Radiolabeled bombesin derivative compounds for in vivo imaging of gastrin-releasing peptide receptor (GRPR) and treatment of GRPR-related disorders

Abstract

There is provided a derivative compound of frombesin of the formula Ia (RX-L-Xaa1-Gln-Trp-Ala-Val-Xaa2-His-Xaa3-psi-Xaa4-NH2), as well as a preparation method and application of the derivative compound of frombesin. RX comprises a radionuclide chelating agent or a prothetic group containing trifluoroborate. L is a linker. Xaa1 is D-Phe, Cpa (4-chlorophenyl alanine), D-Cpa, Tpi (2, 3, 4, 9-tetrahydro-1H-pyrido [3, 4b] indole-3-carboxylic acid), D-Tpi, Nal (naphthyl alanine), or D-Nal, or a pharmaceutically acceptable salt thereof. Xaa2 is Gly, N-methyl-Gly, or D-Ala, or a pharmaceutically acceptable salt thereof. And Xaa3 is Leu, Pro, D-Pro, or Phe. And Xaa4 is Pro, Phe, Tac (thiazolidine-4-formic acid), Nle (n-leucine) or 4-oxa-L-Pro (oxazolidine-4-formic acid). The symbol psi represents a reduced peptide bond between Xaa3 and Xaa4, where psi is CH2-N when Xaa4 is Pro, Tac or 4-oxa-L-Pro, or psi is CH2N (R) when Xaa4 is Phe or Nle, where R is H or a C1-C5 linear or branched alkyl group. Also provided are uses of such compounds as imaging agents or therapeutic agents.

Description

Technical Field

[0001] The present invention relates to radiolabeled compounds for in vivo imaging or treatment of diseases or disorders characterized by gastrin-releasing peptide receptor expression. Background Art

[0002] The gastrin-releasing peptide receptor (GRPR) is a G-protein coupled receptor of the bombesin (BBN) receptor family (1-3). GRPR, together with its endogenous ligand gastrin-releasing peptide (GRP), is involved in synaptic plasticity, mood and feeding behavior, hormone secretion, smooth muscle contraction, and cell proliferation (1-3). Under normal conditions, the expression of GRPR is restricted to the central nervous system, pancreas, adrenal cortex, and gastrointestinal tract (4). GRPR has also been associated with tumor progression, and overexpression of GRPR has been reported in many cancer subtypes, including lung cancer, head and neck cancer, colon cancer, kidney cancer, ovarian cancer, breast cancer, and prostate cancer (5). This ectopic expression in cancer makes it an attractive target for personalized therapy.

[0003] BBN is a 14-amino acid GRPR-binding peptide (7-14). BBN derivatives have been radiolabeled for single photon emission computed tomography (SPECT), positron emission tomography (PET) imaging, and have also been radiolabeled for β and α emitter therapy (6-8). Generally, the radiolabeled group is attached directly to the structure or via a linker at the N-terminus, while modifications at the C-terminus determine agonist / antagonist properties. To target GRPR, antagonists are preferred because agonists have been shown to induce gastrointestinal adverse events (10). Examples of GRPR antagonists evaluated in clinical practice include: 68 Ga-RM2, 68 Ga-SB3, 68 Ga-NeoBOMB1, 68 Ga-RM26, 18 F-BAY-864367, and 64 Cu-CB-TE2A-AR06 (9, 11-16).

[0004] It has been reported that reduction of the peptide bond between residues 13 and 14 of BBN to -CH 2 -NH- yielded a potent antagonist (17). Based on this work, others reported a series of pseudononapeptide BBN antagonists with a reduced bond (CH 2 -NH or CH 2 -N) between amino acid residues 13 and 14 of BBN (Leu 13 ψTac 14)(18). Several of those exhibit picomolar binding affinities for murine GRPR, and some are capable of inhibiting the growth of prostate, breast, colon, lung, liver, pancreatic, ovarian, renal, and glioma cancers in preclinical models (5, 19 - 23).

[0005] In the field of improved tracers for non-invasive in vivo imaging of GRPR, there remains an unmet need. Such tracers can be used to diagnose conditions associated with abnormal / ectopic expression of GRPR, including but not limited to cancer (e.g., prostate cancer). There also remains an unmet need for improved radiotherapeutic agents for treating diseases / disorders associated with abnormal / ectopic expression of GRPR, including but not limited to cancer (e.g., prostate cancer).

[0006] It is not necessarily intended to admit, nor should it be construed that any of the foregoing information constitutes prior art against the present invention. SUMMARY OF THE INVENTION

[0007] Various embodiments of the present disclosure relate to compounds of formula Ia (R X -L-Xaa 1 -Gln-Trp-Ala-Val-Xaa 2 -His-Xaa 3 -ψ-Xaa 4 -NH 2 ), wherein: R X comprises a radionuclide chelator or a boron trifluoride-containing auxochrome; L is a linker; Xaa 1 is D-Phe, Cpa (4-chlorophenylalanine), D-Cpa, Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid), D-Tpi, Nal (naphthylalanine), or D-Nal; Xaa 2 is Gly, N-methyl-Gly, or D-Ala; Xaa 3 is Leu, Pro, D-Pro, or Phe; Xaa 4 is Pro, Phe, Tac (thiazolidine-4-carboxylic acid), Nle (norleucine), 4-oxa-L-Pro (oxazolidine-4-carboxylic acid); and ψ represents a reduced peptide bond between Xaa 3 and Xaa 4 , wherein, when Xaa 4 is Pro, Tac or 4-oxa-L-Pro, ψ is or when Xaa 4 is Phe or Nle, ψ is -CH 2 N(R)-, wherein, R is H or C 1 -C 5A straight-chain or branched-chain alkyl group.

[0008] In some embodiments, R X comprises a radionuclide chelator. The radionuclide chelator may be selected from the group consisting of: DOTA and derivatives; DOTAGA; NOTA; NODAGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and optionally DTPA analogs selected from CHX-A”-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; optionally sarcophagine and sarcophagine derivatives selected from SarAr, SarAr-NCS, diamSar, AmBaSar and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; H2-macropa or its derivatives; H 2 dedpa, H 4 octapa, H 4 py4pa, H 4 Pypa, H 2 azapa, H 5 dedpa, and other picolinic acid derivatives; CP256; PCTA; C-NETA; C-NE3TA; HBED; SHBED; BCPA; CP256; YM103; deferoxamine (DFO) and DFO derivatives; H 6 phospa; trithiol chelates; mercaptoacetyl; hydrazinonicotinamide; dimercaptosuccinic acid; 1,2-ethylenediyldi-L-cysteine diethyl ester; methylenediphosphonate; hexamethylpropyleneamine oxime; and hexakis (methoxyisobutyl isonitrile). In certain embodiments, the radionuclide chelator is selected from DOTA and DOTA derivatives.

[0009] In some embodiments, R X further comprises a radioactive metal, a radionuclide-binding metal or a prosthetic group containing a radionuclide-binding metal, and wherein the radioactive metal, the radionuclide-binding metal or the prosthetic group containing a radionuclide-binding metal chelates with the radionuclide-chelator complex. The radioactive metal, the radionuclide-binding metal or the prosthetic group containing a radionuclide-binding metal may be: 68 Ga, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 111 In, 44 Sc, 86 Y, 89Zr, 90 Nb, 177 Lu, 117m Sn, 165 Er, 90 Y, 227 Th, 225 Ac, 213 Bi, 212 Bi, 72 As, 77 As, 211 At, 203 Pb, 212 Pb, 47 Sc, 166 Ho, 188 Re, 186 Re, 149 Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb, 142 Pr, 114m In, 94m Tc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, or 18 F]AlF. In certain embodiments, the radioactive metal, the radionuclide-binding metal, or the moiety containing the radionuclide-binding metal is: 68 Ga, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 111 In, 44 Sc, 86 Y, 177 Lu, 90 Y, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 225 Ac, 213 Bi, or 212 Bi.

[0010] In some embodiments, R X comprises one or more moieties containing trifluoroborate. R X may comprise one or more R 1 R 2 BF 3group, wherein: each R 1 is independently wherein, each R 3 is independently absent, each R 2 BF 3 is independently: wherein, each R 4 is independently C 1 -C 5 a straight-chain or branched-chain alkyl group, and each R 5 is independently C 1 -C 5 a straight-chain or branched-chain alkyl group,

[0011] wherein, each pyridine-substituted -OR, -SR, -NR-, -NHR or -NR 2 the R in is independently a branched or straight-chain C 1 -C 5 alkyl group. R X may contain one or more than one R 1 R 2 BF 3 wherein: each R 1 is independently wherein, each R 3 is independently absent, each R 2 BF 3 is independently: wherein, each R 4 is independently C 1 -C 5 a straight-chain or branched-chain alkyl group, and each R 5 is independently C 1 -C 5 a straight-chain or branched-chain alkyl group,

[0012]

[0013] wherein, each pyridine-substituted -OR, -SR, -NR-, -NHR or -NR 2 the R in is independently a branched or straight-chain C 1 -C 5 alkyl group. In certain embodiments, R X contains a single R 1 R 2 BF 3group. In certain embodiments, R X comprises two R 1 R 2 BF 3 group. One or more trifluoroborate-containing auxiliaries may comprise 18 F.

[0014] In some embodiments, the linker is a peptide linker (Xaa 5 ) 1-4 , wherein each Xaa 5 is independently a proteinogenic amino acid residue or a non-proteinogenic amino acid residue. In some embodiments, the linker is a peptide linker (Xaa 5 ) 1-4 , wherein each Xaa 5 is independently a proteinogenic amino acid residue or a non-proteinogenic amino acid residue, wherein each peptide backbone amino group is independently optionally methylated, and wherein each non-proteinogenic amino acid residue is independently selected from the group consisting of: D-amino acids of proteinogenic amino acids, N ε ,N ε ,N ε -trimethyl-lysine, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine (Orn), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp), β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), sulfopropylamine, hyaluronic acid, p-aminomethylaniline-diglycolic acid (pABzA-DIG), 4-amino-1-carboxymethyl-piperidine (Pip), NH 2 (CH 2 ) 2 O(CH 2 ) 2 C(O)OH, NH 2 (CH 2 ) 2 [O(CH 2 ) 2 2 C(O)OH (dPEG2), NH 2 (CH 2 ) 2 [O(CH 2 ) 2 3 C(O)OH, NH 2 (CH​​2 ) 2 [O(CH 2 ) 2 4 C(O)OH, NH 2 (CH 2 ) 2 [O(CH 2 ) 2 5 C(O)OH, and NH 2 (CH 2 ) 2 [O(CH 2 ) 2 6 C(O)OH. In some embodiments, the linker is p - aminomethylbenzoic acid - diglycolic acid (pABzA - DIG), 4 - amino - (1 - carboxymethyl) piperidine (Pip), 9 - amino - 4,7 - dioxanonanoic acid (dPEG2), or 4 - (2 - aminoethyl) - 1 - carboxymethyl - piperazine (Acp). In certain embodiments, the linker is pABzA - DIG or Pip.

[0015] In some embodiments, Xaa 1 is D - Phe. In some embodiments, Xaa 2 is Gly. In some embodiments, Xaa 3 is Leu. In some embodiments, Xaa 4 is Pro, Tac, or 4 - oxa - L - Pro. In some embodiments, Xaa 4 is Pro. In some embodiments, Xaa 1 is D - Phe, Xaa 2 is Gly, Xaa 3 is Leu, and Xaa 4 is Pro.

[0016] Various embodiments of the present disclosure relate to a compound having the following chemical structure or a salt or solvate thereof, optionally chelated with a radionuclide X:

[0017] . In some embodiments, X is: 68 Ga, 64 Cu, 67 Cu, 67 Ga, 111 In, 177 Lu, 90 Y, or 225 Ac. In other embodiments, X is: 68 Ga or 177 Lu.​​​

[0018] Various embodiments of the present disclosure relate to a compound having the following chemical structure or a salt or solvate thereof, optionally chelated with a radionuclide X:

[0019] . In some embodiments, X is: 68 Ga, 64 Cu, 67 Cu, 67 Ga, 111 In, 177 Lu, 90 Y, or 225 Ac. In other embodiments, X is: 68 Ga or 177 Lu. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The features of the invention will become apparent from the following description with reference to the accompanying drawings, in which:

[0021] Figure 1 Chemical structures of the prior art compound RC-3950-II (top) and Ga-NeoBOMB1 (bottom) are shown.

[0022] Figure 2 Graph showing intracellular calcium efflux in PC-3 cells. Cells were incubated with 50 nM of Ga-ProBOMB1, H-3042 ([D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14)), bombesin, ATP, or buffer control. p ≤ 0.001 compared to buffer control. *** p≤0.001.

[0023] Figure 3 Maximum intensity projections of PET / CT and PET alone of (A) 68 Ga-NeoBOMB1 and (B) 68 Ga-ProBOMB1 obtained in mice bearing PC-3 tumor xenografts at 1 or 2 h P.i. Blockade was performed with co-injection of 100 μg of [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14). Scale bar is in units of 0 to 15 %ID / g (% injected dose / gram of tissue), where white at the bottom of the bar represents 0 %Id / g and black at the bottom of the bar represents 15 %Id / g: t = tumor; l = liver; p = pancreas; b = intestine; bl = bladder.

[0024] Figure 4 shows 68 Ga-NeoBOMB1 and 68 the biodistribution of Ga-ProBOMB1 in selected tissues at multiple time points ( * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

[0025] Figure 5 shows the biodistribution of Ga-ProBOMB1 at 60 min p.i. with or without co-injection of 100 μg of [D-Phe 6 ,Leu-NHEt 13 ,des-Met 14 bombesin(6-14) ([[]] 68 p ≤ 0.001). *** p ≤ 0.001).

[0026] Figure 6 shows 68 the HPLC chromatogram indicating the plasma stability of Ga-ProBOMB1 at 5 min p.i. A minor metabolite peak M1 was observed at t R = 2.72 min on the HPLC chromatogram.

[0027] Figure 7 shows 68 Ga-NeoBOMB1 and 68 the plot of absorbed dose per unit injection activity of Ga-ProBOMB1 in mice.

[0028] Figure 8 shows 125 I-Tyr 4 bombesin produced by [D-Phe 6 ,Leu-NHEt 13 ,des-Met 14 bombesin(6-14) (H3042), Ga-NeoBOMB1 and Ga-ProBOMB1 representative displacement curves.

[0029] Figure 9 is a plot representing the FLIPR calcium 6 release assay in PC-3 cells. The cells were incubated with Ga-ProBOMB1, H-3042 ([D-Phe 6 ,Leu-NHEt 13 ,des-Met 14Incubate with bombesin(6 - 14), bombesin, ATP, or PBS control. The y-axis is relative fluorescence units (RFU), and the x-axis is time (sec).

[0030] Figure 10 is a composite graph of plots showing the 68 time-dependent uptake of Ga-NeoBOMB1 in the pancreas, blood, kidney, and PC-3 tumor. The total number of decays per unit injected dose was calculated by multiplying the area under the curve by the phantom organ mass. The y-axis is the percentage of injected dose per gram of tissue (%ID / g), and the x-axis is time (h).

[0031] Figure 11 is a composite graph of plots showing the 68 time-dependent uptake of Ga-ProBOMB1 in the pancreas, blood, kidney, and PC-3 tumor. The total number of decays per unit injected dose was calculated by multiplying the area under the curve by the phantom organ mass. The y-axis is the percentage of injected dose per gram of tissue (%ID / g), and the x-axis is time (h).

[0032] Figure 12 shows the maximum intensity projections of PET / CT and of PET alone of Ga-ProBOMB2 obtained in mice bearing PC-3 tumor xenografts at 1 h, 2 h, and 1 h post-injection (p.i.) with a blockage carried out by co-injection of 100 μg of [D-Phe 68 , Leu-NHEt 6 , des-Met 13 , des-Met 14 bombesin(6 - 14). The scale bar is in units of 0 to 5 %ID / g (% injected dose / gram of tissue), where white at the bottom of the bar represents 0 %Id / g and black at the bottom of the bar represents 5 %Id / g.

[0033] Figure 13 shows the biodistribution of Ga-ProBOMB2 in mice bearing PC-3 prostate cancer xenografts at 60 min and 120 min p.i. 68

[0034] Figure 14 shows indicating 68 the in vivo plasma stability of Ga-ProBOMB2 at 5 min and 15 min p.i. by HPLC chromatogram. A minor metabolite peak M1 was observed at t R = 2.7 min on the HPLC chromatogram.

[0035] Figure 15 shows the 125 I-Tyr 4 ​Composite plot of representative displacement curves of bombesin produced by Ga-ProBOMB2 (left) and Lu-ProBOMB2 (right).

[0036] Figure 16 Showing murine GRPR on Swiss 3T3 cells 125 I-Tyr 4 Composite plot of representative displacement curves of bombesin produced by Ga-ProBOMB2 (left) and Lu-ProBOMB2 (right).

[0037] Figure 17 is 68 Time-activity curves of Ga-ProBOMB2 for blood, kidney, muscle, bone, and PC-3 tumor. These curves are obtained from 68 Dynamic PET imaging scans of Ga-ProBOMB2 in mice bearing PC-3 tumors. Detailed Description

[0038] As used herein, the terms "comprising," "having," "including," and "containing" and their grammatical variants are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps, even if the features / components defined as part thereof consist of or consist essentially of the specified one or more features / components. When used herein in connection with a compound, composition, use, or method, the term "consisting essentially of" means that additional elements and / or method steps may be present, but these additions do not materially affect the manner in which the recited compound, composition, method, or use functions. When used herein in connection with a feature of a compound, composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps in that feature. Compounds, compositions, uses, or methods described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments and consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically recited. Uses or methods described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments and consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically recited.

[0039] Elements recited by the indefinite article "a" do not exclude the possibility of more than one element being present, unless the context clearly dictates one and only one element. Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. When used in conjunction with the term "comprising" herein, the use of the word "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0040] In the present disclosure, the recitation of a numerical range by endpoints includes all numbers within that range, including all integers, all integers and, where appropriate, all fractional intermediates (e.g., 1 to 5 can include 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.).

[0041] Unless otherwise specified, "certain embodiments," "various embodiments," "embodiments," and like terms include one or more specific features described for that embodiment, whether directly or indirectly referenced to any other embodiment or embodiments described herein, and regardless of whether the feature or embodiment is described in the context of a method, product, use, composition, compound, etc.

[0042] As used herein, the terms "treat," "treatment," "therapeutic," etc. include alleviating symptoms, slowing disease progression, improving prognosis, and reducing recurrence.

[0043] As used herein, the term "diagnostic agent" includes "imaging agent." Thus, "diagnostic radionuclide" includes radionuclides suitable for use in imaging agents.

[0044] The term "subject" refers to an animal (e.g., a mammalian or non-mammalian animal). A subject can be a human or non-human primate. A subject can be a laboratory mammal (e.g., a mouse, rat, rabbit, hamster, etc.). A subject can be an agricultural animal (e.g., a horse, sheep, cow, pig, camel, etc.) or a domestic animal (e.g., a dog, cat, etc.). In some embodiments, the subject is a human.

[0045] The compounds disclosed herein can also include their free base forms, solvates, salts, or pharmaceutically acceptable salts. Unless otherwise specified or indicated, the compounds claimed and described herein are intended to include all racemic mixtures and all individual enantiomers or combinations thereof, whether or not explicitly shown herein.

[0046] The compounds disclosed herein may be shown to have one or more charged groups, may be shown to have ionizable groups in an uncharged (e.g., protonated) state, or may be shown to have no assigned formal charge. Those skilled in the art will understand that the ionization state of certain groups in a compound (e.g., but not limited to CO 2 H, etc.) depends particularly on the pKa of the group and the pH at that position. For example, but not limited to, carboxylic acid groups (i.e., COOH) are understood to be generally deprotonated (and negatively charged) at neutral pH and up to physiological pH values, unless the protonated state is stable.

[0047] As used herein, the terms “salt” and “solvate” have their ordinary meanings in chemistry. Thus, when a compound is a salt or solvate, it is combined with a suitable counterion. How to prepare salts or exchange counterions is well known in the art. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (e.g., but not limited to Na, Ca, Mg, or K hydroxides, carbonates, bicarbonates, etc.), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or in an organic solvent or in a mixture of both. The counterion can be changed, for example, by ion exchange techniques (such as ion exchange chromatography). All zwitterions, salts, solvates, and counterions are contemplated, unless a particular form is specifically indicated.

[0048] In certain embodiments, the salt or counterion can be pharmaceutically acceptable for administration to a subject. As used herein, “pharmaceutically acceptable” means suitable for use in a subject, and is not necessarily limited to therapeutic uses and also includes diagnostic uses. More generally, with respect to any pharmaceutical composition disclosed herein, non-limiting examples of suitable excipients include any suitable buffering agent, stabilizer, salt, antioxidant, complexing agent, tonicity agent, cryoprotectant, lyoprotectant, suspending agent, emulsifying agent, antimicrobial agent, preservative, chelating agent, binder, surfactant, wetting agent, non-aqueous carrier (such as a fixed oil or a polymer for sustained release or controlled release). See, for example, Berge et al. 1977 (J. Pharm. Sci. 66: 1-19), or Remington - The Science and Practice of Pharmacy, 21st Edition (edited by Gennaro et al., Lippincott Williams & Wilkins Philadelphia), each of which is incorporated by reference in its entirety.

[0049] As used herein, in the context of an alkyl group of a compound, the term "straight-chain" may be used as commonly understood by one of ordinary skill in the art and generally refers to a chemical entity that includes a backbone or main chain that does not break into more than one continuous chain. Non-limiting examples of straight-chain alkyl groups include methyl, ethyl, n-propyl, and n-butyl.

[0050] As used herein, the term "branched-chain" may be used as commonly understood by one of ordinary skill in the art and generally refers to a chemical entity that includes a backbone or main chain that breaks into more than one continuous chain. The portion of the backbone or main chain that breaks in more than one direction may be straight-chain. Non-limiting examples of branched-chain alkyl groups include tert-butyl and isopropyl.

[0051] As used herein, when referring to a chemical entity, the term "saturated" may be used as commonly understood by one of ordinary skill in the art and generally refers to a chemical entity that includes only single bonds and may include straight-chain and / or branched-chain groups. Saturated straight-chain or branched-chain C 1 -C 5 alkyl group non-limiting examples include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, 1,2-dimethylpropyl, and 2-ethylpropyl.

[0052] Without changing the definition of the structure on the opposite side of the wavy line, in a chemical formula (e.g., in the definition of R 2 BF 3 in formula Ia), the wavy line shown by a bond or at the end of a bond symbol is intended to define the R group on one side of the wavy line. Where the R group is bonded on two or more sides (e.g., R 1 and R 3 in formula 1a), any atom shown outside the wavy line is intended to clarify the orientation of the R group. Thus, only the atoms between the two wavy lines constitute the definition of the R group. When an atom is not shown outside the wavy line or for a chemical group that does not have a wavy line but does have bonds on multiple sides (e.g., -C(O)NH-, etc.), the chemical group should be read from left to right, matching the orientation in the chemical formula to which the group pertains (e.g., for the formula -R d -R e -R f -, the definition of R e as -C(O)NH- will be incorporated into the formula as -R d -C(O)NH-R f - rather than -R d -NHC(O)-R f - unless another orientation is explicitly intended.

[0053] In the structures provided herein, hydrogen may or may not be shown. In some embodiments, hydrogen (whether shown or implied) may be protium (i.e., 1 H), deuterium (i.e., 2 H), or a combination of 1 H and 2 H. Methods for exchanging 1 H with 2 H are well known in the art. For solvent-exchangeable hydrogen, the exchange of 1 H with 2 H occurs readily in the presence of a suitable deuterium source without any catalyst. The use of an acid, base, or metal catalyst, in combination with conditions of increased temperature and pressure, can facilitate the exchange of non-exchangeable hydrogen atoms, typically resulting in the exchange of all 1 H in the molecule to 2 H.

[0054] The term "Xaa" refers to an amino acid residue in a peptide chain or an amino acid otherwise as part of a compound. Amino acids have an amino group and a carboxylic acid group, either or both of which can be used for covalent attachment. When attached to the remainder of the compound, the amino and / or carboxylic acid group(s) can be converted to an amide or other structure; for example, when bonded to the amino group of a second amino acid, the carboxylic acid group of the first amino acid is converted to an amide (i.e., a peptide bond). Thus, Xaa can have the formula -N(R a )R b C(O)-, where R a and R b are R-groups. R a will typically be hydrogen or methyl, or R a and R bA cyclic structure can be formed. The amino acid residues of the peptide can contain typical peptide (amide) bonds and can also contain bonds between side chain functional groups and side chain or main chain functional groups of another amino acid. For example, the side chain carboxylate of one amino acid residue (e.g., Asp, Glu, etc.) in the peptide can bind to the amine of another amino acid residue (e.g., Dap, Dab, Orn, Lys) in the peptide. Further details are provided below. Unless otherwise specified, "Xaa" can be any amino acid, including proteinogenic or non-proteinogenic amino acids. Non-limiting examples of non-proteinogenic amino acids are shown in Table 1 and include: D-amino acids (including but not limited to any D-form of the following amino acids), ornithine (Orn), 3-(1-naphthyl)alanine (Nal), 3-(2-naphthyl)alanine (2-Nal), α-aminobutyric acid, norvaline, norleucine (Nle), homonorleucine, β-(1,2,3-triazol-4-yl)-L-alanine, 1,2,4-triazol-3-alanine, Phe(4-F), Phe(4-Cl), Phe(4-Br), Phe(4-I), Phe(4-NH 2 )、Phe(4-NO 2 ), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), Β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, 2-amino-3-(anthracen-2-yl)propanoic acid, 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3-(pyren-1-yl)propanoic acid, Trp(5-Br), Trp(5-OCH 3 ), Trp(6-F), Trp(5-OH) or Trp(CHO), 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), propargylglycine (Pra), homopropargylglycine (Hpg), β-homopropargylglycine (Bpg), 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), azidolysine (Lys(N 3 ), azido-ornithine (Orn(N 3 ), 2-amino-4-azidobutyric acid (Dab(N 3 ), Dab(N 3 ), 2-(5'-azidopentyl)alanine, 2-(6'-azidohexyl)alanine, 4-amino-1-carboxymethyl-piperidine (Pip), 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp), and hyaluronic acid. If not specified as an L-amino acid or a D-amino acid, the amino acid should be understood as an L-amino acid.

[0055] Table 1. List of non-limiting examples of non-proteinogenic amino acids.

[0056]

[0057] Compounds of formula Ia are disclosed:

[0058] R X -L-Xaa 1 -Gln-Trp-Ala-Val-Xaa 2 -His-Xaa 3 -ψ-Xaa 4 -NH 2

[0059] (Ia)

[0060] Wherein:

[0061] R X comprises a radionuclide chelator or a boron trifluoride-containing auxochrome;

[0062] L is a linker;

[0063] Xaa 1 is D-Phe, Cpa (4-chlorophenylalanine), D-Cpa, Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4b]indole-3-carboxylic acid), D-Tpi, Nal (naphthylalanine), or D-Nal;

[0064] Xaa 2 is Gly, N-methyl-Gly, or D-Ala;

[0065] Xaa 3 is Leu, Pro, D-Pro, or Phe;

[0066] Xaa 4 is Pro, Phe, Tac (thiazolidine-4-carboxylic acid), Nle (norleucine), 4-oxa-L-Pro (oxazolidine-4-carboxylic acid); and

[0067] ψ represents a reduced peptide bond between Xaa 3 and Xaa 4 .

[0068] In some embodiments, Xaa 1 is D-Phe. In other embodiments, Xaa 1 is Cpa. In other embodiments, Xaa 1 is D-Cpa. In other embodiments, Xaa 1 is Tpi. In other embodiments, Xaa 1is D-Tpi. In other embodiments, Xaa 1 is Nal. In other embodiments, Xaa 1 is D-Nal. It has been shown that D-Cpa, Tpi, D-Tpi, and D-Nal at the Xaa 1 position retain strong binding affinity for GRPR (e.g., see Tables 1 and 3 of Cai et al., 1994 Proc. Nat1. Acad. Sci. USA 91: 12664-12668; RC-3965-II published by Reile et al. in International Journal of Oncology 7: 749-754 in 1995). Since both L-Tpi and D-Tpi retain binding affinity, the L-isomers of D-Nal and D-Cpa will also retain strong binding affinity for GRPR.

[0069] In some embodiments, Xaa 2 is Gly. In other embodiments, Xaa 2 is N-methyl-Gly. In other embodiments, Xaa 2 is D-Ala. It has been shown that N-methyl-Gly and D-Ala at the Xaa 2 position retain strong binding affinity for GRPR (e.g., see Table 4 of Horwell et al., 1996 Int. J. Peptide Protein Res. 48: 522-531; Table 3 of Lin et al., 1995 European Journal of Pharmacology 284: 55-69).

[0070] In some embodiments, Xaa 3 is Leu. In other embodiments, Xaa 3 is Pro. In other embodiments, Xaa 3 is D-Pro. In other embodiments, Xaa 3 is Phe. It has been shown that D-Pro and Pro at the Xaa 3 position have strong binding affinity for GRPR (e.g., see Table 1 of Leban et al., 1993, Proc. Natl. Acad. Sci. USA 90: 1922-1926). Similarly, Xaa 3The Phe at the position is supported by the Phe at the same position in ranatensin and litorin, which have very strong binding affinities for GRPR (Heimbrook et al., 1991 J. Med. Chem. 34:2102-2107; Lin et al., 1995 European Journal of Pharmacology 294:55-69).

[0071] In some embodiments, Xaa 4 is Pro. In other embodiments, Xaa 4 is Phe. In other embodiments, Xaa 4 is Tac. In other embodiments, Xaa 4 is Nle. In other embodiments, Xaa 4 is 4-oxa-L-Pro. The Phe and Nle at the Xaa 4 position have been shown to have strong binding affinities for GRPR (see, for example, Table 1 in Leban et al., 1993 Proc. Natl. Acad. Sci. USA 90:1922-1926). Based on different peptides (5, 19-23) having Tac at this position and the examples disclosed herein exemplifying Pro at Xaa 4 , Tac and 4-oxa-L-Pro at the Xaa 4 position will also have strong binding affinities for GRPR.

[0072] As indicated by the symbol “ψ”, there is a reduced peptide bond between Xaa 3 and Xaa 4 , meaning that when Xaa 4 is Pro, Tac or 4-oxa-L-Pro, the backbone amide (e.g., -C(O)NH-) formed between consecutive amino acids in the peptide is replaced, or when Xaa 4 is Phe or Nle, it is replaced by -CH 2 N(R)-, where R is H or C 1 -C 5 a straight or branched alkyl. In some embodiments, R is H. In other embodiments, R is methyl. In other embodiments, R is C 1 -C 5 a straight or branched alkyl. In alternative embodiments, R can be methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, 1,2-dimethylpropyl, or 2-ethylpropyl.

[0073] In some embodiments, Xaa 4 is Phe, and ψ is -CH 2 NH-. In other embodiments, Xaa 4 is Phe, and ψ is -CH 2 N(R)-, where R is methyl.

[0074] In some embodiments, Xaa 4 is Nle, and ψ is -CH 2 NH-. In other embodiments, Xaa 4 is Nle, and ψ is -CH 2 N(R)-, where R is methyl.

[0075] The linker can be any suitable linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a linear peptide linker. In some embodiments, the peptide linker is a branched peptide linker, wherein the amino acid residues can be connected by a combination of backbone amide (peptide) bonds and "side chain" to "backbone" or "side chain" to "side chain" bonds. For example, a branched peptide can be connected by one or more of the following: a backbone (main chain) peptide (amide) bond, a'main chain'-side chain amide bond (between an amino group and a carboxylic acid group), and / or a 1,2,3-triazole bond (the reaction product between an azide and an alkyne). In some such embodiments, the peptide linker is (Xaa 5 ) 1-4 , wherein each Xaa 5 is independently a proteogenic or non-proteogenic amino acid residue connected together as a linear or branched peptide linker. In some embodiments, (Xaa 5 ) 1-4 is a linear peptide linker. In some embodiments, (Xaa 5 ) 1-4 is a branched peptide linker.

[0076] In some embodiments, each Xaa 5 is independently -N(R a )R b C(O)-, where: R a can be H or methyl; R b can be an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene or heteroalkynylene of 1 to 30 atoms, including linear, branched and / or cyclic (aromatic or non-aromatic and monocyclic, polycyclic or fused ring) structures; or N, R a and R b together can form a heteroalkylene or heteroalkenylene of 5 to 7 atoms.

[0077] In some embodiments, (Xaa5 ) 1-4 consists of a single amino acid or residue. In some embodiments, (Xaa 5 ) 1-4 is a dipeptide, wherein each Xaa 5 can be the same or different. In some embodiments, (Xaa 5 ) 1-4 is a tripeptide, wherein each (Xaa 5 ) 1-4 can be the same, different, or in combination. In some embodiments, (Xaa 5 ) 1-4 consists of 4 amino acid residues linked by peptide bonds, wherein each (Xaa 5 ) 1-4 can be the same, different, or in combination. In some embodiments, each Xaa 5 is independently selected from the proteinogenic and non-proteinogenic amino acids listed in Table 1, wherein each peptide backbone amino group of the peptide linker is independently optionally methylated. In some embodiments, all peptide backbone amino groups of the peptide linker are methylated. In other embodiments, only one peptide backbone amino group of the peptide linker is methylated. In other embodiments, only two peptide backbone amino groups of the peptide linker are methylated. In other embodiments, no peptide backbone amino group of the peptide linker is methylated.

[0078] In some embodiments, each Xaa 5 is independently a proteinogenic amino acid residue or a non-proteinogenic amino acid residue, wherein each peptide backbone amino group is independently optionally methylated, and wherein the amino acid residues are independently selected from the group consisting of: proteinogenic amino acids, N ε ,N ε ,N ε -trimethyl-lysine, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine (Orn), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp), β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), sulfopropylamine acid, hyaluronic acid, p-aminomethylaniline-diglycolic acid (pABzA-DIG), 4-amino-1-carboxymethyl-piperidine (Pip), NH 2 (CH 2 ) 2 O(CH 2 )2 C(O)OH, NH 2 (CH 2 ) 2 [O(CH 2 ) 2 2 C(O)OH(dPEG2), NH 2 (CH 2 ) 2 [O(CH 2 ) 2 3 C(O)OH, NH 2 (CH 2 ) 2 [O(CH 2 ) 2 4 C(O)OH, NH 2 (CH 2 ) 2 [O(CH 2 ) 2 5 C(O)OH, NH 2 (CH 2 ) 2 [O(CH 2 ) 2 6 C(O)OH, and the D - amino acids of any of the foregoing amino acids. In some embodiments, all of the peptide backbone amines of the peptide linker are methylated. In other embodiments, only one peptide backbone amine of the peptide linker is methylated. In other embodiments, only two peptide backbone amines of the peptide linker are methylated. In other embodiments, none of the peptide backbone amines of the peptide linker are methylated.

[0079] In some embodiments, the linker is pABzA - DIG. In other embodiments, the linker is Pip. In other embodiments, the linker is dPEG2. In other embodiments, the linker is Acp.

[0080] In other embodiments, R X ​​​​​is or comprises Acp. A radionuclide chelator can be any chelator suitable for binding a radioactive metal, a metal-binding radionuclide, or a cofactor containing a metal bound to a radionuclide, and the cofactor is attached to the linker by forming an amide bond (between an amino group and a carboxylic acid group) or a 1,2,3-triazole (reaction between an azide and an alkyne) or by reaction between a maleimide and a thiol group. Many suitable radionuclide chelators are known, such as Price and Orvig, Chem. Soc. Rev., 2014, 43, 260-290. In some embodiments, but not limited to, the radionuclide chelator is selected from the group consisting of: DOTA and DOTA derivatives; DOTAGA; NOTA; NODAGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogs optionally selected from CHX-A”-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; sarcophagine and sarcophagine derivatives optionally selected from SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; H2-macropa or its derivatives; H 2 dedpa, H 4 octapa, H 4 py4pa, H 4 Pypa, H 2 azapa, H 5 dedpa, and other picolinic acid derivatives; CP256; PCTA; C-NETA; C-NE3TA; HBED; SHBED; BCPA; CP256; YM103; deferoxamine (DFO) and DFO derivatives; H 6 phospa; trithiol chelates; mercaptoacetyl; hydrazinonicotinamide; dimercaptosuccinic acid; 1,2-ethylenediyldi-L-cysteine diethyl ester; methylenediphosphonate; hexamethylpropyleneamine oxime; and hexakis(methoxyisobutylisonitrile). In some embodiments, the radionuclide chelator is DOTA or a DOTA derivative.

[0081] Exemplary non-limiting embodiments of radionuclide chelators and exemplary radionuclides that can be chelated by these chelators are shown in Table 2. In alternative embodiments, R X is or comprises a radionuclide chelator selected from those listed above or in Table 2. However, it should be noted that one of ordinary skill in the art can substitute any of the chelators listed herein with another chelator.

[0082] Table 2: Exemplary Chelating Agents and Exemplary Radionuclides That Bind to the Described Chelating Agents

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] In some embodiments, R X further comprises a radioactive metal, a radionuclide-binding metal, or a radionuclide-binding metal-containing moiety, and the radioactive metal, the radionuclide-binding metal, or the radionuclide-binding metal-containing moiety chelates with the radionuclide-chelator complex. In some embodiments, the radioactive metal, the radionuclide-binding metal, or the radionuclide-binding metal-containing moiety is: 68 Ga, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 111 In, 44 Sc, 86 Y, 89 Zr, 90 Nb, 177 Lu, 117m Sn, 165 Er, 90 Y, 227 Th, 225 Ac, 213 Bi, 212 Bi, 72 As, 77 As, 211 At, 203 Pb, 212 Pb, 47 Sc, 166 Ho, 188 Re, 186 Re, 149 Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb,142 Pr, 114m In, 94m Tc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, or 18 F]AlF. In other embodiments, the radioactive metal, radionuclide-binding metal or radionuclide-binding metal-containing prosthetic group is: 68 Ga, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 111 In, 44 Sc, 86 Y, 177 Lu, 90 Y, 225 Ac, 213 Bi, or 212 Bi. In some embodiments, the chelating agent is a chelating agent from Table 2, and the chelated radionuclide is the radionuclide indicated as the binder of the chelating agent in Table 2.

[0090] In some embodiments, the chelating agent is: conjugated with 177 Lu, 111 In, 213 Bi, 68 Ga, 67 Ga, 203 Pb, 212 Pb, 44 Sc, 47 Sc, 90 Y, 86 Y, 225 Ac, 117m Sn, 153 Sm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 165 Er, 213 Bi, 224 Ra, 212 Bi, 212 Pb, 225 Ac, 227 Th, 223 Ra, 47 Sc, 64 Cu or 67 Cu-conjugated DOTA or its derivatives; conjugated with 225 H2-MACROPA conjugated with Ac;227 Conjugated Me-3,2-HOPO; and 225 Ac,[[]] 227 Th or 177 Lu-conjugated H 4 py4pa; and 177 Lu-conjugated H 4 pypa; and 68 Ga-conjugated NODAGA; and 111 In-conjugated DTPA; or and 89 Zr-conjugated DFO.

[0091] In some embodiments, the chelating agent is TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-icosane-1,8-diamine), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TRAP (1,4,7-triazacyclononane-1,4,7-tri[methyl(2-carboxyethyl)phosphinic acid]), HBED (N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid), 2,3-HOPO (3-hydroxypyridin-2-one), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadec-1(15),11,13-triene-3,6,9,-triacetic acid), DFO (deferoxamine), DTPA (diethylenetriaminepentaacetic acid), OCTAPA (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), or another picolinic acid derivative.

[0092] In some embodiments, R X is or comprises a chelating agent for use with 99m Tc,[[]] 94m Tc,[[]] 186 Re or 188 Re radiolabeled chelating agents such as mercaptoacetyl, hydrazinonicotinamide, dimercaptosuccinic acid, 1,2-ethylenediylbis-L-cysteine diethyl ester, methylenediphosphonate, hexamethylpropyleneamine oxime, and hexakis(methoxyisobutylisonitrile), etc. In some embodiments, R X is or comprises a chelating agent, wherein the chelating agent is mercaptoacetyl, hydrazinonicotinamide, dimercaptosuccinic acid, 1,2-ethylenediylbis-L-cysteine diethyl ester, methylenediphosphonate, hexamethylpropyleneamine oxime, or hexakis(methoxyisobutylisonitrile). In some of these embodiments, the chelating agent is bound to a radionuclide. In some such embodiments, the radionuclide is 99m Tc,[[]] 94m Tc,[[]] 186Re, or 188 Re.

[0093] In some embodiments, R x is or comprises a chelating agent that can bind 18 F-fluorinated aluminum ( 18 F]AlF), such as, 1,4,7-triazacyclononane-1,4-diacetate (NODA), etc. In some embodiments, the chelating agent is NODA. In some embodiments, the chelating agent is bound by 18 F]AlF.

[0094] In some embodiments, R X is or comprises a chelating agent that can bind 72 As or 77 As, such as, trithiol chelates, etc. In some embodiments, the chelating agent is a trithiol chelate. In some embodiments, the chelating agent is conjugated to 72 As. In some embodiments, the chelating agent is conjugated to 77 As.

[0095] In certain embodiments, R X is or comprises a prosthetic group containing a radioisotope capable of 18 F / 19 F exchange radiolabeled trifluoroborate (BF 3 ). In some of these embodiments, R X is R 1 R 2 BF 3 , where: R 1 is wherein L is a linker, and R 3 is absent, the group -R 2 BF 3 can be one of those listed in Table 3 (below) or Table 4 (below), or is wherein, R 4 and R 5 are independently C 1 -C 5 linear or branched alkyl.

[0096] In certain embodiments, R X is or comprises more than one (e.g., 2, 3, or 4) prosthetic groups each containing a radioisotope capable of 18 F / 19 F exchange radiolabeled trifluoroborate (BF 3 ). In some of these embodiments, R X comprises more than one R 1 R 2BF 3 , wherein each R 1 is independently , wherein L is a linker, and each R 3 is independently absent, each -R 2 BF 3 can independently be one of those listed in Table 3 (below) or Table 4 (below), or is , wherein each R 4 is independently C 1 -C 5 a straight-chain or branched-chain alkyl group, and each R 5 is independently C 1 -C 5 a straight-chain or branched-chain alkyl group. In some embodiments, R X is or exactly contains two R 1 R 2 BF 3 groups attached to the linker. In some such embodiments, the linker is a branched peptide linker, wherein each R 1 R 2 BF 3 group is attached to the linker by forming an amino amide bond that binds to the linker. For example, when the linker is (Xaa 5 ) 1-4 , the R 1 R 2 BF 3 group can be bonded to the N-terminus of the N-terminal Xaa 5 , and / or the R 1 R 2 BF 3 group can be bonded to any other free amino group of Xaa 5 . Non-limiting examples of amino acid residues having a side chain capable of forming an amide with the R 1 R 2 BF 3 group include Lys, Orn, Dab, Dap, Arg, homo-Arg, etc. In some embodiments, the R 1 R 2 BF 3 is bonded to the N-terminus of the N-terminal Xaa 5 . In some embodiments, the first R 1 R 2 BF 3 group can be bonded to the N-terminus of the N-terminal Xaa 5 , and the second R 1 R 2 BF 3 group can be bonded to Xaa 5Side chain functional groups (e.g., amino groups). Alternatively, each of the two R 1 R 2 BF 3 groups can be bonded to different Xaa 5 side chains or other functional groups.

[0097] For Tables 3 and 4 below, each R in pyridine substituted with -OR, -SR, -NR-, -NHR or -NR 2 group is independently C 1 -C 5 a straight-chain or branched-chain alkyl group. In some embodiments, the -R 2 BF 3 group is selected from those listed in Table 3. In some embodiments, the -R 2 BF 3 group is selected from those listed in Table 4. One or more trifluoroborate-containing auxiliaries can contain 18 F. In some embodiments, one of the fluorines in -R 2 BF 3 is 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 19 F.

[0098] Table 3: Exemplary -R 2 BF 3 groups.

[0099]

[0100]

[0101] Table 4: Exemplary -R 2 BF 3 groups.

[0102]

[0103]

[0104]

[0105] In some embodiments, each -R 2 BF 3 can independently form

[0106]

[0107]

[0108] Wherein, each R (when present) in the pyridine-substituted -OR, -SR, -NR-, -NHR or -NR 2 is independently a straight-chain or branched C 1 -C 5 alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is isopropyl. In some embodiments, R is n-butyl. One or more trifluoroborate-containing auxiliaries may comprise 18 F. In some embodiments, one fluorine in -R 2 BF 3 is 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 19 F.

[0109] In some embodiments, each -R 2 BF 3 can independently form

[0110] Wherein, each R (when present) in the pyridine-substituted -OR, -SR, -NR-, -NHR or -NR 2 is independently a straight-chain or branched C 1 -C 5 alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is isopropyl. In some embodiments, R is n-butyl. In some embodiments, -R 2 BF 3 is In some embodiments, all three fluorines in -R 2 BF 3 are 18 F. In some embodiments, one fluorine in -R 2 BF 3 is 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 19F.

[0111] In some embodiments, each -R 2 BF 3 is independently wherein, R 4 and R 5 are independently C 1 -C 5 a straight-chain or branched-chain alkyl. In some embodiments, R 4 is methyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is propyl. In some embodiments, R 4 is isopropyl. In some embodiments, R 4 is butyl. In some embodiments, R 4 is n-butyl. In some embodiments, R 4 is pentyl. In some embodiments, R 5 is methyl. In some embodiments, R 5 is ethyl. In some embodiments, R 5 is propyl. In some embodiments, R 5 is isopropyl. In some embodiments, R 5 is butyl. In some embodiments, R 5 is n-butyl. In some embodiments, R 5 is pentyl. In some embodiments, R 4 and R 5 are both methyl. The trifluoroborate-containing auxiliary group may comprise 18 F. In some embodiments, one of the fluorines in -R 2 BF 3 is 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 18 F. In some embodiments, all three fluorines in -R 2 BF 3 are 19 F.

[0112] In certain embodiments, the compound is conjugated to a radionuclide for positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging of tumors that express GRPR, wherein the compound is conjugated to a radionuclide that is a positron emitter or a gamma emitter. Non-limitingly, the radionuclides that emit positrons or gamma rays are 68 Ga, 67 Ga, 61 Cu,64 Cu, 67 Ga, 99m Tc, 110m In, 111 In, 44 Sc, 86 Y, 89 Zr, 90 Nb, 152 Tb, 155 Tb, 18 F, 131 I, 123 I, 124 I, and 72 As.

[0113] In certain embodiments, the compound is conjugated to a therapeutic radionuclide. This includes radioisotopes such as, 165 Er, 212 Bi, 211 At, 166 Ho, 149 Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb, 142 Pr, 177 Lu, 111 In, 213 Bi, 203 Pb, 212 Pb, 44 Sc, 47 Sc, 90 Y, 225 Ac, 117m Sn, 153 Sm, 149 Tb, 161 Tb, 165 Er, 213 Bi, 224 Ra, 212 Bi, 212 Pb, 225 Ac, 227 Th, 223 Ra, 47 Sc, 77 As, 64 Cu, or 67 Cu.

[0114] The compound may have the following chemical structure or a salt or solvate thereof, optionally chelated with a radionuclide X:

[0115]

[0116] (ProBOMB1). In an alternative embodiment, X is 177 Lu, 111 In, 213 Bi, 68 Ga, 67 Ga, 203 Pb, 212 Pb, 44 Sc, 47 Sc, 90 Y, 86 Y, 225 Ac, 117m Sn, 153 Sm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 165 Er, 213 Bi, 224 Ra, 212 Bi, 212 Pb, 225 Ac, 227 Th, 223 Ra, 47 Sc, 64 Cu, or 67 Cu. In some embodiments, X is 68 Ga. In some embodiments, X is 64 Cu. In some embodiments, X is 67 Cu. In some embodiments, X is 67 Ga. In some embodiments, X is 111 In. In some embodiments, X is 177 Lu. In some embodiments, X is 90 Y. In some embodiments, X is 225 Ac.

[0117] The compound may have the following chemical structure or a salt or solvate thereof, optionally chelated with the radionuclide X:

[0118]

[0119] (ProBOMB2). In an alternative embodiment, X is 177 Lu, 111 In, 213 Bi, 68 Ga, 67 Ga, 203 Pb, 212 Pb,44 Sc, 47 Sc, 90 Y, 86 Y, 225 Ac, 117m Sn, 153 Sm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 165 Er, 213 Bi, 224 Ra, 212 Bi, 212 Pb, 225 Ac, 227 Th, 223 Ra, 47 Sc, 64 Cu, or 67 Cu. In some embodiments, X is 68 Ga. In some embodiments, X is 64 Cu. In some embodiments, X is 67 Cu. In some embodiments, X is 67 Ga. In some embodiments, X is 111 In. In some embodiments, X is 177 Lu. In some embodiments, X is 90 Y. In some embodiments, X is 225 Ac.

[0120] Also disclosed is a compound / composition having the chemical formula Ib:

[0121] (Radionuclide-chelator complex)-(linker)-AA1-Gln-Trp-Ala-Val-AA2-His-

[0122] AA3-ψ-AA4-NH 2

[0123] (1b)

[0124] In some embodiments, the compound / composition having the chemical formula 1b is 68 Ga-ProBOMB1( 68 Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH 2; see the structure of ProBOMB1 above). The compound can be used for in vivo PET imaging of tissues expressing GRPR. Thus, this and other compounds / compositions disclosed herein can be used for diagnosing and detecting diseases or disorders characterized by abnormal / ectopic expression of GRPR, including but not limited to various forms of cancer.

[0125] In some embodiments, 68 the radionuclide in Ga-ProBOMB1 68 Ga can be replaced by other trivalent radioactive metals (such as, 90 Y or 177 Lu), and these radiopharmaceuticals can form stable complexes with DOTA. These novel compositions (representing the theranostic pair with 68 Ga-ProBOMB1) include new radiotherapeutic agents for treating disorders or diseases characterized by abnormal / ectopic expression of GRPR (including but not limited to cancer).

[0126] In some embodiments, 68 the chelator DOTA in Ga-ProBOMB1 can be replaced / substituted by other suitable chelators, which include but are not limited to other radioactive metal chelators (such as, DOTAGA, NOTA, or NOTAGA, or trifluoroborate) for radiolabeling with fluorine-18 ( 18 F).

[0127] In some embodiments, 68 the linker p-aminomethylaniline-diglycolic acid (pABzA-DIG) in Ga-ProBOMB1 can be replaced / substituted by other suitable linkers, including but not limited to Pip (4-amino-(1-carboxymethyl)piperidine) or dPEG2 (9-amino-4,7-dioxanonanoic acid).

[0128] In some embodiments, 68 the AA1D-Phe in Ga-ProBOMB1 can be replaced / substituted by other suitable amino acids, including but not limited to D-Cpa (4-chlorophenylalanine), Cpa, Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid), D-Tpi, Nal, or D-Nal.

[0129] In some embodiments, 68 the AA2Gly in Ga-ProBOMB1 can be replaced / substituted by other suitable amino acids, including but not limited to N-methyl-Gly or D-Ala.

[0130] In some embodiments, 68The AA3 Leu in Ga-ProBOMB1 can be substituted / replaced by other suitable amino acids, including but not limited to D-Pro, Pro or Phe.

[0131] In some embodiments, 68 The AA4 Pro in Ga-ProBOMB1 can be substituted / replaced by other suitable amino acids, including but not limited to Phe, Tac (thiazolidine-4-carboxylic acid) or N-methyl-Leu.

[0132] Referring to the compounds of Formula 1a and Formula 1b, when the radiolabeling group (i.e., R in Formula 1a x or the radionuclide-chelator complex or trifluoroborate of Formula 1b contains a diagnostic radionuclide or is conjugated to a diagnostic radionuclide. The compounds disclosed herein (i.e., the compounds of Formula 1a, Formula 1b or their salts or solvates) are disclosed for use in the preparation of a radiolabeled tracer for imaging tissues expressing GRPR in a subject. Also disclosed is a method for imaging tissues expressing GRPR in a subject, wherein the method comprises: administering to the subject a composition of certain embodiments comprising a compound (i.e., a compound having Chemical Formula 1a, Chemical Formula 1b or its salt or solvate) and a pharmaceutically acceptable excipient; and imaging the tissues of the subject using, for example, PET or SPECT. When the tissue is diseased tissue (e.g., cancer expressing GRPR), then a GRPR-targeted therapy can be selected for treating the subject.

[0133] Referring again to the compounds of Formula 1a and Formula 1b, when the radiolabeling group (i.e., R in Formula 1a x or the radionuclide-chelator complex or trifluoroborate of Formula 1b contains a therapeutic radionuclide, certain embodiments of the compounds (or their pharmaceutical compositions) are disclosed for use in the treatment of conditions or diseases expressing GRPR (e.g., cancer in a subject, etc.). Accordingly, there is provided the use of the compounds disclosed herein (i.e., Formula 1a, Formula 1b or their salts or solvates) in the preparation of a medicament for treating conditions or diseases expressing GRPR in a subject. There is also provided a method for treating a disease expressing GRPR in a subject, wherein the method comprises: administering to the subject a composition comprising a compound (i.e., a compound of Formula 1a, Formula 1b or its salt or solvate) and a pharmaceutically acceptable excipient. By way of example but not limitation, the disease can be cancer expressing GRPR.

[0134] Aberrant or ectopic GRPR expression has been detected in a variety of disorders and diseases, including mental / neurological disorders, inflammatory diseases, and cancer (5, 19 - 23, and 39 - 43). Thus, by way of example and not limitation, a disorder or disease in which GRPR is expressed can be a mental disorder, neurological disorder, inflammatory disease, prostate cancer, lung cancer, head and neck cancer, colon cancer, kidney cancer, ovarian cancer, liver cancer, pancreatic cancer, breast cancer, glioma, or neuroblastoma. In some embodiments, the cancer is prostate cancer.

[0135] The compound - binding peptides presented herein can be synthesized by any of a variety of methods established in the art. This includes, but is not limited to, synthesizing liquid - phase peptides and solid - phase peptides using 9 - fluorenylmethoxycarbonyl (Fmoc) and / or tert - butyloxycarbonyl (Boc) chemistry and / or other synthetic methods.

[0136] Solid - phase peptide synthesis methods and techniques are well - known in the art. For example, a peptide can be synthesized by sequentially and continuously incorporating amino acid residues of interest. In such methods, peptide synthesis is typically initiated by attaching the C - terminal amino acid of the peptide of interest to a suitable resin. Prior thereto, the reactive side chains and α - amino groups of these amino acids are protected from reaction by suitable protecting groups, allowing only the α - carboxyl group to react with a functional group on the solid support, such as an amine group, a hydroxyl group, or an alkyl halide group. After coupling the C - terminal amino acid to the support, the protecting groups on the side chains and / or the α - amino group of the amino acid are selectively removed, allowing the coupling of the next amino acid of interest. This process is repeated until the desired peptide is fully synthesized, at which point the peptide can be cleaved from the support and purified. A non - limiting example of an instrument for solid - phase peptide synthesis is the Aapptec Endevaor 90 peptide synthesizer.

[0137] To allow for the coupling of additional amino acids, the Fmoc protecting group can be removed from the amino acid on the solid support, for example, under mild basic conditions such as piperidine (20 - 50% v / v) in DMF. The amino acid to be added must also be activated for coupling (e.g., at the α-carboxylate). Non-limiting examples of activation reagents include, but are not limited to, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP). Racemization is minimized by using triazoles such as 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Coupling can be carried out in the presence of a suitable base such as N,N-diisopropylethylamine (DIPEA / DIEA), etc.

[0138] In addition to forming typical peptide bonds to extend the peptide, the peptide can be extended in a branched manner by attachment to side-chain functional groups (e.g., carboxylic acid groups or amino groups): either side-chain to side-chain; or side-chain to the main-chain amino or carboxylate. Coupling to amino acid side chains can be carried out by any known method and can be carried out on-resin or off-resin. Non-limiting examples include: forming an amide between an amino acid side chain containing a carboxyl group (e.g., Asp, D-Asp, Glu, D-Glu, etc.) and an amino acid side chain containing an amino group (e.g., Lys, D-Lys, Orn, D-Orn, Dab, D-Dab, Dap, D-Dap, etc.) or the peptide N-terminus; forming an amide between an amino acid side chain containing an amino group (e.g., Lys, D-Lys, Orn, D-Orn, Dab, D-Dab, Dap, D-Dap, etc.) and an amino acid side chain containing a carboxyl group (e.g., Asp, D-Asp, Glu, D-Glu, etc.) or the peptide C-terminus; and by click chemistry between an amino acid side chain containing an azide group (e.g., Lys(N 3 )、D-Lys(N 3) etc.) and alkynyl groups (e.g., Pra, D-Pra, etc.) to form 1,2,3-triazoles between the amino acid side chains. The protecting groups on the appropriate functional groups must be selectively removed before amide bond formation, while the reaction of forming 1,2,3-triazoles between alkynyl groups and azide groups through click reaction does not require selective deprotection. Non-limiting examples of the protecting groups that can be selectively removed include 2-phenylisopropyl ester (O-2-PhiPr) (e.g., on Asp / Glu) and 4-methyltrityl (Mtt), allyloxycarbonyl (alloc), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) (e.g., on Lys / Orn / Dab / Dap). The O-2-PhiPr and Mtt protecting groups can be selectively deprotected under mild acidic conditions, e.g., 2.5% trifluoroacetic acid (TFA) in DCM. The Alloc protecting group can be selectively deprotected using tetrakis(triphenylphosphine)palladium(0) and phenylsilane in DCM. The Dde and ivDde protecting groups can be selectively deprotected using 2-5% hydrazine in DMF. For example, by using the above coupling reaction conditions, the deprotected side chains of Asp / Glu (L- or D-form) and Lys / Orn / Dab / Dap (L- or D-form) can then be coupled. The above provides means for including multiple BF 3 groups.

[0139] The peptide backbone amide can be N-methylated (i.e., α-aminomethylated). This can be achieved by directly using Fmoc-N-methylated amino acids during peptide synthesis. Alternatively, N-methylation can be carried out under Mitsunobu conditions. First, the free primary amine group is protected using a solution of 4-nitrobenzenesulfonyl chloride (Ns-Cl) and 2,4,6-trimethylpyridine (collidine) in NMP. Then N-methylation can be achieved in the presence of triphenylphosphine, diisopropyl azodicarboxylate (DIAD), and methanol. Subsequently, N-deprotection can be carried out using 2-mercaptoethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in NMP. To couple the protected amino acid to the N-methylated α-amino group, HATU, HOAt, and DIEA can be used.

[0140] The non-peptide moiety (e.g., radiolabeling group and / or linker) can be coupled to the N-terminus of the peptide, while the peptide is attached to a solid support. This is facilitated when the non-peptide moiety includes an activated carboxylic acid ester (and if necessary, protected groups) such that coupling can be carried out on the resin. For example but not limited to, a bifunctional chelator such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tris(tert-butyl ester) can be activated in the presence of N-hydroxysuccinimide (NHS) and N,N'-dicyclohexylcarbodiimide (DCC) for coupling to the peptide. Alternatively, the non-peptide moiety can be incorporated into the compound via a copper-catalyzed click reaction under liquid or solid-phase conditions. The copper-catalyzed click reaction is well established in the art. For example, 2-azidoacetic acid is first activated by NHS and DCC and coupled to the peptide. Then, the alkyne-containing non-peptide moiety can be clicked to the azide-containing peptide in the presence of Cu 2+ and sodium ascorbate in water and organic solvents such as acetonitrile (ACN) and DMF, etc.

[0141] The synthesis of radiometal chelators is well known and many chelators are commercially available (e.g., from Sigma-Aldrich TM / Milipore Sigma TM and other companies). The protocols for conjugating radiometals to chelators are also well known (e.g., see Example 1 below).

[0142] The R 1 R 2 BF 3 component of the compound can be achieved according to previously reported procedures (Liu et al., Angew Chem Int Ed 2014 53:11876-11880; Liu et al., J Nucl Med 2015 55:1499-1505; Liu et al., Nat Protoc 2015 10:1423-1432; Kuo et al., J Nucl Med, 2019 60:1160-1166; each incorporated by reference in its entirety). Generally, the BF 3 -containing motif can be coupled to the linker via click chemistry by forming a 1,2,3-triazole ring between the azido (or alkyne) group containing BF 3 and the alkyne (or azide) group on the linker, or by forming an amide bond between the carboxylic acid ester containing BF 3 and the amino group on the linker. To prepare the azide, alkyne or carboxylic acid ester containing BF 3 , the borate ester is first converted to BF 2 in a mixture of HCl, DMF and KHF 3To prepare azides, alkynes or carboxylates containing borate esters. For alkyl BF 3 , azides, alkynes or carboxylates containing borate esters can be prepared by coupling an alkyl halide containing a borate ester (such as pinacol iodomethyl borate) with an azide, alkyne or carboxylate containing an amine (such as N,N-dimethylpropargylpropylamine). For aryl BF 3 , the borate ester can be prepared by Suzuki coupling using an aryl halide (iodine or bromine) and bis(pinacolato)diboron.

[0143] Via 18 F- 19 F isotope exchange reaction for BF 3 compound 18 F-fluorination can be achieved according to a previously disclosed procedure (Liu et al., Nat Protoc 2015 10:1423-1432, incorporated herein by reference in its entirety). Generally, about 100 nmol of the compound containing BF 3 is dissolved in a mixture of 15 μL of pyridazine-HCl buffer (pH = 2.0 - 2.5, 1 M), 15 μL of DMF and 1 μL of 7.5 mM KHF 2 aqueous solution. 18 F-fluoride solution (in saline, 60 μL) is added to the reaction mixture, and the resulting solution is heated at 80 °C for 20 min. At the end of the reaction, the desired product can be purified by solid-phase extraction or by reverse high-performance liquid chromatography (HPLC) using a mixture of water and acetonitrile as the mobile phase.

[0144] When the peptide has been fully synthesized on a solid support, the desired peptide can be cleaved from the solid support using suitable reagents (such as TFA, triisopropylsilane (TIS) and water). At the same time, side-chain protecting groups are removed (i.e., deprotected), such as Boc, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trityl (Trt) and tert-butyl (tBu). The crude peptide can be precipitated and collected from the solution by adding cold ether followed by centrifugation. The peptide can be purified and characterized by standard separation techniques (such as high-performance liquid chromatography (HPLC)) based on the size, charge and polarity of the peptide. The identity of the purified peptide can be confirmed by mass spectrometry or other similar methods.

[0145] Exemplary compounds ProBOMB1 and ProBOMB2 and their 68 Ga and 177Synthetic scheme for the conjugation of Lu. The following examples show that the compounds of the present invention can have nanomolar affinity and high stability in vivo, and can produce high-contrast images (e.g., PET) with good tumor uptake and extremely low pancreatic uptake, which is superior to the prior art tracers derived from BBN.

[0146] The present invention will be further illustrated in the following examples.

[0147] Example 1: ProBOMB1

[0148] 1.1 Materials and methods

[0149] ProBOMB1 (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ(CH 2 N)-Pro-NH 2 ) was synthesized by solid-phase peptide synthesis. The polyaminocarboxylate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminus and separated from the GRPR targeting sequence by a p-aminomethylaniline-diglycolic acid (pABzA-DIG) linker. A cell-based competition assay was used to determine the binding affinity for GRPR, while a calcium efflux assay was used to determine agonist / antagonist properties.

[0150] For 68 Ga-ProBOMB1, ProBOMB1 was radiolabeled with 68 GaCl 3 . For 177 Lu-ProBOMB1, ProBOMB1 was radiolabeled with 177 LuCl 3 . PET imaging and biodistribution studies were performed in male immunodeficient mice bearing PC-3 prostate cancer xenografts. Blocking experiments were performed with co-injection of [D-Phe6, Leu-NHEt13, des-Met14]bombesin (6-14). Dose calculations were performed using OLINDA software.

[0151] All reagents and solvents were purchased from commercial sources and used without further purification. [D-Phe 6 , Leu-NHEt 13 , des-Met 14Bombesin(6 - 14) and bombesin were purchased from Bachem and Anaspec, respectively. Other peptides were synthesized on an AAPPTec Endevaor 90 - peptide synthesizer. High - performance liquid chromatography (HPLC) was performed on an Agilent 1260 infinity system (1200 - type quaternary pump, 1200 - type UV absorbance detector set at 220 nm, Bioscan NaI scintillation detector). The HPLC columns used were a semi - preparative column (Luna C18, 5 μ, 250×10 mm) and an analytical column (Luna, C18, 5 μ, 250×4.6 mm) from Phenomenex. Mass analysis was performed using an AB SCIEX 4000 QTRAP mass spectrometer with an ESI ion source. 68 Ga was eluted from the iThemba Labs generator and purified using a DGA resin column from Eichrom Technologies LLC according to a previously published procedure (24). 68 The radioactivity of the

[0152] 1.1.1 Chemistry and radiolabeling. The synthetic procedures for radiolabeled precursors and non - radioactive standards are shown below.

[0153] 1.1.1.1 68 Ga - ProBOMB1 and 68 Ga - NeoBOMB1. Purified 68 GaCl 3 (289 - 589 MBq, in 0.6 mL of water) was added to 0.6 mL of HEPES buffer (2 M, pH 5.3) containing ProBOMB1 or NeoBOMB1 (25 μg). The mixture was heated by a microwave oven (Danby DMW7700WDB; power setting 2; 1 min). HPLC purification was used to separate the 68 Ga - labeled product from the unlabeled precursor (semi - preparative column; for 68 Ga - ProBOMB1, 23% acetonitrile and 0.1% TFA in water; for 68 Ga - NeoBOMB1, 35% acetonitrile and 0.1% HCOOH in water; flow rate: 4.5 mL / min). Retention times: 23.7 min ( 68 Ga - ProBOMB1); 11.0 min (68Ga - NeoBOMB1). Fractions containing 68Fractions of Ga-ProBOMB1 or 68 Ga-NeoBOMB1 were diluted with water (50 mL) and passed through a C18 Sep-Pak column. The 68 Ga-ProBOMB1 or 68 Ga-NeoBOMB1 captured on the column was eluted with ethanol (0.4 mL) and diluted with phosphate-buffered saline (PBS). Quality control was performed using an analytical column: 24% acetonitrile and 0.1% TFA in water ( 68 Ga-ProBOMB1); 35% acetonitrile and 0.1% TFA in water ( 68 Ga-NeoBOMB1); flow rate: 2 mL / min. Retention time: 7.9 min ( 68 Ga-ProBOMB1); 9.4 min ( 68 Ga-NeoBOMB1).

[0154] 1.1.1.2 177 Lu-ProBOMB1. 473.6 - 932.4 MBq of 177 3 177 3 Lu]LuCl was added to 25 μg of ProBOMB1 in 0.5 mL of sodium acetate buffer (0.1 M; pH 4.5), and the mixture was incubated at 95 °C for 15 minutes. Thereafter, the mixture was injected into HPLC to separate the radioligand from unreacted

[0155] 1.1.2 Synthesis of Fmoc-p-aminomethylaniline. FmocOSu (10.12 g, 30 mmol) in 60 mL of acetonitrile was added dropwise to a solution of 4-aminobenzylamine (3.67 g, 30 mmol) and triethylamine (2.79 mL, 30 mmol) in 30 mL of acetonitrile and stirred overnight. Water (100 mL) was added to the reaction mixture, and the precipitate was collected after filtration. The precipitate was washed three times with ethanol / ether (1:1, 50 mL) and dried under vacuum to obtain the product as a white powder (yield: 5.5 g, 53%). 1 H NMR (300 MHz, DMSO-d 6 ​​​) δ 7.89 (d, J = 7.4 Hz, 2H, Ar), 7.70 (d, J = 7.4 Hz, 2H, Ar), 7.42 (t, J = 7.4 Hz, 2H, Ar), 7.32 (t, J = 7.5 Hz, 2H, Ar), 6.89 (d, J = 8.2 Hz, 2H, Ar), 6.50 (d, J = 8.2 Hz, 2H, Ar), 4.94 (s, 2H, NH 2 ),4.31 (d, J = 6.9 Hz, 2H, OCH 2 ),4.21 (t, J = 6.8 Hz, 1H, CH 2 CH), 4.00 (d, J = 6.0 Hz, 2H, NHCH 2 ). ESI-MS: For Fmoc-p-aminomethylaniline C 22 H 20 N 2 O 2 , [M + H] + Calculated value is 345.2; measured value is 345.2.

[0156] 1.1.3 Synthesis of Fmoc-p-aminomethylaniline diglycolate. Diethylglycolic anhydride (1.09 g, 9.4 mmol) was added to a suspension of Fmoc-p-aminomethylaniline (2.94 g, 8.6 mmol) in dichloromethane (30 mL). The reaction mixture was stirred for 2 hours and filtered. The collected solid was washed three times with dichloromethane (50 mL) and dried under vacuum to obtain the product as a white powder (yield: 2.87 g, 73%). 1 H NMR (300 MHz, DMSO-d 6 ) δ 9.87 (s, 1H, NH), 7.89 (d, J = 7.4 Hz, 2H, Ar), 7.80 (t, J = 6.0 Hz, 1H, NHCH 2 ), 7.69 (d, J = 7.4 Hz, 2H, Ar), 7.57 (d, J = 8.4 Hz, 2H, Ar), 7.42 (t, J = 7.3 Hz, 2H, Ar), 7.32 (t, J = 7.3 Hz, 2H, Ar), 7.15 (d, J = 8.4 Hz, 2H, Ar), 4.35 (d, J = 6.8 Hz, 2H, OCH 2 ), 4.27 - 4.22 (m, 1H, CH 2 CH), 4.22 - 4.19 (m, 2H, NHCH 2 ), 4.18 - 4.08 (m, 4H, O(CH 2 )) 2 . ESI-MS: For Fmoc-p-aminomethylaniline C 26 H24 N 2 O 6 , [M+H] + The calculated value is 461.2; the measured value is 461.3.

[0157] 1.1.4 Synthesis of ProBOMB1. ProBOMB1 was synthesized on solid phase using the Fmoc-based method. Rink amide-MBHA resin (0.3 mmol) was treated with 20% piperidine in N,N-dimethylformamide (DMF) to remove the Fmoc protecting group. Fmoc-Pro-OH pre-activated with HATU (3 eq), HOAt (3 eq) and N,N-diisopropylethylamine (DIEA, 6 eq) was coupled to the resin. After removal of the Fmoc protecting group, Fmoc-Leu-aldehyde (10 eq) synthesized according to the published procedure was coupled to the resin by reductive amination in the presence of excess sodium cyanoborohydride (33 eq) in 5 mL DMF (1% acetic acid). Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Phe-OH (pre-activated with HATU (3 eq), HOAt (3 eq), DIEA (6 eq)), Fmoc-protected pABzA-DIG linker (pre-activated with HATU (3 eq) and DIEA (6 eq)), and DOTA (pre-activated with HATU (3 eq) and DIEA (6 eq)) were sequentially coupled to the resin. At room temperature, the peptide was deprotected and cleaved from the resin with a mixture of 81.5% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS), 5% water, 2.5% 1,2-ethanedithiol (EDT), 5% benzyl methyl sulfide and 5% phenol within 4 hours. After filtration, the peptide was precipitated by adding cold diethyl ether, collected by centrifugation, and purified by HPLC (semi-preparative column; 23% acetonitrile and 0.1% TFA in water, flow rate: 4.5 mL / min). The isolated yield was 1.1%. Retention time: 11.0 min. ESI-MS: For ProBOMB1 C 79 H 113 N 20 O 19 , [M+H] + The calculated value is 1645.8; the measured value is 1645.8.

[0158] 1.1.5 Synthesis of NeoBOMB1. NeoBOMB1 was synthesized on solid phase using the Fmoc-based method. The BAL resin (1% DVB, 0.3 mmol) was swollen in DMF, drained, and activated by shaking in 4 mL of a 47.5:47.5:5 methanol / DMF / acetic acid solution for 10 minutes. 2,6-Dimethylheptan-4-amine (10 eq) in 2 mL of a 1:1 methanol / DMF solution was added, and the mixture was shaken for 1 h. Sodium cyanoborohydride (10 eq) was added and the mixture was shaken for 16 h. The reaction vial was drained and washed with dichloromethane and DMF. Then, Fmoc-His(Trt)-OH (3 eq) pre-activated in DMF (6 mL) with HATU (3 eq), HOAt (3 eq), and DIEA (8 eq) was added to the reaction vial and shaken for at least 1 h. Fmoc-deprotection was carried out using 20% piperidine in DMF. Using a similar procedure, Fmoc-Gly-OH (HATU and HOAt replaced by HBTU and HOBt), Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Phe-OH, Fmoc-protected pABzA-DIG linker, and DOTA were then coupled to the peptide sequence. The peptide was cleaved with a mixture of 82.5 / 5 / 2.5 / 5 / 5 TFA / water / EDT / thioanisole / phenol and purified by HPLC (Agilent 1260 Infinity II preparative system) using a preparative column ( 5μm NX-C18 LC column 50 x 30 mm; 29 - 30.5% acetonitrile in water and 0.1% TFA for 10 minutes, and then held at 30.5% acetonitrile and 0.1% TFA; flow rate: 30 mL / min). The isolated yield was 39%. Retention time: 9.0 min. ESI-MS: For NeoBOMB1 C 77 H 111 N 18 O 18 , [M+H] + Calculated value is 1575.8; Measured value is 1576.0.

[0159] 1.1.6 Synthesis of non-radioactive standards. ProBOMB1 (1.3 mg, 0.79 μmol) and GaCl 3(0.284 M, 13.9 μL, 3.90 μmol) was incubated at 80 °C for 15 min and purified by HPLC using a semi-preparative column (23% acetonitrile and 0.1% TFA in water; flow rate: 4.5 mL / min). The isolated yield was 67%. Retention time: 15.7 min. ESI-MS: For Ga-ProBOMB1 C 79 H 110 N 20 O 19 Ga, [M+H] + Calculated value was 1711.8; measured value was 1711.7. NeoBOMB1 (2.0 mg, 1.17 μmol) and GaCl 3 (0.265 M, 47 μL, 12.46 μmol) were incubated at 80 °C for 15 min and purified by HPLC using a preparative column (30% acetonitrile and 0.1% TFA in water; flow rate: 30 mL / min). The isolated yield was 38%. Retention time: 13.0 min. ESI-MS: For Ga-NeoBOMB1C 77 H 109 N 18 O 18 Ga, [M+H] + Calculated value was 1643.7; measured value was 1644.0.

[0160] 1.1.7 LogD 7.4 Measurement. The LogD value of the radiolabeled peptide was measured using the shake flask method as previously reported (24). 7.4 Value (24).

[0161] 1.1.8 Cell culture. The PC-3 prostate adenocarcinoma cell line (ATCC-CRL-1435) was cultured in a humidified incubator (5% CO 2 ; 37 °C) in F-12K medium supplemented with 20% fetal bovine serum (Sigma-Aldrich), 100 I.U / mL penicillin, and 100 μg / mL streptomycin (Life Technologies).

[0162] 1.1.9 Competitive binding assay. The in vitro competitive binding assay was modified from a previously published procedure (25). PC-3 cells were seeded at 2 × 10 5Cells were seeded at [number] cells / well in 24-well poly-D-lysine plates. The growth medium was replaced with 400 μL of reaction medium. The cells were incubated at 37 °C for 30 - 60 minutes. Fifty microliters of serially diluted concentrations (from 10 μM to 1 pM) and 50 μL of 0.011 nM 125 I-Tyr 4 of the non-radioactive peptide of bombesin were added to the wells. The cells were incubated at 27 °C for 1 hour with gentle agitation, washed three times with ice-cold PBS, harvested by trypsinization, and assayed for activity on a gamma counter. Data were analyzed using nonlinear regression (one-binding-site model for competition assays) with GraphPad Prism 7.

[0163] 1.1.10 Fluorescent calcium release assay. The calcium release assay was performed using the FLIPR Calcium 6 assay kit (Molecular Devices) according to the published protocol (26). Briefly, 5 × 10 4 PC-3 cells were seeded overnight in 96-well clear-bottom black plates. The growth medium was replaced with loading buffer containing a calcium-sensitive dye and incubated at 37 °C for 30 minutes. The plate was placed in a FlexStation 3 microplate reader (Molecular Devices), and the baseline fluorescence signal was acquired within 15 seconds. Five or 50 nM of Ga-ProBOMB1, [D-Phe 6 Leu-NHEt 13 des-Met 14 bombesin (6 - 14), bombesin, adenosine triphosphate (ATP, positive control), or PBS (negative control) was added to the cells, and the fluorescence signal was acquired for 105 seconds. Relative fluorescence units (RFU = Max - Min) were used to determine agonist / antagonist properties.

[0164] 1.1.11 Animal models. Animal experiments were approved by the Animal Ethics Committee of Columbia University. Male NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl / SzJ (NRG) mice obtained from the in-house colony were subcutaneously inoculated with 5 × 10 6 PC-3 cells (100 μL; 1:1 PBS / matrigel), and the tumors were allowed to grow for 2 - 3 weeks.

[0165] 1.1.12 PET / CT imaging. Mice bearing PC-3 tumors were anesthetized and intravenously injected with a radioactive tracer (4.67 ± 0.91 MBq) with or without 100 μg of [D-Phe 6 Leu-NHEt 13 des-Met 14 bombesin (6 - 14).2 In 2.5% isoflurane in []. The mice were sedated and scanned (Siemens Inveon microPET / CT) using a heating pad to maintain body temperature. CT scans (80 kV; 500 μA; 3 bed positions; 34% overlap; 220° continuous rotation) were obtained 1 or 2 hours post-injection (p.i.) of the radioactive tracer, followed by a 10-min static PET. PET data were acquired in list mode, reconstructed using 3D ordered subset expectation maximization (2 iterations), and then reconstructed using a fast maximum a priori algorithm (18 iterations) with CT-based attenuation correction. Images were analyzed using Inveon Research Workplace software (Siemens Healthineers).

[0166] 1.1.13 Biodistribution. In the presence or absence of 100 μg [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14), mice bearing PC-3 tumors were anesthetized (in 2.5% isoflurane in []) and injected intravenously with the radioactive tracer (1.84 ± 0.99 MBq). At 30 min, 1 h, and 2 h p.i, the mice were sacrificed by inhalation of CO 2 . Blood was collected by cardiac puncture. Organs / tissues were harvested, rinsed with PBS, blotted dry, and weighed. The activity in the tissues was determined by a gamma counter and expressed as percentage of injected dose per gram of tissue (%ID / g). 2

[0167] 1.1.14 In vivo stability. 68 Ga-ProBOMB1 (16.1 ± 2.9 MBq) was injected intravenously into two male NRG mice. At 5 min post-uptake, the mice were sedated / euthanized and blood was collected. Plasma was separated and analyzed by radio-HPLC (24% acetonitrile and 0.1% TFA in water; flow rate: 2.0 mL / min) following a published procedure (26). 68 Retention time of Ga-ProBOMB1: 8.8 min.

[0168] 1.1.15 Dosimetry. The biodistribution data (%ID / g) were decayed to the appropriate time points and fitted to a monoexponential or biexponential model using an in-house developed Python script (Python Software Foundation, v3.5). The choice of fit was based on R 2 ​And the residuals. The resulting time-activity curves were integrated to obtain the residence time, which was multiplied by the model organ mass (25 g MOBY mouse model) to provide input values for OLINDA (Hermes Medical Solution, v2.0) to calculate the dose (27,28).

[0169] 1.1.16 Statistical analysis. One-way ANOVA followed by post hoc t-tests were used to analyze the binding affinity on GraphPad Prism 7. Statistics for biodistribution data were calculated using R (The R Foundation for Statistical Computing, v.3.4.2). Outliers were identified using Grubbs’ test (threshold: p < 0.01). The Shapiro-Wilk test was used to determine whether the distribution was normal (threshold: p > 0.05); if normal, Welch's test was used, otherwise the Wilcoxon test was used. Multiple comparisons were corrected by the Holm method.

[0170] 1.2 Results

[0171] 1.2.1 Chemistry, radiolabeling, and hydrophilicity. The radiolabeled precursors ProBOMB1 and NeoBOMB1 were obtained with yields of 1.1% and 39%, respectively. The non-radiolabeled standards Ga-ProBOMB1 and Ga-NeoBOMB1 were obtained with yields of 67% and 38%, respectively. Ga-ProBOMB1 with a decay-corrected isolated yield of 48.2% ± 10.9% and a molar activity of 121 ± 46.9 GBq / μmol and a radiochemical purity of 96.9% ± 1.4% (n = 6) was obtained. Ga-NeoBOMB1 with a decay-corrected isolated yield of 34.0% ± 11.8% and a molar activity of 239 ± 87.3 GBq / μmol and a radiochemical purity of 96.4% ± 0.8% (n = 3) was obtained. 68 Ga-ProBOMB1. 68 Ga-NeoBOMB1. 68 Ga-ProBOMB1 and 68 Ga-NeoBOMB1 had LogD 7.4 values of -2.34 ± 0.05 and -0.88 ± 0.02 (n = 3), respectively.

[0172] 1.2.2 Binding affinity and agonist / antagonist characterization

[0173] The binding affinities of [D-Phe 6 、Leu-NHEt 13 、des-Met 14 bombesin (6-14), Ga-ProBOMB1, and Ga-NeoBOMB1 for GRPR were measured in PC-3 cells ( Figure 8)。The compound successfully displaced 125 I-Tyr 4 the binding of bombesin. [D-Phe 6 、Leu-NHEt 13 、des-Met 14 bombesin (6-14), Ga-ProBOMB1 and Ga-NeoBOMB1 had K i values of 10.7 ± 1.06 nM, 3.97 ± 0.76 nM and 1.71 ± 0.28 nM, respectively. The differences in binding affinities between the compounds were statistically significant (p < 0.05).

[0174] Measurement of intracellular calcium release in PC-3 cells of Ga-ProBOMB1 ( Figure 2 and Figure 9 ). Compared with 18.3 ± 5.4 RFU for the buffer control, calcium release induced by bombesin (5 and 50 nM) and ATP (50 nM) corresponded to 535 ± 52.0 RFU, 549 ± 58.7 RFU, 511 ± 45.5 RFU. The differences were statistically significant (p < 0.001). For [D-Phe 6 、Leu-NHEt 13 、des-Met 14 bombesin (6-14) (5 and 50 nM), 22.3 ± 16.8 RFU and 42.0 ± 20.4 RFU were observed, while for Ga-ProBOMB1 (5 and 50 nM), 22.3 ± 14.4 RFU and 16.0 ± 3.7 RFU were observed. The differences compared with the buffer control were not statistically significant.

[0175] 1.2.3 PET imaging

[0176] Biodistribution and stability. Representative maximum intensity projection PET / CT images (1 and 2 h p.i.) are shown in Figure 3 . 68 Ga-ProBOMB1 and 68 Ga-NeoBOMB1 enabled clear visualization of PC-3 tumor xenografts. 68 Ga-NeoBOMB1 was excreted via both the hepatobiliary and renal pathways, while 68 Ga-ProBOMB1 was mainly cleared through the renal pathway. For 68 Ga-ProBOMB1, the highest activity was observed in the bladder, followed by the tumor. For 68 Ga-NeoBOMB1, activity was observed in the tumor, liver, pancreas, intestine, and bladder. Compared with 68 Ga-NeoBOMB1,68 Faster clearance of Ga-ProBOMB1 results in higher contrast images. [D-Phe 6 、Leu-NHEt 13 、des-Met 14 Co-injection of 68 gastrin-releasing peptide (6-14) decreased the mean uptake of Ga-ProBOMB1 in tumors by 62%.

[0177] For biodistribution, the uptake (%ID / g) of selected organs of 68 Ga-NeoBOMB1 and 68 Ga-ProBOMB1 was compared ( Figure 4 ). Within thirty minutes after injection, 68 the PC-3 tumor uptake of Ga-ProBOMB1 (4.62 ± 2.13) was lower than that of 68 Ga-NeoBOMB1 (9.60 ± 0.99) (p < 0.001). 68 The tumor uptake of Ga-ProBOMB1 was 8.17 ± 2.13 at 60 minutes and 8.31 ± 3.88 at 120 min, and 68 the tumor uptake of Ga-NeoBOMB1 was 9.83 ± 1.48 at 60 min and 12.1 ± 3.72 at 120 min (no significant difference). At all time points, 68 the uptake of Ga-ProBOMB1 in blood, liver, pancreas, and kidney was lower than that of 68 Ga-NeoBOMB1 in blood, liver, pancreas, and kidney (p < 0.05). Specifically, compared with 68 Ga-NeoBOMB1 (correspondingly: 95.7 ± 12.7, 122 ± 28.4, 139 ± 26.8), 68 the pancreatic uptake of Ga-ProBOMB1 (correspondingly: 10.4 ± 3.79, 4.68 ± 1.26, 1.55 ± 0.49) was significantly lower at 30, 60, and 120 min. At 60 min and 120 min, in 68 Ga-ProBOMB1 compared to 68 Ga-NeoBOMB1, only the muscle uptake was significantly lower (p < 0.01). Except for the seminal vesicles at 60 min, for all other collected organs (Tables 6 and 7), 68 Ga-ProBOMB1 had less uptake than 68 Ga-NeoBOMB1, although this was not always statistically significant. When compared with [D-Phe 6 、Leu-NHEt 13, des-Met 14 Bombesin(6 - 14)( Figure 5 ) when co - injected, 68 the tumor uptake of Ga - ProBOMB1 at 60 min was significantly reduced to 3.12 ± 1.68 (p < 0.01). 68 The injected radiolabeled peptide masses of Ga - NeoBOMB1 (6.01 ± 2.89 pmol) and 68 Ga - ProBOMB1 (20.24 ± 12.9 pmol) were different (p < 0.001), but had overlapping ranges at 30 and 60 min.

[0178] The stability of Ga - ProBOMB1 was measured in plasma at 5 minutes after injection. According to the HPLC results ( 68 ), Figure 6 ) 68 Ga - ProBOMB1 (t R = 8.84 min) was 96.3 ± 2.7% intact. A minor metabolite peak was observed at t R = 2.72 min.

[0179] 1.2.4 Dosimetry

[0180] Based on the kinetic curves obtained from the biodistribution data ( Figure 10 and Figure 11 ), the absorbed doses in mice are shown in Figure 7 and Table 8. The organ receiving the highest dose from 68 Ga - ProBOMB1 was the bladder (10.00 mGy / MBq). Except for the bladder, all other organs received less than 1 mGy / MBq. Higher doses of 68 Ga - NeoBOMB1 were observed in most organs (including the pancreas (8.00 mGy / MBq), kidney (3.29 mGy / MBq), large and small intestines (3.24 and 3.15 mGy / MBq)).

[0181] The estimated absorbed whole - body dose for an average adult male was also calculated (Table 5). Consistent with the mouse model, in all organs except the bladder, 68 Ga - NeoBOMB1 received a higher dose than 68 Ga - ProBOMB1 (5.69×10 -2 vs 6.59×10 -2 mGy / MBq). Notably, the pancreas was expected to receive 2.63×10 68 mGy / MBq for -1 Ga - NeoBOMB1, while for 68Ga-ProBOMB1 receives 1.44×10 -2 mGy / MBq. The kidney is expected to 68 Ga-NeoBOMB1 receives 1.69×10 -2 mGy / MBq, while in contrast 68 Ga-ProBOMB1 receives 4.32×10 -3 mGy / MBq.

[0182] ProBOMB1 and non-radioactive Ga-ProBOMB1 are obtained with yields of 1.1 and 67%, respectively. The K i value of Ga-ProBOMB1 for GRPR is 3.97 ± 0.76 nM. Ga-ProBOMB1 retains antagonist properties after modification. Ga-ProBOMB1 with a molar activity of 121 ± 46.9 GBq / μmol and a radiochemical purity of >95% is obtained with a radiochemical yield of 48.2% ± 10.9% decay-corrected. Imaging / biodistribution studies show 68 that the excretion of Ga-ProBOMB1 is mainly through the renal pathway. At 1 h post-injection (p.i.), PC-3 tumor xenografts are clearly delineated in PET images with excellent contrast. Based on the biodistribution data at 1 h p.i., 68 the tumor uptake of Ga-ProBOMB1 is 8.17 ± 2.57% injected dose per gram (%ID / g), and 68 that of Ga-NeoBOMB1 is 9.83 ± 1.48% ID / g. This corresponds to 68 tumor-to-blood and tumor-to-muscle uptake ratios of 20.6 ± 6.79 and 106 ± 57.7 for Ga-ProBOMB1, and 68 tumor-to-blood and tumor-to-muscle uptake ratios of 8.38 ± 0.78 and 39.0 ± 12.6 for Ga-NeoBOMB1. Blockade with [D-Phe 68 、Leu-NHEt 6 、Leu-NHEt 13 、des-Met 14 bombesin (6-14) causes a significant 62% reduction in the average uptake of Ga-ProBOMB1 in tumors. Compared with 68 Ga-NeoBOMB1, 68 Ga-ProBOMB1 has a lower total absorbed dose in all organs other than the bladder. 68 Ga-ProBOMB1 has a lower total absorbed dose in all organs other than the bladder.

[0183] We report a novel BBN antagonist, 68 Ga-ProBOMB1 ( 68Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH 2 ) synthesis and biological evaluation, based on the previously reported RC-3950-II (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Tac-NH 2 ; Tac: 4-thiazolidinecarboxylic acid; Figure 1 ) sequence.

[0184] Table 5: Estimated absorbed doses in different organs in adult males calculated with OLINDA software.

[0185]

[0186]

[0187] Table 6: Biodistribution of 68 Ga-NeoBOMB1 and tumor-to-organ ratios in mice bearing PC-3 xenografts at selected time points.

[0188]

[0189] Table 7: Biodistribution of 68 Ga-ProBOMB1 and tumor-to-organ ratios in mice bearing PC-3 xenografts at selected time points.

[0190]

[0191] * Mice received co-injection of 100 μg of [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14).

[0192] Table 8: Absorbed doses in different organs in mice calculated with OLINDA software.

[0193]

[0194] 1.3 Discussion

[0195] Due to the overexpression of the GRPR receptor in cancer, the development of radioligands targeting GRPR has been of interest. Estrogen receptor positivity in breast cancer is closely associated with overexpression (29), and cohort studies have shown that GRPR antagonists are effective in detecting primary and metastatic lesions in patients (12, 30). There is a large body of literature supporting the use of GRPR radioligands in patients with prostate cancer (6, 9, 31). Due to tumor heterogeneity, it has been hypothesized that GRPR radiodiagnostics can complement prostate-specific membrane antigen (PSMA) agents to improve prostate cancer management (32, 33).

[0196] Based on the sequence of RC-3950-II, [Leu 13 ψAA 14 BBNBBN derivatives, Ga-ProBOMB1 (17) was synthesized. We replaced the last amino acid Tac 68 with Pro 14 because proline is readily available in our laboratory and shows good structural homology ( 14 ). Compared to the native BBN sequence, RC-3950-II has a D-Phe Figure 1 substitution that enhances binding potency (34) and is present in other antagonists such as RM2 (15) and NeoBOMB1 (13, 35). The radiometal / chelator complex ( 6 Ga-DOTA) was attached at the N-terminus of the GRPR targeting sequence and separated by a pABzA-DIG linker (a modular design parallel to that of 68 Ga-NeoBOMB1). Recently, Nock et al. presented the first-in-human study in four patients with prostate cancer (13). 68 Ga-NeoBOMB1 was well tolerated and generated high-contrast PET images. The tracer successfully localized to primary prostate tumors and distal metastatic sites (lymph nodes, liver, and bone). The authors are exploring 68 the use of 177 Lu-labeled NeoBOMB1 for peptide receptor radionuclide therapy.

[0197] The K i value of Ga-ProBOMB1 for GRPR (3.97 ± 0.76 nM) is approximately two-fold higher than that of Ga-NeoBOMB1. It is also higher than the reported value for RC-3950-II (0.078 nM); however, the latter value was determined using Swiss3T3 cells (17). We continued to investigate the agonist / antagonist properties of Ga-ProBOMB1 using a calcium efflux assay ( Figure 2)。Although BBN and ATP significantly induced intracellular calcium release (>500 RFU) compared to the buffer control (18.3 ± 5.4 RFU), Ga-ProBOMB1 acted as an antagonist and did not significantly induce calcium release (16.0 ± 3.7 RFU). For GRPR, this property is important for tolerability in humans. Additionally, for selected peptide-receptor systems such as somatostatin, there is a paradigm shift in favor of using antagonists rather than agonists for tumor targeting (36).

[0198] PET imaging demonstrated 68 Ga-ProBOMB1 and 68 Ga-NeoBOMB1 were able to detect GRPR-expressing PC-3 prostate cancer xenografts( Figure 3 )。 68 Ga-ProBOMB1 was rapidly cleared via the renal pathway to produce high-contrast images at 1 h p.i. (post-injection). We noted that for 68 Ga-ProBOMB1, tumor uptake was retained at 2 h p.i., along with a further decrease in background activity. This indicates that the optimal imaging window can be extended beyond the 1 h time point without compromising sensitivity or contrast. With [D-Phe 6 、Leu-NHEt 13 、des-Met 14 bombesin (6-14), tumor targeting specificity was successfully blocked.

[0199] Our biodistribution data was consistent with the PET imaging studies( Figure 4 and Figure 5 ). The uptake of 68 Ga-ProBOMB1 in the tumor (%ID / g) increased from 4.62 ± 2.13 at 30 min to 8.31 ± 3.88 at 2 h. Similarly, the uptake of 68 Ga-NeoBOMB1 in the tumor increased from 9.60 ± 0.99 at 30 min to 12.1 ± 3.72 at 2 h. 68 Ga-ProBOMB1 showed slower tumor targeting and accumulation than 68 Ga-NeoBOMB1, but faster clearance from the blood (0.13 ± 0.01 vs 0.45 ± 0.10 at 2 h). Subsequently, for 68Ga-ProBOMB1 showed better contrast ratios. At 1 h p.i., the tumor-to-blood, tumor-to-muscle, tumor-to-kidney, and tumor-to-liver contrast ratios were 20.6 ± 6.79 vs. 8.38 ± 0.78, 106 ± 57.7 vs. 39.0 ± 12.6, 6.25 ± 2.33 vs. 1.66 ± 0.26, and 7.33 ± 2.97 vs. 0.08 ± 0.03, respectively. 68 The slightly lower uptake of Ga-ProBOMB1 in tumor xenografts can be explained by its lower binding affinity for GRPR, while the better contrast can be attributed to differences in hydrophilicity. Interestingly, we observed that compared with 68 Ga-NeoBOMB1 (123 ± 28.4% ID / g and 139 ± 26.8% ID / g at 1 and 2 h), 68 the pancreatic uptake of Ga-ProBOMB1 (4.68 ± 1.26 and 1.55 ± 0.49% ID / g at 1 and 2 h) was significantly lower. In addition to the higher pancreatic uptake noted in our study, 68 the results obtained with Ga-NeoBOMB1 were comparable to those presented by Dalm et al. (35). 68 The high pancreatic uptake of Ga-NeoBOMB1 may potentially be attributed to differences in molar activity and / or mouse strain. Dalm et al. injected 250 pmol of 68Ga-NeoBOMB1 for biodistribution studies, and in nude mice bearing PC-3 tumors, the uptakes in the tumor and pancreas were approximately 10% ID / g and 15% ID / g, respectively (35). From the same paper, when a larger mass of the peptide was injected for 177 Lu-NeoBOMB1 (200 vs. 10 pmol), the pancreatic uptake decreased.

[0200] A general limitation of BBN-based radiopharmaceuticals is their metabolic stability, as BBN is susceptible to enzymatic cleavage by neutral endopeptidase (37, 38). 68 Ga-ProBOMB1 was stable >95% in plasma at 5 min p.i. Although a minor hydrophilic metabolite peak was observed, its identity was not probed in this study. The stability of the compound is promising for translation or for repositioning as a radiotherapeutic agent. The DOTA chelator can form stable complexes with therapeutic trivalent radiopharmaceuticals such as 90 Y or 177 Lu to produce theranostic pairs.

[0201] Dosimetry was calculated in mice and extrapolated to adult males. When compared with 68 Ga-NeoBOMB1, 68The absorbed dose of Ga-ProBOMB1 was low in all organs except the bladder in mice (9.33 vs. 10.00 mGy / MBq). In 68 the case of Ga-ProBOMB1, mice received approximately one-sixth and one-tenth of the estimated absorbed dose to the kidney and pancreas. For the human model, even lower doses were obtained for 68 Ga-ProBOMB1. Thus, it was predicted that the average adult male would receive approximately one-quarter and one-twentieth of the absorbed dose to the kidney and pancreas, respectively.

[0202] 1.4 Conclusion. We synthesized a novel GRPR imaging agent based on the [Leu 13 ψAA 14 BBN family 68 Ga-ProBOMB1. The radiopharmaceutical showed nanomolar affinity for GRPR and high stability in vivo. 68 Ga-ProBOMB1 was able to generate high-contrast PET images with good tumor uptake in a prostate cancer model. Compared with 68 Ga-NeoBOMB1, 68 Ga-ProBOMB1 had a better dosimetry profile (enhanced contrast and lower whole-body absorbed dose).

[0203] Example 2: ProBOMB2

[0204] 2.1 Materials and Methods

[0205] 2.1.1 General overview of methods and procedures. ProBOMB2 (DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ(CH 2 N)-Pro-NH 2 ) was synthesized by solid-phase peptide synthesis. The polyaminocarboxylate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the N-terminus and separated from the GRPR targeting sequence by a 4-amino-(1-carboxymethyl)piperidine (Pip) linker. A cell-based competition assay was used to determine the binding affinity for GRPR, while a calcium flux assay was used to determine agonist / antagonist properties. ProBOMB2 was radiolabeled with 68 GaCl3. PET imaging and biodistribution studies were performed in male immunocompromised mice bearing PC-3 prostate cancer xenografts. Blocking experiments were performed by co-injection with [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14).

[0206] 2.1.2 General methods. All reagents and solvents, except Fmoc-Leu-ψ(CH 2 N)-Tac-OH (synthesized in our laboratory), were purchased from commercial sources and used without further purification. [D-Phe 6 , Leu-NHEt 13 , des-Met 14 Bombesin (6-14) and bombesin were purchased from Bachem and Anaspec, respectively. Other peptides were synthesized on an AAPPTec Endevaor 90 peptide synthesizer. High performance liquid chromatography (HPLC) was performed on an Agilent 1260 infinity system (1200 series quaternary pump, 1200 series UV absorbance detector set at 220 nm, Bioscan NaI scintillation detector). The HPLC columns used were a semi-preparative column (Luna C18, 5 μ, 250×10 mm) and an analytical column (Luna, C18, 5 μ, 250×4.6 mm) from Phenomenex. Mass analysis was performed using an AB SCIEX 4000 QTRAP mass spectrometer with an ESI ion source. 68 Ga was eluted from an iThemba Labs generator and purified using a DGA resin column from Eichrom Technologies LLC according to a previously published procedure (24). Radioactivity of 68 Ga-labeled peptides was measured using a Capintec CRC-25R / W dose calibrator, and radioactivity in tissues collected from biodistribution studies was counted using a Perkin Elmer Wizard2 2480 γ counter.

[0207] 2.1.3 Chemical and radiolabeling. The synthetic procedures for radiolabeled precursors and non-radiolabeled standards are shown below. Purified 68 GaCl 3 (289 - 589 MBq, in 0.6 mL water) was added to 0.6 mL of HEPES buffer (2 M, pH 5.3) containing ProBOMB2. The mixture was heated by microwave oven (Danby DMW7700WDB; power setting 2; 1 min). HPLC purification was used to separate the 68 Ga-labeled product from the unlabeled precursor (semi-preparative column).

[0208] 2.1.4 Fmoc-Leu-ψ(CH 2Synthesis of N)-Tac-OH. Fmoc-leucinol (1.1 g, 3.24 mmol) in 50 mL of dichloromethane was stirred with Dess-Martin periodinane (2.7 g, 6.36 mmol) at room temperature for 4 h. The reaction mixture was concentrated in vacuo, after which hexane (70 mL) and saturated sodium bicarbonate solution (30 mL) were added and stirred for 15 min before filtration. The filtrate was washed with saturated sodium bicarbonate solution (3 x 50 mL), water (3 x 50 mL), and brine (3 x 50 mL). The organic layer was collected and dried over magnesium sulfate, filtered, and evaporated in vacuo to obtain the crude crystalline compound. The isolated solid was dissolved in 36 mL of dichloroethane together with L-proline (410 mg, 3.56 mmol), and the mixture was stirred at room temperature for 48 h. Sodium triacetoxyborohydride (1.7 g, 8.1 mmol) was added to the mixture and further stirred for 16 h. Then the solution was concentrated in vacuo and ethyl acetate and saturated sodium bicarbonate (1:1, 50 mL) were added, and the mixture was stirred for 10 min. The organic layer was washed with saturated sodium bicarbonate solution (3 x 50 mL), water (3 x 50 mL), and brine (3 x 50 mL). The organic layer was dried over MgSO 4 before concentration in vacuo to give a yellow crude solid. The crude material was purified using HPLC (Phenomenex Gemini Prep column, 38% acetonitrile and 0.1% TFA in water, flow rate 30 mL / min). Retention time: 9.8 min. The product peak was collected and lyophilized to obtain the product as a white powder (yield: 436 mg, 31%). ESI-MS: For Fmoc-LeuψTac C 26 H 32 N 2 O 4 , [M+H]+, calculated 437.2; found 437.3.

[0209] 2.1.5 Synthesis of ProBOMB2. ProBOMB2 was synthesized on solid phase using the Fmoc-based method. Rink amide-MBHA resin (0.1 mmol) was treated with 20% piperidine in N,N-dimethylformamide (DMF) to remove the Fmoc protecting group. Fmoc-Leu-ψ(CH 2N)-Pro-OH (shown below) was coupled to the resin. After removal of the Fmoc protecting group, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Phe-OH (pre-activated with HATU (3 eq), HOAt (3 eq) and DIEA (6 eq)), Fmoc-protected Pip linker (pre-activated with HATU (3 eq) and DIEA (6 eq)), and DOTA (pre-activated with HATU (3 eq) and DIEA (6 eq)) were sequentially coupled to the resin. At room temperature, the peptide was deprotected and cleaved from the resin with a mixture of 92.5% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), 2.5% water, 2.5% 2,2'-(ethylenedioxy)diethanethiol (DODT) within 4 hours. After filtration, the peptide was precipitated by addition of cold diethyl ether, collected by centrifugation, and purified by HPLC (semi-preparative column; 20% acetonitrile and 0.1% TFA in water, flow rate: 4.5 mL / min). The isolated yield was 2.4%. Retention time: 16.8 min. ESI-MS: For C 75 H 112 N 20 O 17 GaProBOMB2, [M+2H] + Calculated: 1567.8; Found: 1567.4.

[0210]

[0211] 2.1.6 Synthesis of non-radioactive standards. ProBOMB2 (1.8 mg, 1.15 μmol) in 450 μL sodium acetate buffer (0.1 M, pH 4.2) and GaCl 3 (0.2 M, 28.5 μL, 5.75 μmol) were incubated at 80 °C for 30 min and purified by HPLC using a semi-preparative column (20% acetonitrile and 0.1% TFA in water; flow rate: 4.5 mL / min). The isolated yield was 88%. Retention time: 12.1 min. ESI-MS: For Ga-ProBOMB2 C 75 H 110 N 20 O 19 Ga, [M+H] + Calculated: 1631.7; Found: 1631.9. ProBOMB2 (1.36 mg, 0.869 μmol) in 450 μL sodium acetate buffer (0.1 M, pH 4.2) and LuCl3 (0.2 M, 21.7 μL, 4.3455 μmol) was incubated at 80 °C for 30 min and purified by HPLC using a semi-preparative column (21% acetonitrile and 0.1% TFA in water; flow rate: 4.5 mL / min). The isolated yield was 86%. Retention time: 8.6 min. ESI-MS: for Lu-ProBOMB2C 75 H 110 N 20 O 17 Lu, [M + H] + Calculated value was 1738.7; measured value was 1738.7.

[0212] 2.1.7 Cell culture. Human PC-3 prostate adenocarcinoma and murine Swiss 3T3 fibroblast cell lines were cultured and maintained in F-12K medium and RPMI medium (Life Technologies), respectively, in a humidified incubator (5% CO 2 ; 37 °C) and supplemented with 20% fetal bovine serum, 100 I.U / mL penicillin, and 100 μg / mL streptomycin (Life Technologies).

[0213] 2.1.8 Competitive binding assay. The in vitro competitive binding assay was modified from a previously published procedure (25). PC-3 cells were seeded at 2 × 10 5 cells / well in 24-well poly-D-lysine plates 18 - 24 h before the experiment. The growth medium was replaced with 400 μL of reaction medium. The cells were incubated at 37 °C for 30 - 60 minutes. Fifty microliters of decreasing concentrations (10 μM to 1 pM) and 50 μL of 0.011 nM 125 I-Tyr 4 bombesin non-radioactive peptide were added to the wells.. The cells were incubated at 27 °C for 1 h with gentle agitation, washed three times with ice-cold PBS, harvested by trypsin digestion, and the activity was measured on a gamma counter. Data were analyzed using nonlinear regression (one-binding-site model for competitive assays) with GraphPad Prism 7.

[0214] 2.1.9 Animal model. Animal experiments were approved by the Animal Ethics Committee of Columbia University. Male NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice obtained from an in-house colony were subcutaneously inoculated with 5 × 10 6 PC-3 cells (100 μL; 1:1 PBS / matrigel), and the tumors were allowed to grow for 3 weeks.

[0215] 2.1.10 PET / CT imaging. With or without 100 μg [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14), mice bearing PC-3 tumors were intravenously injected with a radioactive tracer (4.18 ± 0.68 MBq) and anesthetized i.v. (2.5% isoflurane in O 2 ). The mice were sedated and scanned (Siemens Inveon microPET / CT) with body temperature maintained using a heating pad. CT scans (80 kV; 500 μA; 3 bed positions; 34% overlap; 220° continuous rotation) were obtained 1 or 2 h post injection (p.i.) of the radioactive tracer, followed by 10 min of static PET. PET data were acquired in list mode, reconstructed using 3-dimensional ordered subset expectation maximization (2 iterations), and subsequently reconstructed using a fast maximum a priori algorithm (18 iterations) with CT-based attenuation correction. Images were analyzed using Inveon Research Workplace software (Siemens Healthineers).

[0216] 2.1.11 Biodistribution. With or without 100 μg [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14), mice bearing PC-3 tumors were intravenously injected with a radioactive tracer (1.47 ± 1.17 MBq) and anesthetized (2.5% isoflurane in O 2 ). At 1 h and 2 h p.i., the mice were sacrificed by inhalation of CO 2 . Blood was collected by cardiac puncture. Organs / tissues were harvested, rinsed with PBS, blotted dry, and weighed. The activity in the tissues was determined by a gamma counter and expressed as percentage of injected dose per gram of tissue (% ID / g).

[0217] 2.1.12 In vivo stability. 68 Ga-ProBOMB2 (5.9 ± 0.3 MBq) was intravenously injected into two male NSG mice. After 5 min and 15 min uptake periods, two mice were sedated / euthanized at each time point and blood was collected. Plasma was extracted from whole blood with acetonitrile, vortexed, and the supernatant was separated. Plasma was analyzed by radio-HPLC (21% acetonitrile and 0.1% TFA in water); flow rate: 2.0 mL / min. 68 Retention time of Ga-ProBOMB2: 9.3 min.

[0218] 2.2 Results

[0219] 2.2.1 Chemical and radioisotopic labeling. The unnatural amino acid Fmoc-Leu-ψ(CH 2 N)-Pro-OH was obtained in 30% yield. The radiolabeled precursor ProBOMB2 was obtained in 2.4% yield. The non-radiolabeled standards Ga-ProBOMB2 and Lu-ProBOMB2 were obtained in 88% and 86% yields, respectively. Ga-ProBOMB2 was obtained with a decay-corrected isolated yield of 48.2 ± 0.3% and a radiochemical purity of 96%. 68 Ga-ProBOMB2.

[0220] 2.2.2 Binding affinity. The binding affinities of Ga-ProBOMB2 and Lu-ProBOMB2 for human and murine GRPR were measured in PC-3 and Swiss 3T3 cells, respectively ( Figure 15 and Figure 16 ). The compounds successfully displaced 125 I-Tyr 4 bombesin binding in a dose-dependent manner. For human and murine GRPR receptors, the K i values of Ga-ProBOMB2 were 4.58 ± 0.67 nM and 3.97 ± 0.76 nM, respectively. For human and murine GRPR receptors, the K i values of Lu-ProBOMB2 were 7.29 ± 1.73 nM and 7.91 ± 2.60 nM, respectively.

[0221] 2.2.3 PET imaging, biodistribution, and stability

[0222] Representative maximum intensity projection PET / CT images (1 h, 1 h blockade, and 2 h p.i.) are shown in Figure 12 . 68 Ga-ProBOMB2 enabled clear visualization of PC-3 tumor xenografts. 68 Ga-ProBOMB2 was cleared mainly via the renal pathway. Co-injection of [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14) decreased the mean uptake of Ga-ProBOMB2 in tumors by 65%. 68 Ga-ProBOMB2 by 65%.

[0223] The biodistribution and uptake (% ID / g) of 68 Ga-ProBOMB2 in selected organs at 1 h p.i. and 2 h p.i. were compared ( Figure 13 ; Table 9).

[0224] Measured in plasma at 5 min and 15 min p.i68 The stability of Ga-ProBOMB2. According to the HPLC results ( Figure 14 ), 68 Ga-ProBOMB2 (t R = 9.35 min) is 89% intact. A minor metabolite peak was observed at t R = 2.72 min.

[0225] Table 9. Biodistribution of Ga-ProBOMB2 and tumor-to-organ ratios in mice bearing PC-3 xenografts at selected time points. 68

[0226]

[0227]

[0228] * Mice received a co-injection of 100 μg of [D-Phe 6 , Leu-NHEt 13 , des-Met 14 bombesin (6-14).

[0229] List of references not cited in-line

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[0232] 3. Weber HC. Regulation and signaling of human bombesin receptors and their biological effects. Curr Opin Endocrinol Diabetes Obes. 2009;16:66 - 71.

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Claims

1. A compound of formula Ia: R X -L-Xaa 1 -Gln-Trp-Ala-Val-Xaa 2 -His-Xaa 3 -ψ-Xaa 4 -NH 2 (Ia) Wherein, R X comprising a radioactive nuclide chelating agent or a boron trifluoride-containing auxiliary group; L is a linker; Xaa 1 is D-Phe, Cpa (4-chlorophenylalanine), D-Cpa, Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid), D-Tpi, Nal (naphthylalanine), or D-Nal; Xaa 2 is Gly, N-methyl-Gly, or D-Ala; Xaa 3 is Leu, Pro, D-Pro, or Phe; Xaa 4 is Pro, Phe, Tac (thiazolidine-4-carboxylic acid), Nle (norleucine), 4-oxa-L-Pro (oxazolidine-4-carboxylic acid); and ψ represents Xaa 3 and Xaa 4 is a reduced peptide bond therebetween, wherein when Xaa 4 is Pro, Tac or 4-oxo-L-Pro, ψ is or when Xaa 4 is Phe or Nle, ψ is -CH 2 N(R)-, wherein R is H or C 1 -C 5 a straight-chain or branched-chain alkyl group.

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