Diphosphine Compounds and Complexes

Abstract

Disclosed herein is a diphosphine precursor compound of formula (I), its conjugate of formula (II) and its radionuclide conjugate complex.The compounds are advantageous at least because they allow for the easy, one-step, immediate preparation of the corresponding complexes under mild conditions and with high radiochemical yields in clinical settings.Methods for making the compounds and complexes herein are also disclosed, along with their uses.The complexes are particularly useful in the fields of medicine and diagnostics, for example in medical imaging and targeted payload delivery. TIFF2024533992000057.tif4438

Description

[Technical field]

[0001] This application claims the benefit of UK patent application GB2111553.0, filed on August 11, 2021, which is incorporated herein by reference in its entirety.

[0001] This invention relates to compounds and radionuclide complexes, their uses and methods of preparation which are particularly useful in the imaging, diagnosis and treatment of diseases such as rheumatoid arthritis and prostate cancer. [Background technology]

[0002]

[0001] In recent years, there has been a shift towards the development of PET radiotracers in preference to SPECT radiotracers. Clinical PET imaging generally offers superior spatial resolution and sensitivity compared to SPECT. However, SPECT radionuclides are generally more widely available, less expensive, and longer-lived than PET radionuclides, and SPECT technology allows for simultaneous imaging using radionuclides with different emission energies.

[0003]

[0002] However, advances in detector and collimator technology have increased the resolution and sensitivity of commercially available SPECT scanners, bringing SPECT very close to PET in terms of resolution and sensitivity. Gamma scintigraphy and SPECT cameras are generally more readily available than PET facilities (in 2015 / 16, there were 3408 gamma scintigraphy / SPECT cameras and 849 PET scanners in Europe excluding the UK). The number of clinical gamma scintigraphy and SPECT imaging procedures is also now greater than that of PET imaging. For example, in the UK, within the NHS, approximately 440,000 gamma scintigraphy / SPECT scans were performed between February 2018 and February 2019, compared to 170,000 PET scans. New 99m Tc generator production facilities, and 99mThere is also significant international investment in new UK cyclotron technology for Tc. These data are 99m Demonstrate the continuing and future importance of imaging with Tc and other SPECT radionuclides.

[0004]

[0003] However, despite the investment and widespread use of SPECT equipment, over the past 20 years, 99m There has been little parallel development of new kit-based radiopharmaceutical chemistry for modern molecular imaging using Tc. The present invention aims to leverage existing widespread equipment to facilitate access to the benefits of receptor-targeted diagnostic radionuclide imaging by SPECT and gamma scintigraphy.

[0005]

[0004] Existing "one-pot" 99m Tc radiosynthesis requires only a generator-produced technetium-99m, a commercially available "kit" vial containing all non-radioactive materials, syringes, radiation shielding, and a grade A isolator to ensure sterility. The chelator in the kit allows for fast reaction rates with low chelator amounts. 99m They quantitatively coordinate Tc, allowing routine, sterile, and simple radiosynthesis by clinical technicians. In their current form, these chelator complexes are used for conventional functional imaging (perfusion, renal function, pulmonary ventilation) but, crucially, are not suitable for conjugation with peptides.

[0006]

[0005] The new chemical platform described herein allows one-step, kit-based radiolabeling of peptides to provide radiopharmaceuticals targeted to molecular receptors. In the field of nuclear medicine, examples exist of chelation of radionuclides with compounds bearing targeting ligands.

[0007] [ka]

[0008]

[0006] The radiopharmaceutical tetrofosmin is used to image cardiac perfusion. In tetrofosmin (Myoview; compound P1), two bidentate diphosphines coordinate to the Tc(V) metal center, with two oxide ligands occupying the axial positions. However, tetrofosmin uses a very different "diphosphine" chelator, which cannot be attached to peptides or proteins and is therefore not suitable for receptor-targeted imaging of disease.

[0009] [ka]

[0010]

[0007] The webpage (https: / / www.imagingcdt.com / project / bidentate-diphosphine-and-dithiocarbamate-chelators-for-radionuclide-imaging-with-99mtc / ; accessed May 26, 2021) 99m The use of bidentate diphosphine and dithiocarbamate chelators intended for radionuclide imaging with Tc is described. The structures of compounds (I-1) and (II-1-RGD) and formula P1 are described.

[0011]

[0008] Abstracts / Nuclear Medicine and Biology 72-73 / S1 (2019) S1-S67 page #66 99m We describe previous work providing bis(diphosphino)maleic anhydride as a bifunctional chelator for Tc.

[0012]

[0009] Neither of the previous two references describes the substitution of the phosphine atom with a substituted aryl, heteroaryl or cycloalkyl group or the advantages thereof. Only the metals (M) Re and Tc and the peptide RGD are described. The compounds or advantages of the present invention are not described.

[0013] J. Chem. Soc., Dalton Trans., 1997, 855-862, describes the reaction of a chelating diphosphine, 2,3-bis(diphenylphosphino)maleic anhydride, compound (I-1), with CuCl to give a tetrahedral structure.

[0014]

[0011] Chem. Commun. 1996, No. 10, 1093 describes copper(I) bis(diphosphine) complexes as the basis of radiopharmaceuticals for positron emission tomography and targeted radiotherapy.

[0015]

[0012] US20110033379A1 describes a radiolabeled material and methods for making and using it. However, it relies on nitrogen atoms, and optionally sulfur atoms, in a metal chelating moiety to chelate the radionuclide. Diphosphine groups are not described.

[0016]

[0013] WO2003086476A1 describes a technetium-labeled rotenone derivative and its use, particularly in cardiac imaging. However, it relies on a nitrogen atom in a metal chelating moiety to form a complex containing a radionuclide and the rotenone derivative. A diphosphine group is not described.

[0017]

[0014] WO2010108125A2 describes a prostate-specific membrane antigen (PSMA) binding compound. However, it relies on a nitrogen atom in a metal chelating moiety to chelate a radionuclide. A diphosphine group is not described.

[0018]

[0015] The present inventors have identified a new chemical platform that allows for one-step, kit-based radiolabeling of targeting ligands. Summary of the Invention [Problem to be solved by the invention]

[0019] In the broadest sense, the present invention provides a chemical platform to enable one-step, kit-based radiolabeling of targeting ligands. The radiolabeled complexes can then be used in medicine, for example for imaging or disease treatment. Diphosphine compounds are used to combine radioisotopes with biological ligands to simultaneously exploit their advantageous properties. [Means for solving the problem]

[0020] Conjugated Diphosphine Precursor Compounds

[0016] In a first aspect of the present invention, there is provided a method for preparing a conjugated radiolabeled drug (e.g., a conjugated radiolabeled diphosphine complex), comprising the steps of:

[0021] [ka]

[0022] [In the formula, each Z is independently O or S; Y is NH or O; X1, X2, X3 and X4 are each independently a substituted or unsubstituted C5-C8 aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group or a substituted or unsubstituted C3-C8 cycloalkyl group, where each substituent is a C1-C4 alkyl group, a C5-C 12 Aryl or heteroaryl groups, C1-C4 acylamido groups, sulfylhydro groups, C1-C4 alkylthio groups, C1-C4 (di)alkylphosphino groups, hydroxy groups, C1-C4 alkoxy groups, carboxyl groups, C1-C4 (di)alkylamino groups, and C1-C4 alkoxy-(CH2CH2O) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the shortest linear chain of carbon atoms between two Z groups is from 4 to 7; LIG comprises a ligand having a binding motif corresponding to a biological target;

[0023] [ka]

[0024] Provided is a compound which is not:

[0017] Each variable LIG, Z, Y, X1, X2, X3 and X4 in formula (II), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided elsewhere herein. The disclaimer of compound (II-1-RGD) also applies to subformulae of formula (II) herein, which may be formed from the diphosphine precursor compound of the first aspect above.

[0025]

[0018] LIG comprises a binding motif (i.e., a targeting ligand) that is selective for a biological target, such as an enzyme or receptor, due to the formation of a specific interaction with that target. In some cases, LIG ​​comprises a peptide or carbohydrate ligand having a binding motif corresponding to the biological target. In some cases, LIG ​​comprises a prostate-specific membrane antigen targeting ligand (PSMAt), a cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), a pentixafor peptide, a mini-gastrin peptide analog for targeting the cholecystokinin-2 receptor, a c-Met targeting peptide, an alpha-MSH peptide, a bisphosphonate, a folic acid, or a carbohydrate. In some cases, LIG ​​comprises a prostate-specific membrane antigen targeting ligand (PSMAt) or a cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD). PSMAt targets prostate-specific membrane antigen. PSMAt may be provided, for example, as part of a PSMAt1 group described herein. RGD targets the αvβ3-integrin receptor, which is overexpressed in neovasculature, inflammatory processes and cancer cells. Pentixafor peptide targets CXCR-4. Minigastrin peptide analogs target the cholecystokinin-2 receptor. Alpha-MSH targets MCR1 in melanoma. Bisphosphonates target the mineralisation process in bone metastases. Folic acid targets the folate receptor. LIG is preferably PSMAt1. LIG preferably has a molecular weight of 50 g / mol or more, such as 100 g / mol or more or 200 g / mol or more. LIG preferably has a molecular weight of 3,000 g / mol or less, such as 2,000 g / mol or less or 1,000 g / mol or less. LIG is not H, OH, NH2 or NHBn. The LIG preferably comprises 10 or more atoms, such as 15 or more atoms or 20 or more atoms. The LIG preferably comprises 100 or less atoms, such as 75 or less atoms or 50 or less atoms.

[0026] In some cases, LIG ​​is attached through a nitrogen atom that forms an amide bond with a Z group, and thus Z is O. In other cases, LIG ​​is attached through a nitrogen atom that forms a (thio)amide bond with a corresponding Z group, and thus Z is S. LIG may include a PEG linker moiety. LIG may include a terminal moiety having a urea group and three carboxylic acid groups. The carboxylic acid groups may be derived from amino acids. The terminal moiety may be two glutamic acid groups linked by a central urea group; or a lysine group and a glutamic acid group linked by a urea group. The terminal moiety of LIG may be PSMAt.

[0027] In some cases, a conjugated diphosphine precursor compound of formula (IIa) suitable for preparing a conjugated radiolabeled drug is provided:

[0028] [ka]

[0029] [In the formula, each Z is independently O or S; Y is NH or O; X1, X2, X3 and X4 are each independently a substituted or unsubstituted C5-C8 aryl group, where each substituent is selected from the group consisting of a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 (di)alkylamino group and a C1-C4 alkoxy-(CH2CH2O) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The LIG comprises a peptide or carbohydrate ligand having a binding motif corresponding to a biological target.

[0030]

[0021] Each variable group LIG, Z, Y, X1, X2, X3 and X4 in formula (IIa), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided anywhere herein.

[0031] In some cases, conjugated diphosphine precursor compounds of formula (IIb) and / or formula (IIc) suitable for preparing conjugated radiolabeled drugs are:

[0032] [ka]

[0033] [In the formula, X1, X2, X3 and X4 are each independently a substituted or unsubstituted phenyl group, where each substituent is selected from the group consisting of a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 (di)alkylamino group and a C1-C4 alkoxy-(CH2CH2O) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; LIGs include prostate-specific membrane antigen targeting ligands (PSMAt), cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), pentixafor peptides, mini-gastrin peptide analogs for targeting the cholecystokinin-2 receptor, c-Met targeting peptides, alpha-MSH peptides, bisphosphonates, folic acid or carbohydrates.

[0034]

[0023] RGD as used herein is according to the formula: where the wavy line represents an adhesive bond;

[0035] [ka]

[0036]

[0024] The PSMAt herein may be attached to a PEG linker moiety via an amide bond, which is in turn attached to a Z group via an amide. In some cases, the PEG linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units. The PSMAt may be provided as a terminal group in PSMAt1, which is according to the formula:

[0037] [ka]

[0038]

[0025] Each variable group LIG, X1, X2, X3 and X4 in formula (IIb) or formula (IIc), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided anywhere herein.

[0039] In some cases, X1, X2, X3, and X4 are each independently methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, methoxy, ethoxy, ethenyl, dimethylamino, and MeO(CH2CH2O). n and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt) or cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD).

[0040] In some cases, X1, X2, X3, and X4 are each independently methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, methoxy, ethoxy, ethenyl, dimethylamino, and MeO(CH2CH2O). nand n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt) or cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), is provided as a diphosphine precursor compound of formula (IIb) suitable for preparing a conjugated radiolabeled drug.

[0041] In some cases, the conjugated diphosphine precursor compound is of formula (IIb), where X1, X2, X3 and X4 and LIG are according to a row in the following table:

[0042] [Table 1-1]

[0043] [Table 1-2]

[0044] Diphosphine Precursor Compounds In a second aspect, there is provided a diphosphine precursor compound of formula (I) suitable for preparing a conjugated radiolabeled drug (e.g., a conjugated radiolabeled diphosphine complex):

[0045] [ka]

[0046] [In the formula, Ring A is a 5-, 6-, 7- or 8-membered ring; each Z is independently O or S; Y is NH or O; X1, X2, X3 and X4 are each independently a substituted or unsubstituted C5-C8 aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group or a substituted or unsubstituted C3-C8 cycloalkyl group, where the optional substituents are a C1-C4 alkyl group, a C5-C 12 Aryl or heteroaryl groups, C1-C4 acylamido groups, sulfylhydro groups, C1-C4 alkylthio groups, C1-C4 (di)alkylphosphino groups, hydroxy groups, C1-C4 alkoxy groups, carboxyl groups, C1-C4 (di)alkylamino groups, and C1-C4 alkoxy-(CH2CH2O) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0047] [ka]

[0048] Provided is a compound which is not:

[0028] Each variable A, Z, Y, X1, X2, X3 and X4 in formula (I), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided anywhere herein. The disclaimer of compound (I-1) also applies to subformulae of formula (I) herein. A single bond with a dashed line in formula (I) indicates that the bond may be a CC single bond or a C=C double bond.

[0049] One advantage of the present invention is that the A-ring allows for conjugation with a selected ligand moiety via a ring-opening reaction to prepare the compound for binding to a radionuclide immediately prior to clinical use (i.e., in the hospital, radiopharmacology or production department). The diphosphine motif subsequently allows for easy, efficient, immediate, one-step complexation of the selected radioisotope in a physiologically compatible solution shortly prior to clinical use.

[0050] Another advantage is that the presence of a substituted aryl group or a substituted or unsubstituted heteroaryl group provides improved efficiency and radiochemical yields of the corresponding conjugated diphosphine precursor compounds compared to similar known compounds. Radiolabeling is also carried out under milder conditions and can be used without further purification.

[0051] Another advantage of the present invention is that the specific substitution pattern of the phosphine ligands allows for precise electronic tuning to improve the efficiency and radiochemical yield of the corresponding conjugated diphosphine precursor compound taking into account the specific radionuclide or kit used. In particular, it has been found that the choice of electron donating substituents of the X1, X2, X3 and X4 groups provides this advantage. Furthermore, the substitution pattern of the phosphine ligands also allows for tuning the hydrophobicity or hydrophilicity of the final complex and modifying its in vivo properties, such as its biodistribution or pharmacokinetics.

[0052] Another advantage of the present invention is that the stoichiometry of the complex formed by the diphosphine moiety provides two copies of the targeting ligand per complex. This provides higher tumor uptake due to its higher affinity to the target receptor compared to its monomeric analogue. This also means that the complex has a higher affinity to the receptor target than a single uncomplexed targeting ligand. Without being bound by any theory, it is therefore believed that some excess of the targeting ligand does not impair the binding of the tracer complex in vivo, thereby eliminating the need to carry out additional purification steps.

[0053] In some cases, A is a 5- or 6-membered ring. A may be an aryl group. A may be a 5-membered ring. A may be an unsaturated non-aromatic ring. A may be maleic anhydride.

[0054] In some instances, Y is NH or O and each Z is O. In some cases, X1, X2, X3, and X4 are each a substituted aryl group. In other cases, X1, X2, X3, and X4 are each a substituted or unsubstituted heteroaryl group. In some cases, X1, X2, X3, and X4 are each a substituted phenyl group, optionally substituted only in the para position. In some cases, X1, X2, X3, and X4 are each a substituted or unsubstituted cyclohexyl group, optionally substituted only in the para position. In some cases, X1, X2, X3, and X4 donate more electron density to the phosphine than a phenyl group. In some cases, X1, X2, X3, and X4 are each substituted with one or more C1-C4 alkyl groups, optionally where each alkyl group is selected from the list consisting of methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, and cyclobutyl. In some cases, X1, X2, X3 and X4 are each independently substituted with 1 to 3 substituents, 1 or 2 substituents, or only 1 substituent. In some cases, X1, X2, X3 and X4 are each substituted at the same position. In some cases, X1, X2, X3 and X4 each have the same substituent. In some cases, X1, X2, X3 and X4 have the same substituent at the same position. In some cases, X1, X2, X3 and X4 are the same.

[0055] In some cases, a diphosphine precursor compound of formula (Ia) suitable for preparing a conjugated radiolabeled drug is

[0056] [ka]

[0057] [In the formula, each Z is independently O or S; Y is NH or O; X1, X2, X3 and X4 each independently represent a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 (di)alkylamino group and a C1-C4 alkoxy-(CH2CH2O) n and n is a substituted C5-C8 aryl group having one or more substituents selected from the group consisting of:

[0058]

[0037] Each variable group Z, Y, X1, X2, X3 and X4 in formula (Ia), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided anywhere herein.

[0059] In some cases, diphosphine precursor compounds of formula (Ib) and / or (Ic) suitable for preparing conjugated radiolabeled drugs are:

[0060] [ka]

[0061] [In the formula, X1, X2, X3 and X4 each independently represent a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 (di)alkylamino group and a C1-C4 alkoxy-(CH2CH2O) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0062]

[0039] Each variable group X1, X2, X3 and X4 in formula (Ib) or formula (Ic), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided anywhere herein.

[0063] In some cases, X1, X2, X3, and X4 are each independently methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, methoxy, ethoxy, ethenyl, dimethylamino, and MeO(CH2CH2O). n and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0064] In some cases, X1, X2, X3, and X4 are each independently methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, methoxy, ethoxy, ethenyl, dimethylamino, and MeO(CH2CH2O). n and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0065] In some cases, the diphosphine precursor compound is of formula (Ib), where X1, X2, X3, and X4 are according to a row in the following table:

[0066] [Table 2]

[0067] [where o means ortho, m means meta, and p means para]. In some cases, the diphosphine precursor compound is compound (I-2). Radiolabeled conjugated diphosphine complexes In a third aspect, there is provided a radiolabelled diphosphine complex which may be formed from the conjugated diphosphine precursor compound of the first aspect above.

[0068] The complex comprises at least two conjugated diphosphine precursor compounds according to the second aspect of the invention, 99m Tc, 212 Pb, 212 Bi, 213 Bi, 186 Re, 188 Re, 89 Zr, 67 Ga, 68 Ga, 67 Cu, 64 Cu, 62 Cu, 61 Cu, 60 Cu, 62 Zn and 52 Mn as a ligand to coordinate with one or more radionuclides, The complex is

[0069] [ka]

[0070] isn't it. Preferably, the one or more radionuclides are 99m Tc, 186 Re and 188 The radionuclide is also selected from 67 Cu, 64 Cu, 62 Cu, 61 Cu and 60 Cu. At least two of the conjugated diphosphine precursor compounds may be the same. Optionally, the complex has only two of the conjugated diphosphine precursor compounds as ligands. Optionally, the conjugated diphosphine precursor compounds act as bidentate ligands and coordinate the radionuclide via the two phosphine atoms.

[0071] In some cases, the complex is (a) Formula (M-III-trans) or Formula (M-III-cis) or a mixture thereof;

[0072] [ka]

[0073] Formula (M-III-trans) Formula (M-III-cis) [In the formula, M is 99m Tc, 186 Re and 188 Re; or (b) Formula (Cu-III-A) or Formula (Cu-III-B) or mixtures thereof;

[0074] [ka]

[0075] [In the formula, Cu is 67 Cu, 64 Cu, 62 Cu, 61 Cu and 60 Cu] and in each case each of X, Y, Z and LIG is as defined in any of the second aspects of the invention. X is the same in each instance and the X 1 , X 2 , X 3 and X 4 Represents.

[0076] Unless otherwise indicated, references herein to the formula (M-III-cis / trans), and specific compounds thereof, include all isomers.

[0046] Each variable LIG, Z, Y and X in formula (M-III-trans) and formula (M-III-cis), and any subgroups thereof, may also be independently selected from and combined with any of the definitions provided anywhere herein. In particular, X may also be selected from any of the definitions of X1, X2, X3 and X4 provided herein. The disclaimers of compound (Tc-III-1-RGD) and compound (Re-III-1-RGD) also apply to the subformula of formula (M-III-cis / trans) herein.

[0077] The present invention may employ radionuclides singly or in combination. For example, one commonly used combination is 186 / 188 Re. Generally, technetium isotopes are used for imaging purposes, rhenium isotopes are used for therapeutic purposes, and copper isotopes are used for both imaging and therapeutic purposes.

[0078] The isomers of the complex may exist individually or as a mixture. For example: 99m Tc, 186 Re or 188 The mixture of complexes formed with Re is typically about 1:1 cis / trans, although other mixture ratios are envisioned.

[0079] In some cases, the radiolabeled conjugated diphosphine complex is (a) Formula (M-IIIa-trans) or Formula (M-IIIa-cis) or a mixture thereof

[0080] [ka]

[0081] [In the formula, M is 99m Tc, 186 Re and 188 Re; or (b) Formula (M-IIIb-trans) or Formula (M-IIIb-cis) or a mixture thereof

[0082] [ka]

[0083] [In the formula, M is 99m Tc, 186 Re and 188 Re; or (c) according to formula (Cu-IIIc-A) or formula (Cu-IIIc-B) or mixtures thereof;

[0084] [ka]

[0085] [In the formula, Cu is 67 Cu, 64 Cu, 62 Cu, 61 Cu and 60 Cu; or (d) of formula (Cu-IIId-A) or (Cu-IIId-B) or mixtures thereof;

[0086] [ka]

[0087] [In the formula, Cu is 67 Cu, 64 Cu, 62 Cu, 61 Cu and 60 Cu] and, [In the formula, X is a phenyl group having one or more substituents selected from the group consisting of a C1-C4 alkyl group and a C1-C4 alkoxy group; LIGs include prostate-specific membrane antigen targeting ligands (PSMAt), cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), pentixafor peptides, mini-gastrin peptide analogs for targeting the cholecystokinin-2 receptor, c-Met targeting peptides, alpha-MSH peptides, bisphosphonates, folic acid or carbohydrates. Either:

[0088]

[0050] Each variable LIG, Z, Y and X in formula (M-IIIa-trans) and formula (M-IIIa-cis), formula (M-IIIb-trans) and formula (M-IIIb-cis), formula (M-IIIc-A) or formula (M-IIIc-B) or formula (M-IIId-A) or formula (M-IIId-B) and any subgroups thereof may also be independently selected from and combined with any of the definitions provided anywhere herein. In particular, X may also be selected from any of the definitions of X1, X2, X3 and X4 provided herein.

[0089] In some instances, a complex according to (a) formula (M-IIIa-trans) or formula (M-IIIa-cis) or a mixture thereof; or (b) formula (M-IIIb-trans) or formula (M-IIIb-cis) or a mixture thereof is provided, wherein M, X and LIG are according to a row in the following table:

[0090] [Table 3-1]

[0091] [Table 3-2]

[0092] [Table 3-3]

[0093] In some cases, (c) a complex according to formula (Cu-IIIc-A) or formula (Cu-IIIc-B) or a mixture thereof, or (d) a complex according to formula (Cu-IIId-A) or formula (Cu-IIId-B) or a mixture thereof, is provided, wherein X and LIG are according to a row of the following table:

[0094] [Table 4-1]

[0095] [Table 4-2]

[0096] Methods for making diphosphine precursor compounds

[0053] In a fourth aspect, there is provided a method of making a diphosphine precursor compound of formula (I), comprising the step of mixing HPX1X2 and dichloromaleic anhydride in the presence of a base, wherein X1 and X2 each independently follow any of the definitions provided herein.

[0097]

[0054] This approach differs from the prior art in that dichloromaleic anhydride is added to diphenyl(trimethylsilyl)phosphine. One advantage is improved atom economy since the present method does not require the presence of trimethylsilyl groups. Preferably, dichloromaleic anhydride is added to HPX1X2. The addition of dichloromaleic anhydride to HPX1X2 is preferably dropwise.

[0098] The base may be an organic base, such as an amine base, e.g. triethylamine. The organic base is preferably added dropwise. The reaction is preferably carried out in an organic solvent, e.g. diethyl ether. The reaction is preferably carried out at room temperature.

[0099] Methods for making conjugated diphosphine precursor compounds

[0056] In a fifth aspect, there is provided a method for making a conjugated diphosphine precursor compound of formula (II), comprising the step of mixing a compound of formula (I) and LIG-H in the presence of a base, wherein LIG is according to any of the definitions provided herein.

[0100] The base may be an organic base, such as an amine base, for example N,N-diisopropylethylamine. The organic base may be added dropwise. The reaction is preferably carried out in an organic solvent, such as a protic polar solvent, for example N,N-dimethylformamide. The reaction is preferably carried out at room temperature.

[0101] Methods for making radiolabeled conjugated diphosphine complexes

[0058] In a sixth aspect, there is provided a method for making a radiolabeled conjugated diphosphine complex according to the third aspect, the method comprising the step of mixing a compound of formula (II) with a radionuclide in the presence of an intermediate ligand, a reducing agent, a buffer and a solvent.

[0102]

[0059] The radionuclide is 99m Tc, 212 Bi, 213 Bi, 186 Re, 188 Re, 89 Zr, 67 Ga, 68 Ga, 67 Cu, 64 Cu, 62 Cu, 61 Cu, 60 Cu and 52 Mn. Preferably, the radionuclide is selected from one or more of: 99m Tc, 186 Re or 188 The radionuclide is also selected from 67 Cu, 64 Cu, 62 Cu, 61 Cu and 60The intermediate ligand is preferably a polydentate organic ligand, such as sodium tartrate. The reducing agent is preferably a metal salt, such as tin(II) chloride (dihydrate). The buffer is preferably a bicarbonate, such as sodium bicarbonate. The solvent is preferably selected from one or more of water, saline solution, methanol, ethanol, propanol and isopropanol.

[0103] Kits Containing Conjugated Diphosphine Precursor Compounds In a seventh aspect, there is provided a kit for preparing a radiolabelled conjugated diphosphine compound according to the third aspect comprising a mixture of a reducing agent, a buffer, an intermediate co-ligand and a conjugated diphosphine precursor compound of formula (II).

[0104] The reducing agent may be a metal reducing agent, such as tin(II) chloride. The reducing agent may be present in an amount of 0.2 to 2 equivalents, preferably 0.4 to 1.6 equivalents, more preferably 0.6 to 1.2 equivalents relative to the conjugated diphosphine precursor compound.

[0105] The buffering agent may be an inorganic salt, such as sodium bicarbonate. The buffering agent may be present in an amount of 10 to 400 equivalents relative to the conjugated diphosphine precursor compound, preferably 20 to 200 equivalents, more preferably 50 to 100 equivalents.

[0106] The intermediate co-ligand may be a bidentate organic ligand, such as sodium or potassium tartrate. There may be 0.2 to 2 equivalents, preferably 0.4 to 1.6 equivalents, more preferably 0.6 to 1.2 equivalents of the intermediate co-ligand relative to the conjugated diphosphine precursor compound. Alternatively, there may be 1 to 40 equivalents, preferably 10 to 40 equivalents, more preferably 20 to 40 equivalents of the bidentate organic ligand relative to the conjugated diphosphine precursor compound.

[0107]

[0064] In some cases, a kit for preparing a radiolabeled conjugated diphosphine compound comprises a mixture of 0.2 to 2 equivalents of a reducing agent, 10 to 400 equivalents of a buffering agent, 0.2 to 2 equivalents of an intermediate co-ligand and 1 equivalent of a conjugated diphosphine precursor compound of formula (II).

[0108]

[0065] In some cases, a kit for preparing a radiolabeled conjugated diphosphine compound comprises a mixture of 0.2 to 2 equivalents of a reducing agent, 10 to 400 equivalents of a buffering agent, 20 to 30 equivalents of an intermediate co-ligand and 1 equivalent of a conjugated diphosphine precursor compound of formula (II).

[0109] In some cases, the kit comprises: (i) (II-1-RGD) or (II-2-RGD): 1 mg (0.93 μmol); sodium gluconate (NaCH 11 O7): 1 mg (4.6 μmol); SnCl2.2H2O: 50 μg (0.22 μmol) and NaHCO3: 1.8 mg (21.4 μmol); or (ii) (II-1-RGD) or (II-2-RGD): 500 μg (0.47 μmol); sodium tartrate (Na2C4H4O6): 1.05 mg (4.6 μmol); SnCl2.2H2O: 50 μg (0.22 μmol) and NaHCO3: 1.8 mg (21.4 μmol); or (iii) (II-1-RGD) or (II-2-RGD): 125 μg (0.12 μmol); sodium tartrate: 0.26 mg (1.15 μmol); SnCl2.2H2O: 25 μg (0.11 μmol) and NaHCO3: 0.9 mg (10.7 μmol); or (iv) (II-1-RGD) or (II-2-RGD): 63 μg (0.06 μmol); sodium tartrate: 0.26 mg (1.15 μmol); SnCl2.2H2O: 25 μg (0.11 μmol); NaHCO3: 0.9 mg (10.7 μmol); or (v) 110-120 μg of (II-1-PSMAt1) or (II-2-PSMAt1), 0.26 mg (1.15 μmol) of sodium tartrate; 25 μg of SnCl2.2H2O; and 0.9 mg (10.7 μmol) of NaHCO3; or (vi) (II-1-PSMAt1) 85 μg (0.08 μmol), sodium tartrate: 0.53 mg (2.29 μmol), SnCl2.2H2O: 19.0 μg (0.08 μmol), NaHCO3: 0.90 mg (10.71 μmol) Includes.

[0110] The kits may be used by adding a mixture of saline and ethanol to dissolve the conjugated diphosphine precursor compound; kits containing less conjugated diphosphine precursor compound required less ethanol. In some cases, a saline solution is used without ethanol. In some cases, two or more kits are used.

[0111]

[0069] In some cases, the kit mixture is a lyophilized mixture. The kit may be stored at 0 to 4°C prior to use. In some cases, the kit is preferably stored at about -18°C prior to use. The kit may provide a radiochemical yield of about 85% or more, for example about 90% or more or about 95% or more.

[0112] In some cases, the kit comprises: 99m Tc, 212 Bi, 213 Bi, 186 Re, 188 Re, 89 Zr, 67 Ga, 68 Ga, 67 Cu, 64 Cu, 62 Cu, 61 Cu, 60 Cu and 52 The radionuclide preferably comprises a radionuclide selected from: 99m Tc and / or188 Re.

[0113] Uses and Methods

[0071] In another aspect, there is provided the use of a diphosphine precursor compound of formula (I), a conjugated diphosphine precursor compound of formula (II) or a radiolabelled conjugated diphosphine complex according to the third aspect in the preparation of a medicament for the treatment or diagnosis of a disease.

[0114] In another aspect, there is provided a diphosphine precursor compound of formula (I), a conjugated diphosphine precursor compound of formula (II) or a radiolabelled conjugated diphosphine complex according to the third aspect for use in the treatment or diagnosis of disease. One such use is in imaging studies.

[0115]

[0073] In another aspect, there is provided an in vivo method of imaging a tumor comprising administering to a subject a radiolabeled conjugated diphosphine complex according to the third aspect and detecting the radionuclide. In another aspect, there is provided a method of treating or diagnosing a disease comprising administering to a subject a radiolabeled conjugated diphosphine complex according to the third aspect.

[0116]

[0074] The disease may be one or more of cancer (breast cancer, lung cancer, prostate cancer, myeloma, melanoma, ovarian cancer, thyroid cancer, kidney cancer, pancreatic cancer, neuroendocrine cancer or head and neck cancer), autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, graft-versus-host disease, and myasthenia gravis; chronic inflammatory conditions such as psoriasis, asthma and Crohn's disease) or inflammatory disease (vasculitis, particularly Kawasaki disease, cystic fibrosis, chronic inflammatory bowel disease such as ulcerative colitis or Crohn's disease, chronic bronchitis, inflammatory arthritic diseases such as psoriatic arthritis, rheumatoid arthritis, and systemic onset juvenile rheumatoid arthritis (SOJRA, Still's disease)) and bone metastasis.

[0117] In another aspect, there is provided the use of a diphosphine precursor compound of formula (I), a conjugated diphosphine precursor compound of formula (II) or a radiolabelled conjugated diphosphine precursor complex according to the third aspect, optionally non-therapeutic and / or in vitro, in imaging or cell labelling. Preferably, there is provided the use of said compound in SPECT (single photon emission computed tomography) or gamma scintigraphy. Even more preferably, the radionuclide is 64 PET (positron emission spectroscopy) or radionuclide is Cu 188 Re or 186 There is provided a use of said compound in MRT (molecular radiotherapy), where MRT is Re.

[0118]

[0076] The disclaimers applicable to each of formula (I), formula (II) and / or the third aspect herein may apply to all aspects of the invention, such as kits, uses, medical uses and methods, i.e., any of compound (I-1), compound (II-1-RGD), compound (III-1-RGD) and compound (Re-1-RGD) may be independently included in or excluded from any aspect herein.

[0119] general definition

[0077] For any general formula herein, including those set forth in the specific examples, it should be understood that any of the variable definitions provided herein, such as A, Y, Z, X, X1, X2, X3, X4, R1, R2, R3 and R4, may be applied in combination with any of the other variable definitions. Thus, all possible combinations of variable definitions with each general formula are disclosed and may be claimed.

[0120]

[0078] So that the invention may be understood and further aspects and features thereof may be realized, embodiments illustrating the principles of the invention will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0121] [Figure 1]

[0079] FIG. 1A shows that the binding of (Tc-III-1-RGD) (i.e., [99mTcO2(II-1-RGD)2]+) to the αvβ3 integrin receptor can be inhibited by increasing concentrations of peptide (RGD).

[0080] FIG. 1B shows the biodistribution of (Tc-III-1-RGD) in healthy mice at 1 hour post-injection (left bar); co-injection of 400 g of peptide inhibits (Tc-III-1-RGD) uptake into αvβ3 integrin expressing tissues (right bar). Error bars correspond to 95% confidence intervals.

[0081] FIG. 1C shows that accumulation of (Tc-III-1-RGD) in the ankle (cross) and wrist (triangle) correlates with joint swelling in mice with rheumatoid arthritis.

[0082] Figure 1D shows maximum intensity projection of SPECT / CT images of mice with rheumatoid arthritis showing accumulation of (Tc-III-1-RGD) in the arthritic ankle (RA). Bl = bladder, K = kidney, Th = thyroid. [Diagram 2]

[0083] Figure 2A shows the 31P{H}NMR of compounds (II-1-RGD), cis-(natRe-III-1-RGD) and trans-(natRe-III-1-RGD).

[0084] Figure 2B shows the radio HPLC trace of trans- / cis-(Tc-III-1-RGD) (upper line) prepared from 99mTcO4- and aqueous solution of kit 3 (Table 7), and the HPLC trace (λ220) of cis-(natRe-III-1-RGD) (lower dashed line) and trans-(natRe-III-1-RGD) (lower solid line). [Diagram 3]

[0085] Figure 3 shows stability in serum. (Tc-III-1-RGD) was incubated in human serum for 4 hours. C18 analytical radio-HPLC analysis revealed that 0.5% of 99mTc dissociated from (Tc-III-1-RGD) over 1 hour and 3% of 99mTc dissociated from (Tc-III-1-RGD) over 4 hours. [Figure 4]

[0086] Figure 4 shows quantification of radioactivity distribution in the urine of the bladder (inferior triangle), kidney (upper triangle), liver (square) and heart / blood pool (circle) from SPECT / CT imaging of a single healthy Balb / c mouse administered (Tc-III-1-RGD) intravenously. [Diagram 5] FIG. 5 shows the analytical reverse phase C18 UV (254 nm) HPLC trace of compound (II-1-RGD). [Figure 6] FIG. 6 shows radio-HPLC analysis of urine from healthy Balb / c mice administered (Tc-III-1-RGD) intravenously, demonstrating that it is excreted intact. [Figure 7]

[0089] Figure 7 shows that in rheumatoid arthritis-induced mice administered (Tc-III-1-RGD), radioactivity concentration (measured using SPECT / CT image quantification) at the ankle (cross) and wrist (triangle) correlates with the degree of joint swelling (measured using a caliper). For the ankle, y=1.89x+1.587, R2=0.69, and p=0.04 (significance of slope from nonzero). For the wrist, y=1.01*x+1.09, R2 and p=0.12. [Figure 8]

[0090] Figure 8 shows the biodistribution of (Tc-III-1-RGD) in rheumatoid arthritis mice (n=3) 1 hour after injection. Error bars correspond to standard deviation. [Figure 9]

[0091] Figure 9 shows the total 31P{H}NMR of compound (II-1-RGD) (top), cis-(Re-III-1-RGD) (middle) and trans-(Re-III-1-RGD) (bottom). [Figure 10]

[0092] Figure 10 shows SPECT / CT maximum intensity projection images of balb / c mice administered (Tc-III-1-RGD) intravenously. SPECT images were acquired in 30-minute segments over a 4-hour period. Imaging analysis showed that the majority of (Tc-III-1-RGD) was rapidly eliminated via the renal route: at 30 minutes PI (post-injection), 35% of the injected dose of radioactivity was in the bladder, and at 2 hours PI, 56% was in the bladder. [Figure 11]

[0093] Figure 11 shows that geometric isomer 1 and geometric isomer 2, one corresponding to the "cis" geometric isomer of (Tc-III-1-PSMAt1) and the other corresponding to the "trans" geometric isomer, both have nearly identical uptake into PSMA-positive cells. [Figure 12] FIG. 12 shows the in vitro uptake of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) after 60 min incubation into PSMA-positive (DU145-PSMA and LNCAP) and PSMA-negative cell lines (DU-145 and PC-3). From left to right (for each complex), the bars represent DU145-PSMA+, DU145-PSMA+ and PMPA, DU145, LNCAP, LNCAP and PMPA, and PC-3. Uptake was blocked with the PSMA inhibitor 2-phosphonomethylpentanedioic acid (PMPA). Scatter plots represent biological repeats performed in triplicate. [Figure 13]

[0095] Figure 13 shows SPECT images of healthy mice 15 minutes to 4 hours after intravenous injection of (Tc-III-1-PSMAt1) (top) and (Tc-III-2-PSMAt1) (bottom), which shows that (i) both (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) are cleared from the circulation via the renal pathway, which is ultimately favorable for imaging cancer, and (ii) (Tc-III-1-PSMAt1) is cleared from the kidney faster than (Tc-III-2-PSMAt1). [Figure 14]FIG. 14 shows the ex vivo biodistribution of healthy mice 2 hours after injection with either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1): (a) all excised / dissected organs and tissues except kidney, and (b) kidney. Notably, there is a greater amount of (Tc-III-2-PSMAt1) in the kidney 2 hours after injection compared to (Tc-III-1-PSMAt1). Significant amounts of both tracers also accumulate in the spleen, salivary gland, and prostate, tissues known to express PSMA or take up compounds targeted to PSMA. The uptake of (Tc-III-1-PSMAt1) in the spleen and salivary gland is significantly higher than that of (Tc-III-2-PSMAt1) in these organs. [Figure 15]

[0097] Figure 15 shows analytical reversed-phase radio-HPLC chromatograms of urine collected from mice administered either (a) (Tc-III-1-PSMAt1) or (b) (Tc-III-2-PSMAt1). The retention time of each radioactive peak matches that of either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1), indicating that each 99mTc radiotracer is excreted intact and has high metabolic stability. Analytical HPLC conditions: 20 min, linear increase from 100% A to 100% B at 5% min-1 (flow rate 1 ml / min, A=water containing 0.1% TFA (trifluoroacetic acid), B=acetonitrile containing 0.1% TFA, analytical (4.6 x 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column). [Figure 16]

[0098] Figure 16 shows analytical reversed-phase radio-HPLC chromatograms of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) after kit-based radiolabeling reactions performed in 5 min at either room temperature or 100°C. Analytical HPLC conditions: 20 min, linear increase of 5% min-1 from 100% A to 100% B (flow rate 1 ml / min, A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6 x 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column). [Figure 17]

[0099] Figure 17 shows the biodistribution of SCID / beige mice bearing either DU145-PSMA+ or DU145 prostate cancer tumors. Mice were intravenously administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). Three experimental groups of mice (n=5 per group) were used to determine the specificity of radiotracer uptake. In the first group, mice bearing DU145-PSMA+ prostate cancer tumors were administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). In the second group, mice bearing DU145-PSMA+ prostate cancer tumors were co-administered with 2-phosphonomethylpentanedioic acid (PMPA) (to inhibit PSMA receptor uptake of the radiotracer) and either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). In the third group, mice bearing DU145 prostate cancer tumors that do not express PSMA receptors were administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). All animals were sacrificed 2 hours after injection, organs were removed, weighed, and radioactivity was counted. Separately, mice bearing DU145-PSMA+ prostate cancer tumors were administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) (n=3 per group) and sacrificed 24 hours after injection. (a) Tumor uptake / retention of radiotracer; (b) Kidney uptake / retention of radiotracer; (c) Biodistribution of (Tc-III-1-PSMAt1) in organs / tissues except tumor and kidney; (d) Biodistribution of (Tc-III-2-PSMAt1) in organs / tissues except tumor and kidney. Error bars correspond to standard deviation. In (a) and (b), the leftmost bar is "Tracer, DU145-PSMA+(2 hr)", the leftmost bar is "Tracer, DU145-PSMA+(24 hr)", the rightmost bar is "Tracer+PMPA, DU145-PSMA+(2 hr)", and the rightmost bar is "Tracer, DU145(2 hr)".In (c) and (d), the order of the bars is the same except that "Tracer, DU145-PSMA+(24 hr)" is absent. [Figure 18]

[0100] Figure 18a shows the whole-body SPECT / CT maximum intensity projection of SCID / beige mice bearing either DU145-PSMA+ or DU145 tumors 2 hours after injection, administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). Animals were also administered PMPA to inhibit uptake in DU145-PSMA+ tumors.

[0101] Figure 18b shows the whole-body SPECT / CT maximum intensity projection of SCID / beige mice bearing DU145-PSMA+ tumors 24 hours after injection, administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). [Figure 19] 19 shows reversed-phase radio-HPLC traces of (a) (Re-III-1-PSMAt1), (b) (Re-III-2-PSMAt1) and (c) (Re-III-11-PSMAt1). Analytical HPLC conditions: 100% A to 100% B linear increase in 30 min (flow rate 1 ml / min, A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6×150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column). [Figure 20]

[0103] Figure 20 shows the stability of Tc-III-11-PSMAt1 in serum. Tc-III-11-PSMAt1 was incubated in human serum for 24 hours. C18 analytical radio-HPLC analysis revealed that >95% of Tc-III-11-PSMAt1 remained intact after 24 hours of incubation. Analytical HPLC conditions: 20 min linear increase from 100% A to 100% B (flow rate 1 ml / min, A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6 x 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column). [Figure 21]

[0104] Figure 21 shows the uptake and localization of i) (Tc-III-1-PSMAt1) and ii) (Tc-III-2-PSMAt1) into (a) DU145-PSMA cells and (b) LNCaP cells over time. Data are presented as mean ± SD, n=3 biological repeats performed in triplicate. [Figure 22]

[0105] Figure 22 shows reversed-phase radio-HPLC chromatograms showing the stability of a) (Re-III-1-PSMAt1) and b) (Re-II-2-PSMAt1). Both (Re-III-1-PSMAt1) and (Re-II-2-PSMAt1) are stable for up to 24 hours after incubation in human serum at 37°C. [Figure 23]

[0106] Figure 23 shows the in vitro uptake of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1). Uptake of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1) into PSMA positive (DU145-PSMA+) and PSMA negative (DU-145) cell lines after 60 minutes of incubation. Uptake was blocked with the PSMA inhibitor PMPA. Scatter plots represent biological repeats performed in triplicate. *, p<0.05 **, p<0.01; ***, p<0.001, ****, p<0.0001. [Figure 24]

[0107] Figure 24 shows the uptake of (186Re-III-1-PSMAt1) into PSMA-expressing DU145-PSMA+ and LNCaP prostate cancer cells, and PSMA-negative DU145 prostate cancer cells. (186Re-III-1-PSMAt1) was also co-incubated with excess PSMA inhibitor, PMPA. *, p<0.05 **, p<0.01; ***, p<0.001, ****, p<0.0001; n=2-5. Data are presented as mean ± SD. [Diagram 25]

[0108] Figure 25 shows ex vivo biodistribution in mice (n=4 per group) 2 hours after injection with either (188Re-III-1-PSMAt1) or (188Re-III-2-PSMAt1): (a) all excised / dissected organs and tissues except kidneys and (b) kidneys. [Figure 26]

[0109] Figure 26 shows reversed-phase radio-HPLC chromatograms of urine collected from mice 2 hours after administration of (ai) (188Re-III-1-PSMAt1); (a-ii) (188Re-III-1-PSMAt1); (bi) (188Re-III-2-PSMAt1); and (b-ii) (188Re-III-2-PSMAt1). Analytical HPLC conditions: 30 min, linear increase from 100% A to 100% B (flow rate 1 ml / min, A=water with 0.1% TFA, B=acetonitrile with 0.1% TFA, analytical (4.6 x 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122]

[0110] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, which may be expressed in a specific form or as a means for performing a disclosed function, or as a method or process for obtaining a disclosed result, may be utilized, as appropriate, separately or in any combination of such features, to realize the invention in various of its forms.

[0123]

[0111] While the present invention has been described in conjunction with exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present invention described above are considered to be illustrative and not limiting. Various modifications of the described embodiments may be made without departing from the scope of the present invention.

[0124]

[0112] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0125]

[0113] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0114] Throughout this specification, including in the claims which follow, unless the context otherwise requires, the words "have," "comprise," and "include," as well as variations such as "having," "comprises," "comprising," and "including," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. However, each disclosure herein also includes the option of excluding any other integer or step or group of integers or steps.

[0126]

[0115] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the preceding "about," it will be understood that the particular value forms another aspect. The term "about" with respect to numerical values ​​is optional and means, for example, + / - 10%.

[0127]

[0116] The words "preferred" and "preferably" are used herein to refer to embodiments of the invention that may, under some circumstances, provide certain benefits. It should be recognized, however, that other embodiments may also be preferred, under the same or different circumstances. Thus, the recitation of one or more preferred embodiments does not mean or suggest that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure or from the scope of the claims.

[0128]

[0117] The compounds of the present invention include the isomers, salts, solvates, and chemically protected forms thereof, as described in more detail below.

[0118] In the present invention, the alkyl group is generally a C1-C4 alkyl group. The term "C1-C4 alkyl" as used herein includes a monovalent moiety obtained by removing one hydrogen atom from a C1-C4 hydrocarbon compound having 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and may be saturated, partially unsaturated, or fully unsaturated. The term "C1-C4 alkyl" includes methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, ethenyl, cis / trans-1-propenyl, 2-propenyl, cis / trans-1-butenyl, cis / trans-2-butenyl, and 3-butenyl. In a preferred embodiment, the C1-C4 alkyl group is a saturated alkyl group and / or an acyclic alkyl group. In an even more preferred embodiment, the C1-C4 alkyl group is a methyl or ethyl group, since shorter chain alkyl groups tend to reduce the hydrophobicity of the compounds of the present invention.

[0129]

[0119] In the present invention, the alkoxy group is generally a C1-C4 alkoxy group. The term "C1-C4 alkoxy" as used herein includes a monovalent moiety obtained by removing a hydrogen atom from an oxygen atom of a C1-C4 alcohol compound having 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and may be saturated, partially unsaturated, or fully unsaturated. The term "C1-C4 alkoxy" includes methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, t-butoxy, cyclobutoxy, ethenoxy, cis / trans-1-propenoxy, 2-propenoxy, cis / trans-1-butenoxy, cis / trans-2-butenoxy, and 3-butenoxy. In a preferred embodiment, the C1-C4 alkoxy group is a saturated alkoxy group and / or an acyclic alkoxy group. In an even more preferred embodiment, the C1-C4 alkoxy group is a methoxy or ethoxy group, since shorter chain alkoxy groups tend to reduce the hydrophobicity of the compounds of the invention.

[0130]

[0120] In the present invention, a "heteroaryl group" generally refers to a C5-C 12 Heteroaryl groups, preferably 5- or 6-membered heteroaryl groups, as used herein, are C5-C 12 Heteroaryl refers to a monovalent moiety obtained by removing one hydrogen atom from a ring atom of a heterocyclic compound. Heteroaryl groups may be partially or fully unsaturated. The present invention provides examples of compounds in which one or more pyridyl groups (e.g., one or more 2-pyridyl groups) are present. However, examples of heteroaryl compounds that can be used according to the present invention include the following:

[0131] Imidazole: A five-membered aromatic ring having two nitrogen atoms and three carbon atoms. Triazole: A five-membered aromatic ring having three nitrogen atoms and two carbon atoms, with two ring isomers: 1,2,3,triazole and 1,2,4 triazole.

[0132] Tetrazole: A five-membered aromatic ring having four nitrogen atoms and one carbon atom. Pyridine: A six-membered aromatic ring having one nitrogen atom and five carbon atoms. Diazine: A six-membered aromatic ring having two nitrogen atoms and four carbon atoms, with three ring isomers: 1,2-diazine, 1,3-diazine and 1,4-diazine.

[0133] Triazine: A six-membered aromatic ring having three nitrogen atoms and three carbon atoms, with three ring isomers: 1,2,3-triazine, 1,2,4-triazine and 1,3,5-triazine.

[0134] Tetrazine: A six-membered aromatic ring having four nitrogen atoms and two carbon atoms, with three ring isomers: 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, and 1,2,4,5-tetrazine.

[0135] Fused ring systems such as quinoline, isoquinoline and indole. To facilitate chelation of radionuclides with heteroatoms, sp 2 It is generally preferred that the nitrogen-containing heterocyclic group has a donor electron pair ortho to the methylene bridge of the bisphosphonate compound. A preferred heteroatom is nitrogen, thus giving a pyridyl heteroaryl group.

[0136] In the present invention, "Re" and " 188 "Re" stands for rhenium-188 ( 188 Re), while " 186 "Re" refers to rhenium-186, nat "Re" refers to naturally abundant rhenium. "Tc" and " 99m Tc" stands for technetium-99m ( 99m Tc), while " 99g"Tc" stands for techniutium-99g. nat "Cu" refers to naturally abundant copper.

[0137] Other forms of substituents

[0131] Included above are the well-known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also refers to the anionic (carboxylate) form (-COO - ), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (N + HR 1 R 2 ), a salt or solvate of an amino group, such as a hydrochloride salt, and conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-O - ), a salt or solvate thereof, and conventional protected forms of hydroxyl groups.

[0138] Isomers, Salts, Solvates, Protected Forms, and Prodrugs

[0132] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis and trans forms; E and Z forms; c, t, and r forms; endo and exo forms; R, S, and meso forms; D and L forms; d and l forms; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; synclinal and anticlinal forms; alpha and beta forms; axial and equatorial forms; boat, chair, twist, envelope, and half-chair forms; and combinations thereof (hereinafter collectively referred to as "isomers" (or "isomeric forms")).

[0139]

[0133] It should be noted that, except as discussed below regarding tautomeric forms, the term "isomer" as used herein specifically excludes structural (or constitutional) isomers (i.e., isomers that differ not only in the position of the atoms in space but also in the connections between the atoms). For example, a reference to a methoxy group, -OCH3, should not be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a structural class or general formula includes structural isomeric forms that fall within that class or formula, and all possible conformations and configurations of the compounds herein are intended to be included in the general formula unless expressly stated or indicated.

[0140] The above exclusion does not pertain to tautomeric forms, e.g., keto, enol, and enolate forms, as in, for example, the following tautomeric pairs: keto / enol (exemplified below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, N-nitroso / hyroxyazo, and nitro / aci-nitro.

[0141]

[0135] It should be noted that expressly included in the term "isomer" are compounds having one or more isotopic substitutions. For example, H is: 1 H, 2 H(D), and 3 H(T) may be in any isotopic form, and C may be 12 C. 13 C, and 14 C may be in any isotopic form, including 16 O and 18 It may be in any isotopic form, including O, etc.

[0142]

[0136] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by methods taught herein or by adapting known methods in a known manner.

[0143]

[0137] Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate and protected forms thereof, eg, as discussed below.

[0138] It may be convenient or desirable to prepare, purify, and / or handle corresponding salts of the active compounds, e.g., pharma- ceutically acceptable salts. Examples of pharma- ceutically acceptable salts are discussed in Berge et al., J. Pharm. Sci., 66, 1-19 (1977).

[0144] For example, a compound may have a functional group that is anionic or may be anionic (e.g., COOH is COO - In the case of cations having an acid value of 1, 2 or 3, salts can be formed with suitable cations. Examples of suitable inorganic cations include Na + and K. + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 +Examples of some suitable substituted ammonium ions include, but are not limited to, ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3). 4+ It is.

[0145]

[0140] If the compound is cationic or has a functional group which can be cationic (e.g., NH2 is NH 3+ In the case of phosphate groups, which may be phosphate groups, salts may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, palmitic acid, lactic acid, malic acid, pamoic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, aspartic acid, benzoic acid, cinnamic acid, pyruvic acid, salicylic acid, sulfanilic acid, 2-acetyloxybenzoic acid, fumaric acid, phenylsulfonic acid, toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, oxalic acid, pantothenic acid, isethionic acid, valeric acid, lactobionic acid, and gluconic acid. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

[0146]

[0141] It may be convenient or desirable to prepare, purify, and / or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of a solute (e.g., active compound, a salt of an active compound) and a solvent. When the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, etc.

[0147] It may be convenient or desirable to prepare, purify, and / or handle active compounds in chemically protected form. The term "chemically protected form" as used herein includes compounds in which one or more reactive functional groups are protected from undesired chemical reactions, i.e., in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be carried out without affecting the protected group; the protecting group can usually be removed in a subsequent step without substantially affecting the remainder of the molecule. See, for example, "Protective Groups in Organic Synthesis" (T. Green and P. Wuts, Wiley, 1999).

[0148] For example, a hydroxy group may be protected as an ether (-OR) or ester (-OC(=O)R), e.g., as a t-butyl ether; benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (-OC(=O)CH3, -OAc).

[0149] For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, where the carbonyl group (>C=O) is converted to a diether (>C(OR)2), for example, by reaction with a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

[0150] For example, amine groups may be protected, for example, as amides or urethanes, for example, as methylamides (-NHCO-CH3); benzyloxyamides (-NHCO-OCH2C6H5, -NH-Cbz); t-butoxyamides (-NHCO-OC(CH3)3, -NH-Boc); 2-biphenyl-2-propoxyamides (-NHCO-OC(CH3)2C6H4C6H5, -NH-Bpoc), as 9-fluorenylmethoxyamides (-NH-Fmoc), as 6-nitroveratryloxyamides (-NH-Nvoc), as 2-trimethylsilylethyloxyamides (-NH-Teoc), as 2,2,2-trichloroethyloxyamides (-NH-Troc), as allyloxyamides (-NH-Alloc), as 2(-phenylsulfonyl)ethyloxyamides (-NH-Psec); or, in suitable cases, as N-oxides (>NO).

[0151] For example, the carboxylic acid group may be substituted as an ester, for example, a C1-C7 alkyl ester (e.g., a methyl ester; a t-butyl ester); a C1-C7 haloalkyl ester (e.g., a C1-C7-trihaloalkyl ester); a tri-C1-C7-alkylsilyl-C1-C7-alkyl ester; or a C5-C 20 It may be protected as an aryl-C1-C7-alkyl ester (eg benzyl ester; nitrobenzyl ester); or as an amide, eg methylamide.

[0152]

[0147] It may be convenient or desirable to prepare, purify, and / or handle an active compound in the form of a prodrug. As used herein, the term "prodrug" includes a compound which, when metabolized (e.g., in vivo), yields the desired active compound. Typically, a prodrug is inactive or less active than the active compound, but may offer advantageous handling, administration, or metabolic properties.

[0153] For example, some prodrugs are esters of the active compound (e.g., physiologically acceptable metabolically labile esters). During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters can be formed, for example, by esterifying any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with prior protection, if appropriate, of any other reactive groups present in the parent compound, followed by deprotection, if necessary. Examples of such metabolically labile esters are those in which R is C 1~7 Alkyl (e.g., -Me, -Et); C 1~7 Aminoalkyl (e.g., aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4-morpholino)ethyl); and acyloxy-C1-C7 alkyl (e.g., acyloxymethyl; acyloxyethyl; e.g., pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl; 1-(1-methoxy-1-methyl)ethyl-carbonyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl; 1-isopropoxy cyclohexyl-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl; (4-tetrahydropyranyloxy)carbonyloxymethyl; 1-(4-tetrahydropyranyloxy)carbonyloxyethyl; (4-tetrahydropyranyl)carbonyloxymethyl; and 1-(4-tetrahydropyranyl)carbonyloxyethyl).

[0154]

[0149] Also, some prodrugs are enzymatically activated to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

[0155] Complexes of compounds and their uses The compounds of the present invention can be used for therapy, particularly for the treatment of arthritis and cancer. In addition, the compounds of the present invention can be used to chelate radionuclides, for example to enable them to be used in imaging studies or for therapeutic purposes. Examples of radionuclides that can be chelated by the compounds of the present invention are technetium, rhenium and copper isotopes, e.g. 99m Tc, 186 Re, 188 Re, 67 Cu, 64 Cu, 62 Cu, 61 Cu, 60 The present invention may use radionuclides alone or in combination. For example, one commonly used combination is: 186 / 188 Re. Other combinations are 99m Tc / 188 Re or 99m Tc / 186 Re. Generally, technetium isotopes are used for imaging purposes, rhenium isotopes are used for therapeutic purposes, and copper isotopes are used for both imaging and therapeutic purposes. If no specific isotope is given for an atom, it may be selected as any of the known isotopes or mixtures thereof.

[0156]

[0151] The present invention provides active compounds for use in methods of treating the human or animal body. Such methods may comprise the step of administering to such a subject a therapeutically effective amount of an active compound, preferably in the form of a pharmaceutical composition.

[0157] The term "treatment" as used herein in the context of the treatment of a condition relates to the general treatment and therapy of either humans or animals (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, such as inhibition of the progression of the condition, including reducing the rate of progression, stopping the rate of progression, improving the condition, reducing pain, and curing the condition. Treatment as a preventative measure, i.e., prophylaxis, is also included. By way of example, the compounds and complexes of the present invention may be used for the treatment of arthritis and for the treatment of cancer. The treatment of cancer may involve palliative and / or curative treatment.

[0158]

[0153] As used herein, the term "therapeutically effective amount" includes an amount of an active compound, or a material, composition or dosage form containing an active compound, that is effective to produce some desired therapeutic effect and is commensurate with a reasonable benefit / risk ratio.

[0159] Formulation and Dosage

[0154] While it is possible for the active compounds to be administered alone, it is preferable to present them as a pharmaceutical composition (e.g., a formulation) comprising at least one active compound as defined above, together with one or more pharma- ceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art, and optionally other therapeutic or prophylactic agents.

[0160]

[0155] Thus, the present invention further provides pharmaceutical compositions as defined above, and methods of making pharmaceutical compositions which comprise the step of admixing at least one active compound as defined above with one or more pharma- ceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials as described herein.

[0161]

[0156] As used herein, the term "pharmacologically acceptable" includes compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical textbooks, e.g., "Remington's Pharmaceutical Sciences", 18th Edition, Mack Publishing Company, Easton, Pa., 1990.

[0162]

[0157] For intravenous, cutaneous or subcutaneous injection, or injection into an affected area, the active ingredient is in the form of a parenterally acceptable aqueous solution or suspension that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as, for example, Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0163] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0164]

[0159] It will be appreciated that the appropriate dosage of the active compound and compositions containing the active compound may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against any risk or harmful side effects of the treatment of the present invention. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, condition, general health, and previous medical history of the patient. The amount of compound and the route of administration are ultimately at the discretion of the physician, but generally the dosage is to achieve a local concentration at the site of action that achieves the desired effect without causing substantial risk or harmful side effects.

[0165]

[0160] In vivo administration can be performed in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods for determining the most effective means and dosage of administration are well known to those skilled in the art and vary according to the formulation used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed, and the dose level and pattern can be selected by the treating physician. EXAMPLES

[0166] material and method All chemicals were supplied by Sigma-Aldrich or Fisher Scientific unless otherwise stated. Sodium (pertechnetate) in saline (Na[ 99m TcO4] was supplied by Guy's and St Thomas' Hospital Nuclear Medicine Services. Cyclic RGD peptide (Arg-Gly-Asp-D-Phe-Lys, cyclized via the peptide backbone) and PSMAt peptide were purchased from Peptide Synthetics (Hampshire, UK).

[0167]

[0162] NMR data ( 1 H, 13 C{H} and 31 P{H}1D spectra as well as COSY, TOCSY and HSQC spectra) were acquired on a Bruker Avance III 400 spectrometer equipped with a QNP probe or a Bruker Avance III 700 spectrometer equipped with an AVIII console and a quadruple resonance QCI cryoprobe. High-resolution mass spectrometry (MS) was performed by the King's College London Mass Spectrometry Facilities using a high-resolution Thermo Exactive mass spectrometer in positive electrospray mode. Samples were injected into the ion source at a rate of 10 μl / min using a syringe pump. High-performance liquid chromatography (HPLC) was performed on an Agilent 1200 LC system equipped with Laura software, a Rheodyne sample loop (200 μL) and UV spectroscopic detection at 220 nm or 254 nm. The HPLC was fitted with a LabLogic Flow-Count detector equipped with a sodium iodide probe (B-FC-3200) for radiodetection. Semi-preparative (9.4 × 250 mm, 5 μm) and analytical (4.6 × 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 columns were used with purified water (A) and acetonitrile (B) containing 0.005% and 0.1% TFA as mobile phases for semi-preparative and analytical runs, respectively.

[0168] General HPLC methods used herein include the following; HPLC Method 1 (Semi-Prep): 100 min, linear increase of 1% min-1 from 100% A to 100% B, flow rate = 3 ml min-1 -1 HPLC Method 2 (Analytical): 20 min, 100% A to 100% B for 5% -1 Linear increase in A (flow rate 1 ml / min). HPLC method 3 (semi-preparative): 200 min, 95% A to 100% B in 0.5% increments. -1Linear increase of A (flow rate 3 ml / min). HPLC Method 4 (Analytical): 55 min, 2.5% increase from 100% A to 25% B over 10 min. -1 followed by a linear increase of 0.33% from 25%A to 40%B over 45 minutes. -1 Linear increase in (flow rate 1 mL min -1 ).

[0169] Instant thin layer chromatography (iTLC) used iTLC SGI0001 strips (Varian Medical Systems, Crawley, UK). iTLC plates were scanned on a Perkin Elmer Storage Phosphor System (Cyclone) or a LabLogic miniScan TLC reader equipped with Laura software.

[0170]

[0165] High performance liquid chromatography (HPLC) was performed on an Agilent 1200 HPLC system equipped with Laura software, a Rheodyne sample loop (200 μL) and ultraviolet (UV) spectroscopic detection at 214 nm, 220 nm, 254 nm or 280 nm.

[0171] Example 1 Synthesis of Compound (I-1): 3,4-Bis(bisphenylphosphanyl)furan-2,5-dione (Compound (I-1))

[0172] [ka]

[0173] Diphenylphosphine (2.2 equiv., 5.04 mmol, 0.88 mL) was added to a solution of dichloromaleic anhydride (1 equiv., 2.42 mmol, 404.0 mg) in diethyl ether (15 mL) to give a pale yellow solution. Triethylamine (2.2 equiv., 5.04 mmol, 0.7 mL) was added dropwise and the dark yellow suspension was stirred until a cohesive sludge formed (room temperature, 2 h). The solid containing the product was isolated by filter cannula and washed with ice-cold diethyl ether (3×10 mL). The crude product was redissolved and passed through a silica plug in dichloromethane, after which the solvent was removed under reduced pressure to give a yellow solid. The product was recrystallized from chloroform / diethyl ether to give crystalline yellow needles (390.7 mg, 837.7 μmol, 34.6%).

[0174]

[0167] 1 H NMR (399 MHz, acetonitrile-d3, 298 K): δ (ppm) 7.38-7.42 (m, 12 H, H メタ and H パラ ), 7.34-7.30 (m, 8 H, H オルト );

[0168] 13 C NMR (100 MHz, acetonitrile-d3, 298 K): δ (ppm) 163.22 (m, C カルボニル ), 153.50 (m, C アルケン ), 134.12 (m, C オルト ), 133.00 (m, C 置換 ), 129.84 (m, C パラ ), 128.73 (m, C メタ );

[0169] 31 P{ 1 H} NMR (162 MHz, acetonitrile-d3, 298 K): δ (ppm) -18.37;

[0170] 31 P{ 1 H} NMR (162 MHz, dimethylformamide-d7, 298 K): δ (ppm) -19.07;

[0171] 31 P{ 1 H}NMR (162 MHz, chloroform-d3, 298 K): δ (ppm) -20.53; HR-MS-ESI m / z:[M+H] + 467.0954(C 28 H 21 Calculated value of O3P2: 467.0960);

[0173] IR (solid)λ max (cm -1 )3054(w), 1834(m), 1811(m), 1757(s), 1496(w), 1484(w), 1435(m), 1244(s), 913(s);

[0174] Melting point: 149.6°C.

[0175] Example 2 Synthesis of Compound (I-2): 3,4-Bis(bis-p-tolylphosphanyl)furan-2,5-dione (Compound (I-2))

[0176] [ka]

[0177] Step 1: Bis(p-tolyl)chlorophosphine (1 equiv, 4.02 mmol, 0.9 mL) in diethyl ether (5 mL) was added dropwise to a slurry of lithium aluminum hydride (3.2 equiv, 13.01 mmol, 493.8 mg) in diethyl ether (20 mL) at 0° C. The grey suspension was stirred at 0° C. (30 min) and then in situ 31 P{ 1 The reaction was stirred at room temperature until completion (22 h) as determined by H NMR. (aq) (0.5 mL) and iii) the reaction was quenched by the dropwise addition of degassed water (1.5 mL).

[0178] The white precipitate was removed from the filtrate (containing the product) via a filter cannula. The precipitate was then washed with diethyl ether (2×10 mL) and these washes were combined with the filtrate. The resulting solution was dried over magnesium sulfate, reisolated via a filter cannula, the magnesium sulfate was washed with diethyl ether (2×10 mL) and the filtrate and washes were combined. The solvent was removed under reduced pressure to give the product as a clear liquid (593.4 mg, 2.77 mmol, 68.9%) that crystallized below 20° C. When the reaction scale was doubled, the crude product was purified by distillation at 200° C. and 2.5×10 -1 Purified by distillation at mbar.

[0179]

[0177] 1H NMR (400 MHz, chloroform-d) δ 7.48 - 7.36 (m, 4H, Hb), 7.22 - 7.12 (m, 4H, Hc), 5.25 (d, JH-P = 174.9 Hz, 1H, PH), 2.38 (s, 6H, He);

[0178] 31 P{ 1 H} NMR (162 MHz, chloroform-d) δ -41.93;

[0179] 31 P NMR (162 MHz, chloroform-d) δP -41.92 (d, J = 174.9 Hz).

[0180] Step 2: 3,4-Bis(bis-o-tolylphosphanyl)furan-2,5-dione was prepared from (Tol)2PH by the following method: A solution of ditolylphosphine (1.9 equiv., 0.36 mmol, 77.0 mg) in diethyl ether (0.2 mL) was added dropwise to a solution of dichloromaleic anhydride (1 equiv., 0.19 mmol, 31.0 mg) in tetrahydrofuran (1.3 mL) to give a clear orange solution. Triethylamine (3 equiv., 0.58 mmol, 0.08 mL) was added dropwise and the dark orange suspension was stirred (rt, 2 h). The solids were removed via filter cannula and washed with tetrahydrofuran (3×2 mL). The filtrate and washings (containing the product) were combined and the solvent removed from the resulting product solution under reduced pressure. The crude product was redissolved and passed through a silica plug in dichloromethane and the solvent removed under reduced pressure. The product was dissolved in a minimum amount of chloroform and the solution was layered with diethyl ether The precipitate was collected by filtration and dried to give the product as yellow needles (30.2 mg, 0.06 mmol, 16.1%).

[0180]

[0181] 1 H NMR (500 MHz, chloroform-d) δH 7.21 (dt, J = 8.6, 4.4 Hz, 8H, H オルト ), 7.08 (d, J = 7.7 Hz, 8H, H メタ ), 2.34 (s, 12H, H パラ-メチル );

[0182] 13 C{ 1 H} NMR (125 MHz, chloroform-d): δC (ppm) 162.84 (t, J = 2.86, C カルボニル ), 155.03 (m, C アルケン ), 140.06 (s, C パラ ), 134.24 (m, C オルト ), 129.60 (t, J= 4.40, C メタ ), 129.04 (m, C 置換 ), 21.55 (s, C パラ-メチル );

[0183] 31 P{ 1 H} NMR (162 MHz, chloroform-d) δP -23.08 (s);

[0184] HR-MS-ESI m / z:[M+H] + 523.1602(C 32 H 28 (Calculated value of O3P2: 523.1592).

[0181] Example 3a Synthesis of bis(para-methoxyphenyl)phosphine ((p-MeOC6H4)2PH)

[0182] [ka]

[0183]

[0185] A solution of bis(4-methoxyphenyl)chlorophosphine (1 g, 3.56 mmol) in Et2O (4.5 mL) was added dropwise to a suspension of LiAlH4 (1.24 g, 11.4 mmol, 3.2 equiv) in Et2O (18 mL) at 0 °C. The solution was stirred at 0 °C for an additional 30 min before being allowed to warm to room temperature and then stirred overnight. The reaction mixture was cooled to 0 °C and quenched by careful addition of H2O (0.5 mL), 15% NaOH (0.5 mL) and H2O (2.5 mL). After stirring for 1 h, the solution was isolated by filtration and then concentrated in vacuo to give the title compound (744 mg, 3.02 mmol, 85%) as a white solid.

[0184]

[0186] 1 H NMR (400 MHz, CDCl3): δ H (ppm) 7.46-7.28 (m, 4H, Ar-H), 6.90-6.82 (m, 4H, Ar-H), 5.38-4.98 (br. s, PH), 3.80 (s, 6H, OMe).

[0187] 31 P{ 1 H}NMR (162 MHz, CDCl3): δ P(ppm) -44.2 (s).Spectroscopic data are in accordance with the literature (YY Yan and TV Rajan Babu, Org. Lett., 2000, 2, 4137-4140).

[0185] Example 3b Synthesis of Compound (I-11): 3,4-Bis[bis(4-methoxyphenyl)phosphanyl]furan-2,5-dione

[0186] [ka]

[0187]

[0188] NEt3 (30.4 μL, 2.18 mmol, 2.2 equiv) was added to a solution of (p-MeOC6H4)2PH (50.0 mg, 0.203 mmol, 2.05 equiv) in Et2O (0.5 mL). A solution of 2,3-dichloromaleic anhydride (16.5 mg, 98.8 μmol) in Et2O (0.5 mL) was added dropwise, resulting in an immediate color change from a colorless solution to a deep red solution. 31 Once the reaction had reached completion, as monitored by P NMR spectroscopy, the volatiles were removed in vacuo. The crude product was dissolved in DCM, passed through a silica plug (2% MeOH in DCM) and concentrated to dryness. The remaining (p-MeOC6H4)2PH was purified by high vacuum (approximately × 10 -7 Removal under Torr afforded the title compound (51.4 mg, 87.7 μmol, 89%) as an orange solid.

[0188]

[0189] 31 P{ 1 H}NMR (162 MHz, CDCl3): δ P (ppm) -22.3 (s).

[0190] 1 H NMR (400 MHz, CDCl3): δ H (ppm) 7.28-7.21 (m, 8H, Ar-H), 6.83-6.78 (m, 8H, Ar-H), 3.80 (s, 12H, OMe).

[0191] 13C NMR (101 MHz, CDCl3): δ C (ppm) 163.0 (m, C=O), 161.1 (s, p-ArC), 154.2 (m, C=C), 135.9 (t, 2 J P,C = 12.2, o-ArCH), 123.4 (s, ArC), 114.5 (t, 3 J P,C = 4.9 Hz, m-ArCH), 55.3 (s, OMe). HR-MS (nanospray): m / z C 32 H 29 Calculated value of O7P2 [M+H] + = 587.1389;observed = 587.1395.

[0189] Example 4 Synthesis of PEG-PSMA peptide conjugates (II-1-PSMAt1), (II-2-PSMAt1) and (II-11-PSMAt1)

[0190] [ka]

[0191] [ka]

[0192]

[0192] Under a nitrogen stream, compound (I-1), compound (I-2) or compound (I-11) (5-10 mg, 1 equiv.) in DMF (100 μL, dry, degassed) and Lys-((PEG)4-NH2)-uredo-Glu, 5-10 mg, 1 equiv.) in DMF (100 μL, dry, degassed) were combined and N,N-diisopropylethylamine (DIPEA, 6 μL) was added. The tube was sealed and the solution was agitated at room temperature (15-20 min). The product was purified by semi-preparative C 18-HPLC (mobile phase: 0.01% acetic acid in water (A) and acetonitrile (B); the process starts at 95% A and increases to 100% B; unreacted compound is eluted with 100% acetonitrile). The product-containing fractions were neutralized with aqueous ammonium bicarbonate buffer (0.125 M, 15 μL / mL eluent) and freeze-dried to give the PSMAt1 peptide conjugate (>60.0%) as a solid.

[0193]

[0193] The reaction is reversible under acidic conditions, but this is prevented by simply adding ammonium bicarbonate to a solution of the isolated material.

[0194] Characterization of compound (II-1-PSMAt1):

[0195] (700 MHz, DMF-d 7, 298 K): δ (ppm) 1.382-1.436 (m, 2H, Lys, H γ ), 1.445-1.504 (m, 2H, Lys, H δ ), 1.608-1.660 (m, 1H, Lys, H β ), 1.742-1.795 (m, 1H, Lys, H β ), 1.842-1.893 (m, 1H, Glu, H β ), 1.986-2.039 (m, 1H, Glu, H β ), 2.324-2.364 (m, 1H, Glu, H γ ), 2.386 (t, J = 6.24 Hz, 2H, PEG, H o ), 2.455 (dt, J1= 14.68 Hz, J2= 8.39 Hz, 1H, Glu, H γ ), 2.955-2.983 (m, 2H, PEG, H h ), 3.014-3.045 (m, 2H, PEG, H i ), 3.128 (dd, J1= 12.81 Hz, J2= 6.46 Hz, 2H, Lys, H ε ), 3.417-3.431 (m, 2H, PEG, H j-o), 3.520-3.591 (m, 10H, PEG, H j-o ), 3.683 (t, J = 6.24 Hz, 2H, PEG, H p ), 4.204-4.233 (m, 1H, Lys, H α ), 4.261-4.285 (m, 1H, Glu, H α ), 6.543 (m, 1H, Glu, NH), 6.639 (d, J = 7.48 Hz, 1H, Lys, NH), 7.208-7.257 (m, 12H, DP Ph , H e / f ), 7.431-7.466 (m, 4H, DP Ph , CH d / d’ ), 7.574-7.601 (m, 4H, DP Ph , H d / d’ ), 7.806 (t, J = 5.44 Hz, 1H, PEG, NH), 7.828 (t, J = 5.67 Hz, 1H, Lys, NH ζ ); 13 C NMR (176 MHz, DMF-d7, 298 K): δ (ppm), 23.063 (s, Lys, C γ ), 29.320 (Lys, C δ ), 29.840 (Glu, C β ), 32.208 (s, Glu, C γ ), 32.673 (s, Lys, C β ), 36.718 (s, PEG, C q ), 38.739 (s, PEG, C h ), 38.834 (s, Lys, C ε ), 53.364 (s, Glu, C α ), 53.498 (s, Lys, C α ), 67.398 (s, PEG, C p ), 69.051 (s, PEG, C i ), 70.096 (s, PEG, Cj-o ), 70.217 (s, PEG, C j-o ), 70.292 (s, PEG, C j-o ), 70.419 (s, PEG, C j-o ), 70.430 (s, PEG, C j-o ), 127.814 (d, J = 6.78 Hz, DP Ph , C e / e’ ), 127.870 (d, J = 7.35 Hz, DP Ph , C e / e’ ), 128.150 (s, DP Ph , C f / f’ ), 128.379 (s, DP Ph , C f / f’ ), 134.035 (dd, J1= 19.47 Hz, J2= 5.82 Hz, DP Ph , C d / d’ ), 134.653 (d, J = 20.35 Hz, DP Ph , C d / d’ ), 136.936 (m, DP Ph , C c / c’ ), 137.650 (m, DP Ph , C c / c’ ), Quaternary carbons: 157.065 (s), 170.452 (s), 174.729 (s), 175.006 (s), 175.232 (s), the remaining signals corresponding to quaternary carbons could not be discerned from the noise; 31 P{ 1 H} NMR (283 MHz, DMF-d 7, 298 K): δ (ppm) -13.18 (d, J = 162.7 Hz), -12.15 (d, J = 162.7 Hz).

[0194]

[0196] HR-MS-ESI m / z:[M+H] + 1033.3759(C 51 H 63 O 15 N4P2 calculated value 1033.3760), [M+Na] + 1055.3579(C 51 H 62 O 15 Calculated value for N4P2Na: 1055.3579).

[0195]

[0197] Characterization of compound (II-2-PSMAt1):

[0198] 1 H NMR (700 MHz, DMF-d 7, 298 K): δ (ppm) 1.389-1.444 (m, 2H, Lys, H γ ), 1.453-1.503 (m, 2H, Lys, H δ ), 1.618-1.670 (m, 1H, Lys, H β ), 1.752-1.801 (m, 1H, Lys, H β ), 1.899-1.949 (m, 1H, Glu, H β ), 1.983-2.035 (m, 1H, Glu, H β ), 2.262 (s, 6H, DP Tol , H g / g’ ), 2.293 (s, 6H, DP Tol , H g / g’ ), 2.345-2.384 (m, 1H, Glu, H γ ), 2.389 (t, J = 6.25 Hz, 2H, PEG, H o ), 2.451 (dt, J1= 15.00 Hz, J2= 8.17 Hz, 1H, Glu, H γ ), 2.962-3.008 (m, 4H, PEG, H h / i ), 3.139 (Hidden, 2H, Lys, H ε), 3,407–3,421 (m, 2H, PEG, H j-o ), 3,524–3,591 (m, 10H, PEG, H j-o ), 3,685 (t, J = 6.25 Hz, 2H, PEG, H). p ), 4,217–4,247 (m, 1H, Lys, H α ), 4,268–4,294 (m, 1H, Glu, H α ), 6,559 (d, J = 4.84 Hz, 1H, Glu, NH), 6,631 (d, J = 7.72 Hz, 1H, Lys, NH), 7,005 (d, J = 7.63 Hz, 4H, DP). Ph , H e ), 7,036 (d, J = 7.62 Hz, 4H, DP Ph , H e ), 7.325 (dd, J1 = 14.22 Hz, J2 = 7.36 Hz, 4H, DP Ph , CH d / d’ ), 7,427 (t, J = 7.68 Hz, 4H, DP Ph , H d / d’ ), 7.799–7.824 (m, 1H, PEG, NH), 7.799–7.824 (m, 1H, Lys, NH). ζ ); 13 C NMR (176 MHz, DMF-d7, 298 K): δ (ppm) 20.667 (s, DP Tol , 1999 . C g / g’ ), 20,686 (s, DP). Tol , C g / g’ ), 23,064 ( s , Lys , C γ ), 29,306 (Sweet, Lys, C. 2006). δ ), 29,680 (Spider, Glu, C.S β ), 31,929 ( s , Glu , C γ ), 32,656 ( s , Lys , C β ), 36,726 ( s , PEG , C q), 38.703 (s, PEG, C h ), 38.840 (s, Lys, C ε ), 53.328 (s, Glu, C α ), 53.453 (s, Lys, C α ), 67.389 (s, PEG, C p ), 69.058 (s, PEG, C i ), 70.176 (s, PEG, C j-o ), 70.223 (s, PEG, C j-o ), 70.308 (s, PEG, C j-o ), 70.436 (s, PEG, C j-o ), 70.454 (s, PEG, C j-o ), 128.505 (d, J = 7.07 Hz, DP Tol ,C e / e’ ), 128.568 (d, J = 7.35 Hz, DP Tol , C e / e’ ), 134.197 (d, J = 19.96 Hz, DP Tol , C d / d’ ), 134.675 (d, J = 20.96 Hz, DP Tol , C d / d’ ), 137.682 (s, DP Tol , C c / c’ ), 137.949 (s, DP Tol , C f / f’ ), quaternary carbons: 158.097 (s), 170.435 (s), 174.665 (s), 175.003 (s), 175.179 (s), the remaining signals corresponding to quaternary carbons could not be discerned from the noise; 31 P{ 1 H} NMR (283 MHz, DMF-d 7, 298 K): δ (ppm) -15.776 (d, J = 151.40 Hz), -14.392 (d, J = 151.40 Hz).

[0199] HR-MS-ESI m / z:[M+H] +1089.4373(C 55 H 71 O 15 N4P2 calculated value 1089.4386), [M+Na] + 1111.4193(C 55 H 70 O 15 Calculated value of N4P2Na: 1111.4205), [M+MeOH+H] + 1121.4275(C 56 H 75 O 16 (Calculated value of N4P2: 1121.4648).

[0196]

[0200] Characterization of compound (II-11-PSMAt1):

[0201] 31 P NMR 283 MHz, DMF-d 7, 298 K): δ (ppm) -17.21 (d, J = 139.3 Hz), -19.24 (d, -139.3 Hz).

[0202] HRMS: [M+H] + 1153.4196 (observed value), 1153.4182 (calculated value)

[0197] Example 5 Compound ( nat Preparation and characterization of cis / trans Re complexes of conjugated peptide RGD using Re-III-1-RGD / compound (II-1-RGD)

[0203] The chemical properties of Re and Tc are similar. Tc does not have a stable isotope, so it is known as the compound ( nat Re-III-1-RGD) / [ nat ReO2(II-1-RGD)2] + It was expedient to prepare and obtain a complete characterization.

[0198]

[0204] [ in DMF (100 μL) natA solution of ReO2I(PPh3)2] (3.0 mg, 3.45 μmol) was combined with a solution of compound (II-1-RGD) (3.7 mg, 3.45 μmol) and DIPEA (6 μL) in DMF (200 μL). The resulting dark brown / black solution was stirred at room temperature for 10 min. Ice-cold diethyl ether was added, resulting in the formation of a precipitate. The supernatant was removed and the precipitate was dissolved in DMF (200 μL) and applied to a semi-preparative HPLC column. The reaction components were separated using HPLC method 3. Aqueous ammonium bicarbonate (0.125 M) was added in a ratio of 10 μL ammonium acetate solution:1 mL HPLC eluent to eluate the cis / trans-[ nat ReO2(II-1-RGD)2] + The cis / trans-[ nat ReO2(II-1-RGD)2] + The solution containing was lyophilized. The lyophilized fractions eluted at 65-67 min and 68-70 min were trans-[ nat ReO2(II-1-RGD)2] + (0.8 mg, 0.34 μmol, 9.9%) and cis-[ nat ReO2(II-1-RGD)2] + (0.9 mg, 0.38 μmol, 11.0%).

[0199]

[0205] trans-[ nat ReO2(II-1-RGD)2] + Characterization data

[0206] 11H NMR (700 MHz, DMF-d7, 298 K): δ (ppm) 1.184 (m, 4H, Lys, γ CH2), 1.300 (m, 4H, Lys, δ CH2), 1.511 (m, 2H, Arg, β CH), 1.602 (m, 2H, Lys, β CH), 1.612 (m, 2H, Arg, β CH), 1.681 (m, 2H, Lys, β CH), 1.769 (m, 4H, Arg, γ CH2), 2.261 (m, hidden, Asp, β CH), 2.601 (dd, J1 = 14.51 Hz, J2 = 9.26 Hz, 2H, Phe, β CH), 2.940 (m, hidden, Asp, β CH), 2.943 (hidden, Lys, ε CH2), 3.067 (m, 2H, Arg, δ CH), 3.144 (m, 2H, Arg, δ CH), 3.33 (dd, J1 = 9.26 Hz, J2 = 5.00 Hz, 2H, Phe, β CH), 3.416 (dd, J1 = 16.45 Hz, J2 = 9.05 Hz, 2H, Gly, α CH), 4.307 (dd, J1 = 16.24 Hz, J2 = 2.67 Hz, 2H, Gly, α CH), 4.358 (m, 2H, Asp, α CH), 4.365 (m, 2H, Lys, α CH), 4.543 (m, 2H, Arg, α CH), 4.796 (m, 2H, Phe, α CH), 7.111 (m, PPh2, aromatic CH m ), 7.161 (m, Phe, aromatic CH p ), 7.170 (m, PPh2, aromatic CH m ), 7.204 (m, PPh2, aromatic CH o ), 7.232 (m, Phe, aromatic CH o and CH m ), 7.281 (m, PPh2, aromatic CH o ), 7.436 (m, PPh2, aromatic CH p ), 7.477 (m, PPh2, aromatic CH p), 7.787 (d, J = 8.98 Hz, 2H, Phe, NH), 8.089 (m, 2H, Gly, NH), 8.196 (m, 2H, Lys, NH), 8.294 (m, 2H, Lys, εNH), 8.358 (d, J = 9.32 Hz, 2H, Asp, NH), 8.629 (d, J = 8.98 Hz, 2H, Arg, NH).

[0200]

[0207] 13 C NMR (176 MHz, CD3CN, 298 K): δ (ppm) 15.56 (s, Lys, γ CH2), 24.66 (s, Arg, β CH2), 27.14 (s, Arg, γ CH2), 29.47 (s, Lys, δ CH2), 32.87 (s, Lys, β CH2), 36.86 (s, Phe, β CH2), 38.77 (s, Asp, β CH2), 38.98 (s, Lys, ε CH2), 41.04 (s, Arg, δ CH2), 43.11 (s, Gly, α CH2), 49.12 (s, Asp, α CH), 51.32 (s, Arg, α CH), 53.36 (s, Phe, α CH), 55.59 (s, Lys, α CH), 118.09 (m, PPh2, aromatic C s ), 125.99 (s, Phe, aromatic C p ), 127.55 (m, PPh2, aromatic C m ), 128.02 (s, Phe, aromatic C o またはC m ), 129.31 (s, Phe, aromatic C o またはC m ), 131.130 (m, PPh2, aromatic C p ), 134.63 (m, PPh2, aromatic C o ), 158.21-172.63 (X=CR2の>9のシグナル; XはN、O、またはCである).

[0201]

[0208] 31P NMR (283 MHz, DMF-d7, 298 K): δ (ppm) 23.781 (m), 24.506 (m).

[0209] HR-MS-ESI m / z:[M+H] 2+ 1179.3826(C 110 H 122 N 18 O 22 P4Re + Calculated value 1179.3773), [M+2H] 3+ 786.5921(C 110 H 122 N 18 O 22 P4Re + The calculated value is 786.5906).

[0202]

[0210] cis-[ nat ReO2(II-1-RGD)2] + Characterization data

[0211] 11H NMR (700 MHz, DMF-d7, 298 K): δ (ppm) 1.188 (m, 4H, Lys, γ CH2), 1.300 (m, 4H, Lys, δ CH2), 1.528 (m, 2H, Arg, β CH), 1.599 (m, 2H, Lys, β CH), 1.608 (m, 2H, Arg, β CH), 1.664 (m, 2H, Lys, β CH), 1.785 (m, 4H, Arg, γCH2), 2.283 (m, 2H, Asp, β CH), 2.606 (m, 2H, Phe, β CH), 2.876 (m, 4H, Lys, ε CH2), 2.924 (hidden, Asp, β CH), 3.109 (m, 2H, Arg, δ CH), 3.148 (m, 2H, Arg, δ CH), 3.319 (dd, J1 = 14.51 Hz, J2 = 5.14 Hz, 2H, Phe, β CH), 3.415 (hidden, Gly, α CH), 4.308 (dd, J1 = 16.64 Hz, J2 = 9.05 Hz, 2H, Gly, α CH), 4.367 (m, 2H, Lys, α CH), 4.379 (m, 2H, Asp, α CH), 4.525 (m, 2H, Arg, α CH), 4.789 (m, 2H, Phe, α CH), 7.151 (m, PPh2, aromatic CH m ), 7.158 (m, Phe, aromatic CH p ), 7.175 (m, PPh2, aromatic CH o ), 7.220 (m, Phe, aromatic CH o and CH m ), 7.305 (m, PPh2, aromatic CH o ), 7.439 (m, PPh2, aromatic CH p), 7.798 (d, J = 9.37 Hz, 2H, Phe, NH), 8.087 (m, 2H, Gly, NH), 8.088 (m, 2H, Lys, ε NH), 8.253 (d, J = 7.46 Hz, 2H, Lys, NH), 8.335 (d, J = 8.85 Hz, 2H, Asp, NH), 8.621 (d, J = 8.85 Hz, 2H, Arg, NH).

[0203]

[0212] 13 C NMR (176 MHz, CD3CN, 298 K): δ (ppm) 15.56 (s, Lys, γ CH2), 24.32 (s, Arg, β CH2), 27.09 (s, Arg, γ CH2), 29.54 (hidden, Lys, δ CH2), 32.84 (s, Lys, β CH2), 36.81 (s, Phe, β CH2), 38.63 (s, Asp, β CH2), 38.91 (s, Lys, ε CH2), 41.04 (s, Arg, δ CH2), 43.09 (s, Gly, α CH2), 49.21 (s, Asp, α CH), 51.37 (s, Arg, α CH), 53.37 (s, Phe, α CH), 55.52 (s, Lys, α CH), 118.05 (m, PPh2, aromatic C s ), 125.99 (s, Phe, aromatic C p ), 127.55 (m, PPh2, aromatic C m ), 128.02 (s, Phe, aromatic C o or C m ), 129.31 (s, Phe, aromatic C o or C m ), 131.04 (m, PPh2, aromatic C p ), 134.27 (m, PPh2, aromatic C o ), 143.80 (m, PPh2, aromatic C o), 158.03-185.17 (several signals of X=CR2; X is N, O, or C; too weak to characterize).

[0213] 31 P NMR (283 MHz, DMF-d7, 298 K): δ (ppm) 21.848 (dm, J1= 356.1 Hz), 26.335 (dm, J1= 356.1 Hz).

[0204]

[0214] HRMS-ESI m / z:[M+H] 2+ 1179.3826(C 110 H 122 N 18 O 22 P4Re + Calculated value 1179.3773), [M+2H] 3+ 786.5921(C 110 H 122 N 18 O 22 P4Re + The calculated value is 786.5906).

[0205] Example 6 Compound (Tc-III-1-RGD) / [ 99m TcO2(II-1-RGD)2] + Preparation and characterization of

[0215] Preparation of radiolabeling kit

[0216] A stock solution containing the required amount of sodium bicarbonate, tin(II) chloride dihydrate, sodium gluconate or sodium tartrate dibasic dihydrate was prepared. The pH of this solution was adjusted to 8.5 by dropwise addition of an aqueous solution of sodium hydroxide (0.1 M). An aliquot of the stock solution was mixed with the required amount of compound (II-1-RGD) in ethanol, and the resulting solution was frozen and lyophilized. The lyophilized kits were stored at -18°C prior to use.

[0206]

[0217] 99m Tc radiolabel

[0218] Compound (II-1-RGD) was incubated with a generator in saline solution (0.9% NaCl in water, w / v). 99m TcO4 - For each radiolabel, the radiolabeling kit was thawed and the following was added: 99m TcO4 - and ethanol for a total of 300 μL. The reconstituted kits were heated at 60° C. for 30 min and then analyzed by analytical HPLC (Method 2) and instant thin-layer chromatography (iTLC) using iTLC SGI0001 strips (9 or 10 cm long; Varian Medical Systems, Crawley, UK). iTLC plates were scanned on a Perkin Elmer Storage Phosphor System (Cyclone) or a LabLogic miniScan TLC reader equipped with Laura software.

[0207]

[0219] 99m Tc-colloid, unreacted 99m TcO4 - and two separate iTLC analyses were performed to allow quantification of the compound (Tc-III-1-RGD).

[0220] Unresponsive 99m TcO4 - Acetone was used as the mobile phase to quantify the amount of R f value: 99m TcO4 - >0.9, 99m Tc colloid <0.1, compound (Tc-III-1-RGD) <0.1.

[0208]

[0221] 99m To quantify Tc-colloid formation, a 1:1 mixture of methanol and 2 M aqueous ammonium acetate was used as the mobile phase: 99m TcO4 - >0.9, 99m Tc colloid <0.1, compound ( 99m Tc-III-1-RGD)>0.9.

[0209]

[0222] (Tc-III-1-RGD) and cis / trans-( nat Co-elution with Re-III-1-RGD: 99m TcO2(II-1-RGD)2] + was prepared as above with an RCY of >90%, and cis-[ nat ReO2(II-1-RGD)2] + and separately, trans-[ nat ReO2(II-1-RGD)2] + and co-injected onto a reversed-phase analytical HPLC column (Method 4). Retention times: trans / cis-(Tc-III-1-RGD) 41.0 and 44.1 min (NaI scintillator detection); trans-( nat Re-III-1-RGD) 38.3 min and cis-( nat Re-III-1-RGD) 42.6 min.

[0210]

[0223] Log D (pH 7.4)

[0224] The following procedure was carried out in triplicate. 99m A solution containing Tc-III-1-RGD (1 MBq in 7.5 μL) was combined with phosphate buffered saline (pH 7.4, 500 μL) and octanol (500 μL) and the mixture was stirred for 30 min. The mixture was then centrifuged (10000 rpm, 10 min) and aliquots of the octanol and PBS aqueous solutions were analyzed for radioactivity using a gamma counter. OCT / PBS =-1.64±0.04.

[0211]

[0225] Serum stability:

[0226] A solution (100 μL, 79 MBq) containing the compound (Tc-III-1-RGD) was added to filtered human serum (Sigma-Aldrich, 900 μL) and incubated at 37° C. for 4 h. Aliquots were taken at 1 and 4 h. Each aliquot (300 μL) was treated with ice-cold acetonitrile (300 μL) to precipitate and remove serum proteins. The acetonitrile in the supernatant was then removed by evaporation under a stream of N2 gas (40° C., 30 min). The final solution was then analyzed by reversed-phase analytical HPLC (Method 2).

[0212]

[0227] αvβ3-integrin solid-phase competitive binding assay:

[0228] The affinity of compound (Tc-III-1-RGD) to αvβ3 integrin was determined in solid-phase competitive binding assay. Briefly, the wells of a 96-well plate were coated with 150ng / mL integrin αvβ3 in 100μL of coating buffer (25mM Tris HCl pH7.4, 150mM NaCl, 1mM CaCl2, 0.5mM MgCl2, and 1mM MnCl2) at 4℃ overnight. Then, the wells were washed twice in binding buffer (coating buffer plus 0.1% bovine serum albumin (BSA)) and then blocked with blocking buffer (coating buffer plus 1% BSA) at room temperature for 2 hours. After two further washes in binding buffer, both (Tc-III-1-RGD) (RCY>96%, 1-2 kBq in 50 μL binding buffer, containing 1.2 pmol of compound (II-1-RGD) peptide) and RGD peptide (10.0 pM to 10,000 nM, 50 μL in binding buffer) were added simultaneously to the wells and allowed to incubate for 1 h at room temperature, followed by two washes as before. Finally, the amount of radioactivity bound to the wells was counted.

[0213]

[0229] The binding of compound (Tc-III-1-RGD) to αβ integrin was displaced by RGD peptide in a concentration-dependent manner. A pseudo IC of 8.54 ± 3.45 nM (95% CI: 1.67–15.41 nM) was obtained using a nonlinear regression model (binding / saturation, 1 site-total) in GraphPad Prism. 50 Values ​​were calculated (n=6 from one experiment).

[0214] Example 7 Compound (Tc-III-1-RGD) / [ 99m TcO2(II-1-RGD)2] + Preclinical imaging and in vivo biodistribution studies

[0230] The animal imaging study underwent ethical review and was performed in accordance with the UK Home Office regulations of the Animals (Scientific Procedures) Act 1986 (ASPA) regulating animal experimentation. SPECT / CT imaging was performed using a preclinical nanoScan SPECT / CT Silver Upgrade instrument (Mediso) calibrated for Technetium-99m. All scans were acquired by helical SPECT (four-head scanner with a 4 × 9 [1.4 mm] pinhole collimator) and helical CT using a 1.4 mm aperture collimator. All images acquired were reconstructed using a full 3D Monte Carlo-based iterative algorithm (Tera-Tomo; Mediso) and further processed and analyzed using VivoQuant software (invicro, USA).

[0215]

[0231] SPECT / CT imaging and biodistribution in healthy mice

[0232] Female BALB / c mice (2 months old) were anesthetized (2-3% v / v isofluorane in oxygen), scanned by CT and injected intravenously (tail vein) with compound (Tc-III-1-RGD) (21 MBq containing 22 μg of compound (II-1-RGD) peptide). SPECT images (8 x 30 min images) were acquired over a period of 4 h. At the end of the imaging procedure, mice were sacrificed by cervical dislocation and urine samples were analyzed by reversed-phase HPLC (analytical, method 2).

[0216]

[0233] Female BALB / c mice (2 months old) were anesthetized (2–3% v / v isofluorane in oxygen) and 99m Tc-III-1-RGD (2.7-5.3 MBq containing 5 μg of compound (II-1-RGD)) was injected intravenously (tail vein). For blocking studies, animals were co-injected with RGD peptide (400 μg). Mice were kept under anesthesia for 1 h and then sacrificed (pentabarbitone by intravenous injection). Tissues and organs were removed, weighed, and radioactivity was counted using a gamma counter (Wallac 1282 CompuGamma Universal Gamma Counter).

[0217]

[0234] SPECT / CT imaging and biodistribution in mice with induced rheumatoid arthritis

[0235] The AK / BxN serum transfer arthritis (STA) model of rheumatoid arthritis was used (PA Monach et al., Curr. Protoc. Immunol., 2008, 81, 15.22.1-15.22.12 and C. Imberti et al., Bioconjugate Chem., 2017, 28, 481-495.). On days 0 and 2, female C57Bl / 6J mice (2 months old) were injected intraperitoneally with arthritogenic serum (150 μL, 50 v / v%, serum obtained from arthritic K / BxN transgenic mice) in sterile filtered PBS. Disease severity was assessed in mice throughout the induction period by measuring body weight, swollen paw thickness using a microcaliper, and visual scoring on a scale of 0 to 3 per paw. SPECT / CT imaging and biodistribution were performed on day 7.

[0218]

[0236] Mice were anesthetized (2.5-3% v / v isofluorane) and their paws were measured using a microcaliper. Mice were then intravenously injected with compound (Tc-III-1-RGD) (approximately 5MBq containing 5μg compound (II-1-RGD)) and allowed to recover from the anesthesia. One hour after injection of the radiotracer, mice were sacrificed (sodium pentabarbitone) and subjected to SPECT / CT scanning post-mortem for 60-180 minutes. Finally, tissues and organs were removed, weighed and counted for radioactivity using a gamma counter (Wallac 1282 CompuGamma Universal Gamma Counter). Images were processed to units of %ID and regions of interest (ROIs) were delineated by CT using VivoQuant software (invicro, USA). Radioactivity in the ankle and wrist ROIs was measured as %ID and %ID / cm. -3 The ROI for each ankle was defined as the area between the tibiofibular joint and the base of the fifth phalanx. The ROI for each "wrist" was defined as the area between the narrowest point of the wrist (ulna and radius) and the tip of the forefoot.

[0219] Example 8 Compound (Tc-III-1-PSMAt1) / [ 99mTcO2(II-1-PSMAt1)2] + , compound (Tc-III-2-PSMAt1) / [ 99m TcO2(II-2-PSMAt1)2] + and compound (Tc-III-11-PSMAt1) / [ 99m TcO2(II-11-PSMAt1)2] + Preparation and characterization of

[0237] Kit Preparation: A stock solution containing the required amount of sodium bicarbonate, stannous chloride, and sodium tartrate was prepared in water. The pH was adjusted to either 7.5 or 8-8.5 by dropwise addition of either sodium hydroxide (0.1 M) or hydrochloric acid (0.1 M) in water. An aliquot of the stock solution was mixed with the required amount of (II-1-PSMAt1), (II-2-PSMAt1), or (II-11-PSMAt1) (dissolved in a mixture of water / ethanol (50% / 50%)) to form the kit solution outlined in the table below, which was immediately frozen and lyophilized using a freeze dryer. The lyophilized kits were stored in a freezer prior to use.

[0220] [Table 5]

[0221]

[0238] 99m TcO4 - Radiolabeling of (II-1-PSMAt1), (II-2-PSMAt1), or (II-11-PSMAt1) with

[0239] Using the described lyophilization kit, (II-1-PSMAt1) or (II-2-PSMAt1) were lyophilized in saline solution (0.9% NaCl in water, w / v) using a generator. 99m TcO4 - The radiolabeling reaction mixture was either allowed to react at ambient temperature (approximately 22°C) for 5 min or heated at 100°C for 5 min. Aliquots were analyzed by iTLC and C for analysis. 18The radiochemical yields were determined by HPLC. The species assigned to (Tc-III-1-PSMAt1) eluted at 11.0-12.5 min, and (Tc-III-2-PSMAt1) eluted at 12.5-14.0 min. Analytical HPLC conditions: 20 min, 5% dilution from 100% A to 100% B. -1 of 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6×150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column.

[0222]

[0240] Using the above freeze-drying kit, (II-11-PSMAt1) was prepared by generator generation in saline solution (0.9% NaCl in water, w / v). 99m TcO4 - (200MBq, 300uL). The radiolabeling reaction mixture was heated at 100°C for 5 minutes. An aliquot was analyzed by iTLC and C for analysis. 18 The radiochemical yield was determined by HPLC. The species assigned to (Tc-III-11-PSMAt1) eluted at 9.7-11.7 min. Analytical HPLC conditions: 20 min, 5% from 100% A to 100% B. -1 of 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6×150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column.

[0223]

[0241] 99m Tc-colloid, unreacted 99m TcO4 - And to allow quantification of the complexes, two separate iTLC analyses were performed.

[0242] Unresponsive 99m TcO4 - Acetone was used as the mobile phase to quantify the amount of R f value: 99m TcO4 - >0.9, 99mTc colloids <0.1, complexes <0.1.

[0224]

[0243] 99m To quantify Tc-colloid formation, a 1:1 mixture of methanol and 2 M aqueous ammonium acetate was used as the mobile phase: 99m TcO4 - >0.9, 99m Tc colloid <0.1, complex >0.9.

[0225]

[0244] These kit-based reaction solutions were further purified for in vitro and in vivo studies. Solutions of either (Tc-III-1-PSMAt1), (Tc-III-2-PSMAt1), or (Tc-III-11-PSMAt1) prepared from the kit were run on a SE-HPLC column using an aqueous mobile phase of phosphate-buffered saline. Fractions containing either (Tc-III-1-PSMAt1), (Tc-III-2-PSMAt1), or (Tc-III-11-PSMAt1) (>95% radiochemical purity) eluted at 10–12 min. Other reaction components, including unreacted starting material and impurities, also eluted at distinctly different retention times: unlabeled (II-1-PSMAt1) ligand eluted at 16–17 min, unlabeled (II-2-PSMAt1) eluted at 27–28 min, 99m TcO4 - elutes at 14-15 min, 99m The Tc-colloid was trapped on the column.

[0226]

[0245] Compound ( 99g Tc-III-1-PSMAt1) and ( 99g Preparation of Tc-III-2-PSMAt1)

[0246] 99g Tc(V) precursor N t Bu4[ 99g[TcOCl4] was prepared according to a method previously described (A. Davison, C. Orvig, H.S. Trop, M. Sohn, B.V. Depamphilis and A.G. Jones, Inorg. Chem., 1980, 19, 1988-1992). A solution of either (II-1-PSMAt1) or (II-2-PSMAt1) (1.0 mg, approximately 1 μmol, 2 equiv.) in methanol (300 μL, degassed) was added to N t Bu4[ 99g TcOCl4] (0.25 mg, 0.46 μmol, 1 equiv.) The resulting pale yellow solution was allowed to react at ambient temperature for 15 min.

[0227]

[0247] ( 99g Tc-III-1-PSMAt1):HR-MS-ESI m / z:[M+H] 2+ 1098.8183(C 102 H 125 N8O 32 P4Tc calculated value 1098.8221 (peak at 100% abundance), [M+Na] 2+ 1109.8091(C 102 H 124 N8O 32 P4TcNa calculated value 1109.8130 (100% abundance peak); LR-MS-ESI m / z: [M+H] 2+ 1099.0(C 102 H 125 N8O 32 P4Tc calculated value 1098.5), [M+Na] 2+ 1110.0(C 102 H 124 N8O 32 Calculated value of P4TcNa: 1109.5), [M+K] 2+ 1117.7(C 102 H 124 N8O 32 P4TcK calculated value 1117.5), [M+2H] 3+ 732.7(C 102 H 126 N8O 32 P4Tc calculated value 732.7), [M+H+K] 3+ 745.2(C102 H 126 N8O 32 Calculated value of P4TcK is 745.3).

[0228]

[0248] ( 99g Tc-III-2-PSMAt1):HR-MS-ESI m / z:[M+H] 2+ 1154.8811(C 110 H 141 N8O 32 P4Tc calculated value 1154.8847 (peak at 100% abundance), [M+Na] 2+ 1165.8718(C 110 H 140 N8O 32 P4TcNa calculated value 1165.8756 (100% abundance peak); LR-MS-ESI m / z: [M+H] 2+ 1155.0(C 110 H 141 N8O 32 P4Tc calculated value 1154.5), [M+Na] 2+ 1165.8(C 110 H 140 N8O 32 Calculated value of P4TcNa: 1165.5), [M+K] 2+ 1173.8(C 110 H 140 N8O 32 P4TcK calculated value 1173.5), [M+2H] 3+ 770.3(C 110 H 142 N8O 32 P4Tc calculated value 770.0), [M+H+K] 3+ 782.8(C 110 H 141 N8O 32 Calculated value of P4TcK is 782.7).

[0229] Example 9 Biological evaluation of (Tc-III-1-PSMAt1), (Tc-III-2-PSMAt1), and (Tc-III-11-PSMAt1)

[0249] Compound (Tc-III-1-PSMAt1) / [ 99m TcO2(II-1-PSMAt1)2]+ and compound (Tc-III-2-PSMAt1) / [ 99m TcO2(II-2-PSMAt1)2] + was isolated, purified, and evaluated for stability, affinity for PSMA in vitro and in vivo, and pharmacokinetics.

[0230] [Table 6]

[0231]

[0250] Table 6 shows the dissociated amount of compound (Tc-III-1-PSMAt1) and compound (Tc-III-2-PSMAt1) after incubation in serum. 99m Indicates the amount of Tc.

[0232]

[0251] The stability of compounds (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) was determined in serum over a 24 hour period. Both tracers showed high stability, with over 90% remaining intact over 24 hours as determined by analytical C18 radio-HPLC. 99m With the exception of Tc, no other decomposition products are observed in the HPLC chromatogram. OCT / PBS is -2.45, and the log D of (Tc-III-2-PSMAt1) OCT / PBS was −2.08, suggesting that both are hydrophilic and likely eliminated via the renal route.

[0233]

[0252] A solution (20 μL, 13 MBq) containing the compound (Tc-III-11-PSMAt1) was added to filtered human serum (180 μL) and incubated at 37° C. At 1, 4 and 24 hours, samples were taken and treated with an equal volume of ice-cold acetonitrile to precipitate and remove serum proteins. The acetonitrile in the supernatant was then removed by evaporation under a stream of N2 gas. The final solution was then analyzed by reversed-phase analytical HPLC (FIG. 20). The compound (Tc-III-11-PSMAt1) showed high stability, with more than 95% intact over 24 hours as determined by analytical C18 radio-HPLC.

[0234]

[0253] 99m Tc-DP-peptide radiotracers contain two different isomers. Such isomers are known as "geometric cis / trans" isomers. To show that the isomers have equivalent biological behavior, the "cis" and "trans" geometric isomers of (Tc-III-1-PSMAt1) were separated: both have nearly identical uptake into PSMA-positive cells (Figure 11).

[0235]

[0254] Uptake of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) into DU145, DU145-PSMA, LNCaP, and PC-3 cells

[0255] The following experiments were performed in biological triplicate.

[0236]

[0256] A panel of cell lines was selected that express GCP(II) / PSMA (DU145-PSMA (genetically modified to express PSMA) (see F. Kampmeier, J. D. Williams, J. Maher, G. E. Mullen and P. J. Lower, EJNMMI Res., 2014, 4, 13), and LNCaP (CRL-1740)) or have low GCP(II) / PSMA expression (DU145 (HTB-81) and PC-3 (CRL-1435)). All cell lines, except for PC-3 cells, were cultured in 10% fetal bovine serum, 2 mM L-glutamine, and 100 U.mL-1 Penicillin and 100 μg.mL -1 Cells were cultured in RPMI 1640 medium (R0883, Sigma) containing streptomycin, and PC-3 cells were cultured in low glucose Dulbecco's Modified Eagle Medium (DMEM, D5546, Sigma) supplemented as above. Cells were maintained at 37°C and 5% CO2. Cells were plated at 5 × 10 cells per well in 2 mL of complete medium. 5 The cells were seeded at a density of 100 µg / well into 6-well plates to achieve 70–80% confluency the next day. Before treating the cells, the cell medium (1 mL / well) was replaced. A solution containing either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) (100 kBq, >95% radiochemical purity in 5–12 µL phosphate-buffered saline) was added to each well and the cells were incubated for 1 h at 37 °C. Uptake studies were also performed after a 2 min incubation with the PSMA inhibitor 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA; 30 µL / well of 750 µM PMPA solution). After 60 min of incubation, the plates were placed on ice, the supernatant was removed, and the cells were washed with ice-cold phosphate-buffered saline solution (3 × 0.5 mL). Cells were lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA buffer, 500 μL; 150 mM sodium chloride, 0.1 w / w% sodium dodecyl sulfate (SDS), 0.5 w / w% sodium deoxycholate (NaDOC), 1 w / w% Triton-X) and samples were collected for radioactivity counting. Results in FIG. 12 are depicted as mean values ​​± SD of independent biological experiments (performed on different days with different radiotracer preparations).

[0237]

[0257] Tc-III-1-PSMAt1 and Tc-III-2-PSMAt1 showed uptake into DU145-PSMA+ cells (12.4±2.8%AR [percentage of radioactivity added] and 7.8±1.3%AR, respectively). This uptake was specific: DU145-PSMA+ cell uptake of Tc-III-1-PSMAt1 and Tc-III-2-PSMAt1 could be blocked with PMPA, and there was negligible uptake into parental DU145 cells (FIG. 12).

[0238]

[0258] In LNCaP cells, uptake of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) was measured to be 3.7±1.2%AR and 3.0±0.8%AR, respectively, whereas uptake of both tracers in PC3 cells was measured to be less than 0.3%AR. Uptake in LNCaP cells was also blockable with PMPA (FIG. 12).

[0239]

[0259] In vitro time course and localization of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1)

[0260] To determine the cellular uptake and localization of each tracer over time, DU145-PSMA and LNCAP cells were seeded as described above. Cells were supplemented with complete medium (1 mL) 1 h before the addition of either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) (100 kBq, in 5–7 μL of phosphate-buffered saline, >95% radiochemical purity). Cells were incubated at 37 °C under 5% CO2, with three technical replicates for each condition. After 15, 30, 60 and 120 min of incubation, the supernatant was collected and the cells were washed three times with PBS (1 mL) to determine the unbound fraction, followed by an acid wash (0.5 M glycine, pH 2.5) to determine cell surface-bound radioactivity. Cells were then lysed in cold RIPA buffer (500 μl) to determine the radioactivity internalized by the cells. Radioactivity content was determined by gamma counter. The results are depicted in FIG. 21 as the mean values±SD of independent biological experiments (performed on different days and with different radiotracer preparations).

[0240]

[0261] Uptake of both radiotracers increased over a 2-hour period and was consistent at all time points measured. 99m The majority of the Tc cell-associated radioactivity was present in the internalized cell fraction, suggesting that for both PSMA-expressing cell lines, Tc-III-1-PSMAt1 and Tc-III-2-PSMAt1 are rapidly internalized after binding to PSMA. Uptake of Tc-III-1-PSMAt1 (both surface-bound and internalized radioactivity) was slightly higher than that of Tc-III-2-PSMAt1.

[0241]

[0262] In vivo imaging of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) in healthy mice

[0263] Animal imaging studies were ethically reviewed and conducted in accordance with the UK Home Office regulations of the Animals (Scientific Procedures) Act 1986 (ASPA) regulating animal experimentation. Mice were purchased from Charles River (Margate, UK). Male SCID-beige mice (approximately 3 months old, n=1) were anesthetized (2.5% v / v isofluorane, 0.8-1.0 L / min O2 flow rate) and injected intravenously via the tail vein with either (Tc-III-1-PSMAt1) (100 μL, 26 MBq, >99% RCP, 0-5 μg PSMAt peptide in phosphate-buffered saline) or (Tc-III-2-PSMAt1) (160 μL, 30 MBq, >99% RCP, 0-5 μg PSMAt peptide in phosphate-buffered saline) followed immediately by CT imaging and SPECT scanning. SPECT / CT imaging was performed using a preclinical nanoScan SPECT / CT Silver Upgrade instrument (Mediso) calibrated for Technetium-99m (Figure 13). SPECT scans were acquired by helical SPECT (four-head scanner with 4x9 pinhole collimator) and CT scans by helical CT (55 kVP X-ray source, 1000 ms exposure time with 180 projections over 9 min). A 1.0 mm pinhole collimator was used. SPECT imaging was performed in eight segments: the first segment was acquired 15-30 min after injection (frame time 12 s; imaging time 9 min), followed by seven imaging segments of 30 min each, up to 4 h after injection (frame time 33 s; imaging time 24.75 min). At the end of the imaging procedure, mice were sacrificed by cervical dislocation and urine samples were analyzed by analytical HPLC (Figure 15). SPECT images were reconstructed with the HiSPECT (Scivis GmbH) reconstruction software package using standard reconstruction with 35% smoothing and 9 iterations at an isotropic voxel size of 0.3 mm. CT and SPECT images were further processed and analyzed using VivoQuant software (invicro, USA).

[0242]

[0264] Biodistribution of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) in healthy mice

[0265] Male SCID-beige mice (approximately 3 months old) were weighed, anesthetized (2.0-2.5% v / v isofluorane, O2 flow rate of 1.0-1.5 L / min) and injected with (Tc-III-1-PSMAt1) solution (50 μL, approximately 13 MBq in phosphate buffered saline, n=4) or (Tc-III-2-PSMAt1) solution (80 μL, approximately 15 MBq in phosphate buffered saline, n=4) via intravenous tail vein injection. Mice were kept under anesthesia until sacrificed by cervical dislocation 2 hours after injection. Organs / tissues were dissected, weighed, and analyzed for known morphology. 99m The biodistribution of the tracer was determined by gamma counting with a standard solution of Tc radioactivity. The measured radioactivity for each organ / tissue was normalized to obtain a percentage injected dose per gram (%ID / g) value (Figure 14).

[0243] Example 10 Compound (Tc-III-1-PSMAt1) / [ in mice bearing prostate cancer tumors 99m TcO2(DP Ph -PSMAt)2] + and compound (Tc-III-2-PSMAt1) / [ 99m TcO2(DP Tol -PSMAt)2] + Biodistribution of

[0266] The biodistribution of Tc-III-1-PSMAt1 and Tc-III-2-PSMAt1 was determined in SCID / beige mice bearing DU145-PSMA+ tumors (Figure 17a). Each animal was administered either Tc-III-1-PSMAt1 or Tc-III-2-PSMAt1 and euthanized at either 2 or 24 hours post-injection, followed by removal of organs for ex vivo radioactivity counting. Tc-III-1-PSMAt1 was significantly higher in tumors 2 hours post-injection than Tc-III-2-PSMAt1 (18.0±3.5% ID g -1 , mean difference=29.3%ID g -1, p=0.008), a higher amount of (Tc-III-2-PSMAt1) was measured (29.4±6.3%ID g -1 , [percentage of injected dose per gram]). In other organs, radioactivity concentrations were similar to those observed in healthy SCID / beige mice. At 24 hours post-injection, significant amounts of 99m Tc radioactivity was still present - in fact, 100% of the tumors at 2 and 24 hours post-injection for animals receiving the same tracer. 99m There were no statistically significant differences between the Tc radioactivity concentrations (Figure 17a).

[0244]

[0267] To determine the specificity of each radiotracer, separate groups of animals also bearing DU145-PSMA+ tumors were co-administered with either (Tc-III-1-PSMAt1) and PMPA, or (Tc-III-2-PSMAt1) and PMPA to block PSMA-mediated uptake of the radiotracer. In addition, groups of mice bearing parental DU145 tumors (which do not express PSMA) were also co-administered with these 99m Tc radiotracer was administered. Animals were also euthanized 2 hours after injection, followed by removal of organs for ex vivo radioactivity counting (Figure 17).

[0245]

[0268] In mice bearing DU145-PSMA+ tumors, coadministration of PMPA substantially reduced tumor uptake of both (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) (Figure 17a). For (Tc-III-1-PSMAt1), coadministration reduced tumor uptake by 0.91 ± 0.29% ID g -1 (compared to administration of Tc-III-1-PSMAt1 alone: ​​mean difference = 17.12%ID g -1 , p=4×10 -6 For Tc-III-2-PSMAt1, coadministration reduced tumor uptake by 0.76 ± 0.45% ID g -1(compared to administration of Tc-III-2-PSMAt1 alone: ​​mean difference = 28.62%ID g -1 , p=7×10 -6 Similarly, for animals bearing DU145 tumors that do not express PSMA, tumor uptake of (Tc-III-1-PSMAt1) was 0.24 ± 0.07% ID g -1 The tumor uptake of (Tc-III-2-PSMAt1) was 0.18 ± 0.07%ID g -1 For both (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1), co-administration of PMPA significantly reduced splenic uptake (Fig. 17c, d).

[0246]

[0269] For both radiotracers, the 99m The concentration of Tc radioactivity was high (Figure 17b). Of note, for animals administered (Tc-III-1-PSMAt1), the simultaneous administration of PMPA did not result in a significant increase in the renal 99m In contrast, coadministration of PMPA also reduced the radioactivity concentration in the kidneys for animals injected with (Tc-III-2-PSMAt1), but this effect was much less pronounced.

[0247] SPECT / CT Imaging

[0270] The tumors were clearly visible in the SPECT / CT scans of animals administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) alone at both 2 hours (Figure 18a) and 24 hours (Figure 18b) post-injection. The kidneys and bladder were also clearly visible throughout these time points, consistent with previous data indicating that the radiotracer is excreted via the renal route, and with the ex vivo biodistribution data. SPECT / CT also showed negligible tumor uptake for either (i) animals co-administered with PMPA or (ii) animals bearing DU145 tumors that do not express the PSMA receptor (Figure 18a). The spleen was also identified in the SPECT / CT imaged at 2 hours post-injection for animals administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1). Co-administration of PMPA reduced splenic uptake of both radiotracers.

[0248]

[0271] Preparation of tumor-bearing mice: The GCP(II) / PSMA-negative cell line used in these experiments was DU145, a human carcinoma prostate cancer cell line derived from a brain metastasis site. The GCP(II) / PSMA-expressing cell line used in these experiments was DU145-PSMA+, a genetically modified daughter cell line of DU145. This cell line was previously transduced to express full-length human GCP(II) / PSMA according to F. Kampmeier, JD Williams, J. Maher, GE Mullen and PJ Lower, EJNMMI Res., 2014, 4, 13. These cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin / streptomycin. To prepare for the experiments, the cells were grown at 37°C in an incubator containing humidified air equilibrated with 5% CO2.

[0249]

[0272] Animal studies were conducted under a UK Home Office Project and Personal License and in compliance with the UK Research Council and Medical Research Charity guidelines for the responsible use of animals in bioscientific research. SCID / beige mice (male, 7–12 weeks old) were incubated with 4 × 10 6 Subcutaneous prostate cancer xenografts were generated by injecting DU145-PSMA or DU145 cells into the right shoulder. Imaging was performed once tumors reached 5-10 mm in diameter (3-4 weeks after injection). For imaging purposes, mice were anesthetized, positioned on the scanner, and the tail vein was cannulated. For biodistribution purposes, mice were anesthetized and the radiotracer was injected via the tail vein.

[0250]

[0273] SPECT / CT Scanning: SPECT / CT scans are 99mThe animals were imaged on a dedicated small animal SPECT system, NanoSPECT / CT Silver Upgrade (Mediso Ltd., Budapest, Hungary), calibrated for Tc. Animals (2 mice per group) were cannulated via the tail vein and administered radiotracer (10–26 MBq) while the animals were on the scanner, followed by helical CT scan (45 kVP X-ray source, 1000 ms exposure time with 180 projections over 7.5 min). 15 min after injection, whole body SPECT scans were acquired (30 min × 4, performed consecutively) with a frame time of 33 s (using a 4-head scanner with a collimator of 4 × 9 [1.4 mm] pinholes in helical scanning mode). After this, the animals were allowed to recover and sacrificed at 24 h after injection (by cervical dislocation and tail vein incision to confirm death), and organs / tissues were removed, weighed, and radioactivity was counted using a gamma counter. For each radiotracer, additional animals were administered the tracer, allowed to recover, anesthetized, and subjected to SPECT / CT scanning at 24 h post-injection, followed by sacrifice and ex vivo tissue counting. SPECT / CT images were reconstructed with a 256 × 256 matrix using the reconstruction software package, HiSPECT (ScivisGmbH), and visualized and quantified using VivoQuant VivoQuant v.3.5 software (InVicro LLC., Boston, USA).

[0251]

[0274] Biodistribution studies: 99m Tc radiotracer (7–18 MBq) was administered by tail vein injection under isoflurane anesthesia (five mice per group). Animals were allowed to recover and roam freely in a gridded cage. Animals were euthanized by cervical dislocation 2 h after injection, and organs / tissues were removed, weighed, and counted for radioactivity using a gamma counter. Data were analyzed in GraphPad Prism 9 (version 9.1.1) and expressed as mean ± standard deviation (SD). Statistical significance was determined using the Student's t-test.

[0252] Example 11 ( 99g Preparation and characterization of Tc-III-11-PSMAt1)

[0275] A sample (approximately 1 mg) of compound (II-11-PSMAt1) dissolved in DMF was added to N t Bu4[ 99g TcOCl4] (approximately 0.3 mg) was added, and the solution was analyzed.

[0253]

[0276] LC-MS (ESI, positive mode, low resolution) retention time = 8:04-8:17 min LRMS: [M+H] 2+ 1220 (observed value), 1219 (calculated value).

[0254] Example 12 Compound ( 64 Cu-III-1-PSMAt1) / [ 64 Cu(II-1-PSMAt1)2] + and compound ( 64 Cu-III-2-PSMAt1) / [ 64 Cu(II-2-PSMAt1)2] + Preparation and characterization of

[0255]

[0277] (II-1-PSMAt1) and (II-2-PSMAt1) 64 Cu radiolabel

[0278] 64 Cu was measured using the CTI RDS112 11MeV cyclotron. 64 Ni(p,n) 64 in 0.1M HCl solution, which is produced by Cu nuclear reaction, purified and used for radiolabeling. 64 Cu 2+ (See MS Cooper, MT Ma, K. Sunassee, KP Shaw, JD Williams, RL Paul, PS Donnelly and PJ Lower, Bioconjug. Chem. 2012, 23, 1029-1039). 64 Cu 2+The solution (0.1 M in HCl) was dried by heating at 100° C. under a stream of nitrogen and the residue was redissolved in ammonium acetate solution (0.1 M, pH 7). 64 Cu 2+ An aliquot of ammonium acetate solution (10 MBq, 50-100 μL) containing was added to either (II-1-PSMAt1) (50 μg) or (II-2-PSMAt1) (50 μg) dissolved in aqueous ammonium acetate (0.1 M) to obtain a final radiolabeled solution with a volume of 200 μL. The radiolabeled mixture was allowed to react for 20 min at ambient temperature (approximately 22 °C). The aliquot was analyzed by iTLC and analytical HPLC to determine the radiochemical yield. 18 -By analytical HPLC, ( 64 The species assigned to Cu-III-1-PSMAt1) eluted at 12.0-13.0 min, ( 64 Cu-III-2-PSMAt1) eluted at 13.5-14.5 min, and unreacted 64 Cu 2+ eluted with the solvent front at 2.0–3.5 min.

[0256]

[0279] Unresponsive 64 Cu 2+ To allow quantification of the complexes, iTLC analysis was performed. Citrate buffer (0.1 M, pH 5) was used as the mobile phase: f Value: Unreacted 64 Cu 2+ >0.9, complex <0.1.

[0257]

[0280] ( 64 Cu-III-1-PSMAt1) and ( 64 Cu-III-2-PSMAt1) log D OCT / PBS D

[0281] The following procedure was carried out in triplicate. 64 Cu-III-1-PSMAt1) or ( 64A solution (0.5 MBq in 20 μL) containing either Cu-III-2-PSMAt1 or Cu-III-2-PSMAt2 was combined with phosphate buffered saline (pH 7.4, 480 μL) and octanol (500 μL) and the mixture was vortexed for 30 min. The mixture was then centrifuged (10000 rpm, 10 min) and aliquots of the octanol and phosphate buffered saline solutions were analyzed for radioactivity using a gamma counter. log D OCT / PBS ( 64 Cu-III-1-PSMAt1):-3.30±0.03;log D OCT / PBS ( 64 Cu-III-2-PSMAt1): -3.01±0.06.

[0258]

[0282] ( 64 Cu-III-1-PSMAt1) and ( 64 Serum stability of Cu-III-2-PSMAt1)

[0283] in an aqueous solution of ammonium acetate (20 μL, 0.1 M) 64 Cu-III-1-PSMAt1) (>99.0% RCP, 1.7MBq, 5μg DP Ph -PSMAt ligand) or ( 64 Cu-III-2-PSMAt1) (>99.0% RCP, 1.7MBq, 5μg DP Tol Samples of β-PSMAt ligand were added to filtered human serum (180 μL) from healthy volunteers and incubated at 37°C. Aliquots were taken at 1, 4 and 24 hours. Each aliquot (300 μL) was treated with ice-cold acetonitrile (300 μL) to precipitate and remove serum proteins. The acetonitrile in the supernatant was then removed by evaporation under a stream of N2 gas (40°C, 30 min). The final solution was then analyzed by reversed-phase analytical HPLC (Method 2). Radiochromatograms of serum samples were ( 64 Cu-III-1-PSMAt1) and ( 64 We showed that Cu-III-2-PSMAt1) was still present after 24 h incubation in serum and no other degradation products were detectable.

[0259]

[0284] ( nat Cu-III-1-PSMAt1) and ( nat Preparation of Cu-III-2-PSMAt1)

[0285] A solution of either (II-1-PSMAt1) or (II-2-PSMAt1) (1.0 mg, approximately 1 μmol, 2 equiv.) in saline (500 μL) was diluted with [Cu I The reaction mixture was allowed to react at ambient temperature for 60 min. The product was isolated by semi-preparative HPLC (Method 6) at approximately 46-47 min (( nat Cu-III-1-PSMAt1)) or 56-57 min (( nat The product fractions eluting at any time point of Cu-III-2-PSMAt1) were lyophilized. Yield = 30-40%.

[0260]

[0286] ( nat Cu-III-1-PSMAt1):HR-MS-ESI m / z:[M+H] 2+ 1064.3338(C 102 H 125 O 30 N8P4Cu calcd 1064.3369); LR-MS-ESI+ m / z: [M+H] 2+ 1065.8(C 102 H 125 O 30 N8P4Cu calculated value 1065.3), [M+Na] 2+ 1077.2(C 102 H 124 O 30 N8P4CuNa calculated value 1076.3), [M+K] 2+ 1084.6(C 102 H 124 O 30 N8P4CuK calculated value 1084.3), [M+2H] 3+ 711.0(C 102 H 126 O 30 Calculated value for N8P4Cu: 710.5).

[0261]

[0287] ( nat Cu-III-2-PSMAt1):HR-MS-ESI m / z:[M+H] 2+ 1120.3973(C 110 H 141 O 30 N8P4Cu calcd 1120.3995); LR-MS-ESI+ m / z: [M+H] 2+ 1121.3(C 110 H 141 O 30 N8P4Cu calculated value 1121.4), [M+Na] 2+ 1132.6(C 110 H 140 O 30 N8P4CuNa calculated value 1132.4), [M+2H] 3+ 748.0(C 110 H 142 O 30 N8P4Cu calculated value 747.9), [M+H+K] 3+ 761.3(C 110 H 141 O 30 N8P4CuK calculated value 760.6).

[0262] Example 13 Compound ( nat Re-III-1-PSMAt1) / [ nat ReO2(II-1-PSMAt1)2] + and compound ( nat Re-III-2-PSMAt1) / [ nat ReO2(II-2-PSMAt1)2] + Preparation and characterization of

[0288] ( nat Re-III-1-PSMAt1) and ( nat Preparation of Re-III-2-PSMAt1)

[0289] A solution of either (II-1-PSMAt1) (approximately 5.1 mg, 4.9 μmol, 1 equiv.) or (II-2-PSMAt1) (2.6 mg, 2.4 μmol, 1 equiv.) and DIPEA (6 μL) in DMF was diluted with [ natThe resulting dark brown solution was reacted at room temperature for 2-3 hours. The reaction solution was then mixed with a solution of 1000 mL of 1,000 sulphate (ReO2I(PPh3)2) (approximately 2.2 mg, approximately 2.5 μmol, 0.5 or 1 equivalent, respectively). 18 HPLC (C 18 Purification was performed by semi-preparative HPLC (9.4×250 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column: 90 min, isocratic flow at 95% A for 5 min, then 0.93% A / 5% B to 25% A / 75% B for 90 min. -1 followed by a linear increase of 2.5% from 25%A to 0%A. -1 Linear increase in flow rate of 3 mL min -1 A = water containing 0.005% acetic acid, B = acetonitrile containing 0.005% acetic acid; detection at 214 and 254 nm). The fractions containing the desired product were lyophilized to give the desired product. nat Re-III-1-PSMAt1) (1–2 mg, 0.4–0.8 μmol, 15–30% yield) and ( nat Re-III-2-PSMAt1) (approximately 1-1.5 mg, approximately 0.5 μmol, approximately 20% yield) was obtained as a solid.

[0263]

[0290] A relatively "long" HPLC method (60 min gradient mobile phase; 1 ml min) was used to separate the cis and trans isomers. -1 Flow rate: 100%A / 0%B to 40%A / 60%B in 1% min -1 A = water containing 0.1% TFA, B = acetonitrile containing 0.1% TFA, using an analytical (4.6 x 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column) nat Re-III-1-PSMAt1) eluted at 38.11 and 38.51 min ( nat Re-III-2-PSMAt1) eluted at 45.37 and 46.07 min.

[0264]

[0291] ( nat Re-III-1-PSMAt1):HR-MS-ESI m / z:[M+2H]3+ 761.9001(C 102 H 126 O 32 N8P4Re calcd 761.8990); [M+H+Na] 3+ 769.2274(C 102 H 125 O 32 (Calculated value of N8P4ReNa is 769.2263).

[0265] ( nat Re-III-2-PSMAt1):HR-MS-ESI m / z:[M+2H] 3+ 799.2757(C 110 H 142 O 32 N8P4Re calcd 799.2741); [M+H+Na] 3+ 806.6023(C 110 H 141 O 32 Calculated value of N8P4ReNa is 806.6014).

[0266] Example 14 Preparation and use of radiolabeling kits and their effect on radiochemical yields

[0292] (II-1-RGD), (II-1-PSMAt1), (II-2-PSMAt1), and (II-11-PSMAt1) using "kit" formulations 99m To determine the feasibility of Tc radiolabeling, lyophilized mixtures of (II-1-RGD), (II-1-PSMAt1), (II-2-PSMAt1), or (II-11-PSMAt1), stannous chloride, sodium bicarbonate, and sodium gluconate or sodium tartrate were prepared.

[0267]

[0293] A stock solution containing the required amount of sodium bicarbonate, stannous chloride, and sodium gluconate or sodium tartrate was prepared. The pH was adjusted to either 7.5 or 8-8.5 by dropwise addition of either hydrochloric acid (0.1 M) or sodium hydroxide (0.1 M) in water. An aliquot of the stock solution was mixed with the required amount of (II-1-RGD), (II-1-PSMAt1), (II-2-PSMAt1), and (II-11-PSMAt1) (dissolved in a mixture of water / ethanol (70% / 30%)) to form the kit solution outlined in Table 7, which was immediately frozen and lyophilized using a freeze dryer. The lyophilized kits were stored in a freezer prior to use.

[0268]

[0294] Next, the generator was generated in saline solution. 99m TcO4 - (200MBq) was added to these kits and the mixtures were reacted at ambient temperature (approximately 22°C) for 5 min or heated at 100°C for 5 min and then analyzed by radio-iTLC and radio-HPLC.

[0269]

[0295] A relatively "short" HPLC method (20 min gradient mobile phase; 1 ml min -1 Using a flow rate of 100% A / 0% B increasing linearly from 0% A / 100% B; A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA on an analytical (4.6 × 150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column, the species assigned to (Tc-III-1-PSMAt1) eluted between 11.0 and 12.5 min, and (Tc-III-2-PSMAt1) eluted between 12.5 and 14.0 min.

[0270]

[0296] A relatively "long" HPLC method (60 min gradient mobile phase; 1 ml min) was used to separate the cis and trans isomers. -1 Flow rate: 100%A / 0%B to 40%A / 60%B in 1% min -1A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA. Using an analytical (4.6×150 mm, 5 μm) Agilent Zorbax Eclipse XDB-C18 column, the species assigned as (Tc-III-1-PSMAt1) eluted at 38.89 and 39.25 min, and (Tc-III-2-PSMAt1) eluted at 46.21 and 46.83 min.

[0271]

[0297] 99m Tc-colloid, unreacted 99m TcO4 - and (Tc-III-1-PSMAt1) / (Tc-III-2-PSMAt1), two separate iTLC analyses were performed to allow quantification.

[0272]

[0298] Unresponsive 99m TcO4 - Acetone was used as the mobile phase to quantify the amount of R f value: 99m TcO4 - >0.9, 99m Tc colloid <0.1, (Tc-III-1-PSMAt1) / (Tc-III-2-PSMAt1) <0.1.

[0273]

[0299] 99m To quantify Tc-colloid formation, a 1:1 mixture of methanol and 2 M aqueous ammonium acetate was used as the mobile phase: 99m TcO4 - >0.9, 99m Tc colloid <0.1, (Tc-III-1-PSMAt1) / (Tc-III-2-PSMAt1)>0.9.

[0274]

[0300]

[0275] [Table 7-1]

[0276] [Table 7-2]

[0277] [Table 7-3]

[0278]

[0301] The amounts of tin(II) chloride, sodium bicarbonate, and sodium gluconate reagents used in Kit 1 replicate those in the Tetrofosmin Kit. 99m Addition of TcO (20-55 MBq) to the contents of kit 1, followed by heating at 60 °C for 30 min, led to the formation of compound (Tc-III-1-RGD) in radiochemical yields up to 34%. Replacing sodium gluconate with sodium tartrate in the kit mixture while simultaneously reducing the amount of compound (II-1-RGD) conjugate from 1 mg to 0.5 mg increased the radiochemical yield up to 85% (kit 2).

[0279]

[0302] In kit 3, 45 to 65 MBq 99m Using TcO - and as little as 125 μg of compound (II-1-RGD), a radiochemical yield of ≧90% was consistently achieved (93.0±1.0%, n=4). In kit 3, the amount of sodium tartrate and tin(II) chloride was also reduced. However, further reduction of compound (II-1-RGD) to 63 μg in kit 4 reduced the radiochemical yield to 65%. All radiolabeling reactions were performed in a mixture of saline and ethanol to dissolve compound (II-1-RGD); kits containing less compound (II-1-RGD) required less ethanol.

[0280]

[0303] In kits 5 and 6, substitution of the arylphosphine substituent with an electron donating group, in this case phenyl substituted with a methyl group at the para position, is shown to improve yields at both room temperature and at 100° C. In kit 13, a more electron donating group, in this case phenyl substituted with a methoxy group at the para position, is shown to improve yields at 100° C. even more than the methyl substituent using the same methodology.

[0281] Example 15a 188 ReO4 - Kit radiolabeling of (II-1-PSMAt1), (II-2-PSMAt1), and (II-11-PSMAt1) with:

[0304] In saline solution 188 ReO4 - The sample was 188 W / 188 Obtained from the Re generator. 188 ReO4 - was "preconcentrated": in saline 188 ReO4- solution with Ag + Passed through a cartridge (Dionex OnGuard™ II Ag; pre-conditioned with 10 mL water) onto a QMA cartridge (Sep-Pak® Light (46 mg) Accell™ Plus QMA Carbonate; pre-conditioned with 5 mL EtOH, then 10 mL water) where 188 ReO4 - The QMA cartridge was then washed with water (4 mL) and then 188 ReO4 - was eluted into a small volume of saline (0.9% NaCl in water, w / v). This "preconcentration" process could be combined with the generator elution to facilitate direct concentration of the generator eluate while minimizing handling of radioactivity. Tubing was used to connect the generator outlet to two cartridges (in series), which were then attached to a vacuum pump via two or more receiver vials to achieve direct concentration of the eluate.

[0282]

[0305] 188 ReO4 - A saline solution containing (125 μL, 30-450 MBq) was added to an aqueous solution of sodium citrate (1 M, 50 μL) and stannous chloride (3.75 mg) and heated at 90 °C for 30 min. An aliquot of this solution (50 μL, 10-150 MBq) was then added to the contents of either the two (II-1-PSMAt1) kits, the two (II-2-PSMAt1) kits, or the two (II-11-PSMAt1) kits (listed in Table 5) to obtain a solution of pH 8-8.5, which was then heated at 90 °C for 30 min. An aliquot of the reaction solution was then analyzed by reverse-phase C18 radio-HPLC (30 min method).

[0283] An aliquot of the reaction solution was then subjected to reverse phase C 18 Analyzed by radio HPLC.

[0306] Unresponsive 188 ReO4 - and citric acid 188 Re eluted at 2.0-2.3 min. The species assigned to (Re-III-1-PSMAt1) eluted at 12.7 min in 73% radiochemical yield (Figure 19a). (Re-III-2-PSMAt1) eluted at 17.5 min in 46% radiochemical yield (Figure 19b). (Re-III-11-PSMAt1) eluted at 9.53 min in 90% radiochemical yield (Figure 19c).

[0284] Example 15b Purification and stability of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1):

[0307] The crude reaction mixtures containing either (Re-II-1-PSMAt1) or (Re-II-2-PSMAt1) prepared as above were loaded onto a reverse-phase C18 analytical HPLC column and isolated using the following linear HPLC gradient: 0 min, 100% A / 0% B to 60 min, 40% A / 60% B, 1 mL min -1[Fractions containing either (Re-II-1-PSMAt1) (eluted as a double peak at 38-40 min) or (Re-II-2-PSMAt1) (eluted as a double peak at 46-48 min) were immediately frozen and lyophilized. The resulting samples of (Re-II-1-PSMAt1) or (Re-II-2-PSMAt1) were dissolved in phosphate-buffered saline and were determined to be 95% radiochemically pure (by analytical C18 radio-HPLC and radio-iTLC).

[0285]

[0308] A solution of (Re-II-1-PSMAt1) or (Re-II-2-PSMAt1) in phosphate-buffered saline (20 μL, 0.5–1.5 MBq) was added to a sample of human serum (180 μL) and incubated at 37 °C. At 1 and 24 h, the sample was treated with ice-cold acetonitrile (300 μL) to precipitate and remove serum proteins. The acetonitrile in the supernatant was then removed by evaporation under a stream of N2 gas. The final solution was then analyzed by reverse-phase analytical radio-HPLC (Figures 22a and 22b).

[0286]

[0309] Example 16 Uptake of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1) into prostate cancer cell lines

[0310] A panel of cell lines was selected that either express GCP(II) / PSMA- (DU145-PSMA, genetically modified to express PSMA) [1] or have low GCP(II) / PSMA expression (DU145(HTB-81)). Cell lines were cultured in 10% fetal bovine serum, 2 mM L-glutamine, and 100 U.mL -1 Penicillin and 100 μg.mL -1 Cells were cultured in RPMI1640 medium (R0883, Sigma) containing streptomycin. Cells were maintained at 37°C and 5% CO2. Cells were plated at 5 x 10 cells per well in 2 mL of complete medium. 5The cells were seeded at a density of 1000x in 6-well plates to achieve 70–80% confluency the next day. One hour before cells were treated, cell medium (1 mL / well) was changed. A solution containing either (Re-III-1-PSMAt1) or (Re-III-2-PSMAt1) (50 kBq, >95% radiochemical purity in 5–10 uL phosphate-buffered saline) was added to each well, and cells were incubated for 1 hour at 37 °C. Uptake studies were also performed after a 2-min incubation with the PSMA inhibitor 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA; 30 μL / well of 750 μM PMPA solution). After 1 hour of incubation, the supernatant was removed and cells were washed with cold phosphate-buffered saline solution (3 × 1 mL). Cells were lysed in cold radioimmunoprecipitation assay buffer (RIPA buffer, 500 μL; 150 mM sodium chloride, 0.1 w / w% sodium dodecyl sulfate (SDS), 0.5 w / w% sodium deoxycholate (NaDOC), 1 w / w% Triton-X) and samples were collected for radioactivity counting. Results are depicted as mean ± SD of independent biological experiments (performed on different days with different radiotracer preparations).

[0287]

[0311] (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1) showed uptake into DU145-PSMA+ cells (14.37±2.25% AR [percentage of radioactivity added], and 9.23±1.04% AR, respectively). This uptake was specific: DU145-PSMA+ cell uptake of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt) could be blocked with PMPA, and there was negligible uptake into parental DU145 cells (FIG. 23).

[0288] Example 17 ( 186 Preparation of Re-III-1-PSMAt and Radiolabeling Kit

[0312] 186 Re-III-1-PSMA, 186 ReO4 - It was prepared in two steps from a saline solution containing

[0289]

[0313] SnCl2.2H2O (15 mg) was dissolved in aqueous sodium citrate (100 μL, 1 M). A sample of this solution (25 μL) was 186 ReO4 - The reaction mixture was heated to 90° C. for 30 min to obtain citric acid. 186 Re(V) was obtained in 96% radiochemical yield.

[0290]

[0314] This was followed by citric acid 186 Re(V) (4–5 MBq, 50 μL) was dissolved in sodium carbonate, sodium tartrate, stannous chloride, and DP Ph -Pre-manufactured lyophilized kits containing PSMAt (Table 8, 188 This solution was heated at 90°C for 30 minutes, 186 Re-DP1-PSMA was formed in 5.5% radiochemical yield as determined by radio-HPLC.

[0291] [Table 8]

[0292]

[0315] Prepared from the above kit ( 186 The solution of Re-III-1-PSMAt1) was applied to a reverse-phase C18 analytical HPLC column and isolated using the following linear HPLC gradient: 0 min, 100% A / 0% B to 60 min, 40% A / 60% B, 1 mL min -1 (A = water containing 0.1% TFA, B = acetonitrile containing 0.1% TFA). 186 The fraction containing Re-III-1-PSMAt1 eluted at 39.8 min and was immediately frozen and lyophilized. Analytical reversed-phase HPLC showed that ( 186 The radiochemical purity of the radiolabeled species (Re-III-1-PSMAt1) was shown to be >95%. nat Re-III-1-PSMAt1) standard.

[0293] Example 18 Prostate cancer cell lines 186 Re-III-1-PSMAt1) uptake

[0316] GCP(II) / PSMA expressing cells, DU145-PSMA+ and LNCaP cells were suspended in RPMI medium (5 million cells, 1 mL). 186 Re-III-1-PSMAt1) (10,000 cpm, >95% radiochemical purity in 10 μL phosphate-buffered saline) was added to each cell sample and the cells were incubated for 1 h at 37°C with constant agitation. In addition, nonspecific uptake was also determined by using non-GCP(II) / PSMA expressing cells (DU145) or by blocking PSMA expressing cells (DU145-PSMA+ and LNCaP cells) with the PSMA inhibitor, PMPA (30 μL of 750 uM PMPA solution / 5 million cells). After 60 min of incubation, the supernatant was removed and the cells were washed three times with ice-cold phosphate-buffered saline solution. The cells were treated with ice-cold RIPA buffer (500 μL, 150 mM sodium chloride, 0.1 w / w% sodium dodecyl sulfate (SDS), 0.5 w / w% sodium deoxycholate (NaDOC), 1 w / w% Triton-X) to lyse the cells and collect samples for radioactivity counting. 186 Uptake of Re-DP1-PSMA was measured to be 4.23±0.99% AR [percentage of added radioactivity], which decreased to 0.08±0.14% AR in PSMA-negative DU145 cells and to 0.21±0.16% AR upon co-incubation with excess PMPA. In LNCaP cells, 186 Uptake of Re-DP1-PSMA was measured to be 3.98±0.98% AR, which decreased to 0.55±0.15% AR upon co-incubation with excess PMPA (see FIG. 24).

[0294] Example 19 In mice bearing prostate cancer tumors ( 188 Re-III-1-PSMAt1) and ( 188Biodistribution of Re-III-2-PSMAt1

[0317] ( 188 Re-III-1-PSMAt1) and ( 188 The biodistribution of Re-III-2-PSMAt1) was determined in SCID / beige mice bearing DU145-PSMA+ tumors (Figure 25). 188 Re-III-1-PSMAt1) or ( 188 Mice were administered either 100 mg / kg / day or 100 mg / kg / day of PSMAt1 or 100 mg / kg / day of PSMAt2 or 100 mg / kg / day of PSMAt3 or 100 mg / kg / day of PSMAt4 or 100 mg / kg / day of PSMAt5 or 100 mg / kg / day of PSMAt6 or 100 mg / kg / day of PSMAt7 or 100 mg / kg 188 For animals receiving Re-III-1-PSMAt1), 27.7 ± 6.4% ID g -1 The radioactivity concentration (percentage of injected dose per gram) was measured. 188 For animals receiving Re-III-2-PSMAt1), 19.2 ± 8.6% ID g -1 Radioactivity concentrations of 100 mg / kg / day were measured. Both compounds were cleared from the circulation via the renal route, as evidenced by the high radioactivity concentrations measured in the kidney. Biodistribution data also showed that both compounds had low retention in healthy non-target organs / tissues, with the exception of organs known to express PSMA (spleen and prostate). In this experiment, 100 mg / kg / day was measured at 2 hours post-injection ( 188 Re-III-2-PSMAt1) ( 188 No statistically significant differences were observed between the biodistribution profiles of Re-III-1-PSMAt1).

[0295]

[0318] ( 188 Re-III-1-PSMAt1) or ( 188 Urine was collected from mice receiving either 100 mg of 10 ... 188 Re-III-1-PSMAt1) and ( 188Re-III-2-PSMAt1) were both highly stable, and >94% of the radioactivity was 188 Re-III-1-PSMAt1) or ( 188 Re-III-2-PSMAt1) (Figure 26).

[0296]

[0319] The GCP(II) / PSMA expressing cell line used in these experiments was a genetically modified daughter cell line of DU145, DU145-PSMA+. This cell line was previously transduced to express full-length human GCP(II) / PSMA according to F. Kampmeier, JD Williams, J. Maher, GE Mullen and PJ Lower, EJNMMI Res., 2014, 4, 13. These cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin / streptomycin. To prepare for the experiments, the cells were grown at 37°C in an incubator containing humidified air equilibrated with 5% CO2.

[0297]

[0320] Animal studies were conducted under a UK Home Office Project and Personal License and in compliance with the UK Research Council and Medical Research Charity guidelines for the responsible use of animals in bioscientific research. SCID / beige mice (male, 7–12 weeks old) were incubated with 4 × 10 6 Subcutaneous prostate cancer xenografts were generated by injecting DU145-PSMA or DU145 cells into the right shoulder. Once tumors reached 5-10 mm in diameter (3-4 weeks after injection), biodistribution studies were performed. For imaging purposes, mice were anesthetized, positioned on the scanner, and the tail vein was cannulated. For biodistribution purposes, mice were anesthetized and the radiotracer was injected via the tail vein.

Claims

1. Conjugated diphosphine precursor compounds of formula (II) suitable for preparing conjugated radiolabeled drugs: 【Chemical 1】 [In the formula, each Z is independently O or S; Y is NH or O; X １ , X ２ , X ３ and X ４ are each independently a substituted C 5 -C 8 aryl group, a substituted 5- to 8-membered heteroaryl group, or a substituted C 3 -C 8 cycloalkyl group, where each substituent is １ ~C ４ Alkyl group, C ５ ~C １２ an aryl or heteroaryl group, C １ ~C ４ Acylamide group, sulfylhydro group, C １ ~C ４ Alkylthio group, C １ ~C ４ (Di)alkylphosphino group, hydroxy group, C １ ~C ４ Alkoxy group, carboxyl group, C １ ~C ４ (Di)alkylamino group and C １ ~C ４ Alkoxy-(CH ２ CH ２ O) ｎ wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the shortest linear chain of carbon atoms between two Z groups is 4 to 7; wherein the LIG comprises a ligand having a binding motif corresponding to a biological target; 【Chemistry 2】 Not the compound.

2. A conjugated diphosphine precursor compound according to claim 1 of formula (IIa) suitable for preparing a conjugated radiolabeled drug: 【Chemistry 3】 [In the formula, each Z is independently O or S; Y is NH or O; X １ , X ２ , X ３ and X ４ are each independently a substituted C 5 -C 8 aryl group, where each substituent is C １ ~C ４ Alkyl group, C １ ~C ４ Alkoxy group, C １ ~C ４ (Di)alkylamino group and C １ ~C ４ Alkoxy-(CH ２ CH ２ O) ｎ wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; LIGs include prostate-specific membrane antigen targeting ligands (PSMAt), cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), pentixafor peptides, minigastrin peptide analogs for targeting the cholecystokinin-2 receptor, c-Met targeting peptides, alpha-MSH peptides, bisphosphonates, folic acid, or carbohydrates].

3. 10. The conjugated diphosphine precursor compounds of claim 1, of formula (IIb) and / or (IIc), suitable for preparing conjugated radiolabeled drugs: 【Chemistry 4】 [In the formula, X １ , X ２ , X ３ and X ４ are each independently p-tolyl, m-tolyl, o-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, 2,6-xylyl, 3,4-xylyl, 3,5-xylyl, p-methoxyphenyl, o-methoxyphenyl, 4-(MeO(CH ２ CH ２ O))phenyl, 4-(MeO(CH ２ CH ２ O) ２ ) phenyl, 4-(MeO(CH ２ CH ２ O) ３ ) phenyl or 4-dimethylaminophenyl, and LIG comprises a prostate-specific membrane antigen targeting ligand (PSMAt) or cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD)]. 【Request 4】 【Chemical 5】 2. The compound of claim 1, wherein:

5. Diphosphine precursor compounds of formula (I) suitable for preparing conjugated radiolabeled drugs: 【Chemistry 6】 [In the formula, Ring A is a 5-, 6-, 7-, or 8-membered ring; each Z is independently O or S; Y is NH or O; X １ , X ２ , X ３ and X ４ are each independently a substituted C 5 -C 8 aryl group, a substituted 5- to 8-membered heteroaryl group, where the optional substituents are C １ ~C ４ Alkyl group, C ５ ~C １２ an aryl or heteroaryl group, C １ ~C ４ Acylamide group, sulfylhydro group, C １ ~C ４ Alkylthio group, C １ ~C ４ (Di)alkylphosphino group, hydroxy group, C １ ~C ４ Alkoxy group, carboxyl group, C １ ~C ４ (Di)alkylamino group and C １ ~C ４ Alkoxy-(CH ２ CH ２ O) ｎ wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; 【Chemistry 7】 Not the compound.

6. A diphosphine precursor compound according to claim 5 of formula (Ia) suitable for preparing a conjugated radiolabeled drug: 【Chemistry 8】 [In the formula, each Z is independently O or S; Y is NH or O; X １ , X ２ , X ３ and X ４ are each independently C １ ~C ４ Alkyl group, C １ ~C ４ Alkoxy group, C １ ~C ４ (Di)alkylamino group and C １ ~C ４ Alkoxy-(CH ２ CH ２ O) ｎ a substituted C having one or more substituents selected from the group consisting of ５ ~C ８ aryl group, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ]

7. Diphosphine precursor compounds according to claim 5 of formula (Ib) and / or (Ic) suitable for preparing conjugated radiolabeled drugs: 【Chemistry 9】 [In the formula, X １ , X ２ , X ３ and X ４ are each independently p-tolyl, m-tolyl, o-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, 2,6-xylyl, 3,4-xylyl, 3,5-xylyl, p-methoxyphenyl, o-methoxyphenyl, 4-(MeO(CH ２ CH ２ O))phenyl, 4-(MeO(CH ２ CH ２ O) ２ ) phenyl, 4-(MeO(CH ２ CH ２ O) ３ ) phenyl or 4-dimethylaminophenyl].

8. X １ , X ２ , X ３ and X ４ The compound according to any one of claims 1 to 6, wherein are the same.

9. At least two compounds according to any one of claims 1 to 4 ９９ｍ Tc, ２１２ Pb, ２１２ Bi, ２１３ Bi, １８６ Re, １８８ Re, ８９ Zr, ６７ Ga, ６８ Ga, ６７ Cu, ６４ Cu, ６２ Cu, ６１ Cu, ６０ Cu, ６２ Zn and ５２ A radiolabeled diphosphine complex comprising as a coordinating ligand one or more radionuclides selected from Mn, 【Chemistry 10】 [Wherein, in the compound (Tc-III-1-RGD), Tc is ９９ｍ Tc, and in the compound (Re-III-1-RGD), Re is １８６ Re and １８８ Re] is not the radiolabeled diphosphine complex.

10. (a) Formula (M-IIIa-trans) or Formula (M-IIIa-cis) or a mixture thereof 【Chemistry 11】 wherein M is ９９ｍ Tc, １８６ Re and １８８ or (b) Formula (M-IIIb-trans) or Formula (M-IIIb-cis) or a mixture thereof 【Chemistry 12】 wherein M is ９９ｍ Tc, １８６ Re and １８８ or (c) according to formula (Cu-IIIc-A) or formula (Cu-IIIc-B) or mixtures thereof; 【Chemistry 13】 [Wherein Cu is ６７ Cu, ６４ Cu, ６２ Cu, ６１ Cu and ６０ Cu]; or (d) according to formula (Cu-IIId-A) or formula (Cu-IIId-B) or mixtures thereof; 【Chemistry 14】 [Wherein Cu is ６７ Cu, ６４ Cu, ６２ Cu, ６１ Cu and ６０ Cu] and, [In the formula, X is C １ ~C ４ Alkyl group and C １ ~C ４ a phenyl group having one or more substituents selected from the group consisting of alkoxy groups; The LIG comprises a prostate-specific membrane antigen targeting ligand (PSMAt), cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), a pentixafor peptide, a mini-gastrin peptide analog for targeting the cholecystokinin-2 receptor, a c-Met targeting peptide, an alpha-MSH peptide, a bisphosphonate, folic acid, or a carbohydrate.

10. The radiolabeled conjugated diphosphine complex of claim 9, wherein

11. (a) Formula (M-IIIa-trans) or Formula (M-IIIa-cis) or a mixture thereof 【Chemistry 15】 wherein M is ９９ｍ Tc, １８６ Re and １８８ or (b) Formula (M-IIIb-trans) or Formula (M-IIIb-cis) or a mixture thereof 【Chemistry 16】 wherein M is ９９ｍ Tc, １８６ Re and １８８ and Rb is a radionuclide selected from one or more of Rb, ... and, [In the formula, X is C １ ~C ４ Alkyl group and C １ ~C ４ a phenyl group having one or more substituents selected from the group consisting of alkoxy groups; The LIG comprises a prostate-specific membrane antigen targeting ligand (PSMAt), cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD), a pentixafor peptide, a mini-gastrin peptide analog for targeting the cholecystokinin-2 receptor, a c-Met targeting peptide, an alpha-MSH peptide, a bisphosphonate, folic acid, or a carbohydrate.

11. The radiolabeled conjugated diphosphine complex of claim 10, wherein

12. X is p-tolyl, m-tolyl, o-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, 2,6-xylyl, 3,4-xylyl, 3,5-xylyl, p-methoxyphenyl, o-methoxyphenyl, 4-(MeO(CH ２ CH ２ O))phenyl, 4-(MeO(CH ２ CH ２ O) ２ ) phenyl, 4-(MeO(CH ２ CH ２ O) ３ ) phenyl or 4-dimethylaminophenyl; The LIG comprises a prostate-specific membrane antigen targeting ligand (PSMAt) or cyclic (Arg-Gly-Asp-dPhe-Lys) (RGD); 11. The radiolabeled conjugated diphosphine complex of claim 10.

13. 10. The complex of claim 9 according to formula (M-IIIa-trans) or formula (M-IIIa-cis) or mixtures thereof.

14. 10. The complex of claim 9, which is an approximately 1:1 mixture of cis / trans isomers.

15. A method for preparing a conjugated diphosphine precursor compound, or compound (II-1-RGD), according to any one of claims 1 to 4, comprising mixing a compound, or compound (I-1), according to claim 5 and LIG-H in the presence of a base, wherein LIG comprises a peptide or carbohydrate ligand having a binding motif corresponding to a biological target.

16. 16. The method of claim 15, wherein the base is N,N-diisopropylethylamine added dropwise and the reaction is carried out in N,N-dimethylformamide at room temperature.

17. The radiolabeled conjugated diphosphine complex according to claim 9, the compound (Tc-III-1-RGD), the compound ( １８６ 1. A method for making compound (Re-III-1-RGD) or compound (Re-III-1-RGD), said method comprising the step of mixing the compound of any one of claims 1 to 4 or compound (II-1-RGD) with a radionuclide in the presence of an intermediate ligand, a reducing agent, a buffer and a solvent.

18. The radionuclide is ９９ｍ Tc, ２１２ Bi, ２１３ Bi, １８６ Re, １８８ Re, ８９ Zr, ６７ Ga, ６８ Ga, ６７ Cu, ６４ Cu, ６２ Cu, ６１ Cu, ６０ Cu and ５２ 18. The method of claim 17, wherein the intermediate ligand is selected from one or more of: Mn, Mn(II), ...

19. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 in combination with a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising the complex of claim 9 in combination with a pharmaceutically acceptable carrier.

21. The complex according to claim 9, which comprises a mixture of a reducing agent, a buffer, an intermediate co-ligand and the conjugated diphosphine precursor compound according to any one of claims 1 to 4, or the compound (II-1-RGD), the compound (Tc-III-1-RGD), the compound (II-1-RGD), １８６ Re-III-1-RGD) or a kit for preparing the compound (Re-III-1-RGD).

22. ９９ｍ Tc, ２１２ Bi, ２１３ Bi, １８６ Re, １８８ Re, ８９ Zr, ６７ Ga, ６８ Ga, ６７ Cu, ６４ Cu, ６２ Cu, ６１ Cu, ６０ Cu and ５２ 22. The kit of claim 21, further comprising a radionuclide selected from Mn.

23. The radionuclide is ９９ｍ Tc, １８６ Re, １８８ Re, and ５２ 23. The kit of claim 22, wherein the hydroxybenzoates are selected from the group consisting of hydroxybenzoates, ...

24. A compound according to any one of claims 1 to 7, compound (I-1), compound (II-1-RGD), compound (Tc-III ... １８６ Re-III-1-RGD) or the use of the compound (Re-III-1-RGD).

25. Use of a complex according to claim 9 in the preparation of a medicament for the treatment or diagnosis of a disease.

26. A compound according to any one of claims 1 to 7, compound (I-1), compound (II-1-RGD), compound (Tc-III-1-RGD), compound ( １８６ Re-III-1-RGD) or the compound (Re-III-1-RGD), optionally a non-therapeutic and / or in vitro use.

27. Use of the complex of claim 9 in imaging or cell labeling, optionally a non-therapeutic and / or in vitro use.

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