and two show preference for NMB-R.

The ICS° values obtained from competition experiments using 125I-NMB
and 125I-BBN
L # COMPOUND ICSO (nM) GRP-R GRP-R

~zsl-BBN 'zsI-NMB

lPC3 !C6 NMB-R

na N,N-dimethylglycine-Ser-10 10.4 - yes Cys(Acm)-Gly-SS-WAVGHLM-NHz na N,N-dimethylglycine-Ser-25 7.9 - yes Cys(Acm)-Gly -G-WAVGHLM-NHz na N,N-dimethylglycine-Ser-48 20.2 - yes Cys(Acm)-Gly -GG-WAVGHLM-NHz na N,N-dimethylglycine-Ser-13 6.4 - yes Cys(Acm)-Gly -KK-WAVGHLM-NHz na N,N-dimethylglycine-Ser-2 2.2 - yes Cys(Acm)-Gly -SK-WAVGHLM NHz na N,N-dimethylglycine-Ser-1.9 2.0 yes Cys(Acm)-Gly -SR- -na N,N-dimethylglycine-Ser-7.5 24.1 Cys(Acm)-Gly -KS- yes -WAVGHLM-NHz na N,N-dimethylglycine-Ser-32 60.0 Cys(Acm)-Gly -KE- yes -WAVGHLM-NHz na D03A-monoamide-Aoc- 3.4 3.1 yes WAVGHLM-NH2 _ na D03A-monoamide -Apa3- 36 18. 9 yes WAVGHLM-NHz _ na D03A-monoamide Abu4- 19.8 5.2 yes WAVGHLM-NHz _ J., # COMPOUND ICso (nM) GRP-R GRP-R

~zsl-BBN 'ZSI-NMB &

L3 N,N-dimethylglycine-Ser-70 33 yes Cys(Acm)-Gly - DJ- -WAVGHLM-NHZ

L64 DO3A-monoamide -G- 8.5 3.3 yes Adca3- WAVGHLM-NH2 _ L63 DO3A-monoamide -G- 23 3.8 yes Ahl2ca- WAVGHLM-NH2 _ L67 DO3A-monoamide -G-Akca-5.5 2.3 yes WAVGHLM- NHZ _ na D03A-monoamide -Cha- 22 77 -Cha- WAVGHLM- NH2 yes na D03A-monoamide -Nal1- 30 210.9 -Bi - WAVGHLM- NH2 yes na DO3A-monoamide -Cha- 8 66.5 -Na11- WAVGHLM- NH2 yes na D03A-monoamide -Nall- 17 89.9 -B a4- WAVGHLM- NH2 yes L301 D03A-monoamide -Amb4- 10 6.8 yes Nall- WAVGHLM- NH2 L147 D03A-monoamide -G- 4 32 Mo3abz4-QWAVGHLM- yes -L24I D03A-monoamide -G- 4 0.8 Cl3abz4 WAVGHLM- NH2 - yes L242 D03A-monoamide -G- 5 2.2 M3abz4-QWAVGHLM- - yes L243 DO3A-monoamide -G- 14 9.9 Ho3abz4-QWAVGHLM- - yes L202 D03A-monoamide -G- 13 2.7 - yes H bz4- WAVGHLM- NH2 L204 D03A-monoamide -Abz4- 50 1.2 - yes L233 D03A-monoamide -G- 4.8 1.6 - yes Abz3- WAVGHLM- NHZ

L235 D03A-monoamide -G- 7 1.5 Nmabz4-QWAVGHLM- - yes NHa L147 D03A-monoamide - 3.5 1.2 Mo3amb4-QWAVGHLM- - yes L71 D03A-monoamide -Amb4- 7.2 0. 2 - yes WAVGHLM- NHa L73 D03A-monoamide -Aeb4- 5 1.8 - yes I~ # COMPOUND ICso (nM) GRP-R GRP-R

izsl_BBN 'ZSI-NMB &

L208 D03A-monoamide -Dae- 8 0.9 - yes T a- WAVGHLM- NHZ

L206 D03A-monoamide -G- 5 1.3 A4m2biphc4- - yes WAVGHLM- NHZ

L.207 D03A-monoamide -G- 3 15.1 A3biphc3-QWAVGHLM- - yes L72 D03A-monoamide -Amc4-8.2 2.6 - yes L107 D03A-monoamide -Amc4-5 0.3 - yes Amc4- WAVGHLM- NH2 L89 D03A-monoamide -Aepa4-23 114 yes -L28 N,N-dimethylglycine-Ser-25 13 Cys(Acm)-Gly - Aepa4-S- - yes L74 D03A-monoamide -G-Inp-6.5 3.4 - yes L36 N,N-dimethylglycine-Ser-7 12.1 Cys(Acm)-Gly - Pial-J- - yes L82 D03A-monoamide -Ckbp-8 1.7 -yes na D03A-monoamide -Aoc- 11 14 -yes WAVGHL-Nle-NH2 L70 D03A-monoamide -G- 4.5 1.5 -yes Abz4- WAVGHLM- NHz na D03A-monoamide - 366 >250 No selective WAVGHLM- NHS reference na QWAVGHLM- NHZ 369 754 No selective reference na WAVGHLM -NHS >800 >800 No selective reference L204 DO3A-monoamide -Abz4->50 1.2 preference to NMB-na GNLWATGHFM-NH2 >500 0.7 preference to NMB-R

L227 D03A-monoamide -G- 28 0.8 - Ye Abz4-LWATGHFM -NHa s In the above Table "na" indicates "not applicable" (e.g., the compound does not contain a linker of the invention and thus was not assigned an L#).

[00417] Based on the above, several results were observed. The receptor binding region alone (BBN~_~4 or BBN$_~4) did not show any preference to GRP-R or NMB-R. The addition of a chelator alone to the receptor binding region did not contribute to the affinity of the peptide to GRP-R or NMB-R (D03A-monoamide -QWAVGHLM-NH2). Coupling the chelator to the peptide through a linker did contribute to the affinity of the peptide towards the receptor. However, depending on the type of linker this affinity varied from being dual (preference for both NMB-R and GRP-R) to GRP-R (preferring GRP-R).
[00418] The cn-Aminoalkanoic acids tested (8-Aminooctanoic acid in ~~SLu- D03A-monoamide-Aoc-QWAVGHLM-NH2 and D03A-monoamide -Aoc-QWAVGHL-Nle-NH2 , 3-aminopropionic acid in D03A-monoamide -Apa3-QWAVGHLM-NH2 and 4-aminobutanoic acid in D03A-monoamide-Abu4-QWAVGHLM-NH2) as linkers, conferred the peptide with dual affinity for both GRP-R and NMB-R. Replacement of 'Met' in I~SLu-DOTA-Aoc-QWAVGHLM NH2 by 'Nle' did not change this dual affinity of the peptide.
[00419] Cholic acid containing linkers (3-aminocholic acid in L64, 3-amino-12-hydroxycholanic in L63 and 3-amino-12-ketocholanic in L67 conferred the peptides with dual affinity for both GRP-R and NMB-R. Cycloalkyl and aromatic substituted alanine containing linkers (3-cyclohexylalanine in DO3A-monoamide -Cha-Cha-QWAVGHLM-NH2, 1-Naphthylalanine in D03A-monoamide-Cha-Nal 1-QWAVGHLM-NH2, 4-Benzoylphenylalanine in DO3A-monoamide -Nall-Bpa4-QWAVGHLM-NH2 and Biphenylalanine in DO3A-monoamide -Nall-Bip-QWAVGHLM-NHZ) imparted the peptides with selective affinity towards GRP-R. A linker containing only 4-(2-Aminoethylpiperazine)-1 also contributed to the peptides with GRP-R
selectivity (L89).
[00420] Introduction of G-4-amino benzoic acid linker to NMB sequence conferred the compound with an affinity to GRP-R in addition to its inherent NMB-R affinity (L227 vs GNLWATGHFM-NHz). Shifting the position of Gly around the linker altered the affinity of L70 from its dual affinity to a selective affinity to NMB-R (L204). 3-methoxy substitution in 4-aminobenzoic acid in L70 (as in L240) changed the dual affinity to a selective affinity to GRP-R.
[00421 ] It is apparent from the preceding data that the linker has a significant effect on the receptor subtype specificity. Three groups of compounds can be identified:

~:~ Those that are active at the GRP-R
[00422] These compounds provide information specific to this receptor in vitro and in vivo, which can be used for diagnostic purposes. When these compounds are radiolabeled with a therapeutic radionuclide, therapy can be performed on tissues containing only this receptor, sparing those that contain the NMB-R
~:~ Those that are active at the NMB-R
[00423] These compounds provide information specific to this receptor in vitro and in vivo, which can be used for diagnostic purposes. When radiolabeled with a therapeutic radionuclide, therapy can be performed on tissues containing only this receptor, sparing those that contain the GRP-R
~:~ Those that are active at both the GRP-R and the NMB-R
[00424] These compounds provide information on the combined presence of these two receptor subtypes in vitro and in vivo, that can be used for diagnostic purposes. Targeting both receptors may increase the sensitivity of the examination at the expense of specificity. When these compounds are radiolabeled with a therapeutic radionuclide, therapy can be performed on tissues containing both receptors, which may increase the dose delivered to the desired tissues.
Example LXI - Competition studies of modified Bombesin (BBN) analogs with lzsl-BBN
for GRP-R in human prostate cancer (PC3) cells [00425] To determine the effect of replacing certain amino acids in the BBN ~-~4 analogs, peptides modified in the targeting portion were made and assayed for competitive binding to GRP-R in human prostate cancer (PC3) cells. All these peptides have a common linker conjugated to a metal chelating moiety (DOTA-Gly-Abz4-). The binding data (ICSO nM) are given below in Table 13.
A. Materials and Methods:
1. Cell Culture:
[00426] PC3 cell lines were obtained from ATCC (CRL-1435) and cultured in RPMI
1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 glL Sodium bicarbonate, 10 mM HEPES and 10% FBS. Cultures were maintained in a humidified atmosphere containing 5% C02/95% air at 37 °C. PC3 cells for the assays were plated at a concentration of 2.0 x 104 cells/ well in a 96-well white/clear bottom plates (Falcon Optilux-I). Plates were used for the assays on day 2 of the post-plating.
2. Binding buffer:
[00427] RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0 mM AEBSF (CAS # 3087-99-7) and 0.1 % Bacitracin (CAS # 1405-87-4), pH
7.4.
3. ~2sI-Tyr4-Bombesin [~ZSI-BBNI
[00428] ~2sI-BBN (Cat # NEX258) was obtained from PerkinElmer Life Sciences.
C. In vitro Assay:
[00429] Competition assay with ~ZSI-BBN for GRP-R in PC3 cells:
[00430] All compounds tested were dissolved in binding buffer and appropriate dilutions were also done in binding buffer. PC3 cells for assay were seeded at a concentration of 2.0 x 104 /well either in 96-well black/clear collagen I
cellware plates (Beckton Dickinson Biocoat). Plates were used for binding studies on day 2 post- plating. The plates were checked for confluency (>90%
confluent) prior to assay. For competition assay, N,N-dimethylglycyl-Ser-Cys(Acm)-Gly-AvaS-QWAVGHLM-NH2 (control) or other competitors at concentrations ranging from 1.25x 10-9 M to 500 x 109 M, was co-incubated with lasl-BBN (25,000 cpmlwell). The studies were conducted with an assay volume of 75 ~l per well. Triplicate wells were used for each data point.
After the addition of the appropriate solutions, plates were incubated for 1 hour at 4°C. Incubation was ended by the addition of 200 uL of ice-cold incubation buffer. Plates were washed 5 times and blotted dry. Radioactivity was detected using either a LIMB CompuGamma counter or a microplate scintillation counter. The bound radioactivity of ~2sI-BBN was plotted against the inhibition concentrations of the competitors, and the concentration at which l2sl-BBN binding was inhibited by 50% (ICso) was obtained from the binding curve.
TABLE 13: Competition studies with lzsl-BBN for GRP-R in PC3 cells ICso L # PEPTIDES [nM]

N,N-dimethylglycyl-Ser-Cys(Acm)-Gly-AvaS-Ref na QWAVGHLM-NHZ 2.5 1 L70 D03A-monoamide-G-Abz4-QWAVGHLM-NHZ 4.5 2 L214 D03A-monoamide-G-Abz4-fQWAVGHLM-NHZ 18 3 L215 D03A-monoamide-G-Abz4-QRLGNQWAVGHLM-NH2 6 4 L216 D03A-monoamide-G-Abz4-QRYGNQWAVGHLM-NH2 4.5 L217 D03A-monoamide-G-Abz4-QKYGNQWAVGHLM-NHZ 10 >EQ-[K(D03A-monoamide-G-Abz4)-LGNQWAVGHLM-7 L219 D03A-monoamide-G-Abz4-fQWAVGHLM-NH-CSH~Z 75 8 L220 DO3A-monoamide-G-Abz4-QWAVaHLM-NH2 13 9 L221 D03A-monoamide-G-Abz4-fQWAVGHLL-NHZ 340 L222 DO3A-monoamide-G-Abz4-yQWAV-Ala2-HF-Nle-NH246 11 L223 D03A-monoamide-G-Abz4-FQWAV-Ala2-HF-Nle-NH252 12 L224 D03A-monoamide-G-Abz4-QWAGHFL-NH2 >500 13 L225 D03A-monoamide-G-Abz4-LWAVGSFM-NH2 240 14 L226 D03A-monoamide-G-Abz4-HWAVGHLM-NH2 5.5 L227 D03A-monoamide-G-Abz4-LWATGHFM-NH2 39 16 L228 D03A-monoamide-G-Abz4-QWAVGHFM-NHZ 5.5 17 na GNLWATGHFM-NHZ >500 18 na yGNLWATGHFM-NH2 450 19 L300 D03A-monoamide-G-Abz4-QWAVGHFL-NH2 2.5 5 [00431 ] Results/Conclusions: Analysis of the binding results of various peptides modified in the targeting portion indicated the following:

[00432] Neuromedin analogs (GNLWATGHFM-NHZ, yGNLWATGHFM-NH2) are unable to compete for the GRP-R except when conjugated to D03A-monoamide-G-Abz4 (L227).
They are, however, effective NMB competitors. This is similar to the requirement for derivatisation of the amino end of the bombesin sequence as reflected in QWAVGHLM-NHZ, D03A-monoamide-QWAVGHLM-NHS & L70. Replacement of the histidine (L225) reduces competition at the GRP-R.
[00433] Reversal of the two linker components in L70 to give L204 changes the subtype specificity to favor the NMB subtype. L~3F substitution in the bombesin sequence maintains GRP-R activity. (L228).

ICso L Number Sequence C6/NMB- PC3/GRP
R -R

na GNLWATGHFM-NHZ 0.69 >500 na yGNLWATGHFM-NH2 0.16 884.6 L227 D03A-monoamide-G-Abz4-LWATGHFM-NH2 0.07 28.0 L225 D03A-monoamide-G-Abz4-LWAVGSFM-NH2 - 240 na WAVGHLM-NH2 >800 >800 na QWAVGHLM-NHZ 369 754 na D03A-monoamide-QWAVGHLM-NHZ 161 366 L70 D03A-monoamide-G-Abz4-QWAVGHLM-NH2 4.5 1.5 L204 D03A-monoamide-Abz4-GQWAVGHLM-NH2 1.19 >50 L228 ~ D03A-monoamide-G-Abz4-QWAVGHFM-NH2 ~ 5.5 ~

[00434] As seen here, Fl3Mia to F13L14 substitution in L228 produces a compound (L300) with high activity at the GRP-R. The removal of the methionine has advantages as it is prone to oxidation. This benefit does not occur if the L13F substitution is not also performed 1 S (L221) . Removal of V 1° resulted in complete loss of binding as seen in L224.

Number Sequence C6/NMB- PC3/GRP-R R

L300 D03A-monoamide-G-Abz4-QWAVGHFL-NHZ - 2.5 L221 D03A-monoamide--G-Abz4-f WAVGHLL-NH2- 340 L224 D03A-monoamide-G-Abz4-QWA GHFL-NH2 - >500 As seen in Table 16, various substitutions are allowed in the BBN2-6 region (L214-L217, L226) Number Sequence IC50 R R

pEQRYGNQWAVGHLM-NH2 3.36 2.2 na L214 D03A-monoamide-G-Abz4-fQWAVGHLM-NHZ - 18 D03A-monoamide-G-Abz4-D03A-monoamide-G-Abz4-L216 QRYGNQWAVGHLM-NH2 - 4.5 D03A-monoamide-G-Abz4-L217 QKYGNQWAVGHLM-NHa - 10 L226 D03A-monoamide-G-Abz4-HWAVGHLM-NH2 - 5.5 As expected, results from Table 17 show that the universal agonists (L222 &
L223) compete reasonably well at ~ 50 nM level.

Name Number Sequence IC50 R R

Universal D03A-monoamide-G-Abz4-a onist L222 y WAV-Ala2-HF-Nle-NH2 - 46 Universal D03A-monoamide-G-Abz4-a onist L224 F WAV-Ala2-HF-Nle-NH2 - 52 EXAMPLE LXI - NMR Structural Comparison of ~~sLu-L70 and I~sLu-D03A-monoamide-Aoc-QWAVGHLM-NHZ
[00435} The purpose of this NMR study was to provide complete structural characterization of Lu-L70 and compare it to the structure of I~SLu-DOTA-Aoc-QWAVGHLM. L70 and ~~SLu- DOTA-Aoc-QWAVGHLM are both bombesin analogues (see Figures 60 and 61), differing only in the linker between the chelating group and the targeting peptide. In L70 there is a glycyl-4-aminobenzoyl group, whereas in I~SLu-DOTA-Aoc-QWAVGHLM there is an S-aminooctanoyl group. However, the biological data of these two compounds is strikingly different. Detailed NMR studies were performed to explain this difference.
A. EXPERIMENTAL
1. Materials [00436} 5 mg of I~SLu-D03A-monoamide-Aoc-QWAVGHLM-NH2 was dissolved in 225 uL of DMSO-d6 (Aldrich 100% atom %D).
5 mg of l~sLu-L70 was dissolved in 225 uL of DMSO-d6 (Aldrich 100% atom %D).
2. Acquisition of NMR Data [00437} All NMR experiments were performed on a Varian Inova-500 Fourier Transform NMR spectrometer equipped with a 3 mm broad-band inverse (z-axis gradient) probe. The chemical shifts were referenced to the residual CH peaks of DMSO-d6 at 2.50 ppm for the proton and 40.19 ppm for 13C. The sample temperatures were controlled by a Varian digital temperature controller. The data were processed using NMRPipe, VNMR, PROSA, and VNMRJ software on the Sun Blade 2000 Unix computer and analyzed using NMRView and SPARI~Y software on the Linux computer. The modeling of the peptides was performed .
employing CYANA software on the Linux computer and further analyzed using MOLMOL
software on a Compaq Deskpro Workstation.
B. RESULTS and DISCUSSION
[00438 The proton chemical shifts of ~~SLu-L70 were assigned as follows. A
quick survey of the methyl region (0.5 to 2.5 ppm) in the 1D spectrum allowed the identification of a sharp singlet at 2.02 ppm as the methyl peak of methionine. In the same region of the TOCSY
spectrum, the chemical shift at 1.16 ppm which correlates to only one peak at 4.32 ppm indicates that they belong to alanine. The methyl peaks at 0.84 and 0.85 ppm which correlate to two peaks at 1.98 and 4.12 ppm must belong to valine. The remaining methyl peaks at 0.84 and 0.88 ppm which correlate to peaks at 1.60, 1.48, and 4.23 ppm belong to leucine.
These chemical shifts and the chemical shifts of other amino acids are also present in the "fingerprint" region (see Wuthrich, K. "NMR of Proteins and Nucleic Acids,"
John Wiley &
Sons, 1986) - the backbone NH-aH region of the TOCSY spectrum (see FIG. 52).
All the chemical shifts belonging to a spin system of an amino acid will align themselves vertically.
After a careful examination of the spectrum, all chemical shifts were assigned. The chemical shifts were further verified by reviewing other spectra such as COSY (see FIG.
53) and NOESY (see FIG. 54). After the proton chemical shifts were assigned, their carbon chemical shifts were identified through the gHSQC spectrum (see FIG. 55) and further verified by reviewing the gHMBC (see FIG. 56) and gHSQCTOCSY (see FIG. 57) spectra. The chemical shifts of Lu-L70 are listed in Table 19 (the atom numbers are referenced to FIG.
60).
[00439] Interestingly, in the TOCSY spectrum of l~SLu-L70, the chemical shift of the NH
proton at 14.15 ppm shows strong correlations to two other peaks of the histidine ring, and also to a water molecule. This water molecule is not freely exchanging and is clearly seen in the NMR timeframe. To see which proton of the histidine interacts more strongly with the water molecule, a selective homo-decoupling experiment was performed on the ~~SLu-L70 at 15 °C. When the water peak was selectively saturated with a low power, the intensities of the NH peaks of histidine at 14.16 and 14.23 ppm were dramatically reduced while the intensities of the two remaining peaks of histidine at 7.32 and 8.90 ppm were partially reduced (see FIG. 58). The observation of the water protons on the NMR time scale suggests a rigid confirmation.
[00440] A proposed chemical structure of ~~$Lu-L70 with a water molecule can be seen in FIG. 62. A water molecule occupies a ninth coordination site by capping the square plane described by the coordinated oxygens. This has other precedents. Coordination of water at the ninth site of Lu in Na[Lu(DOTA)H20)]~4Hz0 was observed in an x-ray structure, as shown by Aime et al, Inorg. Chim. Acta 1996, 246, 423-429, which is incorporated by reference.
[00441] In contrast, in the TOCSY spectrum of'~SLu-DO3A-monoamide-Aoc-QWAVGHLM-NH2, the chemical shift of the NH proton only shows strong correlations to two other peaks of the histidine ring, but not to the water molecule (see FIG.
59). This indicates that there is no water molecule simultaneously coordinating both the I~SLu and the His-NH in'~SLu-D03A-monoamide-Aoc-QWAVGHLM-NH2. Thus, the difference between the two molecules is significant. In the I~SLu-L70 a secondary structure of the peptide is stabilized via the bound water molecule, and this may be responsible for increased in vivo stability.
TABLE 19 - Chemical Shifts (ppm) of ~~SLu-L70 in I?MSO-d6 at 25 °C
Position Chemical Shift Assi nment Proton Carbon -17 3.69/3.62 22 9.95/9.73 23 4.04/4.16 (43.57) 26 10.47 28/32 7.62 (118.9) 29/31 7.79 (128.7) Position Chemical Shift Assi nment Proton Carbon 35a 8.54 36 4.29 (54.26) 39 1.83/1.91 (27.26) 40 2.16 (32.08) 47 6.84/7.3 0 43 7.97 44 4.54 (53.37) 48 2.98/3.12 (27.74) SO 7.12 (123.9) 51 10.79 53 7.53 (118.7) 54 6.93 ( 118.6) 55 7.03 (121.3) 56 7.28 (111.7) 58 8.09 59 4.32 (48.71) 62 1.16 (17.86) 63 7.65 64 4.12 (58.28) 67 1.98 (30.96) 68/73 0.84 (18.42) 0.85 (19.52) 69 8.19 70 3.70/3.74 (42.45) 74 8.10 75 4.60 (51.85) 78 2.95/3.08 (27.50) 80 14.15 81 8.91 83 7.32 84 8.14 Position Chemical Shift Assi nment Proton Carbon 85 4.23 (51.93) 86 1.48 (40.6) 87 1.60 (24.61 88/91 0.84 (21.8) 0.88 (23.41) 92 8.04 93 4.25 (52.25) 96 1.76/1.92 (32.16) 97 2.4I (29.91 ) 99 2.02 (15.13)

Claims (27)

1. A compound selected from the group consisting of:
DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-QWAVaHLM

DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-f-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-f-WAVGHLL

DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-f-QWAVGHL
NH-pentyl DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-y-QWAV-Bala-H-F-Nle-NH2 DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-f-QWAV-Bala-H-F-Nle-NH2 DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-QWAVGHFL-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-QWAVGNMeH-L-M-NH2 DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-LWAVGSF-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-LWAGHFM-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-DO3A-monoamide-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-Pglu-Q-Lys (DO3A-monoamide)-Gly-(3.beta.,5.beta. 7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic acid-DO3A-monoamide-Gly-3-amino-3-deoxycholic acid- QRLGNQWAVGHLM-NH2 DO3A-monoamide-Gly-3-amino-3-deoxycholic acid- QRYGNQWAVGHLM-NH2 DO3A-monoamide-Gly-3-amino-3-deoxycholic acid- QKYGNQWAVGHLM-NH2; and Pglu-Q-Lys(D03A-monoamide- G-3-amino-3-deoxycholic acid)-LGNQWAVGHLM-NH2.

2. A compound selected from the group consisting of:
DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFL-NH2 DO3A-monoamide- 4-aminomethylbenzoic acid-L-1-Naphthylalanine-QWAVGHLM-NH2;
and DO3A-monoamide-G-4-aminobenzoic acid-QWAVGNMeHisLM-NH2.

3. A compound of the general formula:
M-N-O-P-G
wherein M is DO3A, 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;
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 8-amino-3,6-dioxaoctanoic acid; and wherein the GRP receptor targeting peptide is selected from the group consisting of QWAVGHLM-NH2, QWAVGNMeHLM-NH2, QWAVGHFL-NH2, LWATGSFM-NH2 and QWAVaHLM-NH2.

4. A compound of the general formula:
M-N-O-P-G
wherein M is DO3A, 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 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 (3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid; and wherein the GRP receptor targeting peptide is selected from the group consisting of QWAVGHLM-NH2, QWAVGNMeHLM-NH2, QWAVGHFL-NH2 LWATGSFM-NH2 and QWAVaHLM-NH2.

5. A compound of the general formula:
M-N-O-P-G
wherein M is DO3A, 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;
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 4-aminobenzoic acid; and wherein the GRP receptor targeting peptide is selected from the group consisting of QWAVGHLM-NH2, QWAVGNMeHLM-NH2, QWAVGHFL-NH2, LWATGSFM-NH2, QWAVaHLM-NH2, Nme-QWAVGHLM-NH2, Q-.PSI.[CSNH]WAVGHLM-NH2, Q-.PSI.[CH2NH]-WAVGHLM-NH2, Q-.PSI.[CH=CH]WAVGHLM-NH2, a-MeQWAVGHLM-NH2, QNme-WAVGHLM-NH2, QW-.PSI.[CSNH]-AVGHLM-NH2, QW-.PSI.[CH2NH]-AVGHLM-NH2, QW-.PSI.[CH=CH]-AVGHLM-NH2, Q-a-Me-WAVGHLM-NH2, QW-Nme-AVGHLM-NH2, QWA=.PSI.[CSNH]-VGHLM-NH2, QWA-.PSI.[CH2NH]-VGHLM-NH2, QW-Aib-VGHLM-NH2, QWAV-Sar-HLM-NH2, QWAVG-.PSI.[CSNH]-HLM-NH2, QWAVG-.PSI.[CH=CH]-HLM-NH2, QWAV-Dala-HLM-NH2, QWAVG-Nme-His-LM-NH2, QWAVG-H-.PSI.[CSNH]-L-M-NH2, QWAVG-H-.PSI.[CH2NH]-LM-NH2, QWAVGH-.PSI.[CH=CH]-LM-NH2, QWAVG-a-Me-HLM-NH2, QWAVGH-Nme-LM-NH2, QWAVGH-a-MeLM-NH2, QWAVGHF-L-NH2 and QWAVGHLM-NH2.

6. A compound selected from the group consisting of:
DO3A-monoamide-G-4-aminobenzoic acid-QWAVaHLM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-fQWAVGHLM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-fQWAVGHLL-NH2, DO3A-monoamide-G-4-aminobenzoic acid-fQWAVGHL-NH-pentyl, DO3A-monoamide-G-4-aminobenzoic acid-yQWAV-Bala-HFNle-NH2, DO3A-monoamide-G-4-aminobenzoic acid-fQWAV-Bala-HFNle-NH2, DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFL-NH2, DO3A-monoamide-G-4-aminobenzoic acid-QWAVGNMeHisLM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-LWAVGSFM- NH2, DO3A-monoamide-G-4-aminobenzoic acid-HWAVGHLM- NH2, DO3A-monoamide-G-4-aminobenzoic acid-LWATGHFM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-QRLGNQWAVGHLM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-QRYGNQWAVGHLM-NH2, DO3A-monoamide-G-4-aminobenzoic acid-QKYGNQWAVGHLM-NH2, Pglu-Q-Lys(DO3A-monoamide- G-4-aminobenzoic acid)-LGNQWAVGHLM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QWAVaHLM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-fQWAVGHLM- NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-fQWAVGHLL-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-fQWAVGHL-NH-pentyl, DO3A-monoamide-G-3-amino-3-deoxycholic acid-yQWAV-Bala-HFNle-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-fQWAV-Bala-HFNle- NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QWAVGHFL-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QWAVGNMeHLM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-LWAVGSFM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-HWAVGHLM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-LWATGHFM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QWAVGHFM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QRLGNQWAVGlyHLM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QRYGNQWAVGHLM-NH2, DO3A-monoamide-G-3-amino-3-deoxycholic acid-QKYGNQWAVGHLM-NH2, and Pglu-Q-Lys(DO3A-monoamide-G-3-amino-3-deoxycholic acid)-LGNQWAVGHLM-NH2.

7. A compound of the general formula:
M-N-O-P-G
wherein M is DO3A, 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;
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 8-amino-3,6-dioxaoctanoic acid or (3.beta.,S.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid; and wherein G is selected from the group consisting of Nme-QWAVGHLM-NH2, Q-.PSI.[CSNH]WAVGHLM-NH2, Q-.PSI.[CH2NH]-WAVGHLM-NH2, Q-.PSI.[CH=CH]WAVGHLM-NH2, .alpha.-MeQWAVGHLM-NH2, QNme-WAVGHLM-NH2, QW-.PSI.[CSNH]-AVGHLM-NH2, QW-.PSI.[CH2NH]-AVGHLM-NH2, QW-.PSI.[CH=CH]-AVGHLM-NH2, Q-.alpha.-Me-WAVGHLM-NH2, QW-Nme-AVGHLM-NH2, QWA=.PSI.[CSNH]-VGHLM-NH2, QWA-.PSI.[CH2NH]-VGHLM-NH2, QW-Aib-VGHLM-NH2, QWAV-Sar-HLM-NH2, QWAVG-.PSI.[CSNH]-HLM-NH2, QWAVG-.PSI.[CH=CH]-HLM-NH2, QWAV-Dala-HLM-NH2, QWAVG-Nme-His-LM-NH2, QWAVG-H-.PSI.[CSNH]-L-M-NH2, QWAVG-H-.PSI.[CH2NH]-LM-NH2, QWAVGH-.PSI.[CH=CH]-LM-NH2, QWAVG-.alpha.-Me-HLM-NH2, QWAVGH-Nme-LM-NH2, QWAVGH-.alpha.-MeLM-NH2, QWAVGHF-L-NH2 and QWAVGHLM-NH2.

8. A method for targeting the gastrin releasing peptide receptor (GRP-R) and neuromedin-B receptor (NMB-R), said method comprising administering a compound of the general formula:
M-N-O-P-G
wherein M is an optical label or 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;
P is 0, an alpha or non-alpha amino acid or other linking group;
G is a GRP receptor targeting peptide; and wherein at least one of N, O or P is a non-alpha amino acid.

9. The method of claim 8, wherein at least one of N, O or P is a non-alpha amino acid with a cyclic group.

10. The method of claim 9, wherein N is Gly, O is 4-aminobenzoic acid and P is 0.

11. A method of targeting the GRP-R and the NMB-R, said method comprising administering a compound of the general formula:

M-N-O-P-G

wherein M is an optical label or a metal chelator, optionally complexed with a radionuclide;
N is 0, an alpha amino acid, a substituted bile acid or other linking group;
O is an alpha amino acid or a substituted bile acid;
P is 0, an alpha amino acid, a substituted bile acid or other linking group;
G is a GRP receptor targeting peptide; and wherein at least one of N, O or P is a substituted bile acid.

12. The method of claim 11, wherein N is Gly, O is (3.beta.,5.beta.,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid, and P is 0.

13. The method of any one of claims 8, 9 or 12, wherein the GRP receptor targeting peptide is selected from the group consisting of:
Nme-QWAVGHLM- NH2, Q-.PSI.CSNH]WAVGHLM-NH2, Q-.PSI.[CH2NH]-WAVGHLM-NH2, Q-.PSI.[CH=CH]WAVGHLM-NH2, .alpha.-MeQWAVGHLM-NH2, QNme-WAVGHLM-NH2, QW-.PSI.[CSNH]-AVGHLM- NH2, QW-.PSI.[CH2NH]-AVGHLM-NH2, QW-.PSI.[CH=CH]-AVGHLM-NH2, Q-.alpha.-Me-WAVGHLM-NH2, QW-Nme-AVGHLM-NH2, QWA=.PSI.[CSNH]-VGHLM- NH2, QWA-.PSI.[CH2NH]-VGHLM-NH2, QW-Aib-VGHLM-NH2, QWAV-Sar-HLM-NH2, QWAVG-.PSI.[CSNH]-HLM-NH2, QWAVG-.PSI.[CH=CH]-HLM-NH2, QWAV-Dala-HLM-NH2, QWAVG-Nme-His-LM-NH2, QWAVG-H-.PSI.[CSNH]-L-M-NH2, QWAVG-H-.PSI.[CH2NH]-LM-NH2, QWAVGH-.PSI.[CH=CH]-LM-NH2, QWAVG-.alpha.-Me-HLM-NH2, QWAVGH-Nme-LM-NH2, and QWAVGH-.alpha.-MeLM-NH2.

14. A method of improving the in vivo activity of a compound of any one of claims 1 through 7, comprising the step of modifying the GRP receptor targeting peptide so as to reduce proteolytic cleavage of said peptide.

15. The method of claim 14, wherein the modified GRP-R targeting peptide is an agonist.

16. A method of reducing proteolytic cleavage of a gastrin releasing peptide (GRP) analogue of any one of claims 1 through 7, said method comprising the step of modifying the peptide bond in the GRP-R targeting moiety.

17. The method of claim 16, wherein the modified GRP-R targeting peptide is an agonist.

18. A method of reducing proteolytic cleavage of a gastrin releasing peptide (GRP) analogue having a gastrin releasing peptide receptor (GRP-R) targeting moiety that is an agonist, said method comprising the step of modifying the peptide bond in the GRP-R
targeting moiety.

19. The method of any one of claims 14, 16 or 18, wherein the GRP-R targeting moiety is selected from the group consisting of:

Nme-QWAVGHLM- NH2, Q-.PSI.[CSNH]WAVGHLM-NH2, Q-.PSI.[CH2NH]-WAVGHLM-NH2, Q-.PSI.[CH=CH]WAVGHLM-NH2, .alpha.-MeQWAVGHLM-NH2, QNme-WAVGHLM-NH2, QW-.PSI.[CSNH]-AVGHLM- NH2, QW-.PSI.[CH2NH]-AVGHLM-NH2, QW-.PSI.[CH=CH]-AVGHLM- NH2, Q-.alpha.-Me-WAVGHLM-NH2, QW-Nme-AVGHLM-NH2, QWA=.PSI.[CSNH]-VGHLM- NH2, QWA-.PSI.[CH2NH]-VGHLM-NH2, QW-Aib-VGHLM-NH2, QWAV-Sar-HLM-NH2, QWAVG-.PSI.[CSNH]-HLM-NH2, QWAVG-.PSI.[CH=CH]-HLM-NH2, QWAV-Dala-HLM-NH2, QWAVG-Nme-His-LM-NH2, QWAVG-H-.PSI.[CSNH]-L-M-NH2, QWAVG-H-.PSI.[CH2NH]-LM-NH2, QWAVGH-.PSI.[CH=CH]-LM-NH2, QWAVG-.alpha.-Me-HLM-NH2, QWAVGH-Nme-LM-NH2, and QWAVGH-.alpha.-MeLM-NH2.

20. A compound according to any one of claims 1 through 7, wherein G is a GRP
receptor targeting peptide that has been modified so as to reduce proteolytic cleavage.

21. A method of conferring specificity for the GRP-R and/or the NMB-R on a compound comprising an optical label or metal chelator optionally complexed with a radionuclide and a GRP-R targeting peptide, comprising including in such compound a linker of the general formula:

N-O-P

wherein N is 0, an alpha or non-alpha amino acid or other linking group;
O is an alpha or non-alpha amino acid;
P is 0, an alpha or non-alpha amino acid or other linking group; and wherein at least one of N, O or P is a non-alpha amino acid.

22. A method of conferring specificity for the GRP-R and/or the NMB-R on a compound comprising an optical label or metal chelator optionally complexed with a radionuclide and a GRP-R targeting peptide, comprising including in such compound a linker of the general formula:

N-O-P

wherein N is 0, an alpha amino acid, a substituted bile acid or other linking group;
O is an alpha amino acid or a substituted bile acid;
P is 0, an alpha amino acid, a substituted bile acid or other linking group; and wherein at least one of N, O or P is a substituted bile acid.

23. A method of conferring specificity for the GRP-R and/or the NMB-R on a compound comprising an optical label or metal chelator optionally complexed with a radionuclide and a GRP-R targeting peptide, comprising including in such compound a linker of the general formula:

N-O-P

wherein N is 0, an alpha amino acid, a non-alpha amino acid with a cyclic group or other linking group;

O is an alpha amino acid or a non-alpha amino acid with a cyclic group;
P is 0, an alpha amino acid, a non-alpha amino acid with a cyclic group or other linking group; and wherein at least one of N, O or P is a non-alpha amino acid with a cyclic group.

24. A method of improving the in vivo activity of a compound comprising an optical label or metal chelator optionally complexed with a radionuclide and a GRP-R
targeting peptide, comprising including in such compound a linker of the general formula:

N-O-P

wherein N is 0, an alpha or non-alpha amino acid or other linking group;
O is an alpha or non-alpha amino acid;
P is 0, an alpha or non-alpha amino acid or other linking group; and wherein at least one of N, O or P is a non-alpha amino acid.

25. A method of improving the in vivo activity of a compound comprising an optical label or metal chelator optionally complexed with a radionuclide and a GRP-R
targeting peptide, comprising including in such compound a linker of the general formula:

N-O-P

wherein N is 0, an alpha amino acid, a substituted bile acid or other linking group;
O is an alpha amino acid or a substituted bile acid;
P is 0, an alpha amino acid, a substituted bile acid or other linking group; and wherein at least one of N, O or P is a substituted bile acid.

26. A method of improving the in vivo stability of a compound comprising an optical label or metal chelator optionally complexed with a radionuclide and a GRP-R
targeting peptide, comprising including in such compound a linker of the general formula:

N-O-P

wherein N is 0, an alpha amino acid, a non-alpha amino acid with a cyclic group or other linking group;

O is an alpha amino acid or a non-alpha amino acid with a cyclic group;
P is 0, an alpha amino acid, a non-alpha amino acid with a cyclic group or other linking group; and wherein at least one of N, O or P is a non-alpha amino acid with a cyclic group.

27. A compound having the following structure: