Radiopharmaceuticals, radioimaging agents and their uses

Abstract

This invention relates to compounds used as radiopharmaceuticals and radioimaging agents having radionuclide chelating agents. These coordination compounds can be used in radiotherapy and diagnostic imaging. The invention also relates to methods for diagnosis, prognosis, and treatment using the non-coordinating and radiolabeled compounds of this invention.

Description

[0001] This application is a divisional application of the invention patent application filed on June 5, 2018, with application number CN 201880037177.5 and entitled "Radiopharmaceuticals, Radioimaging Agents and Their Uses Thereof". The entire contents of the original parent application are incorporated herein by reference. Technical Field

[0002] This invention relates to compounds used as radiopharmaceuticals and radioimaging agents containing radionuclide chelating agents. These coordination compounds can be used in radiotherapy and diagnostic imaging. The invention also relates to methods for diagnosis, prognosis, and treatment using the non-coordinating and radiolabeled compounds of this invention. Technical Background

[0003] Prostate cancer is a leading cause of cancer-related death in men, with mortality often attributed to the difficulty in detecting and treating the disease. Prostate-related tumors typically exhibit increased expression of prostate-specific membrane antigen (PSMA), an enzyme normally expressed in prostate tissue but often upregulated in some prostate cancers. This means that PSMA is a good biomarker or target for imaging, diagnosis, and prognostic purposes. However, successful imaging of prostate cancer is challenging because PSMA is also expressed in other tissues, both normal and malignant.

[0004] If a radiolabeled compound can bind sufficiently to the desired site and can also deliver a radionuclide to the same site for imaging or therapeutic purposes, then the radiolabeled compound can be used as a radiopharmaceutical or radioimaging agent.

[0005] Compounds or ligands containing urea-based motifs, such as glutamic acid-substituted ureas below, are known to bind to the catalytic site of PSMA with good affinity.

[0006]

[0007] Although compounds containing this motif or similar motifs have been synthesized, problems related to their stability or binding behavior in vivo have been observed. For a compound to be usable in radioimaging or radiotherapy, the resulting complex containing the radionuclide must be stable in vivo. One known problem associated with radiolabeled compounds is that the complexes formed with the radionuclide are not strong enough, and the radionuclide “leaks” from the complex and is not delivered to the intended site. Other problems caused by radionuclide leakage include the radionuclide spreading to unwanted sites. This can potentially damage healthy tissue due to the reactivity of the radionuclide. Furthermore, radionuclide spread results in poorer image quality because the contrast between the true binding site (indicating the location of the tumor) and the site containing the unwanted radionuclide is reduced.

[0008] Other problems associated with radiolabeled compounds include the possibility of radioactive decomposition, in which the radioactive isotope itself causes the destruction of the compound and subsequent radionuclide diffusion. Spontaneous decay of a radionuclide leads to radiolysis, releasing energy that causes bond breakage in ligands and destruction of the complex. Radiolysis also causes the radionuclide to diffuse to unwanted sites, further exacerbating the aforementioned problems.

[0009] Because the compound is intended for use in individuals in need, it must also be non-toxic to the subject. Another issue related to the use of radiolabeled compounds for diagnosis, imaging, and treatment is binding affinity. A low binding affinity to the target, i.e., PSMA in this case, may prevent the complex from binding and being excreted, or may only show limited binding. When the complex is excreted immediately, this results in reduced overall efficacy. However, in cases where the complex exhibits limited excretion and limited binding, it can diffuse throughout the subject and cause the aforementioned adverse effects. Furthermore, limited binding of the complex may reduce the time available for acceptable imaging or treatment of the tumor.

[0010] There is a need for compounds that provide the required binding affinity to tumors associated with prostate cancer and also possess the ability to provide the necessary imaging properties. These compounds also need to be sufficiently stable and not decompose during use. Summary of the Invention

[0011] This invention relates to novel compounds with improved binding affinity for PSMA. The inventors have discovered that amino acid-substituted ureas, which bind to sarcophagine via specific linkers, provide compounds that bind to PSMA and offer good / improved imaging performance when complexed with radionuclides.

[0012] On the one hand, the present invention provides compounds of formula (I) or their salts, complexes, isomers, solvates or prodrugs:

[0013]

[0014] in:

[0015] X is selected from H, OH, halogen, cyano, NO2, NH2, or optionally substituted C1-C. 12 Alkyl, optionally substituted amino, optionally substituted amide, and optionally substituted aryl groups;

[0016] Y is an optional C1-C. 12 Alkylene, wherein one or more methylene groups in the alkylene group may be further optionally substituted with groups selected from amide, carbonyl, urea and thiourea;

[0017] m is 0, 1, or 2;

[0018] n is 0, 1, or 2.

[0019] In one embodiment, the compound or its salt, complex, isomer, solvate, or prodrug has the following formula:

[0020]

[0021] In another embodiment, the compound or its salt, complex, isomer, solvate, or prodrug has the following formula:

[0022]

[0023] In another embodiment, the compound or its salt, complex, isomer, solvate, or prodrug has the following formula:

[0024]

[0025] In another aspect, the present invention provides a composition comprising a compound according to the foregoing aspects and a pharmaceutically acceptable excipient.

[0026] In another aspect, the present invention provides an aqueous composition for parenteral administration comprising the compounds described above, wherein the composition further comprises: ethanol, gentianic acid or a salt thereof, and sodium chloride.

[0027] In another aspect, the present invention provides a method for treating or preventing a condition in a subject who requires such treatment, the method comprising administering a therapeutically effective amount of a compound or a composition according to the foregoing. Attached Figure Description

[0028] Figure 1 Use in 30 minutes, 2 hours and 22 hours64 PET imaging of NSG mice carrying LNCaPs treated with Cu-Sar-PSMA.

[0029] Figure 2 : Shown in NSG mice carrying LNCaPs 64 A graph showing the biodistribution of Cu-Sar-PSMA relative to blood levels at 22 hours (right).

[0030] Figure 3 : In 1 and 6 hours, use 64 PET imaging of NSG mice carrying LNCaPs treated with Cu-Sar-PSMA.

[0031] Figure 4 :show 64 Biodistribution of Cu-Sar-PSMA in LNCaP of NSG-carrying mice at 1 and 6 hours.

[0032] Figure 5 : A graph showing the biodistribution of various radiolabeled complexes in LNCaPs xenograft mice, represented as the proportion of tumor:kidney uptake at 1 hour.

[0033] Figure 6 : Shown in LNCaPs xenograft mice 64 Cu-Sar-PSMA and 68 A graph showing the preclinical biodistribution of Ga-labeled complexes, representing the proportion of tumor:kidney uptake at 1 hour.

[0034] Figure 7 : A graph showing the biodistribution of various radiolabeled complexes in LNCaPs xenograft mice, expressed as the proportion of uptake in the tumor: kidney.

[0035] Figure 8 The structure of existing PSMA ligand targets.

[0036] Figure 9 Detected using UV at 220nm 64 HPLC chromatograms of Cu-Sar-PSMA (RT: 12.43 min) compared to nat Cu-Sar-PSMA (RT: 12.38 min).

[0037] Figure 10 : 64 Radio-HPLC chromatogram of Cu-CoSar(PSMA)2 (RT: 13.9 min).

[0038] Figure 11Analytical HPLC chromatogram of CoSar(PSMA)2 (RT: 10.3 min), detected by UV at 220 nm. Detailed description

[0039] As described and shown herein, the inventors have discovered that compounds comprising amino acid-substituted ureas linked to a sarcophagus via a linker group can bind to PSMA. Not wishing to be bound by theory, the combination of the amino acid-urea fragment, the linker, and the sarcophagus is considered to provide the advantages observed and discussed below.

[0040] The complexes described herein are radiolabeled with radionuclides or radioisotopes that undergo spontaneous decay, wherein these decay byproducts are detected by various methods, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT). The quality of the obtained images and the confidence in any subsequent diagnosis based on these images depend on the ability of the radiolabeled complex to specifically bind to the prostate cancer site.

[0041] As used herein, the term "sarcophagus" refers to a nitrogen-containing macrocyclic ligand having the formula 3,6,10,13,16,19-hexaazabicyclo[6.6.0]eicosane.

[0042] The term "optionally substituted" as used throughout the specification means that the group may or may not be further substituted or fused (to form a fused polycyclic system) by one or more non-hydrogen-substituted groups. In some embodiments, the substituent is one or more independently selected from halogens, =O, =S, -CN, -NO2, -CF3, -OCF3, alkyl, alkenyl, ynyl, haloalkyl, haloalkenyl, haloynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxy Cycloalkyl, alkoxyheteroalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, heterocyclic alkoxy, heterocyclic alkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkoxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, sulfinyl, alkylsulfinyl, arylsulfinylaminoalkyl, -C(=O)OH, -C(=O)R a -C(=O)OR a C(=O)NR a Rb C(=NOH)R a C(=NR) a )NR b R c NR a R b NR a C(=O)R b NR a C(=O)OR b NR a C(=O)NR b R c NR a C(=NR b )NR c R d NR a SO2R b -SR a SO2NR a R b -OR a OC (=O)NR a R b OC(=O)R a and acyl group, wherein R a R b R c and R d Each is independently selected from H, C1-C 12 Alkyl, C1-C 12 Haloalkyl, C2-C 12 alkenyl, C2-C 12 alkynyl group, C2-C 10 Heteroalkyl, C3-C 12 cycloalkyl, C3-C 12 Cycloalkenyl, C2-C 12 Heterocyclic alkyl, C2-C 12 Heterocyclic alkenyl, C6-C 18 Aryl, C1-C 18 heteroaryl and acyl, or R a R b R c and R d Any two or more of them, together with the atoms they are attached to, form a heterocyclic system with 3 to 12 ring atoms.

[0043] In some embodiments, each optional substituent is independently selected from: halogen, =O, =S, -CN, -NO2, -CF3, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkoxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, -COOH, -SH and acyl.

[0044] Examples of particularly suitable optional substituents include F, Cl, Br, I, CH3, CH2CH3, OH, OCH3, CF3, OCF3, NO2, NH2, COOH, COOCH3, and CN.

[0045] The term "alkenyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond. It can be straight-chain or branched, preferably having 2-12 carbon atoms on the positive chain, more preferably 2-10 carbon atoms, and most preferably 2-6 carbon atoms. The group can contain multiple double bonds on the positive chain, each oriented independently as either E or Z. Exemplary alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, and nonenyl.

[0046] Unless otherwise stated, "alkyl" as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group, preferably C1-C. 12 Alkyl, more preferably C1-C 10 Alkyl groups, preferably C1-C6. Examples of suitable straight-chain and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, hexyl, etc.

[0047] "Alynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond, which can be straight-chain or branched, preferably having 2-12 carbon atoms on its positive chain, more preferably 2-10 carbon atoms, and even more preferably 2-6 carbon atoms. Exemplary structures include, but are not limited to, ethynyl and propynyl.

[0048] "Aryl" as a group or part of a group means: (i) an optionally substituted monocyclic or fused polycyclic aromatic carbocyclic ring (having a ring structure in which all ring atoms are carbon), each ring preferably having 5 to 12 atoms, examples of aryl include phenyl, naphthyl, etc.; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety, wherein the phenyl and C 5-7cycloalkyl or C 5-7 Cycloalkenyl groups fuse together to form cyclic structures, such as tetrahydronaphthyl, indenyl, or indenyl. Typically, aryl groups are C6-C. 18 Aryl.

[0049] Unless otherwise stated, "cycloalkyl" refers to a saturated monocyclic, fused, or spirocyclic polycyclic carbon ring, preferably containing 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. It includes monocyclic systems (e.g., cyclopropyl and cyclohexyl), bicyclic systems (e.g., decahydronaphthalene), and polycyclic systems (e.g., adamantane). Cycloalkyl groups are typically C3-C9 cycloalkyl groups.

[0050] "Halogen" represents chlorine, fluorine, bromine, or iodine.

[0051] "Heteroalkyl" refers to a straight-chain or branched alkyl group preferably having 2 to 12 carbons in the chain, more preferably 2 to 6 carbons, wherein one or more carbon atoms (and any associated hydrogen atoms) are each independently substituted with a heteroatom group selected from S, O, P, and NR', wherein R' is selected from H, and optionally substituted C1-C 12 Alkyl, optionally substituted C3-C 12 Cycloalkyl, optionally substituted C6-C 18 Aryl and optionally substituted C1-C 18 Heteroalkyl groups. Exemplary heteroalkyl groups include alkyl ethers, secondary and tertiary alkylamines, amides, alkyl sulfides, etc. Examples of heteroalkyl groups also include: hydroxy C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, amino C1-C6 alkyl, C1-C6 alkylamino C1-C6 alkyl, and di(C1-C6 alkyl)amino C1-C6 alkyl.

[0052] A "heteroaryl" group, either alone or as part of a group, refers to a group containing an aromatic ring (preferably a 5- or 6-membered aromatic ring), wherein the aromatic ring has one or more heteroatoms as ring atoms, and the remaining ring atoms are carbon atoms. Suitable heteroatoms include nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzoisothiazole, naphtho[2,3-b]thiophene, furan, isoindoleazine, xanthine, phenothiazine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazolium, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthidine, quinoxaline, cyclophosphine, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazine, phenothiazine, oxazole, isoxazole, furazine, phenothiazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5- or 8-quinolinyl, 1-, 3-, 4- or 5-isoquinolinyl, 1-, 2- or 3-indoleyl, and 2- or 3-thiophene. Heteroaryl groups are typically C1-C 18 Mixed aromatic compounds.

[0053] As used in this article, the term "C1-C" 12 "Alkylene" refers to a divalent straight-chain or branched aliphatic hydrocarbon group, wherein the group has 1 to 12 carbon atoms in the chain.

[0054] In one embodiment, X is an optionally substituted C1-C 12 alkyl.

[0055] In one embodiment, X is C1-C 12 alkyl.

[0056] In one embodiment, X is an optionally substituted C1-C3 alkyl group.

[0057] In one embodiment, X is a C1-C3 alkyl group.

[0058] In one embodiment, X is a methyl group.

[0059] In one embodiment, X is CH3.

[0060] In one embodiment, X is an optionally substituted amino group, such as –NCH3.

[0061] In one embodiment, X is an amino group.

[0062] In one embodiment, X is an optionally substituted amide. As used herein, the term "amide" refers to a functional group consisting of a carbonyl group attached to a nitrogen atom. Thus, the term "optionally substituted amide" refers to an amide functional group with further substitution.

[0063] In one embodiment, X is an optionally substituted amide, for example,

[0064]

[0065] In one embodiment, Y is a substituted alkylene group.

[0066] In one embodiment, Y is an unsubstituted alkylene group.

[0067] In one embodiment, Y is CH2.

[0068] In one embodiment, Y is a carbonyl group.

[0069] In one embodiment, Y is a substituted C1-C 12 Alkylene groups, wherein one or more of the methylene groups are further substituted with amide groups, for example...

[0070] In one embodiment, n is 1.

[0071] In one embodiment, n is 2.

[0072] In one embodiment, n is 0.

[0073] In one embodiment, m is 1.

[0074] In one embodiment, m is 2.

[0075] In one embodiment, m is 0.

[0076] In one embodiment, the present invention provides compounds of the following formula:

[0077]

[0078] In another embodiment, the present invention provides compounds of the following formula:

[0079]

[0080] In this compound, two phenylalanine residues are D-Phe, yielding the MeCOSar-D-Phe-D-Phe-AOC-Lys-urea-Glu ligand. The inventors have discovered that incorporating phenylalanine residues into specific D-stereochemistry can produce compounds with improved metabolic stability. Furthermore, the inventors have determined that using two D-Phe residues in the ligand can increase the hydrophobicity of the compound and the potential π-π interactions between the ligand and the binding pocket of the target enzyme.

[0081] In another embodiment, the present invention provides compounds of the following formula:

[0082]

[0083] In another embodiment, the present invention provides compounds of the following formula:

[0084]

[0085] This compound contains two linkers and two urea motifs that bind to PSMA. Not wanting to be bound by theory, it seems that the compound with two urea motifs may exhibit a further improved binding affinity to PSMA. It has also been suggested that this dual derivative, i.e., the compound with two linkers and two urea motifs, may provide a better signal-to-noise ratio when used for imaging purposes compared to the corresponding single compound with a single linker and urea motif. Upon administration, the dual compound also shows further improvement in renal clearance. When comparing the single and dual compounds, this may be attributed to differences in overall charge and charge separation and distribution.

[0086] In another embodiment, the present invention provides compounds of the following formula:

[0087]

[0088] In another embodiment, the present invention provides compounds of the following formula:

[0089]

[0090] In another embodiment, the present invention provides compounds of the following formula:

[0091]

[0092] In one embodiment, the compound coordinates with a metal ion.

[0093] In one embodiment, the metal ions are Cu, Tc, Gd, Ga, In, Co, Re, Fe, Au, Mg, Ca, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Zn, Cd, Mn, Ru, Pd, Hg, Ti, Lu, Sc, Zr, Pb, Ac, and Y.

[0094] In one embodiment, the metal ion is a radioactive nuclide.

[0095] In some embodiments, the metal in the metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, Co, In, Fe, and Ti. The compounds of the present invention have been found to be particularly suitable for binding copper ions. In some embodiments, the metal in the metal ion is selected from... 60 Cu、 61 Cu、 62 Cu、 64 Cu and 67 Radionuclides belonging to the group consisting of Cu. In some embodiments, the metal in the metal ion is... 60 Cu. In some embodiments, the metal in the metal ion is... 61 Cu. In some embodiments, the metal in the metal ion is... 62 Cu. In some embodiments, the metal in the metal ion is... 64 Cu. In some embodiments, the metal in the metal ion is... 67 Cu.

[0096] The compound of formula (I) comprises a coffin macrocyclic ligand and a Lys-urea-Glu moiety targeting PSMA. The compound also comprises an intermediate portion connecting the coffin and the PSMA-targeting moiety. In formula (I), these include a propyl linker group linked to two amide groups, two phenylalanine residues, and an aminooctanoic acid (AOC) group. The propyl linker group, phenylalanine residues, and aminooctanoic acid group together act as spacer groups to separate the coffin and the PSMA-targeting moiety. A certain degree of separation between the coffin and the PSMA-targeting moiety is desired to ensure that the activities of the two groups do not interfere with each other; however, it is important that the two groups are not too far apart so that, in the case of bound radionuclides in the coffin, the radionuclide complex can be delivered to the target site defined by the PSMA-targeting moiety. The PSMA-targeting moiety comprises a Lys-urea-Glu moiety having three carboxyl functional groups that provide the total negative charge and contribute to the zinc-binding region. The aminooctanoic acid group adjacent to the PSMA-targeting moiety is designed to provide a length of approximately [missing information]. The linking group separates the charge between the PSMA-targeting moiety and the rest of the molecule. The two D-phenylalanine residues are inherently hydrophobic and allow for π-π binding interactions with the active site. These residues also contribute to the metabolic stability of the compound. The propenyl group located between the two amide functional groups also provides the necessary distance between the macrocyclic ligand (chelating positively charged Cu ions) and the PSMA-targeting moiety. The inventors have discovered that compounds according to the invention comprise various segments (i.e., macrocyclic ligands, linking groups, and PSMA-targeting moieties) that together provide a PSMA-binding ligand with the necessary stability and binding affinity.

[0097] In one embodiment, the present invention provides a composition comprising the compound as described above and a pharmaceutically acceptable excipient.

[0098] In another aspect, the present invention provides a method for treating or preventing a condition in a subject who requires such treatment, the method comprising administering a therapeutically effective amount of the above-described compound or a combination thereof.

[0099] In one embodiment, the condition is cancer.

[0100] In one embodiment, the disease is breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, head and / or neck cancer, or kidney, stomach, pancreatic cancer, brain cancer, hematologic malignancies such as lymphoma or leukemia.

[0101] In one embodiment, the condition is prostate cancer.

[0102] In another aspect, the present invention provides a method for performing radiographic imaging on a subject, the method comprising administering an effective amount of the compound or a combination thereof as described above.

[0103] Ideally, the radiopharmaceutical should be retained at the predetermined target site and not anywhere else, and any unbound radiopharmaceutical should be removed from the circulatory system. This will then allow for images with sufficient contrast, which in turn allows for more accurate analysis and diagnosis. For this purpose, the radiolabeled complex should possess physical and chemical properties such that the bound complex remains bound at the desired site for a sufficient time to allow for the necessary imaging; however, any unbound complex should be completely removed from the subject to prevent any background radiation caused by unbound complexes, which could interfere with and reduce the contrast of the acquired images.

[0104] The compounds of the present invention exhibit more favorable distribution characteristics in vivo. Figure 2 and Figure 4 This indicates that administering medication to tumor-bearing mice... 64 Cu-Sar-PSMA causes the compound to be localized in tumors rather than in any major organ or bloodstream. Minimizing the binding of the radiolabeled complex to other tissues reduces damage to healthy tissues. The complex shows relatively little accumulation in the bloodstream, which also demonstrates the high binding affinity of the Sar-PSMA complex to PSMA-expressing tumors.

[0105] Complexes with other known radiolabeled substances (see Figure 5 Compared to, for example 68 Ga-PSMA-617 177 Lu-PSMA-I&T and 68 Ga-DOTAGA-ffk(PSMA), 64 The Cu-Sar-PSMA complex exhibited greater tumor uptake. Furthermore, when considering renal uptake (meaning compound excretion), it showed better renal uptake compared to other compounds that exhibited similar binding to tumor sites. 64 Cu-Sar-PSMA showed significantly less renal uptake. Figure 6 Showing 64 Cu-Sar-PSMA complexes and various 68 A similarity comparison between Ga complexes indicates that using 64 Cu radionuclides can also be used in the same way... 68 The complex of Ga radionuclides binds as well as or is comparable to the use of 68 Complexes of Ga radionuclides bind more effectively to tumors. Furthermore, they are more compatible with... 68 Compared to complexes of Ga radionuclides. 64 The Cu-Sar-PSMA complex showed reduced renal uptake.

[0106] Subsequently, the inventors discovered that using Sar-PSMA ligands and 64 The Cu radionuclide exhibits better affinity for tumor sites and better clearance from the kidneys after administration. These advantages allow for better imaging results; the higher affinity for tumor sites provides images with better contrast because the radionuclide is primarily located at the target site, and unbound ligands are better removed from circulation, thus reducing background accumulation. This can then improve the diagnosis of tumors such as prostate cancer. The increased affinity for tumor-binding sites also indicates less diffusion of the radionuclide into other tissues, thus improving the quality of the acquired images. Furthermore, minimizing the diffusion of the radionuclide into non-tumor sites means that less radiolabeled complex is required for administration, and any harmful effects of the radiocomplex are localized, leaving healthy tissue unaffected.

[0107] The inventors have discovered that the compounds of this invention can be used as diagnostic and therapeutic compounds. This diagnostic and therapeutic approach allows the same compound to be used for both diagnosis and treatment of an indication, offering advantages over using one compound for diagnosis and another for treatment. Overall, this can improve the efficiency of diagnosis and treatment of specific diseases. This contrasts with conventional methods, where a ligand with a specific isotope may be suitable for diagnosing a disease, but the same combination of ligand and isotope may not be suitable for treating the disease. This then requires modification of the ligand, isotope, or both to treat the disease.

[0108] Figure 7 The study showed the accumulation of radiolabeled complexes in tumors and kidneys over time. (During administration...) 64 In the case of Cu-Sar-PSMA complexes, the ratio of complexes located in the tumor to complexes located in the tumor continued to increase over 24 hours. Figure 7 The tumor site was shown after 1 hour. 64 The Cu-Sar-PSMA complex has a greater absorption than other radiolabeled complexes, indicating that... 64 The Cu-Sar-PSMA complex is absorbed more rapidly than other comparative complexes. Furthermore, the ratio of the complex in the tumor to that in the kidney does not increase until 24 hours, indicating a favorable binding affinity for tumors. The resulting advantage is that, due to the longer binding time, the subject can be imaged during this period, resulting in better quality and higher contrast images. This, in turn, allows for more accurate disease diagnosis.

[0109] 64Cu-Sar-PSMA complexes exhibit higher binding affinity than other radiolabeled complexes, suggesting that the same complexes could be used for therapy. Since the therapeutic properties of the complexes depend on the delivery of the radionuclide to the target site, i.e., the tumor, good specificity and affinity for the tumor site are essential. This allows the radionuclide to deliver the radiotherapy effect to the desired site and prevent damage to other tissues. Furthermore, the ability of radiolabeled complexes to maintain binding to the target site allows for prolonged therapeutic effects, increasing the efficiency of the treatment method.

[0110] The inventors have now stated that, 64 The Cu-Sar-PSMA radiolabeled complex has sufficient binding specificity and affinity to the target PSMA site, making it possible to use the radiolabeled complex in therapeutically effective amounts for the treatment of prostate cancer.

[0111] The inventors have now also demonstrated that copper isotope-labeled Sar-PSMA complexes can be used for diagnostic and therapeutic purposes. For example, 64 Cu-Sar-PSMA radiolabeled complexes can be used for diagnostic and therapeutic purposes. 67 Cu-Sar-PSMA radiolabeled complexes can also be used for diagnostic and therapeutic purposes.

[0112] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological activity required for the compounds described above, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of compounds of formula (I) can be prepared from inorganic or organic acids. Examples of such inorganic acids are hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, alicyclic, aromatic, heterocyclic carboxylic acids, and sulfonic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkyl sulfonic acids, and aryl sulfonic acids. Further information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. In the case of solid reagents, those skilled in the art will understand that the compounds, reagents, and salts of the present invention can exist in different crystalline or polymorphic forms, all of which should be within the scope of the present invention and the specific chemical formulas.

[0113] The term "therapeutic effective dose" or "effective dose" is a dose sufficient to produce a beneficial or desired clinical outcome. An effective dose can be administered once or multiple times. An effective dose is generally sufficient to alleviate, improve, stabilize, reverse, slow, or delay the progression of a disease state. An effective dose for radiological imaging is generally sufficient to identify radionuclides in a subject's body.

[0114] Monitoring the location of radiolabeled material on a subject typically provides analysts with information about the location of the radiolabeled material, thereby informing the location of any material targeted by molecular recognition (e.g., cancerous tissue). The effective amount of the compounds of this invention will depend on many factors and necessarily involves a balance between the amount of radioactivity required to achieve the desired radioimaging effect and the general benefit of avoiding exposure of the subject (or its tissues or organs) to any unnecessary harmful radiation levels.

[0115] The treatment method of the present invention includes administering a compound of formula (I) that has been conjugated with a radionuclide. The compound of formula (I) is capable of delivering the radionuclide to the desired location in the body where its mode of action is required.

[0116] The attending physician can easily determine the effective dose of treatment using standard techniques and by observing results obtained in similar situations. Many factors must be considered when determining the effective dose, including but not limited to the animal's species, size, age, and overall health; the specific disease involved; the severity of the disease; the patient's response to treatment; the specific radiolabeled compound administered; the route of administration; the bioavailability of the formulation; the chosen dosing regimen; the use of other medications; and other relevant circumstances.

[0117] Furthermore, treatment regimens typically involve multiple cycles of radiation therapy, which continue until the symptoms are relieved. Again, the optimal number of cycles and the intervals between each treatment cycle will depend on many factors, such as the severity of the condition being treated, the health (or lack thereof) of the subject being treated, and their response to radiation therapy. Generally, the optimal dosage and treatment regimen can be readily determined by those skilled in the art using well-known techniques.

[0118] When using the compounds of the present invention, they can be administered in any form or manner to make the compound available for the desired application (imaging or radiotherapy). Those skilled in the art who prepare such formulations can readily select a suitable form and route of administration, depending on the specific characteristics of the compound chosen, the condition being treated, the stage of the condition being treated, and other relevant circumstances. We provide readers with information from Remington's Pharmaceutical Sciences, 19 th More information on the edition, Mack Publishing Co. (1995).

[0119] The compounds of the present invention can be administered alone or in combination with pharmaceutically acceptable carriers, diluents, or excipients in the form of pharmaceutical compositions. Although the compounds of the present invention are effective on their own, they are usually formulated and administered in the form of their pharmaceutically acceptable salts because these forms are generally more stable, easier to crystallize, and have increased solubility.

[0120] However, the compounds are typically used in the form of pharmaceutical compositions, which are formulated according to the desired route of administration. These compositions are prepared in a manner well known in the art.

[0121] In other embodiments, the present invention provides a pharmaceutical package or kit comprising one or more containers containing one or more components of the pharmaceutical composition of the present invention. In such a package or kit, at least one container containing a unit dose of the pharmaceutical agent may be found. Conveniently, in the kit, a single dose may be provided in a sterile vial so that a clinician can use the vial directly, wherein the vial will contain the desired amount and concentration of the compound and the radioactive nucleotide, which may be mixed prior to use. Associated with such containers may be various written materials, such as instructions for use, or notices in the form prescribed by government agencies regulating the production, use, or sale of pharmaceuticals, imaging agents, or biological products, reflecting approval for human administration by the agency of production, use, or sale.

[0122] The compounds of the present invention can be used or administered in conjunction with one or more additional drugs, said additional drugs being anticancer drugs and / or methods for treating said dysfunction / disease (e.g., surgery, radiation therapy). The components can be administered in the same formulation or in separate formulations. If administered in a separate formulation, the compounds of the present invention can be administered sequentially or simultaneously with other drugs.

[0123] In addition to being able to be administered in combination with one or more other drugs, including anticancer drugs, the compounds of the present invention can also be used for combination therapy. After this is done, the compounds are typically administered in combination with each other. Therefore, one or more compounds of the present invention can be administered simultaneously (as a combination formulation) or sequentially to achieve the desired effect. This is particularly desirable when each compound has different therapeutic properties, thus the combined action of the two drugs provides an improved therapeutic effect.

[0124] The pharmaceutical compositions of the present invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injections or dispersions prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or mediators include: water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Appropriate flowability can be maintained, for example, by using a coating material such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0125] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolic sorbic acid. Isotonic agents, such as sugars and sodium chloride, may also be required. Absorption of injectable drug forms can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0126] If needed, and for more efficient distribution, compounds can be incorporated into slow-release or targeted delivery systems, such as polymer matrices, liposomes, and microspheres.

[0127] Injectable formulations can be sterilized, for example, by filtering through a bacterial retention filter, or by incorporating a sterilizing agent into a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0128] In one embodiment, the present invention provides an aqueous composition of a compound of formula (I) or a salt thereof:

[0129]

[0130] Wherein: X is selected from H, OH, halogen, cyano, NO2, NH2, or optionally substituted C1-C. 12 Alkyl, optionally substituted amino, optionally substituted amide, and optionally substituted aryl groups;

[0131] Y is an optional C1-C. 12 Alkylene, wherein one or more methylene groups in the alkylene are optionally substituted with groups selected from amides, carbonyl groups, ureas, and thioureas;

[0132] m is 0, 1, or 2;

[0133] n is 0, 1, or 2;

[0134] Among them, the compound of formula (I) is complexed with Cu ions;

[0135] The composition also includes ethanol, gentianic acid or its salt, and sodium chloride.

[0136] The inventors have discovered that the use of gentian acid and ethanol in compositions of compounds of formula (I) having complex Cu ions can help prevent or minimize the radiodegradation of radiolabeled complexes.

[0137] In the above embodiments, the compositions of the present invention comprise ethanol as a component. The ethanol used in the composition may be anhydrous ethanol. Alternatively, the ethanol used in the composition may not have undergone a drying process and may be hydrated. The ethanol is preferably pharmaceutical grade ethanol. The presence of ethanol in the composition can help prevent radiodegradation of the radiolabeled complex of formula (I).

[0138] In the above embodiments, the compositions of the present invention further include sodium chloride as a component. The sodium chloride in the formulations of the present invention can be provided in the form of a salt solution. A salt solution is defined as an aqueous solution of sodium chloride. For example, physiological saline is defined as an aqueous solution of sodium chloride with a concentration of 0.9% (w / v). In one embodiment of the present invention, the sodium chloride in the formulation is provided by a salt solution.

[0139] In the above embodiments, the compositions of the present invention comprise gentianic acid or a pharmaceutically acceptable salt thereof and / or hydrate as a component. Gentianic acid is also known as 2,5-dihydroxybenzoic acid, 5-hydroxysalicylic acid, or hydroquinone carboxylic acid. Salts of gentianic acid may include sodium salts and sodium salt hydrates. Where applicable, any reference to gentianic acid may include a reference to its salts. The inventors have determined that gentianic acid or its salts in the compositions of the present invention can help prevent or minimize the radiodegradation of radiolabeled complexes of (I).

[0140] Oral solid dosage forms include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is combined with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or fillers, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, dosage forms may also include buffers.

[0141] Similar types of solid compositions can also be used as fillers in soft and hard filled gelatin capsules, using excipients such as lactose or lactose and high molecular weight polyethylene glycol.

[0142] Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. They may optionally contain light-blocking agents and may also have compositions that optionally release the active ingredient in a delayed manner only or preferably in certain portions of the intestine. Examples of encapsulation compositions that can be used include polymeric substances and waxes.

[0143] If needed and for more efficient distribution, compounds can be incorporated into slow-release or targeted delivery systems, such as polymer matrices, liposomes, and microspheres.

[0144] If appropriate, the active compound may also be in microencapsulated form with one or more of the above excipients.

[0145] Oral liquid dosage forms include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and savory tinctures. In addition to the active compound, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and esters and mixtures of fatty acid sorbitol.

[0146] In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and flavoring agents.

[0147] In addition to active compounds, suspensions may also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth gum and mixtures thereof.

[0148] As described above, the compounds of the embodiments can be used to treat and / or detect proliferative diseases. Examples of such proliferative diseases or conditions include cancers (including any metastatic cancers), psoriasis, and smooth muscle cell proliferative diseases such as restenosis. The compounds of the present invention can be used in particular to treat and / or detect tumors such as breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, head and / or neck cancer, or kidney, stomach, pancreatic cancer, and brain cancer, as well as hematologic malignancies such as lymphoma and leukemia. Additionally, the compounds of the present invention can be used to treat and / or detect proliferative diseases that are refractory to other anticancer drugs. They can also be used to treat and / or detect diseases of excessive proliferation, such as leukemia, psoriasis, and restenosis. In other embodiments, the compounds of the present invention can be used to treat and / or detect precancerous conditions or hyperplasia, including familial adenomatous polyposis, colonic adenomatous polyps, medullary dysplasia, endometrial dysplasia, endometrial hyperplasia with dysplasia, cervical dysplasia, vaginal intraepithelial hyperplasia, benign hyperplasia, laryngeal papilloma, actinic and actinic keratosis, seborrheic keratosis, and keratoacanthoma.

[0149] Synthesis of the compounds of the present invention

[0150] The reagents of various embodiments can be prepared using the reaction routes and synthetic schemes described below, employing techniques available in the art and readily available starting materials. The preparation of specific compounds of the embodiments is described in detail in the following examples; however, those skilled in the art will recognize that the described chemical reactions can be readily adapted to prepare many other reagents of various embodiments. For example, non-exemplary compounds can be successfully synthesized by modifications obvious to those skilled in the art, such as by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by conventional modifications to the reaction conditions. A list of suitable protecting groups in organic synthesis can be found in TW Greene's Protective Groups in Organic Synthesis, 3 rd Edition, John Wiley & Sons, 1991. Alternatively, other reactions disclosed herein or known in the art will be considered suitable for the preparation of other compounds of the various embodiments. Reagents for synthesizing compounds can be obtained or prepared according to techniques known in the art.

[0151] Example 1

[0152] Synthesis of Sar-PSMA

[0153] Scheme 1 outlines the synthetic route for Sar-PSMA1 compounds.

[0154] MeCOSar-D-Phe-D-Phe-Aoc-Lys-urea-Glu ligand 1, wherein...

[0155] Aoc=8-aminooctanoic acid (i.e., Sar-PSMA) was prepared via solid-phase peptide synthesis. The glutamate-urea-lysine binding motif was synthesized by reacting imidazole-activated and protected glutamate with a protected L-lysine residue immobilized on Wang resin. Using the standard Fmoc protocol, the peptide linker was attached to the ε-amine of lysine via solid-phase peptide synthesis. This was achieved by using (tBoc) 4-5 MeCOSar is coupled as a chelating agent by reacting with a urea-linking group protected on a solid support via a side chain. Sar-PSMA is cleaved from the resin while simultaneously deprotecting it (TFA / TIPS / H2O).

[0156] 64 Radiolabeling of Sar-PSMA by Cu

[0157] At room temperature, in an aqueous solution (0.1 M NH4OAc, pH 8, 1-10 nmol Sar-PSMA) 64 Cu II The Sar-PSMA ligand 1 was radiolabeled. 64Cu-Sar-PSMA was obtained by elution from a solid-phase column (Phenomenex Strata-X RP 60 mg / mL) with a yield (ndc) >94% and a radiochemical yield (7.95–21.9 GBq / μmol) >97%.

[0158]

[0159] Synthesis of activated Glu intermediate 3

[0160] Literature: Duspara, PA; Islam, MS; Lough, AJ; Batey, RA, Synthesis and reactivity of N-alkyl carbamoylimidazoles: development of N-methylcarbamoylimidazole as a methyl isocyanate equivalent. J Org Chem 2012, 77(22), 10362-8.

[0161] A 1:5 mixture of DMF / MeCN (50 mL) was added to a flask containing L-Bis(tBu)GluHCl2 (3.56 g, 12.04 mmol, 1.0 eq) and carbonyl diimidazole (2.15 g, 13.24 mmol, 1.1 eq). The reaction was stirred overnight at room temperature. After stirring, the solvent was removed under vacuum, and the remaining crude mixture was dissolved and purified by rapid chromatography (mobile phase: 7:3:1 petroleum solvent oil / chloroform / methanol, Rf: ~0.24, 30:1 silica / crude oil mass ratio) to give a white semi-crystalline powder product (2.25 g, 52.9% yield).

[0162] Fmoc-Lys(DDiv)-OH loading in Wang resin

[0163] A mixture of pre-activated Fmoc-Lys(DDiv)-OH (2.038 g, 3.55 mmol, 3.0 eq), HCTU (1.33 g, 3.5 mmol, 2.96 eq), DIPEA (1.24 mL, 7.09 mmol, 6 eq), and DMAP (43.3 mg, 0.355 mmol, 0.3 eq) in DMF was added to a 50 mL Falcon tube containing Wang resin (1.028 g, 1.15 mmol, 1.18 mmol). The resin was placed on a shaker for 2 hours to allow it to react. Then, acetic anhydride (223 μL, 2.36 mmol, 2 eq) and pyridine (190 μL, 2.36 mmol, 2 eq) were added to the reaction mixture to cover the remaining functional groups of the resin and the mixture was stirred for 30 minutes. The resin was then filtered and washed with DMF×3, DCM×3, MeOH×2, and Et2O×2, dried, and weighed to determine the final resin loading (0.759 mmol / g). The resin loading was determined as follows:

[0164]

[0165] Synthesis of resin-protected KuE4

[0166] Sar-PSMA ligands were synthesized from the KuE motif on the resin under standard Fmoc solid-phase peptide synthesis conditions.

[0167] Typical Fmoc deprotection experimental protocol

[0168] The resin-bound peptide 4 was treated three times with a DMF solution containing 20% ​​piperidine for 5 minutes each. The resin was then washed sequentially with DMF × 3 and DCM × 3.

[0169] TNBSA test for confirming coupling / deprotection reactions

[0170] Qualitative testing was performed on each coupling / deprotection step using the TNBSA (trinitrobenzenesulfonic acid) test. A small amount of resin (approximately 20 beads) was placed in an Eppendorf tube. TNBSA (10 μL of 5% DMF solution) and DIPEA (10 μL of 5% DMF solution) were added, and the mixture was stirred for 2 minutes. If no color change was observed in the resin, the test indicated the absence of a primary amine, while an orange color in the resin indicated the presence of a free primary amine.

[0171] After deprotection, activated Glu intermediate 3 (0.95 g, 2.69 mmol, 2.0 eq) and a DMF (5 mL) solution of DIPEA (240 μl, 1.38 mmol, 1.0 eq) were added to the resin. The resin was manually stirred for 24 hours and washed with DMF×3 and DCM×3. After confirmation by TNBSA coupling and cleavage / MS testing, the DDiv group was deprotected by treatment with DMF×3 containing 2% hydrazine hydrate to obtain 5.

[0172] General experimental protocol for coupling Fmoc-amino acids with resin

[0173] Fmoc amino acids were activated using a DMF solution containing HCTU (0.96 eq relative to AA) and DIPEA (2 eq relative to AA) (3 eq). After 5 minutes, the solution was added to the resin and stirred intermittently. After 20 minutes, the resin was filtered and washed sequentially with DMF×1, DCM×3, and DMF×3 to obtain 6.

[0174] Coupling MeCOSar to a PSMA ligand on the resin yielded 7

[0175] BocMeCOSar (0.464 g, 0.5 mmol, 1.2 eq) was activated using a DMF solution containing HCTU (0.207 g), HOBt (67.6 mg), and DIPEA (174 μL). After 5 minutes, the solution was added to resin 6 (0.4 mmol) with intermittent stirring. After 24 hours, the resin was filtered and washed sequentially with DMF×1, DCM×3, and DMF×3.

[0176] The resin pyrolysis experimental protocol was designed to produce 1

[0177] Resin 7 was washed several times with DCM and then transferred to a 50 mL Falcon tube. 95:2.5:2.5 TFA / TIPS / H2O (15 mL) was added to the resin and stirred at room temperature for 2 h. The resin was filtered and washed twice with 3 mL of TFA. The filtrate was collected, and the TFA was evaporated under N2 flow. Excess cold Et2O was added to precipitate the crude peptide, and the mixture was centrifuged. The Et2O was discarded, and this process was repeated three times. The precipitated crude peptide was dried, weighed, and purified by preparative HPLC.

[0178] HPLC purification

[0179] The crude peptide (818 mg) was dissolved in 6.4 mL of H₂O containing 22% MeCN and subjected to RP-HPLC (24% isocratic elution for 60 min) on a C18 21.2 × 150 mm semi-preparative column packed with a 5 μl 100A AXIA resin, at a flow rate of 5 mL / min. The fraction containing the product was separated and lyophilized to give product 1 (58.5 mg, 16.1% based on the resin used) as a fluffy white powder.

[0180] Radioactive labeling

[0181] Divide into equal parts 64 Cu II (30-200 MBq, 0.1 M NH4OAc, pH 6) added to: Sar-PSMA1 (5 μg, 4.3 × 10⁻⁶) -3 The reaction mixture was prepared in MilliQ water (μmol), NH4OAc (pH 5, final concentration: 0.05 M), ethanol (10%), and gentic acid in MilliQ water (final concentration: 0.056%), and the pH was measured (pH: 5). The reaction was incubated at room temperature for 30 minutes. After 30 minutes, aliquots were analyzed by RP-HPLC to determine the product with >98% radiochemical purity. 64 Cu-Sar-PSMA.

[0182] plasma stability

[0183] Add to 200 μL of fresh human plasma at 37°C 64 A saline solution of Cu-Sar-PSMA (100 μL, ~8.8 MBq, <10% EtOH) was prepared and the mixture was incubated at 37 °C for 24 hours. After 24 hours, cold acetonitrile (600 μL) was added. The precipitated serum proteins were separated by centrifugation (13000 rpm), and 300 μL of supernatant was removed and concentrated by evaporation. The solution was diluted with water (100 μL) and the product was analyzed by RP-HPLC.

[0184] Tumor imaging in LNCaP tumor-bearing mice

[0185] The study was conducted in NSG (NOD SCID Gamma) mice carrying LNCaP tumors at 1 hour, 6 hours, and 22 hours post-injection. 64 Biodistribution of CuSarPSMA in vivo. At 1 hour, 64 CuSarPSMA showed the highest uptake in the kidneys, resulting in a low tumor / kidney ratio. Figure 5 However, biodistribution data showed that at later time points, renal clearance was rapid, with moderate tumor preservation. Despite moderate preservation within the tumor... 64 Cu-Sar-PSMA, however, showed significant contrast due to rapid clearance from circulation and minimal background accumulation after 6 hours. Furthermore, low uptake in other PSMA-positive tissues (lung, spleen) allowed for… 64 Cu-Sar-PSMA has a high tumor-to-background ratio.

[0186] Example 2

[0187] Synthesis of CoSar(PSMA)2

[0188] Synthesis of active glutamate intermediates

[0189] Literature: Duspara, PA; Islam, MS; Lough, AJ; Batey, RA, Synthesis and reactivity of N-alkyl carbamoylimidazoles: development of N-methylcarbamoylimidazole as a methyl isocyanate equivalent. J Org Chem 2012, 77(22), 10362-8.

[0190] A 1:5 mixture of DMF / MeCN (50 mL) was added to a flask containing L-Bis(tbu)GluHCl (3.56 g, 12.04 mmol, 1.0 eq) and carbonyl diimidazole (2.15 g, 13.24 mmol, 1.1 eq). The reaction was stirred overnight at room temperature. After stirring, the solvent was removed under vacuum, and the remaining crude mixture was dissolved and purified by rapid chromatography (mobile phase: 7:3:1 petroleum solvent oil / chloroform / methanol, RF: ~0.24, 30:1 silica / crude oil mass ratio) to give a white semi-crystalline powder product (2.25 g, 52.9% yield).

[0191] Synthetic protected urea

[0192]

[0193] A solution of activated Glu intermediate (3.71 g, 10.5 mmol, 1.0 eq) and DIPEA (1.83 mL, 10.5 mmol, 1 eq) in DCM (30 mL) was added to a flask containing H-Lys(Fmoc)-OtBu·HCl (4.84 g, 10.5 mmol, 1.0 eq) and stirred overnight at room temperature. The reaction mixture was washed with water × 3 and brine, dried over MgSO4, loaded onto an 80 g Reveleris HP Silica column, and purified using a Biotage Isolera automated rapid chromatography system (mobile phase: 70:30:2.5 petroleum solvent oil / chloroform / methanol). Analysis by TLC (mobile phase: 7:3:1 petroleum solvent / chloroform / methanol, RF: ~0.30), combined, and the solvent was removed under vacuum to give 5.06 g of a yellow oily product (68%).

[0194] ESIMS + [M+H + m / z 710.386 (experiment), m / z 710.401 (calculation).

[0195] Fmoc urea cleavage

[0196]

[0197] A 20% solution of diethylamine in MeCN (100 mL) was added to a flask containing protected urea (5.06 g, 7.13 mmol, 1 eq), and the reaction mixture was stirred at room temperature for 7 hours. Aliquots were periodically taken out for MS analysis to determine the reaction completion. After 7 hours, the diethylamine and MeCN were reduced to 5 mL under vacuum, and an additional 50 mL of MeCN was added to azeotropically evaporate the diethylamine three times using a rotary evaporator. The reaction mixture was again reduced to 5 mL, 200 mL of a 50 / 50 water / MeCN solution was added, and the reaction mixture was lyophilized. Due to impurities resulting from incomplete removal of the dibenzofullerene moiety, the final product was usable without further purification. (Estimated purity: 70%) ESIMS + [M+H + m / z (experiment), m / z 488.333 (calculation).

[0198] Synthesis of 8-Aoc-ff connectors on resin

[0199] The 8-Aoc-ff linker was prepared using the following Fmoc experimental protocol.

[0200] Typical Fmoc deprotection experimental protocol

[0201] The resin-bound peptides were treated with a 20% piperidine DMF solution for 15 minutes × 3. The resin was then washed sequentially with DMF × 3 and DCM × 3.

[0202] Experimental protocol for loading Fmoc-aminooctanoic acid (Fmoc-8-Aoc-OH) onto 2-CT resin

[0203] 80 mL of DCM containing Fmoc-8-Aoc-OH (5.00 g, 13.1 mmol, 1.75 eq) and DIPEA (2 eq relative to AA) was added to 2-CT resin (7.50 g, 1 mmol / g, 1 eq) and stirred. After 2 hours, 8 mL of MeOH was added, and the resin was stirred for another 30 minutes. The resin was filtered and washed successively with DCM×3, DMF×3, DCM×3, MeOH×2, and Et2O×2 and dried. The resin loading was calculated using the following equation, yielding 0.628 mmol / g (total 6.16 mmol).

[0204]

[0205] Experimental scheme for coupling Fmoc-D-Phe-OH with resin

[0206] Fmoc-D-Phe-OH (2 eq) was activated using an NMP solution containing HATU (0.96 eq relative to AA) and DIPEA (2 eq relative to AA). After 5 minutes, the solution was added to the resin and stirred for at least 12 hours. The resin was filtered and washed sequentially with DMF×1, DCM×3, and DMF×3. The coupling was then repeated as described above for at least 12 hours, followed by filtration and washing sequentially with DMF×1, DCM×3, and DMF×3.

[0207] Resin pyrolysis scheme

[0208] The resin was washed several times with DCM and then transferred to two 50 mL Falcon tubes. A 75 mL solution of 5% TFA in DCM was added to the resin and stirred at room temperature for 2 h. The resin was filtered and washed twice with 15 mL of 5% TFA in DCM. The filtrate was collected and the solvent was reduced under N2 flow. The crude peptide was redissolved in 50 / 50 water / MeCN and lyophilized to obtain the crude peptide. The crude peptide was ready for use without further purification.

[0209] Trifluoroacetamide protection

[0210]

[0211] To a flask containing crude peptide linker (1.35 g, or 2.98 mmol, 1 eq if pure), add 10 mL of MeOH solution containing ethyl trifluoroacetate (0.532 mL, 4.47 mmol, 1.5 eq) and DIPEA (1.04 mL, 5.96 mmol, 2 eq). Stir the reaction mixture overnight at room temperature, monitoring complete conversion of the starting material by MS and analytical HPLC. Reduce MeOH under vacuum, and add EtOAc / 0.01 M HCl to the reaction mixture. Separate the organic layer, wash with 0.01 M HCl × 3 and brine, dry over MgSO4, remove solvent under vacuum, and dry to obtain crude product (0.985 g), which is ready for use without further purification. Orbitrap-MS + [M+H + m / z 550.253 (experimental), m / z 550.252 (calculated), [2M+H + m / z 1099.498 (experiment), m / z 1099.497 (calculation).

[0212] Urea connector coupling

[0213]

[0214] A DMF solution (5 mL) of HATU (0.608 g, 1.6 mmol, 0.89 eq) and DIPEA (0.56 mL, 3.2 mmol, 1.79 eq) was added to a flask containing a crude TFA-protected connector (0.985 g, if pure, 1.79 mmol, 1 eq) and stirred at room temperature. After 5 minutes, deprotected urea (approximately 1.5 mmol) was added, and the reaction was monitored overnight by MS and analytical HPLC. After 24 hours, an aqueous solution of K₂CO₃ was added, and the reaction was heated to 60 °C overnight, with monitoring for the removal of the trifluoroacetamide protecting group. The reaction was diluted with water (150 mL) and extracted with Et₂O × 3. The ether fractions were combined, washed with water, 0.01 M HCl × 3, and brine, and dried over MgSO₄. The aqueous layer was acidified with 0.1 M HCl, extracted with Et₂O, washed with water, 0.01 M HCl, and brine, and dried over MgSO₄. The ether layers were combined, the solvent was removed under vacuum, and the crude product was dissolved in 80% MeCN in water and analyzed by RP-HPLC (60-77% within 35 minutes) on a Phenomenex Luna 5μm precipitator. Purification was performed on a C18 21.2×250 mm semi-preparative column at a flow rate of 8 mL / min. The fraction containing the product was collected and lyophilized to obtain a fluffy white powder (49.6 mg, 98%+ purity). Orbitrap-MS + [M+H +m / z 923.586 (experimental), [M+2H + m / z 462.297 (calculated), [M+2H + m / z 462.297 (experiment), m / z 462.296 (calculation).

[0215] (tBoc) 4-5 Synthesis of CoSar-Plus

[0216] (tBoc) 4-5 CoSar-Plus can be prepared according to the steps outlined in Ma, MT; Cooper, MS; Paul, RL; Shaw, KP; Karas, JA; Scanlon, D.; White, JM; Blower, PJ; Donnelly, PS. Inorg Chem 2011, 50, 6701.

[0217] Synthesis of CoSar-(PSMA)2

[0218]

[0219] Add (tBoc) to the Eppendorf tube. 4-5 COSar-Plus (25.3 mg, 0.027 mmol, 1 eq), HATU (20.4 mg, 0.054 mmol, 2 eq), and DIPEA (18.7 μL, 0.107 mmol, 4 eq) in NMP solution (500 μL) were added. The mixture was shaken for 10 minutes to activate it, and then added to a 2 mL microwave-safe vial containing pure PSMA connector-urea (49.6 mg, 0.054 mmol, 2 eq) in NMP solution (400 μL). The reaction mixture was stirred in a microwave reactor at 60 °C for 10 minutes, cooled, and analyzed by MS and analytical HPLC to show complete consumption of the starting material. 1.8 mL of 72% MeCN aqueous solution was added to the reaction mixture, and the mixture was analyzed by RP-HPLC (60-90% within 60 minutes) on a Phenomenex Luna 5 μL filter. Purification was performed at a rate of 8 mL / min on a C182 1.2 × 250 mm semi-preparative column. The fraction containing the product was collected and lyophilized to give a fluffy white powder of the protected product (14.0 mg, 5.08 μmol, 18.9% yield). The protected product was dissolved in 95% TFA in water overnight, diluted with 50 / 50 MeCN / water, and lyophilized to give a white powder of tri-trifluoroacetate monohydrate (12.1 mg, 5.08 μmol) (room temperature: 10.3 min, purity 96.4%). Orbitrap-MS: [M+2H+] m / z 1009.065 (experimental), m / z 1009.066 (calculated); [M+3H+] m / z 673.046 (experimental), m / z 673.046 (calculated).

[0220] Radioactive labeling

[0221] Will 64 Cu II An aliquot of sample (100-400 MBq, 0.01 M HCl) was added to a solution containing 0.1 M NH4OAc, pH 5.5 (500 μL), ethanol (100 μL), MilliQ water (300 μL), and gentian acid (1.2 mg, 10 mg / mL in MilliQ water), and the pH was measured (pH: 5). CoSar-(PSMA)2 (20 μg, 8.4 × 10⁻⁶) was added to this solution. -3 The concentration of the sample was μmol (1 mg / mL in MilliQ), and the reaction was incubated at room temperature for 30 minutes. After 30 minutes, the aliquots were analyzed by RP-HPLC to determine the product with a radiochemical purity >97%. 64 Cu-CoSar(PSMA)2 (rt: 13.9min).

[0222] Throughout the specification and the subsequent claims, unless the context otherwise requires, the word “comprising” and variations such as “comprising” and “including” shall be understood to imply inclusion of the said integer or step or a set of integers or steps, but not to exclude any other integer or step or a set of integers or steps.

[0223] In this specification, any reference to any prior publication (or information derived therefrom) or any known issues is not, and should not be construed as, an acknowledgment or endorsement of any part of the common general knowledge of such prior publication (or information) or the field covered by this specification, or any implied meaning of any kind.

Claims

1. A method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof: in: X is selected from H, OH, halogen, cyano, NO2, NH2, or optionally substituted C1-C. 12 Alkyl, optionally substituted amino, optionally substituted amide, and optionally substituted aryl groups; Y is an optional C1-C. 12 Alkylene, wherein one or more methylene groups in the alkylene group may be further optionally substituted with groups selected from amides, carbonyl groups, ureas, and thioureas; m is 0, 1, or 2; and n is 0, 1, or 2; The method includes the step of coupling a compound of formula (A) or a protected form thereof with a compound of formula (B) or a protected form thereof: The compound of formula (I) has one of the following structures:

2. The method of claim 1, further comprising one or more deprotection steps.

3. The method of claim 1, further comprising a purification step by HPLC.

4. The method according to claim 3, wherein, The purification step performed by HPLC is carried out under reversed-phase conditions.

5. The method according to claim 1, wherein, The compound of formula (A) or its protected form thereof is produced by subjecting a compound having the structure of formula (C) or its protected form thereof to one or more peptide coupling steps.

6. The method according to claim 1, wherein, The coupling step is carried out under solid-phase or solution-phase peptide synthesis conditions.

7. The method according to claim 1, wherein, The coupling step is performed under solid-phase peptide synthesis conditions.

8. A method for preparing a metal-ligand complex, wherein the metal-ligand complex comprises: Ligands having the structure of a compound of formula (I), and copper ions; The method includes the step of combining a solution containing a compound of formula (I) with a solution containing copper ions in a buffer solution; in: X is selected from H, OH, halogen, cyano, NO2, NH2, or optionally substituted C1-C. 12 Alkyl, optionally substituted amino, optionally substituted amide, and optionally substituted aryl groups; Y is an optional C1-C. 12 Alkylene, wherein one or more methylene groups in the alkylene group may be further optionally substituted with groups selected from amides, carbonyl groups, ureas, and thioureas; m is 0, 1, or 2; and n is 0, 1, or 2; The compound of formula (I) has one of the following structures:

9. The method according to claim 8, wherein, Copper ions are selected from 60 Cu、 61 Cu、 62 Cu、 64 Cu or 67 The radioactive isotope of Cu.

10. The method according to claim 8 or 9, wherein, The solution containing copper ions is a hydrochloric acid solution.

11. The method according to claim 8, wherein, The solution containing copper ions further comprises ethanol and gentianic acid or its salt.

12. A kit for preparing metal-ligand complexes, the metal-ligand complexes comprising: a ligand having the structure of a compound of formula (I), and copper ions; in: X is selected from H, OH, halogen, cyano, NO2, NH2, or optionally substituted C1-C. 12 Alkyl, optionally substituted amino, optionally substituted amide, and optionally substituted aryl groups; Y is an optional C1-C. 12 Alkylene, wherein one or more methylene groups in the alkylene group may be further optionally substituted with groups selected from amides, carbonyl groups, ureas, and thioureas; m is 0, 1, or 2; and n is 0, 1, or 2; The kit includes: i) A container containing a compound of formula (I) or a pharmaceutically acceptable salt thereof; ii) Containers containing copper ion solutions; and iii) Description of the preparation of the metal-ligand complex; The compound of formula (I) has one of the following structures:

13. The kit according to claim 12, wherein, Copper ions are selected from 60 Cu、 61 Cu、 62 Cu、 64 Cu or 67 The radioactive isotope of Cu.

14. The kit according to claim 12, wherein, The description includes adding a solution of the compound of formula (I) to a copper ion solution.

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