Metal / radiometal-labeled PSMA inhibitors for PSMA-targeted imaging and radiotherapy

Abstract

To provide a compound useful for imaging, diagnosing, or treating one or more prostate-specific membrane antigen (PSMA) tumors or cells.SOLUTION: The following compound or a pharmaceutically acceptable salt thereof is used. In the formula, Q is H, or a protecting group; X1 and X2 are NH or O; R1 is C1-C4 alkyl substituted with a halogen-substituted aryl group; R2 and R4 are H or C1-C4 alkyl; each R3 is H, C1-C6 alkyl or C6-C12 aryl; W is O or S; Y is, for example, -NH-; L is a linker; Ch is a chelating moiety that can comprise one or more non-radioactive metals or radioactive metals.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 989,428, filed May 6, 2014, and No. 62 / 117,603, filed Feb. 18, 2015, each of which is hereby incorporated by reference in its entirety.

[0002] Research or Development Sponsored by the Federal Government This invention was made with government support under NIH grants K25CA148901 - 01A1 and U54CA1346751. The government has certain rights in this invention.

Background Art

[0003] Prostate-specific membrane antigen (PSMA) is being recognized as a highly promising target for imaging and therapy of prostate and other types of cancer (Ghosh and Heston, 2004; Milowsky et al., 2007; Olson et al., 2007). PSMA is significantly overexpressed in prostate cancer and metastases, especially with regard to hormone-resistant types (Ghosh and Heston, 2004; Milowsky et al., 2007). PSMA is also known to be expressed in most solid tumors and tumor vasculature (Haffner et al., 2012; Haffner et al., 2009). Imaging of PSMA can provide insights into androgen signaling (Evans et al., 2011) and response to taxane therapy (Hillier et al., 2011). Previous studies have performed PSMA-targeted radionuclide imaging using functionalized cysteine-glutamate or lysine-glutamate ureas in experimental models of prostate cancer (Schulke et al., 2003; Mease et al., 2013; Banerjee et al., 2010) and in clinics (Cho et al., 2012; Kulkarni et al., 2014; Zechmann et al., 2014). Large molecular fragments, such as radioactive metals ( 99m Tc, 68 Ga, 111 In, 86 Y, 203 Pb, 64To attach Cu complexes (Banerjee, Pullambhatla, Shallal, et al., 2011; Banerjee, Pullambhatla, Byun, et al., 2011; Banerjee et al., 2008) and nanoparticles (Chandran et al., 2008; Kam et al., 2012), PSMA target binding was maintained by placing a long linker between the large molecule and the target urea. Without wishing to be bound by a particular theory, PSMA was considered suitable as a biomarker for MR molecular imaging because the ligand binding site is extracellular and the receptor concentration per cell is high (about 3.2 μM / cell volume).

[0004] MR imaging is a clinically relevant non-invasive diagnostic tool for performing high-resolution anatomical and functional imaging. Molecular MR imaging enables visualization of biological markers in vivo (Artemov, Mori, Okollie et al., 2003; Artemov, Mori, Ravi, Bhujwalla, et al., 2003; Konda et al., 2001; Lanza et al., 2004; Huang, et al., 2013). Gd(III)-based contrast agents are widely accepted by clinicians because they are easy to administer and have a T 1This is because positive contrast can be enhanced. Although the design of contrast agents with high relaxation has advanced, sensitivity remains a limiting factor for molecular MR imaging. For use in molecular imaging applications (specifically, imaging of receptor or protein expression), Gd(III)-based contrast agents rarely exceed the detection limit (Artemov, Mori, Okollie et al., 2003; Artemov, Mori, Ravi, Bhujwalla, et al., 2003; Konda et al., 2001; Lanza et al., 2004; Huang, et al., 2013). Using signal amplification methods, MR may become a highly sensitive modality for molecular imaging that complements radionuclide-based techniques (Aime et al., 2004; Major et al., 2009; Song et al., 2008; Artemov, 2003). Even if the sensitivity of the target substance can be improved by amplification methods, the pharmacokinetic profile of the substance may change significantly due to the shift from simple low molecular weight compounds to larger complexes (Artemov, Mori, Okollie et al., 2003; Artemov, Mori, Ravi, Bhujwalla, et al., 2003; Konda et al., 2001; Lanza et al., 2004; Huang, et al., 2013). Sherry et al. addressed the sensitivity issue by creating a contrast agent with extremely high binding affinity (K d ) that can minimize the amount of substance required for detection by MR (Hanaoka et al., 2008; De Leon-Rodriguez et al., 2010). As one solution to enable receptor imaging based on MR, it has been proposed to combine a receptor-specific high-affinity ligand with a multimeric Gd(III) for detection (Wu et al.2012).

[0005] Examples of that approach include high longitudinal relaxation rate (r 1)Molecular imaging of VEGFR2 by preparing multimeric Gd-dendrons with values is included (De Leon-Rodriguez et al., 2010). For other multimeric substances, improved r 1 values have been reported, which is because the magnetic field of MR imaging is stronger in both experimental and clinical settings (Mastarone 2011). By optimizing the relaxation at high magnetic fields, advantages such as a larger signal-to-noise and contrast-to-noise ratio (SNR / CNR) are obtained, along with the benefits of higher spatial resolution and shorter scan times (Rooney 2007). The combination of these concepts, namely using a highly sensitive multimeric contrast agent with a high-affinity targeting moiety, provides a logical basis for investigating targeted MR imaging of cells and tissues expressing prostate-specific membrane antigen (PSMA).

[0006] Furthermore, it is thought that urea-based substances may also be used for the radiotherapy of lesions containing PSMA using radionuclides. In fact, 131 I]MIP1095 ((S)-2-(3-((S)-1-carboxy-5-(3-(4- 131 I]iodophenyl)ureido)phenyl)ureido)pentaanedioic acid) (Zechmann et al., 2014) and 177Clinical studies using that approach with the Lu-labeled PSMA targeting agent (Kulkarni et al., 2014) are underway for the treatment of castration-resistant prostate cancer. This approach will be similar to radioimmunotherapy (RIT), which has proven to be extremely effective in the treatment of lymphoma with two commercially available products routinely incorporated into clinical practice. However, because radiolabeled antibodies are used for imaging, RIT is associated with difficulties, including long circulation times, unpredictable biological effects, and in some cases the need for pretargeting methods. In addition, antibodies may be less able to access tumors than pharmacologically manipulable low molecular weight substances. Therefore, there is still a need for low molecular weight compounds with high binding affinity for PSMA for tumor imaging and radiotherapy.

[0007] Positron-emitting radionuclide 86 Y (half-life [t 1 / 2 = 14.74 hours, β + = 33%, E β+ = 664 keV) is an isotope attractive for molecular imaging (Nayak and Brechbiel, 2011). Yttrium-86 can be easily prepared on a small medical cyclotron using the 86 Sr(p,n) 86 Y nuclear reaction (Yoo et al., 2005). High-energy β - emitter 90 Y (t 1 / 2 = 64.06 hours, β - = 72%, E β- = 2.288 MeV) is widely used in internal radiotherapy (Witzig et al., 2003; Bodei et al., 2004), so 86 Y is 90 ideal for dosimetry estimation of 86 Y-labeled radiotherapy (Helisch et al., 2004). 90 Antibodies and peptides radiolabeled with 90The absorbed dose can be accurately estimated for Y (Nayak and Brechbiel, 2011; Palm et al., 2003). 177 Lu has 90 a shorter β-particle range than Y (t 1 / 2 = 6.7 days, E β- = 0.5 MeV), but because it has similar chelation properties, 90 not only those radiolabeled with Y, but also potential 177 Y is proposed as a suitable imaging alternative for investigating Lu-based radiotherapy. Similar rationale has also been applied to substances for peptide receptor radionuclide therapy of neuroendocrine targets (Chen et al., 2012). Using a similar approach, potential matched-pair imaging radioisotopes suitable for SPECT imaging 86 Pb (half-life, 51.9 hours, E 203 = 279-keV γ-rays, 81%) can be used for the therapeutic radionuclide β- Pb for α-particle therapy (Chappell, et al. 2000; Yong, et al. 2011; Yong, et al. 2012; Yong, et al. 2013). 212 The decay scheme of Pb includes 212 Bi and emits α-particles, two β-particles and some γ-rays upon decay. α-particle emitters are particularly attractive for targeted radiotherapy due to their high linear energy transfer properties such as high local density ionization (resulting in irreparable DNA double-strand breaks and cytotoxicity independent of tissue oxygen content or dose rate) (McDevitt, et al, 1998). 212 Both Pb and 212 Bi are promising future α-particle emitting sources with radiochemical properties that have been well discussed regarding antibody binding, and are also 212 easily obtained with a Ra generator. 224

[0008] ​Radiolabeled carbamate-based PSMA inhibitors that also exhibit high binding affinity for PSMA in vitro have also been developed and, when radiolabeled with the positron emitter F-18, have shown high uptake in PSMA-positive mouse tumor xenografts and rapid clearance from normal tissues. The pharmacokinetic profiles of compounds in this class are favorable (i.e., low non-specific binding, not metabolized in vivo, and reasonable tumor residence times), and imaging studies have extended to molecular radiotherapy. Additionally, high binding affinity for PSMA can be maintained by coupling carbamate-based inhibitors to metal chelating substances using linker functionality, similar to urea-based metal / radiometal-based substances. Therefore, metal or radiometal conjugate carbamate scaffolds can also be utilized for imaging and treatment of PSMA-expressing cells and tissues. SUMMARY OF THE INVENTION

[0009] In some aspects, the subject matter of the present disclosure is a compound of formula (I):

[0010]

Chemical Formula

[0011] or a pharmaceutically acceptable salt thereof, wherein Z is tetrazole or CO 2 Q, where Q is H or a protecting group, X 1 and X 2 are each independently NH or O, a is an integer selected from the group consisting of 1, 2, 3, and 4, c is an integer selected from the group consisting of 0, 1, 2, 3, and 4, each R 1 , R 2 and R 4 are independently H or C 1 -C 4 alkyl, and each R 3 is independently H, C 1 -C 6 alkyl or C 2 -C 12Aryl, W is independently O or S, Y is -NH- and may be present or absent, L is a linker, and the linker is selected from the group consisting of

[0012] [Chemical formula]

[0013] selected from the group consisting of, where m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8, and each R 5 is independently H or each R 6 is independently H or C 1 -C 6 alkyl -COOR 6 where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, p is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8, and Ch is a chelating moiety that may contain one or more metals or radioactive metals.

[0014] In another aspect, the subject matter of the present disclosure provides a method for imaging or treating one or more prostate-specific membrane antigen (PSMA) tumors or cells, the method comprising contacting one or more tumors or cells with an effective amount of a compound of formula (I) to form an image.

[0015] Particular aspects of the subject matter of the present disclosure that have been addressed in whole or in part have been described above, and other aspects will become apparent as the description proceeds in conjunction with the accompanying examples and drawings, which will be described in detail later. Accordingly, the subject matter of the present disclosure has been generally described, and reference is now made to the accompanying drawings. The drawings are not necessarily to scale. Brief Description of the Drawings

[0016]

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DETAILED DESCRIPTION OF THE INVENTION

[0017] The subject matter of the present disclosure will now be described in more detail with reference to the accompanying examples and drawings. These illustrate only some, not all, embodiments of the subject matter of the present disclosure. The subject matter of the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, those skilled in the art to which the present disclosure pertains will envision many modifications and other embodiments of the subject matter of the present disclosure that are within the merits of the description and the teachings presented in the relevant examples and drawings. Accordingly, it is to be understood that the subject matter of the present disclosure is not limited to the particular embodiments disclosed and that modifications and other embodiments are included within the scope of the appended claims.

[0018] I. Metal / Radioactive Metal-Labeled PSMA Inhibitors for PSMA-Targeted Imaging and Radiotherapy Magnetic resonance (MR) imaging is advantageous because it can simultaneously obtain anatomical, functional, and molecular information. MR molecular imaging can combine this established and widely used clinical modality and its high spatial resolution with molecular profiling in vivo. However, because the sensitivity of MR is inherently low, high local concentrations of biological targets are required to generate recognizable MR contrast.

[0019] Without wishing to be bound by any particular theory, PSMA was considered to be a good target for MR molecular imaging agents. This is because the target concentration per cell is high (about 3 μM / cell volume) and the ligand binding site is extracellular. The approach of the present disclosure aims to improve the binding affinity (minimum K d ) of contrast agents for specific molecular or cellular targets so that less of the agent is required for MR detection. Accordingly, in the approach of the present disclosure, a high-binding-affinity receptor-specific ligand is combined with a multimeric Gd(III) species as one possible solution for MR-based molecular imaging.

[0020] Previously, radioactive metal-based PET ( 64 Cu) and SPECT ( 111 In and 99m Tc) imaging has been successfully performed in mice using radiolabeled urea-based PSMA inhibitors. A three-molecule strategy has been developed that includes (i) a PSMA targeting moiety, (ii) a linker for pharmacological modulation, and (iii) a chelating agent that enables attachment of a radionuclide. This strategy is for PET imaging and serves as a model for radiotherapy using the corresponding 90 Y-labeled substance and includes a 86 Y-labeled DOTA conjugate. Since DOTA is a strong chelating agent for many metals, the same DOTA conjugate can be used with other radionuclides for radiotherapy, such as Lu-177, Ac-225, Bi-213, Bi-212, Pb-212, Cu-67, and Sc-47. In the subject matter of the present disclosure, the same urea linker construct was used, and the number of Gd chelates was increased (monomeric, dimeric, and trimeric Gd) to systematically investigate the potential of PSMA-based MR imaging of PCa by optimizing the relaxation time measurement behavior or MR sensitivity as a high-field contrast agent and its binding affinity.

[0021] A. Compounds of formula (I) In some embodiments, the subject matter of the present disclosure is formula (I):

[0022]

Chemical formula

[0023] provides a compound of or a pharmaceutically acceptable salt thereof, wherein Z is tetrazole or CO 2 Q, Q is H or a protecting group, X 1 and X 2 are each independently NH or O, a is an integer selected from the group consisting of 1, 2, 3, and 4, c is an integer selected from the group consisting of 0, 1, 2, 3, and 4, each R 1 , R 2 and R4 is independently H or C 1 -C 4 is alkyl, and each R 3 is independently H, C 1 -C 6 is alkyl or C 2 -C 12 is aryl, W is independently O or S, Y is -NH-, may be present or absent, and L is a linker selected from the group consisting of

[0024] [Chemical formula]

[0025] wherein m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8, and each R 5 is independently H or each R 6 is independently H or C 1 -C 6 is alkyl, -COOR 6 wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, p is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8, and Ch is a chelating moiety that may contain one or more metals or radioactive metals.

[0026] Formula (I) does not include the compounds disclosed in the pamphlets of International Publication No. WO 2009 / 002529, International Publication No. WO 2010 / 108125, and International Publication No. WO 2013 / 082338. In particular, the following compounds are specifically excluded from the composition claims in the present application.

[0027] [Chemical formula]

[0028] In a further specific embodiment, the chelating moiety is

[0029] [Chemical formula]

[0030] selected from the group consisting of, wherein q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8.

[0031] In a further particular embodiment, the compound of formula (I) is

[0032]

Chemical formula

[0033] (wherein x is selected from the group consisting of 2 and 3, and M is a metal or a radioactive metal), or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the metal is selected from the group consisting of Gd, Lu, Ac, Bi, Pb, Cu, In, Sc and Y. In a particular embodiment, the metal or radioactive metal is selected from the group consisting of Gd-157, Lu-177, Ac-225, Bi-212, Bi-213, Pb-203 / Pb-212, Cu-67, In-111, Sc-44 / Sc-47 and Y-90. In a further particular embodiment, for MRI applications, the non-radioactive metal is Gd-157 (stable isotope). For radiotherapy applications, the radioactive metal is selected from the group consisting of Lu-177, Ac-225, Bi-203, Pb-210, Cu-67, In-111, Sc-47 and Y-90. For PET imaging, the radioactive metal is selected from the group consisting of Y-86 and Sc-44. Also, for SPECT applications, the radioactive metal is selected from the group consisting of Lu-177 and In-111.

[0035] Method of using a compound of formula (I) for MR imaging and / or treatment of PSMA-expressing tumors or cells In some embodiments, the subject matter of the present disclosure provides a method for imaging or treating one or more prostate-specific membrane antigen (PSMA) tumors or cells, the method comprising contacting one or more tumors or cells with an effective amount of a compound of formula (I) to form an image, wherein the compound of formula (I) is

[0036]

Chemical formula

[0037] (wherein Z is tetrazole or CO 2 Q, where Q is H or a protecting group, X 1 and X 2 are each independently NH or O, a is an integer selected from the group consisting of 1, 2, 3, and 4, c is an integer selected from the group consisting of 0, 1, 2, 3, and 4, each R 1 、R 2 、R 3 and R 4 is independently H or C 1 -C 4 alkyl, W is independently O or S, Y is -NH- and may be present or absent, L is a linker selected from the group consisting of

[0038]

Chemical formula

[0039] wherein m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8, each R 5 is independently H or each R 6 is independently H or C 1 -C 6 alkyl of -COOR 6 and n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, p is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8, and Ch is a chelating moiety that may contain one or more metals or radioactive metals) or a pharmaceutically acceptable salt thereof.

[0040] "Contact" means any action that results in at least one compound containing the imaging agent of the subject matter of the present disclosure coming into physical contact with at least one PSMA-expressing tumor or cell. Contact can include exposing the cell or tumor to the compound in an amount sufficient to result in contact between the at least one compound and the at least one cell or tumor. This method can be performed in vitro or ex vivo by introducing, preferably mixing, the compound and the cell or tumor in a controlled environment (e.g., a culture dish or test tube). This method can be performed in vivo, in which case contact means exposing at least one cell or tumor in a subject to at least one compound of the subject matter of the present disclosure, e.g., any suitable route of administration of the compound to the subject. In the subject matter of the present disclosure, contact can include introducing, exposing, etc., the compound at a location distant from the cell to be contacted, and bringing the compound into contact with the cell or tumor by natural (e.g., diffusion) or artificial (e.g., agitation) movement of the body functions or body fluids of the subject. In some embodiments, the tumor or cell is found in vitro, in vivo, or ex vivo.

[0041] "Forming an image" means generating an image of a cell, tissue, tumor, part of the body, etc. using a magnetic resonance (MR)-based method (a magnet that generates a detectable signal by polarizing and exciting hydrogen nuclei in water molecules in tissue).

[0042] Formula (I) does not include the compounds disclosed in the pamphlets of International Publication No. WO 2009 / 002529, International Publication No. WO 2010 / 108125, and International Publication No. WO 2013 / 082338. In particular, the following compounds are specifically excluded from the imaging claims in the present application.

[0043]

Chemical Formula

[0044] In further specific embodiments, the chelating moiety is

[0045]

Chem.

[0046] selected from the group consisting of, wherein q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8.

[0047] In a further particular embodiment, the compound is

[0048]

Chem.

[0049] (wherein x is selected from the group consisting of 2 and 3, and M is a metal or a radioactive metal), or a pharmaceutically acceptable salt thereof.

[0050] In some embodiments, the metal is selected from the group consisting of Gd, Lu, Ac, Bi, Pb, Cu, In, Sc, and Y. In certain embodiments, the metal or radioactive metal is selected from the group consisting of Gd-157, Lu-177, Ac-225, Bi-203, Pb-210, Cu-67, In-111, 44Sc- / 47Sc, and Y-90. In further particular embodiments, for MRI applications, the non-radioactive metal is Gd-157 (stable isotope). For radiotherapy applications, the radioactive metal is selected from the group consisting of Lu-177, Ac-225, Bi-203, Pb-210, Cu-67, In-111, Sc-47, and Y-90. For PET imaging, the radioactive metal is selected from the group consisting of Y-86 and Sc-44. Also, for SPECT applications, the radioactive metal is selected from the group consisting of Lu-177 and In-111.

[0051] In certain embodiments, the one or more PSMA-expressing tumors or cells are selected from the group consisting of prostate tumors or cells, metastatic prostate tumors or cells, lung tumors or cells, kidney tumors or cells, glioblastoma, pancreatic tumors or cells, bladder tumors or cells, sarcoma, melanoma, breast tumors or cells, colon tumors or cells, germ cells, pheochromocytoma, esophageal tumors or cells, stomach tumors or cells, and combinations thereof. In further particular embodiments, the one or more PSMA-expressing tumors or cells are prostate tumors or cells.

[0052] In some embodiments, the one or more PSMA-expressing tumors or cells are in vitro, in vivo, or ex vivo. In certain embodiments, the one or more PSMA-expressing tumors or cells are present in a subject.

[0053] In some embodiments, the tumor or cell is found in a subject. The subject to be treated by the methods of the present disclosure in many of its embodiments is preferably a human subject. However, it should be understood that the methods described herein are effective for all vertebrate species included in the term "subject". Thus, a "subject" can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or for prophylactic treatment to prevent the onset of a condition or disease, or an animal (non-human) subject for medical, veterinary or developmental purposes. Suitable animal subjects include mammals, including but not limited to primates such as humans, monkeys, apes, etc., bovines such as cattle, bulls, etc., ovines such as sheep, etc., caprines such as goats, etc., porcines such as piglets, grown pigs, etc., equines such as horses, donkeys, zebras, etc., felines (including wild cats and domestic cats), canines (including dogs), lagomorphs (including rabbits, wild rabbits, etc.), rodents (including mice, rats, etc.). The animal can be a transgenic animal. In some embodiments, the subject is a human and includes, but is not limited to, fetal, neonatal, infant, juvenile and adult subjects. Furthermore, a "subject" can include a patient (affected animal) suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient (affected animal)" are used interchangeably herein. In some embodiments, the subject is a human. In other embodiments, the subject is non-human.

[0054] In some embodiments, a subject is administered a detectably effective amount of an imaging agent of the methods of the present disclosure. In the subject matter of the present disclosure, a "detectably effective amount" of an imaging agent is defined as an amount sufficient to obtain an acceptable image using equipment available for clinical use. A detectably effective amount of an imaging agent can be administered by more than one injection. This detectably effective amount of the imaging agent can vary depending on factors such as the degree of sensitivity of the individual, the age, sex and weight of the individual, the idiosyncratic response of the individual, factors such as dosimetry, and factors related to the equipment and film. Optimization of such factors is well within the skill level of one of ordinary skill in the art.

[0055] It is preferred that the compounds of the subject matter of the present disclosure are rapidly excreted from the body tissues. Typically, the compounds of the subject matter of the present disclosure are excreted from the body in less than about 24 hours. More preferably, the compounds of the subject matter of the present disclosure are excreted from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes or 60 minutes.

[0056] In some embodiments, the methods of the present disclosure include clearance of a compound containing an imaging agent from a tumor or cells of a subject. At least one advantage of the methods of the present disclosure is that, in some embodiments, clearance of a compound containing an imaging agent from the kidneys occurs more rapidly than clearance from the tumor of the subject.

[0057] In some embodiments, because the methods of the present disclosure use compounds that are stable in vivo, substantially all, for example, amounts greater than about 50%, 60%, 70%, 80% or more preferably greater than 90% of the injected compound are not metabolized in the body before excretion. In other embodiments, the compounds containing the imaging agent are stable in vivo.

[0058] C. Definitions i. Chemical definitions While those skilled in the art will be able to fully understand the following terms regarding the compounds of formula (I), the following definitions are provided to facilitate the description of the subject matter of the present disclosure. These definitions are intended to supplement and exemplify definitions that will be apparent to those skilled in the art upon consideration of the present disclosure, and are not intended to exclude them.

[0059] Regardless of whether the term "optionally" precedes it, the terms "substituted" and "substituent" as used herein refer to the ability, as will be understood by those skilled in the art, to change one functional group for another as long as the valence of all atoms is maintained. When two or more substituents selected from a specified group can be substituted at two or more positions in a given structure, the substituents can be the same or different at each position. Substituents can be further substituted (e.g., an aryl group substituent can have another substituent, e.g., another aryl group, near it, and this aryl group can be further substituted at one or more positions, e.g., with fluorine).

[0060] When specifying a substituent or a linking group by its conventional chemical formula written from left to right, these groups are equivalent and include chemically identical substituents that result from writing the structure from right to left, e.g., CH 2 O- is equivalent to -OCH 2 - and -C(=O)O- is equivalent to -OC(=O)-, -OC(=O)NR- is equivalent to -NRC(=O)O-, etc.

[0061] In this specification, when a bond comes to the side of an internal substituent (e.g., -NRC(O)-), the order of the atoms is fixed, the direction of the group is not reversed, and it is inserted into the structure in the presented direction. In other words, -NRC(O)- is different from -C(O)NR-. In this specification, the term C(O) (e.g., -NRC(O)-) is used to denote a carbonyl (C=O) group, and oxygen is bonded to carbon by a double bond.

[0062] When the expression "independently selected" is used, the substituents referred to (e.g., the groups R 1 、R 2 such R groups, or variables such as "m" and "n") can be the same or different. For example, R1 and R 2 both may be substituted alkyl, or R 1 may be hydrogen, and R 2 may be substituted alkyl, etc.

[0063] The words indicating the singular (a, an, a(n)) used when referring to a substituent group in this specification mean at least one. For example, when a compound is substituted with "an" alkyl or aryl, the compound is optionally substituted with at least one alkyl and / or at least one aryl. Further, when a moiety is substituted with an R substituent, this group may be referred to as "R-substituted". When a moiety is substituted with R, the moiety is substituted with at least one R substituent, and each R substituent is optionally different.

[0064] Unless otherwise specified, a named "R" or group generally has a structure recognized in the art corresponding to the group having that name. For the sake of illustration, the above specific representative "R" groups are defined below.

[0065] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art. Thus, when a group can be substituted with one or more of a number of substituents, such substitution is selected so that compounds will be obtained that will be understood by those skilled in the art to follow the principles of chemical bonding and not be inherently unstable and / or likely to become unstable under ambient conditions (e.g., aqueous, neutral, and some known physiological conditions). For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule via a ring heteroatom in accordance with the principles of chemical bonding known to those skilled in the art to avoid inherently unstable compounds.

[0066] As used herein, the term "hydrocarbon" refers to all chemical groups containing hydrogen and carbon. Hydrocarbons can be substituted or unsubstituted. As will be appreciated by those skilled in the art, when substitution occurs, all valences must be satisfied. Hydrocarbons can be unsaturated, saturated, branched, unbranched, cyclic, polycyclic or heterocyclic. Examples of hydrocarbons will be defined in detail later, but include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.

[0067] The term "alkyl", as used by itself or as part of another substituent, unless otherwise specified, means a straight-chain (i.e., unbranched) or branched-chain, acyclic or cyclic hydrocarbon group or a combination thereof, which can be fully saturated, monovalent or polyvalent unsaturated, can include divalent and polyvalent groups, and has a specified number of carbon atoms (i.e., C 1 -C 10 means 1 to 10 carbons). In certain embodiments, the term "alkyl" refers to a C 1-20 comprising linear (i.e., "straight-chain"), branched or cyclic saturated or at least partially, optionally fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicals derived from a hydrocarbon moiety containing 1 to 20 carbon atoms by removal of one hydrogen atom.

[0068] Representative saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl and homologs and isomers thereof.

[0069] "Branched" refers to an alkyl group in which a lower alkyl group such as methyl, ethyl or propyl is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having from 1 to about 8 carbon atoms, for example 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms (i.e., C 1-8 alkyl). "Higher alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. In certain embodiments, "alkyl" particularly refers to C 1-8 linear alkyl. In other embodiments, "alkyl" particularly refers to C 1-8 branched-chain alkyl.

[0070] In certain embodiments, the alkyl group is a C 1 -C 6 alkyl group or a C 1 -C 4 alkyl group. The term "C 1 -C 6 alkyl" as used herein means a fully saturated straight-chain, branched or cyclic C 1 -C 6 hydrocarbon and their hybrids, such as (cycloalkyl)alkyl. Examples of C 1 -C 6 alkyl substituents include methyl (Me), ethyl (Et), propyl (n-propyl (n-Pr, n Pr), iso-propyl (i-Pr, 1 Pr) and cyclopropyl (c-Pr, 0 Pr)), butyl (n-butyl (n-Bu, n Bu), iso-butyl (i-Bu, 1 Bu), sec-butyl (s-Bu, s Bu), tert-butyl (t-Bu, 1 Bu) or cyclobutyl (c-Bu, 0 Bu)), etc.

[0071] The alkyl group may optionally be substituted with one or more alkyl group substituents which may be the same or different (a "substituted alkyl"). The term "alkyl group substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo and cycloalkyl. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkyl chain, and this nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl") or aryl.

[0072] Accordingly, as used herein, the term "substituted alkyl" includes an alkyl group as defined herein, and one or more atoms or functional groups of the alkyl group are substituted with another atom or functional group including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate and mercapto.

[0073] The term "heteroalkyl", alone or in combination with another term, means a stable straight-chain, branched-chain or cyclic hydrocarbon group or combinations thereof consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, unless otherwise defined, and the nitrogen, phosphorus and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, P, S and Si may be substituted at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2-S-CH 2 -CH 3 、 -CH 2 -CH 25 -S(O)-CH 3 、 -CH 2 -CH 2 -S(O) 2 -CH 3 、 -CH=CH-O-CH 3 、 -Si(CH 3 ) 3 、 -CH 2 -CH=N-OCH 3 、 -CH=CH-N(CH 3 )- CH 3 、 O-CH 3 、 -O-CH 2 -CH 3 and -CN are included. Up to two or three heteroatoms can be consecutive, for example -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 .

[0074] As described above, the heteroalkyl groups herein include groups that are bonded to the rest of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', -NR'R", -OR', -SR and / or -SO 2 R'. When described as "heteroalkyl" and followed by a specific heteroalkyl group, such as -NR'R etc., it can be seen that the terms heteroalkyl and -NR'R" are neither redundant nor mutually exclusive. Rather, it becomes clear by describing the specific heteroalkyl group. Therefore, in this specification, the term "heteroalkyl" should not be construed as excluding specific heteroalkyl groups, such as -NR'R".

[0075] In the term "(cycloalkyl)alkyl", cycloalkyl and alkyl are as defined above, and the point of attachment is on the alkyl group. This term includes, but is not limited to, cyclopropylmethyl, cyclopentylmethyl and cyclohexylmethyl. The alkyl group can be substituted or unsubstituted.

[0076] "Cyclic" and "cycloalkyl" refer to non-aromatic monocyclic or polycyclic systems of about 3 to about 10 carbon atoms, such as 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The cycloalkyl group can optionally be partially unsaturated. Also, the cycloalkyl group can optionally be substituted with alkyl group substituents, oxo and / or alkylene as defined herein. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms can optionally be inserted along the cyclic alkyl chain, and the nitrogen substituents are hydrogen, alkyl, substituted alkyl, aryl or substituted aryl, so that a heterocyclic group is obtained. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0077] Polycyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphan and noradamantyl, as well as fused ring systems such as dihydro- and tetrahydronaphthalene, etc.

[0078] The term "cycloheteroalkyl" or "heterocycloalkyl" refers to a non-aromatic ring system, an unsaturated or partially unsaturated ring system, such as a 3- to 10-membered substituted or unsubstituted cycloalkyl ring system, which contains one or more heteroatoms selected from the group consisting of the same or different nitrogen (N), oxygen (O), sulfur (S), phosphorus (P) and silicon (Si), and can optionally contain one or more double bonds.

[0079] The cycloheteroalkyl ring may optionally be fused or otherwise attached to another cycloheteroalkyl ring and / or a non-aromatic hydrocarbon ring. The heterocyclic ring includes those having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, and the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized. In certain embodiments, the term "heterocyclic ring" refers to a non-aromatic 5-, 6- or 7-membered ring or polycyclic group in which at least one ring atom is a heteroatom selected from O, S, and N (the nitrogen and sulfur heteroatoms may optionally be oxidized), including, but not limited to, bicyclic or tricyclic groups, including a fused 6-membered ring having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatoms may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative cycloheteroalkyl ring systems include, but are not limited to, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiazadinanyl, tetrahydrofuranyl, etc.

[0080] The terms "cycloalkyl" and "heterocycloalkyl", whether by themselves or in combination with another term, unless otherwise defined, each represent a cyclic version of "alkyl" and "heteroalkyl", respectively. In addition, in the case of heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms "cycloalkylene" and "heterocycloalkylene" refer to divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0081] As used herein, the term "cycloalkylalkyl" refers to a cycloalkyl group as defined above and is attached to the parent molecular moiety via an alkyl group as also defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0082] An unsaturated alkyl group is one having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkyl group limited to hydrocarbon groups is termed "homoalkyl".

[0083] In particular, as used herein, the term "alkenyl" refers to a C having at least one carbon-carbon double bond by the removal of one hydrogen atom 1-20Refers to a monovalent group derived from a linear or branched hydrocarbon moiety. Examples of alkenyl groups include ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, and butadienyl.

[0084] The term "cycloalkenyl" as used herein refers to a cyclic hydrocarbon having at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatriene, and cyclooctenyl.

[0085] The term "alkynyl" as used herein refers to a linear or branched C of a specified number of carbon atoms having at least one carbon-carbon triple bond 1-20 Refers to a monovalent group derived from a hydrocarbon. Examples of "alkynyl" include ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, heptynyl, and arylenyl groups, etc.

[0086] The term "alkylene" refers to a linear or branched divalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, either by itself or as part of another substituent. The alkylene group can be linear, branched or cyclic. Also, the alkylene group can optionally be unsaturated and / or substituted with one or more "alkyl group substituents". One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms can optionally be inserted along the alkylene group (also referred to herein as "alkylaminoalkyl"), and the nitrogen substituents are alkyl as described above. Exemplary alkylene groups include methylene (-CH 2 -); ethylene (-CH 2 -CH 2 -); propylene (-(CH 2 ) 3 -); cyclohexylene (-C 6 H10 -); -CH=CH-CH=CH-; -CH=CH-CH 2 -; -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH=CHCH 2 -, -CH 2 CsCCH 2 -, -CH 2 CH 2 CH(CH 2 CH 2 CH 3 )CH 2 -, -(CH 2 ) q -N(R)-(CH 2 ) r -(where each of q and r is independently an integer from 0 to about 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and R is hydrogen or lower alkyl); methylenedioxy (-O-CH 2 -O-) and ethylenedioxy (-O-(CH 2 ) 2 -O-) are included. The alkylene group can have from about 2 to about 3 carbon atoms and can further have from 6 to 20 carbons. Typically, the alkyl (or alkylene) group has from 1 to 24 carbon atoms, and groups having 10 or fewer carbon atoms are some embodiments of the present disclosure. "Lower alkyl" or "lower alkylene" is a shorter-chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0087] The term "heteroalkylene" means a divalent group derived from heteroalkyl, either by itself or as part of another substituent, for example -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2- but not limited to these. Regarding the heteroalkylene group, the heteroatom(s) may occupy one or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, etc.). Further, in the case of alkylene and heteroalkylene linking groups, the orientation of the linking group is not indicated by the direction in which the formula of the linking group is written. For example, the formula: -C(O)OR'- represents both -C(O)OR'- and -R'OC(O)-.

[0088] The term "aryl", unless otherwise defined, means an aromatic hydrocarbon substituent which can be monocyclic or fused or covalently linked polycyclic (e.g., 1 to 3 rings).

[0089] The term "heteroaryl" refers to an aryl group (or ring) having 1 to 4 heteroatoms selected from N, O, and S (in the case of polycyclic, in separate rings), where nitrogen and sulfur atoms are optionally oxidized and nitrogen atoms are optionally quaternized. The heteroaryl group can be attached to the remainder of the molecule via a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, indazolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the aryl and heteroaryl ring systems listed above are selected from the group of acceptable substituents described below. The terms "arylene" and "heteroarylene" refer to the divalent forms of aryl and heteroaryl, respectively.

[0090] For the sake of brevity, when the term "aryl" is used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl), it includes both aryl and heteroaryl rings as defined above. Thus, the terms "arylalkyl" and "heteroarylalkyl" are taken to include groups in which an aryl or heteroaryl group is bonded to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, etc.) that includes an alkyl group (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.) in which a carbon atom (e.g., a methylene group) is substituted, for example, by an oxygen atom. However, the term "haloaryl" as used herein is taken to include only aryl substituted with one or more halogens within its scope.

[0091] When heteroalkyl, heterocycloalkyl or heteroaryl contains a specific number of members (e.g., "3 to 7 members"), the term "member" refers to a carbon or heteroatom.

[0092] As used herein, the term "alkylaryl" includes an alkyl group as defined above substituted with an aryl group as defined above. The aryl group may be bonded at any position on the alkyl group. The term C 4 -C 16 Alkylaryl includes alkylaryl groups having a total of 4 to 16 carbon atoms, counting together the carbon atoms on the alkyl and aryl groups. Examples of alkylaryl groups include, but are not limited to, benzyl (phenylmethyl), phenylethyl, and naphthylmethyl. The alkylaryl group may be substituted or unsubstituted. The substituents are not counted towards the total number of atoms in the alkylaryl group unless all atoms in the substituent are larger than those in the alkylaryl group.

[0093] Furthermore, in the formulas herein:

[0094]

Chemical formula

[0095] The structure generally represented by is a ring structure, for example, but not limited to, aliphatic and / or aromatic cyclic compounds such as 3-carbon, 4-carbon, 5-carbon, 6-carbon, 7-carbon, etc. (including saturated ring structures, partially saturated ring structures, and unsaturated ring structures), and includes an R group as a substituent. The R group can be present or absent. When present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and the number of R groups are determined by the value of the variable "n", and n is generally an integer having a value from 0 to the number of substitutable carbon atoms on the ring. When there are two or more, each R group is substituted on an available carbon on the ring structure rather than on another R group. For example, for the above structure where n is from 0 to 2,

[0096]

Chemical formula

[0097] includes compound groups and the like including but not limited to these.

[0098] The dashed line representing the bond in the ring structure indicates that the bond can be present or absent in the ring. That is, the dashed line representing the bond in the ring structure indicates that the ring structure is selected from the group consisting of saturated ring structures, partially saturated ring structures, and unsaturated ring structures.

[0099] A substituent having a broken bond as in the example shown below means that the substituent is directly bonded to the molecule at the position where it is shown. An additional methylene (CH 2 ) group is not implied. The symbol

[0100] TIFF2025081334000020.tif8170

[0101] indicates the bonding point of the part to the rest of the molecule.

[0102]

Chemical formula

[0103] Substituents having two broken bonds, such as those in the examples shown below, mean that the orientation of the atoms is as described from left to right and should be inserted into the molecule in the direction shown in the figure. Unless otherwise specified, additional methylene (CH 2 ) groups are not implied.

[0104]

Chemical formula

[0105] When an atom named in an aromatic or heteroaromatic ring is defined as "absent", the atom so named is replaced by a direct bond.

[0106] Each of the above terms (e.g., "alkyl", "heteroalkyl", "cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "phosphonate" and "sulfonate" and their divalent derivatives") is intended to include both substituted and unsubstituted forms of the indicated group. Examples of optional substituents for each type of group are listed below.

[0107] Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl monovalent and divalent derivative groups (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) are not limited to the following, but include -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO 2 R’, -C(O)NR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R’”, -NR”C(O)OR’, -NR-C(NR’R”)=NR’”, -S(O)R’, -S(O) 2 R’, -S(O) 2 NR’R”, -NRSO 2 R’, -CN and -NO 2It can be one or more of a variety of groups of numbers from 0 to (2m'+l) selected from, where m' is the total number of carbon atoms of such groups. R', R", R''', and R'''' are each independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. As used herein, an "alkoxy" group is alkyl bonded to the remainder of the molecule through a divalent oxygen. If the compounds of the present disclosure contain, for example, two or more R groups, each R group is independently selected and is the same as each R', R", R''', and R'''' group when two or more are present. When R' and R" are bonded to the same nitrogen atom, they can be combined to form a 4-, 5-, 6- or 7-membered ring with the nitrogen atom. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above considerations regarding substituents, one of ordinary skill in the art will understand that the term "alkyl" includes groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , etc.).

[0108] Similar to the substituents described above for alkyl groups, examples of substituents for aryl and heteroaryl groups (and divalent derivatives thereof) are diverse and are, for example, halogens, -OR', -NR'R", -SR', -halogen, -SiR'R"R''', -OC(O)R', -C(O)R', -CO 2 R', -C(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R''', -NR"C(O)OR', -NR-C(NR'R"R''')=NR"", -NR-C(NR'R")=NR'''-S(O)R', -S(O)2 R’, -S(O) 2 NR’R”, -NRSO 2 R’, -CN and -NO 2 , -R’, -N 3 , -CH(Ph) 2 , fluoro(C 1 -C 4 ) alkoxo as well as fluoro(C 1 -C 4 ) alkyl, and are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When the compounds of the present disclosure contain, for example, two or more R groups, each R group is independently selected and is the same as each R’, R”, R’” and R”” group when two or more are present.

[0109] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR’) q -U-, where T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be substituted with a substituent of the formula -A-(CH 2 ) r -B-, where A and B are independently -CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR’- or a single bond, and r is an integer from 1 to 4.

[0110] One of the single bonds of the newly formed ring in this way may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be substituted with a substituent of the formula -(CRR’) s -X’-(C”R’”) d -, where s and d are independently integers from 0 to 3, and X’ is -O-, -NR’-, -S-, -S(O)-, -S(O) 2 - or -S(O)2 It is NR’-. The substituents R, R’, R” and R’” can independently be selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0111] As used herein, the term “acyl” refers to an organic acid group in which the —OH of the carboxyl group is replaced by another substituent and has the general formula RC(═O)—, and R is an alkyl, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic or aromatic heterocyclic group as defined herein. Thus, the term “acyl” specifically includes arylacyl groups, such as acetylfuran and phenacyl groups. Specific examples of acyl groups include acetyl and benzoyl.

[0112] The terms “alkoxyl” or “alkoxy” are used interchangeably herein and also refer to a saturated (i.e., alkyl—O—) or unsaturated (i.e., alkenyl—O— and alkynyl—O—) group bonded to the parent molecular moiety through an oxygen atom, and the terms “alkyl”, “alkenyl” and “alkynyl” are as described above, C 1-20 It may include an inclusive linear, branched or cyclic saturated or unsaturated oxo-hydrocarbon chain, such as methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl and n-pentoxyl, neopentoxyl, n-hexoxyl, etc.

[0113] The term “alkoxyalkyl” as used herein refers to an alkyl—O—alkyl ether, such as a methoxyethyl or ethoxymethyl group.

[0114] “Aryloxyl” refers to an aryl—O— group in which the aryl group is as described above including substituted aryl. The term “aryloxyl” as used herein can refer to phenoxyl or hexyloxyl and alkyl, substituted alkyl, halo or alkoxyl-substituted phenoxyl or hexyloxy.

[0115] "Aralkyl" means an aryl-alkyl group in which aryl and alkyl are as described above and which includes substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.

[0116] "Aralkyloxy" means an aralkyl-O-group in which the aralkyl group is as described above. An exemplary aralkyloxy group is benzyloxy.

[0117] "Alkoxycarbonyl" means an alkyl-O-CO-group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl and t-butyloxycarbonyl.

[0118] "Aryloxycarbonyl" means an aryl-O-CO-group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0119] "Aralkoxycarbonyl" means an aralkyl-O-CO-group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0120] "Carbamoyl" means an amide group of the formula -CONH 2 "Alkylcarbamoyl" means an R'RN-CO-group, where one of R and R' is hydrogen and the other of R and R' is alkyl and / or substituted alkyl as described above. "Dialkylcarbamoyl" means an R'RN-CO-group, where each of R and R' is independently alkyl and / or substituted alkyl as described above.

[0121] The term "carbonyldioxy" as used herein refers to a carbonate group of the formula: -O-CO-OR.

[0122] "Acyl oxy" means an acyl-O-group, where acyl is as described above.

[0123] The term "amino" refers to -NH 2 group, and also refers to nitrogen-containing groups known in the art derived from ammonia by substitution of one or more hydrogen radicals by organic radicals. For example, the terms "acylamino" and "alkylamino" refer to specific N-substituted organic radicals having acyl and alkyl substituents, respectively.

[0124] As used herein, "aminoalkyl" refers to an amino group covalently bonded to an alkylene linker. In particular, the terms alkylamino, dialkylamino and trialkylamino as used herein refer to one, two or three alkyl groups, respectively, bonded to the parent molecular moiety via a nitrogen atom as defined above. The term alkylamino refers to a group having the structure -NHR', where R' is an alkyl group as defined above. The term dialkylamino refers to a group having the structure -NR'R", where R' and R" are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure -NR'R"R"', where R', R" and R'" are each independently selected from the group consisting of alkyl groups. In addition, R', R" and / or R'" may together optionally be -(CH 2 ) k - and k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino and propylamino.

[0125] The amino group is -NR'R", where R' and R" are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0126] The terms "alkylthioether" and "thioalkoxyl" refer to saturated (i.e., alkyl-S-) or unsaturated (i.e., alkenyl-S- and alkynyl-S-) groups attached to the parent molecular moiety via a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0127] "Acylamino" refers to an acyl-NH- group, where acyl is as described above. "Aroylamino" refers to an aroyl-NH- group, where aroyl is as described above.

[0128] The term "carbonyl" refers to a -(C=O)- group.

[0129] The term "carboxyl" refers to a -COOH group. Such a group is also referred to herein as the "carboxylic acid" moiety.

[0130] The terms "halo", "halide", or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C 1 -C 4 )alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0131] The term "hydroxyl" refers to an -OH group.

[0132] The term "hydroxyalkyl" refers to an alkyl group substituted with an -OH group.

[0133] The term "mercapto" refers to an -SH group.

[0134] The term "oxo" as used herein means an oxygen atom double-bonded to a carbon atom or another element.

[0135] The term "nitro" refers to the -NO 2 group.

[0136] The term "thio" refers to the aforementioned compounds in which a carbon or oxygen atom is replaced by a sulfur atom.

[0137] The term "sulfate" refers to the -SO 4 group.

[0138] The term thiohydroxyl or thiol in this specification refers to the group of the formula: -SH.

[0139] The term ureido refers to the urea group of the formula: -NH-CO-NH 2 of.

[0140] Unless otherwise explicitly defined, the "substituents" in this specification include functional groups selected from one or more of the following moieties, which are defined herein as follows. (A) -OH, -NH 2 , -SH, -CN, -CF 3 , -NO 2 , oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. The groups of B are (i) oxo, -OH, -NH 2 , -SH, -CN, -CF 3 , -NO 2 , halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl substituted with at least one substituent selected from (a) oxo, -OH, -NH 2 , -SH, -CN, -CF3 , -NO 2 , halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl substituted with at least one substituent selected from, and the group of (b) is oxo, -OH, -NH 2 , -SH, -CN, -CF 3 , -NO 2 , halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl and unsubstituted heteroaryl, and is substituted with at least one substituent selected therefrom.

[0141] As used herein, "lower substitution" or "lower substituent" means a group selected from all of the substituents described above for "substituent", and each substituted or unsubstituted alkyl is substituted or unsubstituted C 1 -C 8 alkyl, each substituted or unsubstituted heteroalkyl is substituted or unsubstituted 2- to 8-membered heteroalkyl, and each substituted or unsubstituted cycloalkyl is substituted or unsubstituted C 5 -C 7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is substituted or unsubstituted 5- to 7-membered heterocycloalkyl.

[0142] As used herein, "size-limited substitution" or "size-limited substituent" means a group selected from all of the substituents described above for "substituent", and each substituted or unsubstituted alkyl is substituted or unsubstituted C 1 -C 20 alkyl, each substituted or unsubstituted heteroalkyl is substituted or unsubstituted 2- to 20-membered heteroalkyl, and each substituted or unsubstituted cycloalkyl is substituted or unsubstituted C 4 -C 8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is substituted or unsubstituted 4- to 8-membered heterocycloalkyl.

[0143] Throughout the specification and claims, any chemical formula or name includes all tautomers, homologs, and optical and stereoisomers, as well as racemic mixtures in which such isomers and mixtures exist.

[0144] It will be apparent to those skilled in the art that certain compounds of the present disclosure may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the present disclosure. As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are rapidly convertible from one isomeric form to the other.

[0145] Unless otherwise specified, the structures described herein are intended to include all stereochemical forms of the structure, i.e., R and S configurations for each chiral center. Accordingly, single stereoisomers of the compounds of the present invention, as well as enantiomeric and diastereomeric mixtures, are within the scope of the present disclosure.

[0146] Certain compounds of the present disclosure have asymmetric carbon atoms (optical or chiral centers) or double bonds. Enantiomers, racemic compounds, diastereomers, tautomers, geometric isomers, stereoisomers that can be defined as (R)- or (S)- from the perspective of absolute stereochemistry or (D)- or (L)- in the case of amino acids, and individual isomers are included within the scope of the present disclosure. The compounds of the present disclosure do not include those known in the art to be too unstable to be synthesized and / or isolated. The present disclosure is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or can be resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other geometrically asymmetric centers, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0147] How to prepare the optically active form is well known in the art and includes, for example, resolution of a racemate (racemic compound), asymmetric synthesis, or synthesis from an optically active starting material. Resolution of a racemic compound can be achieved, for example, by crystallization in the presence of a resolving agent or by conventional methods such as chromatography using, for example, a chiral HPLC column. Many geometric isomers such as olefins, C=N double bonds, etc. may also be present in the compounds described herein, and all such stable isomers are contemplated in the subject matter of the present disclosure. The cis and trans geometric isomers of the compounds of the subject matter of the present disclosure are described and can be isolated as a mixture of isomers or in separated isomeric forms. Unless a specific stereochemistry or isomeric form is specifically indicated, all chiral (enantiomeric and diastereomeric) and racemic forms, as well as all geometric isomeric forms of a structure, are intended.

[0148] The compounds described herein may have one or more charged atoms. For example, the compounds can be zwitterionic but overall neutral. Other embodiments may have one or more charged groups depending on pH and other factors. In these embodiments, the compounds can associate with appropriate counterions. How to prepare salts or exchange counterions is well known in the art. Generally, such salts can be prepared by reacting these compounds in the free acid form with a stoichiometric amount of an appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.) or by reacting these compounds in the free base form with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent or a mixture of the two. The counterions can be changed by ion exchange techniques such as ion exchange chromatography. Unless a specific counterion or salt is specifically indicated, all zwitterions, salts, and counterions are intended. In certain embodiments, the salt or counterion can be pharmaceutically acceptable for administration to a subject. Pharmaceutically acceptable salts are described below.

[0149] As used herein, a "protecting group" is a chemical substituent that can be selectively removed by an easily available reagent that does not attack the regenerated or other functional groups in a molecule. Suitable protecting groups are known in the art and are continuously being developed. Suitable protecting groups can be found, for example, in Wutz et al. ("Greene’s Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). Protecting groups for protecting carboxyl groups as described in Wutz et al. (pp. 533 - 643) are used in certain embodiments. In some embodiments, the protecting group is removable by acid treatment. Specific examples of protecting groups include, but are not limited to, benzyl, p-methoxybenzyl (PMB), tertiary butyl ( t Bu), methoxymethyl (MOM), methoxyethoxymethyl (MEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), benzyloxymethyl (BOM), trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), and triphenylmethyl (trityl, Tr). One of ordinary skill in the art can recognize the appropriate situations where a protecting group is needed and can select a protecting group suitable for use in a particular environment.

[0150] Unless otherwise specified, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the same structure except that hydrogen is replaced with deuterium or tritium, or carbon is replaced with 13 C- or 14 C-enriched carbon are within the scope of this disclosure.

[0151] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes in one or more of the atoms that make up such compounds. For example, the compound may be a radioactive isotope, such as tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14C) It can be radiolabeled. All isotopic variants of the compounds of the present disclosure are included within the scope of the present disclosure, whether radioactive or not.

[0152] ii. Pharmaceutical salts The compounds of the present disclosure may exist as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" is intended to include salts of the active compounds prepared with relatively non-toxic acids or bases depending on the particular substituents found on the compounds described herein. Pharmaceutically acceptable salts are generally well known to those skilled in the art and include, by way of example and not limitation, acetates, benzenesulfonates, besylates, benzoates, bicarbonates, bisulfates, bromides, calcium edetate, carnsylates, carbonates, citrates, edetates, edisylates, estolates, esylates, fumarates, gluceptates, gluconates, glutamates, glycolylarsanilates, hexylresorcinates, hydrabamines, hydrobromides, hydrochlorides, hydroxynaphthoates, iodides, isethionates, lactates, lactobionates, malates, maleates, mandelates, mesylates, mucates, napsylates, nitrates, pamoates (embonates), pantothenates, phosphates / diphosphates, polygalacturonates, salicylates, stearates, basic acetates, succinates, sulfates, tannates, tartrates (including these mixtures including (+)-tartrates, (-)-tartrates or racemic mixtures) or theophyllinates. These salts can be prepared by methods known to those skilled in the art. Other pharmaceutically acceptable salts can be found, for example, in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0153] Base addition salts, such as sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts are also included. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid in a pure or suitable inert solvent. Examples of acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, monohydrogen sulfate, hydroiodic acid or phosphorous acid, etc., and also organic acids, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc.

[0154] Salts of amino acids such as arginate and salts of organic acids such as glucuronic acid or galacturonic acid are also included. See, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present disclosure have both basic and acidic functional groups that allow for the conversion of the compound to a base addition salt or an acid addition salt. The neutral form of the compound can be regenerated by contacting the salt with a base or an acid and isolating the parent compound by conventional methods. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0155] Certain compounds of the present disclosure can exist in unsolvated and solvated forms (including hydrate forms). Generally, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure can exist in various crystalline or amorphous forms. Generally, all physical forms are equivalent for the uses contemplated by the present invention and are considered to be within the scope of the present disclosure.

[0156] iii. General definitions Although certain terms are used in this specification, they are used only in a general and descriptive sense and not for purposes of limitation. For clarity, certain definitions are set forth herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs.

[0157] "Cancer" in an animal refers to the presence of cells having characteristics typical of cancer-causing cells, such as uncontrolled growth, loss of specialized function, immortality, significant metastatic ability, significant improvement in anti-apoptotic activity, rapid growth and proliferation rates, and certain characteristic morphologies and cellular markers. Depending on the situation, cancer cells may take the form of a tumor, and such cells may be present locally within the body of an animal or circulate in the bloodstream as independent cells.

[0158] "Control" means a standard or reference condition.

[0159] "Disease" means any condition or disorder that impairs or interferes with the normal function of a cell, tissue, organ, organism, or subject.

[0160] "Effective amount" of a substance means an amount of that substance sufficient to elicit a desired biological response or measurable difference when compared to a control. As will be understood by those of ordinary skill in the art, the absolute amount of a particular substance effective for treating a disease, disorder, condition, or injury can vary depending on factors such as the substance to be delivered, the mode of administration, the age, weight, and general health of the subject, the desired biological endpoint, the desired therapeutic effect, and the like. Ultimately, the responsible clinician determines the appropriate amount and dosing regimen. For example, an "effective amount" of a substance can be an amount sufficient to generate a measurable image when the compound is used for imaging, or an amount sufficient to improve the symptoms of a disease when the compound is used for treatment. Those of ordinary skill in the art will also understand whether an effective amount of a substance can be administered in a single dose or achieved through multiple divided doses.

[0161] As used herein, the term "administering" means contacting a subject with a substance of the disclosure.

[0162] In accordance with longstanding patent law convention, in this application, singular indefinite and definite articles are used to mean "one or more." Thus, for example, the term "a subject" includes a plurality of subjects, unless the context clearly dictates otherwise (e.g., where multiple subjects are specified), and the same holds true for other terms.

[0163] Throughout this specification and the claims, the term "comprise" is used in a non-exclusive sense, unless the context clearly dictates otherwise. Similarly, the terms "include" and its grammatical variations are non-limiting, such that listing items does not exclude other similar items that could be substituted for or added to the listed items.

[0164] For the purposes of this specification and the appended claims, unless otherwise specified, all numbers expressing quantities, sizes, dimensions, ratios, shapes, compounding ratios, parameters, percentages, parameters, quantities, features, and other numerical values used in the specification and claims are to be understood as being modified by the term "about" in all instances, even if the term "about" is not specifically attached to the value, quantity, or range. Accordingly, unless otherwise specified, the numerical parameters set forth in the following specification and the appended claims are not and need not be exact, and may be approximations and / or may vary as desired depending on the properties sought to be obtained by the subject matter of the present disclosure, reflecting tolerances, conventional elements, rounding, measurement errors, and other elements known to those of ordinary skill in the art. For example, the term "about" when referring to a value may include variations of ±100% in some embodiments, ±50% in some embodiments, ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments of the specified amount. This is because such variations are appropriate for carrying out the disclosed methods or using the disclosed compositions.

[0165] Furthermore, when using the term "about" in relation to one or more numbers or numerical ranges, it should be understood to refer to all such numbers, including all numbers within a given range, and this term modifies the range by expanding the boundaries above and below the recited numerical values. Reciting numerical ranges by endpoints includes all numbers, e.g., integers (including fractions thereof) within that range (e.g., if 1 - 5 is recited, then 1, 2, 3, 4, and 5, and fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc. are included), and any range within that range.

Examples

[0166] The following examples are included to provide guidance to those skilled in the art in practicing representative embodiments of the subject matter of this disclosure. Given the present disclosure and the general state of the art in the field, one of ordinary skill in the art will understand that the following examples are for illustrative purposes only and that numerous changes, modifications, and alterations can be made without departing from the scope of the subject matter of this disclosure. The descriptions and examples regarding the syntheses that follow are for convenience of explanation only and are not to be construed as limiting in any way in generating the disclosed compounds by other methods.

[0167] Example 1 Synthesis and Evaluation of Gadolinium (Gd)-Based Contrast Agents Overview Magnetic resonance (MR) imaging is advantageous because it can simultaneously obtain anatomical, functional, and molecular information. MR molecular imaging can combine this established and widely used clinical modality and its high spatial resolution with molecular profiling in vivo. However, because the sensitivity of MR is inherently low, high local concentrations of biological targets are required to generate recognizable MR contrast. The inventors hypothesized that prostate-specific membrane antigen (PSMA), an attractive target for prostate cancer imaging and therapy due to its high concentration in target cells, limited expression in non-target tissues, and accessibility on the cell surface, could serve as a suitable biomarker for MR-based molecular imaging. For this purpose, three high-affinity low-molecular-weight gadolinium (Gd)(III)-based PSMA-targeted contrast agents, classified as Gd1, Gd2, and Gd3, respectively, with 1 - 3 Gd chelates per molecule were synthesized (Figure 1A). The purpose of this study was to evaluate the PSMA binding affinity and longitudinal relaxivity (r 1 ) of the synthesized substances. Cellular uptake of the substances in PSMA-expressing cells (isogenic) and non-expressing control cells was evaluated by inductively coupled plasma mass spectrometry (ICP-MS). Finally, the ability of the substances to distinguish PSMA-expressing cells from control cells was evaluated by MR imaging both in vitro and in vivo.

[0168] Materials and Methods (21S,25S)-8,15,23-Trioxo-1-((4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)amino)-1-thioxo-2,7,16,22,24-pentaazapentacosan-21,25,27-tricarboxylic acid, Gd1 Compound Gd1 was prepared according to recent reports. Compound 1 was prepared in three steps described below. Commercially available N-Boc-1,4-diaminobutane (68 mg, 0.36 mmol, in 0.5 ml of DMSO) was mixed with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 2-[(4-isothiocyanatophenyl)methyl] (p-SCN-Bn-DOTA) (192 mg, 0.28 mmol, in 2.5 mL of DMSO) and DIEA (132 μl, 0.75 mmol) and stirred at 40 °C for 4 hours. The solvent was evaporated and the solid residue was purified by reverse phase C 18 Flash chromatography (5.5 g, Agilent SF10) using water and acetonitrile (0.1% TFA each) gave Boc-protected 7 after lyophilization. Yield: 146 mg, about 55%. ESI-MS 740 [M+H] + The compound obtained in that step was then treated with ice-cold TFA / CH 2 Cl 2 (1 / 1) solution and stirred at ambient temperature for 2 hours. The solvent was evaporated, the residue was dried under vacuum and purified by reverse phase flash chromatography (5.5 g, Agilent SF10) to give 7 in moderate yield. The solvent was evaporated, the residue was dried under vacuum and purified by reverse phase flash chromatography (5.5 g, Agilent SF10) to give 7. Yield about 104 mg, 40%. 1 1H NMR (DMSO-d 6) δ: 8.80 - 8.64 (m, 1H), 8.12 - 7.90 (m, 2H), 7.75 - 7.10 (bm, 4H), 4.65 - 4.63 (m, 1H), 4.17 - 2.59 (m, 27H), 2.40 - 1.11 (m, 6H). ESI-MS: 640 [M+1] + . A solution of 7 (110 mg, 0.17 mmol, in 3 mL of distilled water) was added to a solution of Gd 2 (CO 3 ) 3 (85 mg, 0.17 mmol), and the mixture was stirred at 60 °C for 14 h. ESI-MS: Calculated for C 48 H 77 N 10 O 17 S, 797.5183 [M+H] + , found: 797.5212. Next, the compound was purified by HPLC. Method 1: Solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile), flow rate 8 mL / min. The elution gradient was 100% A and 0% B for 5 min, 100% A - 80% A, 0% B - 20% B for 5 - 25 min, and 80% A - 20% A and 20% B - 80% B for 25 - 30 min.

[0169] (30S,34S)-2,9,17,24,32-Pentaoxo-1-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-8-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)-3,10,16,25,31,33-hexaazaoctatriacontane-11,30,34,36-tetracarboxylic acid, digadolinium(III) salt, Gd2 The compound was prepared according to Scheme 2. Fmoc-Lys(Boc)-OH (2.49 g, 6.7 mmol) was added to a solution of N-bis-Boc-L-lysine NHS (3 g, 6.7 mmol, in 10 mL of DMF), and the solution was sonicated at room temperature until a clear solution was obtained in 1 h. The solution was stirred at room temperature for 4 h, and the solvent was removed under vacuum to give 4 in almost quantitative yield. Compound 4 was further purified by silica gel column with 3 / 97 MeOH / CH2 Cl 2 was used as an eluent for purification. 1 H NMR (CDCl 3 ) δ: 8.01 (d, 2H), 7.89 (m, 2H), 7.78 - 7.44 (m, 4H), 6.82 (m, 1H), 6.15 (m, 1H), 5.58 (m, 1H), 5.01 - 4.03 (m, 5H), 3.75 - 3.32 (m, 6H), 2.22 - 1.31 (m, 30H). ESMS m / Z: 696 [M + H] + . Compound 4 (2 g, 2.9 mmol) was dissolved in 10 mL of 1 / 1 TFA / CH 2 Cl 2 solution and stirred at room temperature for 2 hours. After evaporation of the solvent, the solid residue was washed with 3 x 3 mL of diethyl ether and dried under vacuum to give 5 as the TFA salt. Compound 5 was obtained in quantitative yield and used without further purification after lyophilization. 1 H NMR (D 2 O) δ: 8.01 (d, 2H), 7.89 (m, 2H), 7.78 - 7.44 (m, 4H), 4.78 - 4.75 (m, 2H), 4.32 (m, 1H), 4.11 - 4.09 (m, 1H), 4.01 - 3.98 (t, 1H), 3.50 - 3.11 (m, 3H), 3.10 - 2.99 (m, 2H), 2.01 - 1.01 (m, 12H). To a solution of DOTA - NHS (100 mg, 0.13 mmol, in 0.5 mL of DMSO) were added 5.2 TFA (32 mg, 0.04 mmol) and DIEA (0.78 mmol, 136 μL) portionwise at room temperature over 45 minutes. The solution was then stirred for a further 2 hours and the completion of the reaction was monitored using HPLC. After completion of the reaction, the reaction mixture was purified by HPLC to give 6. Compound 6 was treated with a 20% piperidine solution for the Fmoc group and purified by C 18 flash chromatography using 90 / 10 H 2 O / CH 3 CN (each 0.1% TFA) solution and lyophilized. ESIMS: 1046 [M + H] + . The lyophilized compound (50 mg, 0.047 mmol) was dissolved in distilled water (2 mL), and Gd 2 (CO 3 )3 (0.26 mmol, in 3 mL of water) was added and stirred at 60 °C for 12 hours. Compound 7 was added to C 18 Gradient 90 / 10 - 80 / 20 of H by flash chromatography 2 O / CH 3 CN (each 0.1% TFA) solution was used for purification and freeze-dried. 7 (40 mg, 0.003 mmol) was slowly added to a solution of 3 in DMSO (25 mg, 0.004 mmol) over 30 minutes and stirred at room temperature until the reaction was complete for about 2 hours. The completion of the reaction was monitored by HPLC. After completion, the reaction mixture was subsequently purified by HPLC and the product was freeze-dried. ESI-MS: 1813.08 [M+H] + , Measured value: 1813.08. Next, the compound was purified by HPLC. Method 1: C 64 H 103 Gd 2 N 15 O 26 Theoretical value for, 1813.5681 [M] + , Measured value 1813.5681 [M+1]. Solvent A (0.1% TFA, in water) and solvent B (0.1% TFA, in acetonitrile), flow rate 8 mL / min. The elution gradient was 100% A and 0% B in 5 minutes, 100% A - 80% A and 0% B - 20% B in 5 - 25 minutes, 80% A - 20% A and 20% B - 80% B in 25 - 30 minutes.

[0170] (3S,7S)-5,13,20,28-Tetraoxo-32-(2,4,6-tris(1-(2-hydroxy-3-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)propyl)-1H-1,2,3-triazol-4-yl)phenoxy)-4,6,12,21,27-pentaazadotriacontane-1,3,7,22-tetracarboxylic acid, trigadolinium(III) salt, Gd3 Gd3 was prepared by a multi-step synthesis shown in Scheme 3. Compound 8 was prepared according to the previous report.

[0171] 2,5-Dioxopyrrolidin-1-yl 5-(2,4,6-triethynylphenoxy)pentanoate, 9 To a solution of 8 (300 mg, 1.13 mmol, in 5 mL of DMF), TSTU (440 mg, 1.47 mmol) and TEA (541 μL, 3.39 mmol) were added, and the resulting solution was stirred at room temperature for 4 hours until the reaction monitored by TLC was complete. The solvent was removed under high vacuum, and the residue was dissolved in CH 2 Cl 2 and purified by silica gel column using a 40 / 60 - 50 / 50 EtOAc / hexane solution as the eluent. The fractions containing the product were combined and evaporated to give the desired product as a colorless solid. Yield: about 310 mg. NMR (CDCl 3 ): δ 7.56 (s, 2H), 4.26 (t, 2H), 3.39 (s, 2H), 3.04 (s, 1H), 2.78 (s, 4H) 2.48 (t, 2H), 2.01 - 1.80 (m, 4H).

[0172] (3S,7S)-26-Amino-5,13,20-trioxo-4,6,12,21-tetraazapentacosan-1,3,7,22-tetracarboxylic acid 2,2,2-trifluoroacetate, 10 Compound 10 was prepared according to the previous report. Briefly, to a solution of tris-t-Bu protected 3 (100 mg, 0.135 mmol, in 1.35 ml of DMF), H-Lys(Boc)(O-t-Bu) (59.5 mg, 0.175 mmol) was added, followed by DIEA (70.7 μL, 0.135), and the clear solution was stirred overnight at room temperature. Next, the solution was concentrated under vacuum until a clear oily residue was obtained. The residue was dissolved in 2:1 MeCN / water (6 mL) and lyophilized to give a clear foamy product. Yield. The product was used without further purification. Yield: 117 mg, 0.126 mmol, 93%. ESI-MS: 928[M + H] + . The compound was dissolved in 2 ml of TFA / CH 2 Cl 2It was dissolved in the ice-cold solution, and then TES (278 μL, 1.7 mmol) was added dropwise. The clear solution was stirred for 5 hours and concentrated under vacuum. The residue was dissolved in 5 mL of water and purified by reverse-phase flash chromatography. The product was eluted using 80 / 20 water / CH 3 CN (0.1% TFA for each). ESI-MS: 603 [M+H] + .

[0173] (3S,7S)-5,13,20,28-Tetraoxo-32-(2,4,6-triethynylphenoxy)-4,6,12,21,27-pentaazadotriacontane-1,3,7,22-tetracarboxylic acid, 11 Compound 9 (132 mg, 0.362 mmol) was added in one portion to a solution containing (3S,7S)-26-amino-5,13,20-trioxo-4,6,12,21-tetraazapentacosane-1,3,7,22-tetracarboxylic acid 2,2,2-trifluoroacetic acid (260 mg, 0.362 mmol), triethylamine (0.202 mL, 1.44 mmol) and DMF (3.62 mL). The mixture was stirred at room temperature for 4 hours and concentrated until a yellowish-brown residue was obtained. The residue was dissolved in 1 / 1 water / acetonitrile (3 mL), and purified by reverse-phase flash chromatography with a step gradient consisting of 100% water, 0.1% TFA, followed by 80 / 20, 60 / 40 water / acetonitrile (0.1% TFA for each). Each gradient step consisted of a solvent volume of about 144 mL. The flow rate was 40 mL / min. The fractions containing the desired product were concentrated until a residue was obtained and lyophilized, and (3S,7S)-5,13,20,28-tetraoxo-32-(2,4,6-triethynylphenoxy)-4,6,12,21,27-pentaazadotriacontane-1,3,7,22-tetracarboxylic acid was obtained as a white solid. 169 mg, yield 54%. ESI-MS, C 18 H 43 H 57 N 5 O 13 [M+H] + The theoretical value for, 852.4, measured value 851.9. 11H NMR (400 MHz, DMSO-d 6 ) 12.12 (bs, 4H), 8.01 (d, 1H), 7.76 (m, 2H), 7.57 (s, 2H), 6.33 (m, 2H), 4.47 (s, 2H), 4.28 (s, 1H), 4.09 - 4.15 (m, 4H), 3.00 (m, 4H), 2.21 - 2.27 (m, 2H), 2.10 (m, 4H), 2.02 (t, 2H), 1.89 - 1.94 (m, 1H), 1.22 - 1.69 (m, 24H). 13 13C NMR (100 MHz, DMSO-d 6 ) δ 175.0, 174.6, 174.3, 174.1, 172.8, 172.3, 172.1, 162.1, 158.9, 158.5, 157.7, 137.6, 118.0, 117.5, 86.6, 82.0, 81.5, 78.6, 74.1, 52.7, 52.1, 38.7, 38.6, 35.8, 35.5, 32.2, 31.1, 30.3, 29.7, 29.3, 29.2, 28.9, 28.8, 27.9, 25.7, 25.6, 23.3, 23.0, 22.1.

[0174] Gd3 (3S,7S)-5,13,20,28 - Tetraoxo - 32-(2,4,6 - triethynylphenoxy)-4,6,12,21,27 - pentaazadotriacontane - 1,3,7,22 - tetracarboxylic acid (12 mg, 0.14 mmol), Compound 002 (28 mg, 0.046 mmol) and t - butanol (0.1 mL) in a mixture were added with water (0.05 mL), followed by TBTA (0.15 mg, 0.3 μmol) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.11 mg, 0.3 μmol). The mixture was stirred at 65 °C for 18 h. The reaction mixture was dissolved in 2.5 mL of 0.1% sodium bicarbonate and filtered. The solution thus obtained was purified by HPLC using a Phenomenex, Luna, 10 micron, 10 x 250 mm column and a gradient consisting of 0 - 95% acetonitrile:water over 20 min. The desired product (003) eluted at 6.1 - 7.1 min. The fractions containing 003 were combined, concentrated and lyophilized to give a white solid. 13 mg, yield 34%. ESI - MS C94 H 141 Gd 3 N 26 O 34 [M-H] - The theoretical value for this is 2650.7 and the measured value is 2648.9.

[0175] Scheme 1

[0176]

Chem.

[0177] Scheme 2

[0178]

Chem.

[0179] Scheme 3

[0180]

Chem.

[0181] Results Typical PSMA contrast agents containing Lys-Glu urea as the target moiety: The structures of the monomers, dimers, and trimers of Gd (Gd1, Gd2, and Gd3) are shown in Figure 1A. The target compounds were prepared by devising a multi-step liquid-phase synthesis method. This method is outlined in Schemes 1 - 3.

[0182] For all three compounds, the chelator DOTA was used because it can form complexes with high thermodynamic and kinetic stability. Gd1 contains DOTA-Bn-SCN to obtain a high relaxation rate. The structure of Gd1 is based on a positron emission tomography (PET) lead-86Y labeled imaging agent reported in recent years, and this imaging agent has demonstrated high specific tumor accumulation in preclinical models (Banerjee, et al. 2015). Gd2 was prepared by conjugating both the ∝- and ε-amines of lysine with DOTA-NHS using a solution-based peptide synthesis method. Under the same conditions, when the reaction was carried out in a sonic bath at room temperature, the yield of the coupling reaction was significantly improved. Gd3 contains a phenol core to which three Gd(III)-DOTA are bound via a rigid triazole bond as previously reported by Mastarone et al. A PSMA targeting functional group was conjugated to the core via the oxygen of the phenol. Gd3 showed a relatively high relaxation rate as a result of the more rigid triazole linker moiety. The compounds were purified by reverse-phase HPLC and characterized by LCMS. To confirm whether the Gd(III)-containing moiety of the substance has no potentially negative effect on the binding affinity of the probe, the values of the PSMA inhibition constant (Ki) for Gd1, Gd2, and Gd3 were determined using a fluorescence-based PSMA inhibition assay and are shown in Table 1.

[0183] [Table 1]

[0184] The known high-affinity PSMA inhibitor N-[[[(S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ43) (Olszewski et al., 2004) was used as the reference ligand. As expected, all compounds showed high binding affinity, with the highest Ki value for Gd1 (0.45 nM), followed by Gd3 (7.19 nM), and the lowest for Gd2 (18.18 nM). When imaging at 9.4 T and 25 °C, the solution phantom was 3.0 - 6.2 mM-1 s -1 (Gd(III)), 3.0 - 12.5 mM -1 s -1 (Contrast agent) showed the r1 relaxation rate in PBS that varies among them (Table 1). As expected, at 25 °C, the relaxation rate of Gd1 was the lowest, followed by Gd2 and Gd3. To confirm the selectivity and specificity of the substances, human prostate cancer cells genetically engineered to express a large amount of PSMA (PC3 PIP) and the corresponding wild-type PSMA non-expressing cells (PC3 flu) were selected as negative controls (Banerjee, Angew., 2001). After incubation with Gd1 or Gd2, the pellet-shaped PSMA+PC3 PIP and PSMA-PC3 flu cells showed no change in T 1 Enhanced MR contrast or R 1 showed no change. Conversely, the T 1 enhanced images of both cell lines incubated with Gd3 showed significant MR contrast enhancement in PSMA+PC3 PIP cells as shown in Figure 4A compared to unlabeled cells and PSMA-PC3 flu cell pellets. MRI enhancement and T 1 measurements of cell pellets in the presence and absence of 50 μM Gd3 showed high enhancement and a difference in T 1 relaxation rate, ΔR 1 between Gd3-treated PIP cells and control PIP cells compared to Gd3-treated flu cells and control flu control cells after removal of Gd3 from the cell lines by washing in standard medium (Figures 4B and 4C).

[0185] The selective blockade experiment by co-incubation of Gd3 and ZJ43 actually showed a significant blockade of T 1 enhancement. Cells incubated with Gd3 in the presence of ZJ43 showed no change in T 1showed only slight changes in value. This indicates that ZJ43 was able to specifically block the binding of Gd3. These results show that Gd3 exhibits receptor-specific cell binding to PSMA+PC3 PIP cells, indicates that PSMA-mediated contrast enhancement is seen, and proves the concept of receptor-mediated endocytosis. Indeed, ICP-MS analysis of the cells after post-image analysis showed that the amount of Gd(III) associated with the PC3 flu cell pellet was negligible, while the PC3 PIP cell pellet had a high amount of Gd (Figures 2 and 3). The PSMA+PC3 PIP cell pellet had an estimated intracellular Gd(III) concentration of approximately 22.82 μM for Gd3, followed by approximately 12.5 μM and approximately 7.2 μM for Gd2 and Gd1, respectively (Figure 2). Therefore, the difference in ΔR 1 between PIP cells and that of flu cells reflects the change due to specific Gd3 binding to PSMA in PIP cells.

[0186] The cellular internalization assay revealed that the percentage of the incubated amount that underwent internalization in PSMA+PC3 PIP cells for Gd1 and Gd2 was 9.06±0.31 and 21.63±3.51, respectively, after 4 hours of incubation, at which time only 2.42±0.11 and 3.51±1.32%ID were associated with the cell surface (Figure 3). Furthermore, slightly higher non-specific uptake was associated with Gd2 in PSMA-PC3 flu cells, which is thought to be related to its lower K i value compared to Gd1. To further investigate cellular uptake and internalization, a dual-modal Gd monomer contrast agent labeled with Rhodamine-Red TM -X was prepared to confirm PSMA-mediated internalization of this class of contrast agents (Figure 5D). As expected, this substance showed specific and high accumulation only in PSMA+PC3 PIP cells (Figures 5A - 5C). These results show that cell receptors expressed at this level can be detected by MRI using these simple targeting substances.

[0187] Time-dependent internalization studies were performed for Gd3 at 1, 4, and 24 hours after incubation (Figures 6A and 6B). Intracellular uptake at 1 and 4 hours was high and specific, being 28.30 ± 0.47 and 39.92 ± 3.59%ID, respectively, in PSMA+PC3 PIP cells, and approximately 89.69 ± 3.90%ID was observed 24 hours after incubation. Similar amounts of Gd (approximately 33 - 37%ID) were associated with the cell membrane at the same time points. These results show a strong correlation between the detectable T 1 contrast enhancement and the highly specific accumulation of Gd3 in PSMA+PIP cells.

[0188] Prior to the evaluation of Gd3 in imaging of live mice, its biocompatibility was investigated using a cell proliferation assay. Various concentrations of Gd3 were incubated with PSMA+PC3 PIP and PSMA-PC3 flu cells for 24 hours. Gd3 had no significant effect on the viability of PSMA-flu cells up to a Gd(III) concentration of 1 mM (i.e., approximately 90% viability) (Figure 7). However, Gd(III) concentrations >1 mM affected PSMA+PC3 PIP cell viability (Figure 8). The observed levels of PSMA+PC3 PIP cell death are thought to be due to the high intracellular translocation of Gd3 and the long incubation time (24 hours) used.

[0189] In vivo MR imaging was performed on male NOD / SCID mice bearing PSMA+PC3 PIP (right) and PSMA-PC3 flu (left) tumors subcutaneously implanted in the right lower and left lower abdominal regions, respectively, after intravenous injection of Gd3 (0.05 mmol / Kg dose) at 9.4T. No non-specific uptake occurred in either PIP or flu tumors during the first 20 minutes after injection. In all tissues, T 1A sharp decrease in value was observed, following by 0.63 s for the highest PSMA flu, 0.57 s for the PIP tumor, and 0.261 s for the muscle. Importantly, rapid clearance of the contrast agent from the muscle and flu tumors was observed. The contrast enhancement in the PIP tumor was highest at 36% 40 minutes after injection and maintained a high 30% until 1.5 hours after injection. After 3 hours, the T 1 values were confirmed to have returned to the initial values, and the T 1 values of the PIP tumor showed no significant change.

[0190] In vivo MR imaging of Gd3 was also performed on mice with subcutaneous PSMA+PC3 PIP and PSMA-PC3 flu tumor xenografts in the right lower and left lower abdominal regions, respectively, after a single bolus intravenous injection (0.06 mmol / kg). Figure 9A shows the quantitative contrast enhancement mapping of 1 mm slices for both tumors from 40 to 160 minutes after injection (ΔR 1 ). The contrast enhancement remained constant within the PSMA+PC3 PIP tumor for at least 3 hours, but decreased rapidly in the PSMA-PC3 flu tumor and muscle tissue. The change in the T 1 value of the PSMA+PC3 PIP tumor (Figures 10A and 10B) reached a minimum of 1819±76 ms (mean±SD, with an average 36% enhancement in the R 1 value, n = 4) in the first 40 - 60 minutes, remained constant at 29% until 90 minutes, and slowly decreased to 24% 190 minutes after injection. For the PSMA-PC3 flu tumor, the highest contrast enhancement was approximately 24% 20 minutes after injection, and subsequently the contrast enhancement decayed rapidly (ΔR 1 <20% at 40 minutes). These results demonstrate specific contrast enhancement (P≤0.05) for the PSMA+PC3 PIP tumor at 80 and 120 minutes after injection. As shown in Figure 9B, these results were directly compared with those of other mice administered a trimeric Gd probe without a targeting moiety in the same manner (no tumor enhancement was seen) (Mastarone, 2011).

[0191] Under the same experimental conditions, in the control study using physiological saline (PBS), no change in T was observed for PIP and flu tumors (Figure 12). 1 No change in value was observed (Figure 12).

[0192] Although not intending to be bound by a particular theory, when binding to PSMA, the rotational correlation time of Gd3 increased compared to the unbound state. The binding also changed the hydration number and water exchange rate for each substance, and it is considered that the relaxation value changed from that expected for the free contrast agent (Caravan et al., 2007). Also, at high magnetic fields, when the rotational correlation time is extended, the relaxation can be slightly reduced due to the interaction between the contrast agent and cell components (Caravan, P., et al. 2009; De Leon-Rodriguez, L.M., et al. 2010; Geninatti-Crich, S. 2011). By incorporating a sensitive multimeric Gd(III) complex in combination with an established PSMA-targeting small molecule, PSMA-targeted MR molecular imaging was performed in vitro and in vivo.

[0193] In summary, the Gd-based contrast agent Gd3 could be used for in vivo PSMA-specific MR imaging using mouse xenografts. Optimization of the construct is underway assuming parallel use in prostate cancer and other cancers.

[0194] Example 2 Receptor concentration of PSMA Number of receptors per cell (N.R.C.) = 4.9x10 6 , r 細胞 = 8.75 μm

[0195]

Number

[0196]

Number

[0197] Therefore, for a 0.05 second -1 change (considering tissue t 0 1 = 2 seconds, there is an enhancement of about 10%) to be seen, relaxation is necessary (if the receptor:contrast is 1:1).

[0198]

Number

[0199] Example 3 Preclinical evaluation of a prostate-specific membrane antigen 86 Y-labeled inhibitor for dosimetry estimation Overview 86 Y (half-life = 14.74 hours, 33% β + ) is included in a new class of positron-emitting isotopes with a relatively long physical half-life that enables long-time imaging of biological processes. Preparation and biodistribution studies of three low molecular weight 86 Y-labeled PSMA-binding ureas (Figure 14) in rodent experimental models, and imaging of the most pharmacokinetically favorable substances in non-human primates for dosimetry in preparation for clinical trials with the corresponding 90 Y- and 177 Lu-labeled substances have been reported.

[0200] 86 Y]-4 to 6 were prepared using multi-step synthesis. The PSMA inhibition constant was evaluated by a competitive binding assay. In vivo characterization using male mice with tumors was performed by PET / CT for 86 Y]-4 to 6, and also for 86 Y]-4 and 86 Y]-6 from 24 hours after injection by biodistribution studies. By recording quantitative whole-body PET scans, the kinetics of 14 organs in male baboons were 86 measured using

[0201] Compound​86 Y]-4~6 was obtained in high radiochemical yield and purity, with a specific activity exceeding 83.92 GBq / μmol. From PET imaging and biodistribution studies using mice bearing PSMA1 / 2-positive PC-3 PIP and PSMA-negative PC-3 flu tumors, 86 Y]-4~6 was shown to exhibit high site-specific uptake starting 20 minutes after injection in PSMA-positive PC-3 PIP tumors and remained high at 24 hours. Compound 86 Y]-6 showed the highest tumor uptake and retention, being 32.17 ± 7.99 and 15.79 ± 6.44% injected dose / g (%ID / g) at 5 hours and 24 hours, respectively. The low radioactivity concentrations were associated with blood and normal organs other than the kidney, which is a PSMA-expressing tissue. PET imaging in baboons revealed that all organs had two-phase (rapid and slow) clearance, with the highest uptake in the kidney at 25 minutes (8%ID / g). Radiation doses were calculated using the OLINDA / EXM software with individual absolute uptake kinetics. The highest mean absorbed dose was observed in the renal cortex, 1.9 mGy / MBq 86 Y]-6.

[0202] Materials and Methods Solvents and chemicals obtained from commercial sources were of analytical grade or higher and were used without further purification. 9-Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids (including Fmoc-Lys(Boc)-Wang resin), 1-hydroxybenzotriazole monohydrate, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were all purchased from Chem Impex International Inc. (Wood Dale, Illinois). Carrier-free 86 Y](NO 3 ) 3It was obtained from the National Cancer Institute of the National Institutes of Health (Bethesda). DOTA-tris(t-butyl ester)-monoacid and p-SCN-Bn-DOTA (B-205) were purchased from Macrocyclics, Inc. (Dallas, Texas). Yttrium(III) nitrate, triethylsilane (Et 3 SiH), diisopropylethylamine (DIEA), and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All other chemicals were purchased from Thermo Fisher Scientific (Pittsburgh, Pennsylvania) unless otherwise specified. Analytical thin-layer chromatography (TLC) was performed using Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1 plates and visualized with ultraviolet light (254 nm), I 2 in EtOH, and 1% ninhydrin. Flash chromatography was performed using silica gel MP SiliTech 32-63 D60A purchased from Bodman (Aston, Pennsylvania). All experiments were performed in duplicate or triplicate to ensure reproducibility. 1 1H NMR spectra were recorded on a Bruker Ultrashield TM 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm, downfield, relative to the proton resonance arising from incomplete deuteration of the NMR solvent. Low-resolution ESI mass spectra were obtained from a Bruker Daltonics Esquire 3000 Plus spectrometer (Billerica, Massachusetts). High-resolution mass spectra were obtained from the University of Notre Dame Mass Spectrometry & Proteomics Facility (Notre Dame, Indiana) using ESI, by direct injection on a Bruker micrOTOF-II or via a C 18Obtained by LC elution using an ultra-high pressure Dionex RSLC equipped with a column.

[0203] Purification of 4 - 6 and 89 Y]4 - 6 was performed using a Phenomenex C 18 Luna 10x250mm 2 column together with a Waters 486 variable wavelength UV / Vis detector, and both were controlled by Empower software (Waters Corporation, Milford, Massachusetts) (Figs. 15A and 15B, Figs. 16A and 16B, Figs. 17A, 17B and 17C). HPLC was performed using solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) according to the following methods. Method 1: The elution gradient was 75% A and 25% B (5 minutes), 75% A to 60% A and 25% B to 40% B (5 - 25 minutes), 60% A to 75% A and 40% B to 25% B (25 - 30 minutes), and the flow rate was 8 mL / min. Method 2: The flow rate was 8 mL / min. The elution gradient was 100% A and 0% B (0 - 5 minutes), 100% A to 45% A and 0% B to 55% B (5 - 45 minutes). 86 Purification of Y]4 - 6 by HPLC was performed using a Varian Prostar system (Palo Alto, California) equipped with a Model 490 UV absorbance detector and a Bioscan NaI scintillation detector connected to a Bioscan Flow-count system (Bioscan, Washington D.C., USA). 86 For the HPLC purification of Y]4 - 6, a Waters Novapak C 18 150x3.9mm 2 column was used. HPLC was performed using solvent A (0.1% TFA in water) and solvent B (0.1% TFA in CH 3 CN) with a flow rate of 1 mL / min. An isocratic method of 85% A and 15% B for 25 minutes was 86It was used for the purification of Y]4. The gradient method (78% A and 22% B from 0 to 5 minutes, 78% A to 58% A and 22% B to 42% B from 5 to 25 minutes) was 86 used for Y]5. The gradient method (88% A and 12% B from 0 to 5 minutes, 88% A to 68% A and 12% B to 32% B from 5 to 25 minutes) was 86 used for the purification of Y]6. The specific radioactivity was calculated as the radioactivity eluting at the retention time of the product during preparative HPLC divided by the mass corresponding to the area under the UV absorption curve. All final compounds were obtained with >95% radiochemical purity as measured by HPLC. Compound 1 was prepared according to a previous report (Banerjee, Pullambhatla, Byun, et al., 2011). Compounds 4 and 5 were prepared by the same general method previously reported for 4 (Banerjee et al., 2010) and briefly described below for 5.

[0204] Synthesis and radiochemistry (13S,27S,31S)-4,7,10-Tribenzyl-2,5,8,11,18,21,29-heptaoxo-1-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-3,6,9,12,17,22,28,30-octaazatritriacontane-13,27,31,33-tetracarboxylic acid, 5 Compound 5 was prepared according to a previous report outlined in Scheme 4 ((Banerjee et al., 2010). Compounds 3 and 4 were prepared according to the solid-phase peptide method. Fmoc-Lys(Boc)-Wang resin (100 mg, 0.43 mM) was swollen with CH 2 Cl 2 (3 mL), followed by DMF (3 mL). A 20% piperidine solution in DMF (3 x 3 mL) was added to the resin and then gently shaken at ambient temperature for 30 minutes on a mechanical shaker. The resin was washed with DMF (3 x 3 mL) and CH 2 Cl 2(3 x 3 mL) was washed. The formation of free amine was evaluated by the Kaiser test (Kaiser et al., 1970). After swelling the resin in DMF, a solution of Fmoc-Phe-OH (3 eq), HBTU (3 eq), HOBt (3 eq) and DIPEA (4.0 eq) in DMF was added and gently shaken for 2 hours. Next, the resin was washed with DMF (3 x 3 mL) and CH 2 Cl 2 (3 x 3 mL). The coupling efficiency was evaluated by the Kaiser test. The above sequence was repeated for two more coupling steps with Fmoc-Phe-OH and DOTA-(t-butyl ester) 3 -CO 2 H. The final compound was cleaved from the resin using TFA / CH 2 Cl 2 (1 / 1) and concentrated under vacuum to give 3. The concentrated product was purified using a C 18 SepPak Vac 2g column. The product was eluted with a solution of 70 / 30 water / acetonitrile (each 0.1% TFA) and lyophilized. ESI-MS: 974 [M+H] + . To a solution of 3 (15 mg, 15.4 μmol, in 1 mL of DMSO), 1 (15 mg, 26.18 μmol) and TEA (30 μL) were added and left standing at ambient temperature for 2 hours. After removal of the solvent, compound 5 was purified by HPLC (method 1). 1 H NMR (DMSO-d 6 ) δ: 8.64 (m, 1H), 8.44 (m, 1H), 8.29 - 8.18 (m, 2H), 7.77 - 7.75 (m, 2H), 7.30 - 7.17 (m, 15H), 6.35 - 6.33 (m, 2H), 4.65 - 4.63 (m, 2H), 4.17 - 2.59 (m, 26), 2.40 - 1.11 (m, 30H). 13 C NMR (DMSO-d 6)δ: 175.00, 174.64, 173.82, 173.52, 172.11, 172.02, 171.05, 170.95, 158.20, 157.88, 157.39, 137.79, 137.67, 137.52, 129.52, 129.34, 129.27, 126.35, 54.01, 53.61, 52.36, 51.74, 38.37, 38.31, 37.65, 35.52, 31.88, 29.98, 28.95, 27.61, 25.33, 22.92, 22.73. ESI-MS: 1431 [M+H] + , HRESI+-MS: C 69 H 96 N 12 O 21 The theoretical value for, 1431.7042 [M+H] + , Measured value: 1431.7064.

[0205] (21S,25S)-8,15,23-Trioxo-1-((4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)amino)-1-thioxo-2,7,16,22,24-pentaazaoctacosane-21,25,27-tricarboxylic acid, 6 Compound 6 was prepared in the following three steps. Commercially available N-Boc-1,4-diaminobutane (27 mg, 0.15 mmol, in 0.5 mL of DMSO) was mixed with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 2-[(4-isothiocyanatophenyl)methyl] (p-SCN-Bn-DOTA) (100 mg, 0.15 mmol, in 1.5 mL of DMSO) and DIEA (132 μL, 0.75 mmol) and stirred at 40 °C for 4 hours. The solvent was evaporated and the solid residue was purified by reverse phase C 18 flash chromatography (5.5 g, Agilent SF10) with water and acetonitrile (each with 0.1% TFA) to give Boc-protected 7 after lyophilization. Yield: approximately 55%. ESI-MS 740 [M+H] + . The compound obtained in this step was then treated with ice-cold TFA / CH 2 Cl 2(1 / 1) Treated with the solution and stirred at ambient temperature for 2 hours. The solvent was evaporated, the residue was dried under vacuum, and purified by reverse-phase flash chromatography (5.5 g, Agilent SF10) to obtain 7 in a moderate yield. 1 H NMR (DMSO-d 6 ) δ: 8.80 - 8.64 (m, 1H), 8.12 - 7.90 (m, 2H), 7.75 - 7.10 (bm, 4H), 4.65 - 4.63 (m, 1H), 4.17 - 2.59 (m, 27H), 2.40 - 1.11 (m, 6H). ESI-MS: 640 [M+1] + To a solution of 7 (11 mg, 17 μmol, in 400 μL of DMSO) was added 1 (10 mg, 17.4 μmol, in 200 μL of DMSO) and DIEA (27 μL, 170 μmol), and the mixture was allowed to stand at ambient temperature for 2 hours. After evaporation of the solvent, the residue was dissolved in water and purified by HPLC (Method 2) to obtain 6. R t , 22.5 minutes. 1 H NMR (DMSO-d 6 ) δ: 8.88 (m, 1H), 8.44 (m, 1H), 8.21 - 7.98 (m, 2H), 7.77 - 7.75 (m, 2H), 6.35 - 6.33 (m, 2H), 4.65 - 4.63 (m, 2H), 4.17 - 2.59 (m, 29H), 2.40 - 1.11 (m, 30H). HRESI-MS: C 48 H 77 N 10 O 17 Theoretical value for S, 1097.5183 [M+H] + , Measured value: 1097.5212.

[0206] 89 Y]4 To a solution of 4 (10 mg, 9.11 μmol, in 500 μL of 0.5 M NaOAc, pH 6.8) was added 50 μL of YNO 3 (0.5 M), and the mixture (pH 6.1) was incubated at 90 °C for 30 minutes. A solution of EDTA (200 μL, 30 mM, pH 6.0) was added, and the reaction mixture was incubated at 40 °C for 10 minutes to complex unreacted yttrium(III). The resulting compound was purified by HPLC (Method 2, R t ​, purified at 21 min, concentrated by evaporation, and lyophilized. ESI-MS: 1370 [M+H] + . C 60 H 87 N 11 O 20 Theoretical value for Y, 1370.5187; measured value 1370.5435.

[0207] 89 Y]5. Method 2 (R t , purification by HPLC for 26 min. ESI-MS: 1517 [M+H] + . C 69 H 96 N 12 O 21 Theoretical value for Y, [M+H] + 1516.5793; measured value 1516.5793

[0208] 89 Y]6 HPLC, method 2, R t , 23 min. HRESI+-MS. C 48 H 77 N 10 O 17 Theoretical value for SY, 1183.4007 [M+H] + ; measured value 1183.4020.

[0209] Radiochemistry: 86 Y]4 - 5 and 86 Y]6 radiolabeling was carried out by the same general method as described for 86 Y]6.

[0210] 86 Y]6 Radiolysis was prevented by adding a solution of freshly prepared ascorbic acid (50 μL, 220 μg) to a solution of 86 YNO 3 (111 - 148 MBq (3 - 4 mCi), in 500 μL of 0.1 M nitric acid). Approximately 50 - 70 μg of 6 (in 0.3 M NaOAc) (N 2 ​​​Next, purge was added to the solution over 2 - 3 minutes and neutralized by adding 60 μL of 3M NaOAc until pH was about 5.5 - 6. Subsequently, the mixture was vortexed briefly and then incubated at 95 °C for 20 minutes. The reaction mixture was diluted with 1 mL of water. Complexation was monitored by injecting an aliquot of 10 - 15 μL of the solution into HPLC. The radiolabeled product 86 Y]6, when measured by ITLC (Gelman ITLC strip, 10 mM EDTA), was obtained with a radiochemical yield of about 90 - 95% and a radiochemical purity > 98%. A broad radioactive peak was at R t , about 13.9 - 14.8 minutes for the desired product as a mixture of isomers, and R t for the free ligand was 15.8 minutes. The specific activity was > 83.92 GBq / μmol (n = 5). The acidic eluate was neutralized with 20 μL of 1M sodium carbonate solution and the volume of the eluate was reduced until dry under vacuum. For biodistribution and imaging studies, the solid residue was diluted with saline to the desired radioactivity concentration. Interestingly, after neutralization and evaporation of the elution peak, when the tracer was reinjected into HPLC, only one peak was isolated around 14.3 minutes. 86 To confirm the isomerization of 86 Y]6, compound 6 was radiolabeled with carrier - added 86 Y and the mixture was analyzed by HPLC. Only one peak was isolated at 14.3 minutes. 86 For 86 Y]4 - 5, a single radiolabeled peak was isolated. 86 The R t of t Y]4 was 14.0 minutes and the R t of non - chelated 4 was 15.5 minutes, 86 and for t Y]5, R t = 16.9 minutes and for non - chelated 5, R t = 19.5 minutes.

[0211] Animal models and assays PSMA inhibitory activity was measured using a fluorescence - based assay (Banerjee, Pullambhatla, Byun, et al., 2011). The enzyme inhibition constant (K iThe value) was determined using the Cheng-Prusoff transformation (Cheng and Prusoff, 1973). Sublines of androgen-independent PC-3 human prostate cancer xenografts were used (Banerjee, Pullambhatla, Byun, et al., 2011). These sublines are genetically engineered to express high levels of PSMA (PC-3 PIP) or produce low levels of native PSMA (PC-3 flu) (Dr. Warren Heston, Cleveland Clinic, Cleveland, Ohio).

[0212] Both PSMA-expressing (PC-3 PIP) and non-expressing (PC-3 flu) cell lines were grown as previously described in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen) and 1% Pen-Strep (Biofluids) (Banerjee, Pullambhatla, Byun, et al., 2011).

[0213] Six- to eight-week-old male non-obese diabetic / severe combined immunodeficient (NOD) / SCID mice (Charles River Laboratories) were subcutaneously (SC) implanted with PSMA+ PC-3 PIP and PSMA- PC-3 flu cells (2x10 6 , in 100 μL of Matrigel) into the right cranial and left flank, respectively. Mouse imaging or biodistribution assays were performed when the diameter of the xenograft reached 5-7 mm.

[0214] For biodistribution assays, NOD / SCID mice bearing PSMA+ PC-3 PIP and PSMA- PC-3 flu xenografts received 0.55 MBq (15 μCi) of 86 Y-4 or 86Y-6 was injected. In each case, four mice were sacrificed by cervical dislocation at 1, 2, 5, and 24 hours after injection. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidneys, muscle, small intestine, large intestine, bladder, and PSMA+PC-3 PIP and PSMA-PC-3 flu tumors were quickly removed. A 0.1 mL blood sample was also collected. Each organ was weighed and the tissue radioactivity was measured with an automatic gamma counter (1282 Compugamma CS, Pharmacia / LKB Nuclear Inc.). The percentage of injected dose per gram of tissue (%ID / g) was calculated using serial dilution samples of the injected radioactivity. All radioactivity measurements were corrected for radioactive decay up to the time of injection.

[0215] Animal imaging Small animal PET and CT For imaging studies, NOD / SCID mice bearing PSMA+PC-3 PIP and PSMA-PC-3 flu tumors were anesthetized at 3% and maintained under 1.5% isoflurane (v / v). The mice ( 86 Y-4 or 86 For Y-4 and 86 Y-6, n = 3, 86 and for Y-5, n = 2) were injected via the tail vein with 3.33 - 6.21 MBq (90 - 168 μCi) of a radiotracer formulated in 100 μL of physiological saline at pH approximately 7. For the binding specificity study, a blocking dose of the known PSMA inhibitor N-[[[(S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ43) (Olszewski et al., 2004) (50 mg / kg) was 86 subcutaneously injected 30 minutes prior to the injection of Y-4, and in another mouse 86Only Y-4 was injected. At different time points, individual anesthetized mice were placed in a prone position on the scanner gantry, fixed with medical tape, and the flow rate of the anesthetic was increased to 0.8 L / min. Images were reconstructed using the FORE / 2D-OSEM method (2 iterations, 16 subsets) and corrected for radioactive decay, scanner dead time, and scattered radiation. Partial volume correction (PVC) was not performed. After each PET scan, a CT scan was performed for anatomical co-registration. A special bed for mice that fit both the PET and CT scanners was used to smoothly align the PET and CT images. The mice were under anesthesia and could not move during transfer between scanners and during both scans. Next, the reconstructed PET and CT images were manually aligned by aligning natural landmarks (e.g., the limbs of the mouse and the outline of the bed) through rigid body transformation using the AMIDE software (sourceforge.net / amide). The data was displayed and analyzed with AMIDE.

[0216] Dynamic whole-body PET and CT images were acquired using an eXplore VISTA small animal PET (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and an X-SPECT mini SPECT / CT system (Gamma Medica Ideas, Northridge, California), respectively.

[0217] 86 PET Imaging of Y-6 in Anubis Baboons (Papio anubis) Male Anubis baboons (8 years old, 27.1 kg) were used 86The biodistribution of Y-6 was studied. During imaging, the monkeys were positioned in the supine position. For attenuation correction, low-dose CT images were acquired immediately before the first and last PET images. PET and CT images were aligned between time points using a Hermes workstation (Hermes Medical Solutions, Greenville, South Carolina). The contours of 14 source organs were delineated on CT with the aid of fused PET / CT images. The decay-corrected mean radioactivity concentration (Bq / g) was extracted from the PET images for each source organ. Contours were drawn on the PET images of the kidneys, renal cortex, and prostate. The decay-corrected total radioactivity per organ quantified within the PET images showed a nearly 1:1 correspondence with the administered radioactivity for the first six time points (within 1 hour), confirming that all of the administered radioactivity was utilized in the PET images, after which the total amount decreased below the administered dose by urinary excretion. Whole-body retention kinetics were obtained using the total radioactivity quantified in each PET image.

[0218] Nine static PET images were acquired at 5, 10, 15, 20, 35 minutes, 1, 2, 3, 5, and 23 hours after intravenous administration of 86 80.7 MBq (2.2 mCi) of Y-6 as a bolus. The images were acquired in 2D mode using a Discovery Rx VCT scanner (GE Healthcare).

[0219] Radiation Dosimetry For each time point, the radioactivity concentration (Bq / cm 3) was measured in each of the 14 imaged organs and multiplied by the volume of the organ to obtain the total radioactivity per organ at each time point. Next, the measured values were decay-corrected and divided by the mass of the baboon organ determined from the CT density and the volume of the delineated contour and the injected radioactivity, to obtain the ratio to the initial radioactivity per gram (FIA / g) for each time point and each organ. Next, the baboon FIA / g values were converted to human FIA (per organ) using the following equation (Schwartz et al., 2011; Woodard et al., 1975).

[0220]

Equation

[0221] where the body mass ヒヒ = 27.1 kg, and the body mass ヒト = 73.7 kg.

[0222] This approach assumes that the radioactivity concentration in a specific tissue relative to the overall concentration throughout the body is conserved across species (i.e., organ concentration / whole body concentration is the same for baboons and humans). The obtained human FIA values were plotted as a function of time for each organ (9 data points) and fit to a biexponential expression.

[0223]

Equation

[0224] where A1, A2, λ1 bio and λ2 bio are fitting parameters. The sum of A1 and A2 gives the ratio at zero time that is back-extrapolated to the administered radioactivity in each organ, and λ1 bio and λ2 biois the biological clearance constant. For each source organ, the equation for the time-integrated activity coefficient (TIAC, previously known as the retention time (Bolch et al., 2009)) is obtained by integrating Equation (2), as the name implies, and introducing the physical decay term λ φ (which depends on the isotope used).

[0225]

Number

[0226] For TIAC 90 Y,[[]] 177 Lu and 86 Y, the corresponding physical decay constants excreted in urine are respectively: 90 Y λφ = 0.01083 h -1 (T 1 / 2 = 64.0 hours); 177 Lu λφ = 0.00429 h -1 (T 1 / 2 = 161.52 hours) and 86 Y λφ = 0.04702 h -1 (T 1 / 2 = 14.74 hours) are used for calculation. The radiation absorbed dose is obtained by converting the time-integrated radioactivity to the absorbed dose according to the MIRD absorption fraction method (Bolch et al., 2009) using the OLINDA / EXM software (Stabin et al., 2005). The TIAC for the bladder is obtained using the MIRD bladder model implemented in OLINDA / EXM. The input to the model requires the whole-body TIAC, which is fitted to the whole-body retention kinetics

[0227]

Number

[0228] It was obtained from the equation. The urination interval was set to 2 hours. Next, TIAC was input into OLINDA / EXM (Stabin et al., 2005), and the absorbed dose per unit radioactivity was obtained for 14 organs. The renal cortex dose value was obtained by using a specific kidney model in OLINDA / EXM. The absorbed dose from the organs outside the kidney was added to the renal cortex dose calculated from the internal kidney model. A special prostate model was used for the prostate self-dose, and the external dose to the bladder was added to the prostate dose as a substitute for the whole body. The self-dose component of the absorbed dose per unit radioactivity for the salivary gland was obtained using 3D-RD Monte Carlo (EGSnrc) and human CT images of the salivary gland. The cross-dose component was obtained assuming that the cross-dose of an organ of the same size (pancreas) was the same.

[0229] The measured radioactivity concentration (Bq / cm 3 ) value for each time point per organ was decay-corrected and divided by the mass of the organ of the baboon obtained from the CT density and the volume of the outlined contour and the injected radioactivity, and the ratio to the initial radioactivity per gram for each time point and each organ was obtained (FIA / g). Next, the baboon FIA / g values were converted using an equation related to human FIA (per organ) (Olszewski et al., 2004; Schwartz et al., 2011). Next, the obtained human FIA values were plotted as a function of time and fitted to a biexponential expression, and the value of the time-integrated activity coefficient (TIAC, previously known as the retention time (Woodard et al., 1975)) for each source organ was calculated. The radiation absorbed dose was obtained by converting the time-integrated activity to the absorbed dose using the MIRD absorption method (Woodard et al., 1975) with OLINDA / EXM software (Bolch et al., 2009).

[0230] Data were calculated using Microsoft Excel (Microsoft Corporation, 2010) and presented as mean ± standard deviation (SD). Statistical significance at the 95% confidence level was determined using Prism software (GraphPAD), and P ≤ 0.05 was considered significant.

[0231] Results Compounds 4 and 5 were prepared using the solid-phase liquid-phase combinatorial peptide synthesis methods shown in Schemes 4 and 5. Compounds 1 and 4 were prepared as previously reported (Banerjee, Pullambhatla, Byun, et al., 2011). The synthesis of the DOTA-conjugated ligand 5 was carried out using standard fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS), starting from Fmoc-Lys(Boc)-Wang resin and following Scheme 5. Three phenylalanine residues were coupled to the resin-bound lysine, followed by conjugation with DOTA, and then the compound was cleaved from the resin with a 1 / 1 mixture of TFA / CH 2 Cl 2 to give 3 in moderate yield (ca. 20%). Next, the free ε-amino group of the lysine in 3 was conjugated with 1 (Davis et al., 2009) to give 5. Compound 6 was synthesized by reacting commercially available DOTA-benzyl-isocyanate and N-Boc-1,4-diaminobutane (in DMSO) at 40 °C for 4 h in the presence of diisopropylethylamine, followed by removal of the Boc group and purification by HPLC to give 7 in moderate yield. Next, conjugation of compound 7 with 1 gave 6 in good yield. Stable yttrium ( 89 Y) complexes were prepared by incubating the conjugates 4 - 6 with an aqueous solution of YNO 3 at 95 °C as shown in Schemes 4 - 5. 86 / 89 Y(III)-labeled compounds 4 - 5 contain three carboxylic acids coordinated to the metal, which renders the compound neutral overall, but 86 / 89It should be mentioned that Y]6 has four coordinating carboxylic acids, thereby becoming a negatively charged compound as a whole. Radiotracer 86 Y]4 - 6 were prepared by using the same general procedure 86 Y]NO 3 in a reaction with a ligand concentration of 10 -6 M in boiling water at pH 5 - 6 for 30 minutes.

[0232]

Chemical Structure

[0233] Scheme 4: Synthesis of 4 - 5 and 86 / 89 Y]4 - 5

[0234]

Chemical Structure

[0235] Scheme 5: Synthesis of 6 and 86 / 89 Y]6

[0236] 86 Y - labeled PSMA - targeting compound, 86 Y - 4, 86 Y - 5 and 86The chemical structure of Y-6 is shown in Figure 14. The radiolabeling of the target compounds proceeded with high yields (about 90 - 97%) and high radiochemical purity (>98%), with high specific radioactivity (>83.92 GBq / μmol (2.27 Ci / μmol)). All compounds showed high binding affinity, and the K i values were 0.10 - 4.69 nM (Table 2).

[0237]

Table 2

[0238] Small animal PET imaging Whole body PET / CT images were 86 obtained for Y-4, 86 Y-5 and 86 Y-6 (Figures 18A, 18B, 18C, 19A, 20A, 20B, and 20C). Visualization was possible for all three radiotracers at 2 hours after injection into the PSMA+PC-3 PIP tumor and the kidney, a known PSMA-expressing organ (Figures 18A, 18B, and 18C). Renal uptake of the radiotracer is due not only to specific uptake by the expression of PSMA in mouse proximal tubules but also in part to the excretion pathways of these substances (Stabin et al., 2005). Substance 86 Y-5 showed non-specific accumulation in the gastrointestinal tract, presumably due to increased hydrophobicity resulting from the three Phe residues on the linker portion. 86 PET-CT images of Y-4 were acquired at 1, 4, and 18 hours after injection, taking into account the short biological half-life of this class of low molecular weight compounds. The presence of the radiotracer in the PSMA+PC-3 PIP tumor and the kidney and bladder was observed up to 4 hours maximum (Figure 19A). The radioactivity in the bladder and kidney disappeared significantly by 18 hours. However, some radioactivity remained in the PSMA+PC-3 PIP tumor. As a further test of in vivo binding specificity, 86The blocking study of Y-4 was conducted by pre-treating animals with 50 mg / kg of the potent and selective PSMA inhibitor ZJ43 (Silver et al., 1997). Figure 19B shows that ZJ43 can block the binding of Y-4 not only in tumors but also in the renal cortex, another PSMA-expressing tissue (Stabin et al., 2005). Figures 20A, 20B, and 20C are PET-CT images of Y-6 at 0.5, 2, and 12 hours after injection. Importantly, 86 Y-6 shows faster radiotracer clearance from normal tissues, and by 12 hours after injection, most of the radioactivity was excreted from the kidneys, resulting in a clean tumor / background contrast. Clear imaging of PSMA+PC-3 PIP tumors was achieved as early as 15 minutes later. In particular, 86 Y-6 86 does not contain the additional phenylalanine moiety of Y-4 and Y-5, and utilizes the p-isothiocyanatobenzyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, which adds additional carboxylates to strongly retain the metal and also reduces lipophilicity. 86 Y-6 86 Y-4 and 86 Based on the imaging results, compounds

[0239] In Vivo Distribution in Mice Y-4 and 86 Y-6 were further evaluated in a standard in vivo distribution assay (Banerjee, Pullambhatla, Byun, et al., 2011). Tables 3 and 4 show the %ID / g uptake values in selected organs at 1, 2, 5, and 24 hours after injection. Both radiotracers showed PSMA-dependent binding in PSMA+PC-3 PIP tumor xenografts, 86 and 86Y-4 showed high tumor uptake as early as 1 hour after injection (29.3±8.7% ID / g), with relatively slow clearance, reaching 15.7±1.7% ID / g at 5 hours and 5.9±0.8% ID / g at 24 hours after injection. The PSMA+PC-3 PIP tumor to PSMA-PC-3 flu tumor uptake ratio was high, ranging from 89 at 1 hour to 229 at 24 hours. Blood and normal tissues, such as the heart, liver, stomach, and pancreas, showed no significant uptake (about 1% ID / g) and decreased to less than 0.02% ID / g at 24 hours. The PSMA+PC-3 PIP tumor to muscle ratio was also high, reaching a maximum of 1046 at 24 hours. Renal uptake was predictably high, peaking at 244.9±8.8% ID / g at 1 hour and decreasing to 1.5±0.7% ID / g by 24 hours.

[0240] Table 4 shows 86 the organ % ID / g uptake values for Y-6. The compound 86 Y-6 accumulated rapidly within 1 hour after injection in PSMA+PC-3 PIP tumors, with an uptake value of 26.6±1.9% ID / g. The radiotracer concentration continued to increase within the PSMA+PC-3 PIP tumor, showing a maximum uptake of 32.2±8.0% ID / g at 5 hours after injection. Tumor uptake remained high until 24 hours after injection. Normal organs, such as blood, heart, liver, spleen, stomach, and pancreas, had low uptake at 1 hour and decreased to less than 0.4% ID / g by 5 hours. 86 Renal uptake for Y-6 was (86.5±13.6% ID / g and 54.0±9.2% ID / g at 1 hour and 2 hours, respectively) 86 much less than that of Y-4.

[0241]

Table 3

[0242]

Table 4

[0243] Hihi PET imaging and 86 Pharmacokinetics of Y-6 Figures 21A and 21B show PET studies where the radiotracer is seen in the liver, salivary glands, kidneys, and bladder. For the whole kidney, renal cortex, and prostate, contours were drawn on each PET image for quantification. All organs showed biphasic (rapid and slow) biological clearance. The kidney showed the highest uptake at approximately 25 minutes after injection (8% ID / g). Sixty-eight percent of the radioactivity seen in the kidney disappeared with a biological half-life of approximately 1 hour (0.84 hours), and the remaining radioactivity disappeared with a biological half-life of 16.6 hours. Most (66%) of the radioactivity in the renal cortex disappeared with a biological half-life of 1.1 hours, and the remaining radioactivity disappeared with a biological half-life of approximately 19 hours. Marked uptake and retention were seen in the liver and salivary glands. However, 68 Ga-labeled PSMA targeting substance and 124 / 131 It was mild compared to the PET scans of patients imaged with I-MIP-1095 (Zechmann et al., 2014). Table 5 summarizes the biological clearance kinetics of all organs. The TIAC used in the dose calculation is shown in Table 6.

[0244]

Table 5

[0245]

Table 6

[0246] Organ absorbed dose Table 7 is expressed in units of mGy / MBq 86 Y, 90 Y / 177This is a detailed list of organ absorbed doses for Lu. For all isotopes, the renal cortex received the highest absorbed dose per unit radioactivity. Therefore, when formulating a patient-specific absorbed dose treatment plan, the renal cortex is considered to be the dose-limiting organ for therapeutic radioactive metals (Baechler et al., 2012; Hobbs et al., 2009), followed by the bladder. Diagnostic isotopes 86 For Y, the effective dose of 0.099 mSv / MBq was also calculated with OLINDA / EXM.

[0247]

Table 7

[0248] Discussion Three 86 Y-labeled PSMA targeting substances were synthesized and evaluated. These compounds contain DOTA- or DOTA mono-amide chelated radioactive metals bound to the same target urea as those previously reported (Banerjee et al., 2010; Banerjee, Pullambhatla, Byun, et al., 2011). DOTA and its derivatives have been noted for their ability to be used in both PET ( 86 Y) and radiopharmaceutical therapy ( 90 Y). Pharmacokinetics have been reported to depend on the radioactive metal chelating agent used, including those for compounds specifically designed to bind to PSMA. Without intending to be bound by a particular theory, this is thought to be mainly due to the total charge of the radioactive ligand and the stability of the metal chelate complex. Specifically, 68 In previous reports on 68 Ga-labeled PSMA-binding DOTA conjugate substances, 86 Ga-4 showed the fastest clearance from normal tissues, including the kidney (Banerjee et al., 2010). However, in this study, 86From the evaluation of Y-6, the desirable low kidney uptake and high tumor retention required for radiotherapy were demonstrated, and it was subsequently selected for quantitative PET imaging in baboons for dosimetry.

[0249] In the binding specificity study (Figure 19B), after 1 hour, 86 It was shown that almost all of the binding of Y-4 in the kidney is specific and not due to excretion. The evidence suggests that the fact that renal parenchyma has more tissue and faster blood flow than tumors may not be the reason for the longer retention in tumors than in the kidney for many of these substances. PSMA binding affinity is one factor thought to determine tumor-to-kidney uptake, but other factors such as lipophilicity, charge, plasma protein binding, and molecular weight are also thought to play important roles. The estimated renal cortex dose of 1.19 mGy / MBq ( 90 Y) and 0.245 mGy / MBq ( 177 Lu) was not inferior to the values of 1.97 mGy / MBq ( 90 Y) and 0.45 mGy / MBq ( 177 Lu) calculated in reports involving peptide receptor radiotherapy (Baechler et al., 2012) (where the renal cortex was the dose-limiting organ).

[0250] The commonly used and clinically utilized chelating agent DOTA was used for all three radiopharmaceuticals. This is because DOTA and many of its derivatives are known to form kinetically and thermodynamically stable complexes. The corresponding Y(III) complexes have been shown to be stable in many cases (a desirable feature for a chelating agent). Importantly, DOTA 86 forms stable complexes with many trivalent metal ions, including lanthanides such as 177 Lu(III) and actinides such as 225 Ac(III), which are chemically heterogeneous from Y(III). Furthermore, the PSMA-binding urea-based substances are stable under the radiolabeling conditions used for DOTA.

[0251] In recent years,90 Y or 177 The Lu-labeled version of the PSMA-targeted monoclonal antibody J591 has shown promising results in phase 1 and phase 2 clinical trials (Bander et al., 2005; Tagawa, Akhtar, et al., 2013; Tagawa, Milowsky, et al., 2013). In those cases, 111 In-labeled antibodies were used for dosimetry calculations (Vallabhajosula et al., 2005). Although those radiolabeled monoclonal antibodies hold promise for tumor detection and treatment, their limited tumor target specificity and relatively high absorbed dose to the red bone marrow act against routine clinical use. As an alternative approach, 131 The initial clinical results using I-labeled PSMA-targeted urea-based small molecules have shown high dose delivery to the lesions (Zechmann et al., 2014). In these published studies, the salivary glands showed the highest absorbed dose (4.62 mGy / MBq), followed by both the liver (1.47 mGy / MBq) and the kidneys (1.45 mGy / MBq) (Zechmann et al., 2014). It is thought that the uptake of free iodine contributes most to this salivary gland absorbed dose, and is also evidenced by a relatively high (0.91 mGy / MBq) thyroid absorbed dose, which does not occur in this study. Generally, the clearance rate from normal organs, except for the kidneys, 86 is faster for Y-6 than the published results (Zechmann et al., 2014).

[0252] In summary, the biodistribution and dosimetry results suggest that 86 Y-6 is a promising candidate for quantitative PET imaging of PSMA-expressing tumors, and that PSMA-targeted 90 Y-, 177 could be a suitable imaging alternative for the planning and monitoring of Lu-based radiopharmaceutical therapy.

[0253] Example 4 For PSMA-based targeted radionuclide therapy 177 Lu-SR-VI-71,203 Pb-SR-VI-71 and 203 Pb-SR-IX-11 0.01 to 10 μCi of 177 Figure 24, which is a cell uptake study using Lu-SRVI71, shows high uptake in PSMA+PIP and very little uptake in PSMA-flu tumors. In addition, internalization studies revealed that approximately 44% of the total cell-bound radioactivity internalizes. Furthermore, incubation with 10 μM of N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ43), a PSMA-specific inhibitor, resulted in approximately 90% blockade in PSMA+ cells, further confirming the excellent specificity of the agent (Figure 23). In vivo evaluation was performed by standard PSMA+PIP and PSMA-flu mouse xenografts and SPECT imaging with the VECT or ultra-high sensitivity mouse collimator disclosed in Figures 25A, 25B, and 25C. The highest accumulation of radioactivity was seen in PSMA+PIP tumors at all time points. The other visible organs were the kidneys and bladder. In vivo distribution studies at 2 hours (59.1 ± 12.8%ID / g) and 24 hours (40.6 ± 5.8 ID / g) (n = 4) revealed high uptake and retention in PSMA+ tumors with high specificity (approximately 180 PIP:flu at 2 hours). Initial uptake in the kidneys was high at 89.3 ± 28.9% at 2 hours, followed by rapid clearance within 24 hours (6.29 ± 3.4%ID / g).

[0254]

Table 8

[0255]

Table 9

[0256]

Table 10

[0257] These results are highly promising regarding the feasibility of preparing such low molecular weight seranostic substances with pharmacokinetics desirable for radionuclide therapy. Furthermore, the data support that low molecular weight substances of this class can effectively translocate internally when bound to PSMA.

[0258] Example 5 Overview of the synthesis and use of ZCP-01 and related substances for PSMA-based targeted radionuclide therapy The preparation and use of PSMA-binding ureas conjugated to chelated radioactive metals via various linking groups for imaging of PSMA-expressing tumors and possible radiotherapy have been described not only in this patent application but also in several patents and literature (Banerjee, et al., 2008; Banerjee, et al., 2010; Banerjee, et al., 2011; Banerjee, et al., Oncotarget 2011; Banerjee, et al., 2013; Banerjee, et al., 2014). Novel lysine-carbamate scaffold oxypentanedioic acid (OPA) corresponding to the carbamate scaffold and amino-pentanedioic acid (NPA) corresponding to the "reverse" carbamate scaffold, including F-18 labeled analogs, have been developed in recent years. The F-18 labeled NPA and OPA compounds showed selective uptake in PSMA-positive tumor xenografts in mice.

[0259] ZCP-01 and DOTA-PEG-conjugated lysine OPA carbamate for complexing radioactive metals for imaging and radiotherapy of PSMA-positive tumors and tissues have been synthesized as examples. By binding the broad-spectrum metal chelating ligands and linkers disclosed so far in WO 2009 / 002529 (A2) and WO 2010 / 108125 (A2) pamphlets for use with urea to the OPA and NPA scaffolds, novel radiolabeled substances for prostate cancer imaging and / or radiotherapy can be obtained.

[0260] Materials and Methods (18S,22S)-2,12,20-Trioxo-1-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-6,9,21-trioxa-3,13,19-triazatetracosan-18,22,24-tricarboxylic acid, ZCP-01 Referring to Scheme 7, diisopropylethylamine (27 μL, 0.255 mmol) was added to a solution of Compound 4 (8.5 mg, 0.015 mmol, in 200 μL of DMSO), followed by slow addition of DOTA-NHS (15.2 mg, 0.023 mmol, in 200 μL of DMSO), and the resulting solution was stirred at room temperature for 2 hours. Next, the solution was diluted with water and purified by HPLC. HPLC method: Phenomenex C 18 Luna, 10 mm * 250 mm, flow rate: 8 ml / min, λ: 200 nm, 220 nm, solvent H 2 O and CH 3 CN (each 0.1% TFA). Gradient method: 0 - 20 minutes, 100 / 0 H 2 O / CH 3 CN ~ 80 / 20 H 2 O / CH 3 CN; 20 - 30 minutes 80 / 20 H 2 O / CH 3 CN ~ 0 / 100 H 2 O / CH 3 CN; 31 minutes 100 / 0 H 2 O / CH 3 CN. HPLC retention time (t r ) = 16 minutes. ESI-MS: 954 (M + H). Yield: 9.4 mg (about 65.7%) after HPLC purification.

[0261] 113 / 115 Preparation of In-ZPC-01 To a solution of ZPC-01 (5 mg, 5.24 μmol, in 500 μL of 0.5 M NaOAc, pH 6.8) was added 50 μL of InNO 3(0.5 M) was added and the mixture (pH 6) was incubated at 90 °C for 30 minutes. A solution of EDTA (200 μL, 30 mM, pH 6.0) was added and the reaction mixture was incubated at 40 °C for 10 minutes to complex unreacted indium(III). The resulting compound was purified by HPLC (same as ZPC-01), concentrated by evaporation, and lyophilized. ESI-MS: 1066 [M+H] + C 39 H 64 InN 7 O 20 Theoretical value for, 1065.79.

[0262] 111 Preparation of In-ZPC-01 1.0 μl of 111 InCl 3 (1 mCi) was added to 1 mM Ourea-PEG-DOTA in 20 μl of 0.2 M NaOAc. The pH of the mixture was approximately 4.0. Next, 20 μl of 0.2 M NaOAc to adjust the pH to approximately 6. The mixture was maintained at 50 °C for 1 hour and purified by radio-HPLC using a gradientless method containing mobile phase 90% water (containing 0.1% TFA) and 10% CH 3 CN (0.1% TFA). Flow rate: 1.0 mL / min; λ: 200 nm and C 18 column (25 x 4.6 mm), Varian microsob-MV 100-5. Radiolabeled 111 In]ZPC-01 eluted at 14.9 minutes and the unlabeled chelating agent eluted at 32 minutes

[0263] Scheme 7

[0264]

Chemical Structure

[0265] Results ZCP-01 and [In]-ZCP-01 showed high binding affinity, with Ki values of 17.82 nM to 58.21 nM and 0.29 μM to 0.92 μM, respectively (Table 8).

[0266]

Table 11

[0267] As shown in FIGS. 34A, 34B, and 34C, in vivo SPECT imaging of [In]-ZCP-01 was performed on mice with PSMA+PC3 PIP and PSMA-PC3 flu tumor xenografts subcutaneously implanted in the right and left flanks, respectively 111 after intravenous injection of [In]-ZCP-01. However, 111 [In]-ZCP-01 enabled visualization of the PSMA+PC3 PIP tumor and the kidney, a known PSMA-expressing organ, 2 and 4 hours after injection, but showed non-specific uptake in the flu tumor. By 24 hours after injection, most of the radioactivity had been excreted from the tumor and the kidney.

[0268] These results are very promising regarding the feasibility of preparing such low-molecular-weight seranostic substances with pharmacokinetics desirable for tumor imaging and radiotherapy.

[0269] References All publications, patent applications, patents, and other references cited herein are indicative of the level of those skilled in the art to which the disclosed subject matter pertains. All publications, patent applications, patents, and other references are hereby incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference were specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references have been cited herein, such citation is not to be construed as an admission that these documents form part of the general knowledge in the art.

[0270] International PCT Patent Application Publication No. PCT / US2008 / 007947 to Pomper, M.G., Ray, S., Mease, R.C., Foss, C. for Labeled inhibitors of prostate specific membrane antigen (PSMA), biological evaluation, and use as imaging agents, published 2008 / 12 / 31 (WO 2009 / 002529 A2). International PCT Patent Application Publication No. PCT / US2010 / 028020 to Pomper, M.G., Mease, R.C.; Ray, S., Chen, Y. for PSMA-targeting compounds and uses thereof, published 2010 / 09 / 23 (WO2010 / 108125 A2). International PCT Patent Application Publication No. PCT / US2008 / 013158 to Chandran, S.S., Ray, S., Denmeade, S.R., Pomper, M.G., Mease, R.C. for Prostate specific membrane antigen (PSMA) targeted nanoparticles for therapy of prostate cancer, published 2009 / 06 / 04 (WO2009 / 070302 A1). Aime, S., et al. Curr Pharm Biotechnol 2004, 5, 509 - 518. Artemov, D. Molecular magnetic resonance imaging with targeted contrast agents. J.Cell.Biochem. Oct 15 2003;90(3):518 - 524. Artemov, D., Mori N., Okollie, B., Bhujwalla, Z.M. Magn Reson Med 2003, 49, 403 - 408. Artemov, D., Mori N., Ravi, R., Bhujwalla, Z.M. Cancer Res 2003, 63, 2723 - 2727. Babich, J.W., Zimmerman, C., Joyal, J., Lu, G. Radiolabeled prostate specific membrane antigen inhibitors. US 2013 / 0034494 A1. Baechler, S., et al. Three - dimensional radiobiological dosimetry of kidneys for treatment planning in peptide receptor radionuclide therapy. Med.Phys.2012;39:6118 - 6128. Bander, N.H., et al. Phase I trial of 177 lutetium-labeled J591, a monoclonal antibody to prostate-specific membrane antigen, in patients with androgen-independent prostate cancer. J.Clin.Oncol. 2005;23:4591-4601. Banerjee, S.R., et al. Synthesis and evaluation of technetium-99m- and rhenium-labeled inhibitors of the prostate-specific membrane antigen (PSMA). J.Med.Chem. 2008;51:4504-4517. Banerjee, S.R., Pullambhatla, M., Byun, Y., et al. 68 Ga-labeled inhibitors of prostate-specific membrane antigen (PSMA) for imaging prostate cancer. J.Med.Chem. 2010;53:5333-5341. Banerjee, S.R., Pullambhatla, M., Shallal, H., et al. Sequential SPECT and Optical Imaging of Experimental Models of Prostate Cancer with a Dual Modality Inhibitor of the Prostate-Specific Membrane Antigen. Angew.Chem.Int.Ed.Engl. Sep 19 2011;50(39):9167-9170. Banerjee, S.R., et al. A modular strategy to prepare multivalent inhibitors of prostate-specific membrane antigen (PSMA). Oncotarget. 2011;2:1244-1253. Banergee, S.R., Pullambhatla, M., Foss, C.A., Falk, A., Byun, Y., Nimmagadda, S., Mease, R.C., Pomper, M.G. Effect of chelators on the pharmacokinetics of 99mTc-labeled imaging agents for the prostate-specific membrane antigen (PSMA). J.Med.Chem. 2013; 56: 6108-6121. Banergee, S.R., Pullambhatla, M., Foss, C.A., Nimmagadda, S., Ferdani, R., Anderson, C.J., Mease, R.C., Pomper, M.G. 64Cu-Labeled inhibitors of prostate-specific membrane antigen for PET imaging of prostate cancer. J.Med.Chem. 2014; 57: 2657-2669. Banerjee, S.R., Foss, C.A., Pullambhatla, M., Wang, Y., Srinivasan, S.R.F.K.E.Hobbs, M.Baidoo, R.C.Brechbiel, Mease, G.Sgouros, M.G.Pomper, Preclinical evaluation of 86 Y-labeled inhibitors of prostate specific membrane antigen for dosimetry estimates. J Nucl Med 2015. Baur, B., et al. Synthesis, radiolabelling and in vitro characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-labelled PSMA ligand, CHX-A’’-DTPA-DUPA-Pep. Pharmaceuticals. 2014;7:517-529. Behnam Azad, B., et al. Nanoscale 2015, 7, 4432-4442. Bodei, L., et al. Receptor radionuclide therapy with 90Y-[DOTA]0-Tyr3-octreotide (90Y-DOTATOC) in neuroendocrine tumours. Eur. J. Nucl. Med. Mol. Imaging. 2004;31:1038-1046. Bolch, W.E., et al., MIRD pamphlet No.21: a generalized schema for radiopharmaceutical dosimetry--standardization of nomenclature. J. Nucl. Med. 2009;50:477-484. Boros, E., et al. J Am Chem Soc 2012, 134, 19858-19868. Caravan, P., et al. Inorg Chem 2007, 46, 6632-6639. Caravan, P., et al. Contrast Media Mol Imaging 2009, 4, 89-100. Chandran, S.S., et al., Characterization of a targeted nanoparticle functionalized with a urea-based inhibitor of prostate-specific membrane antigen (PSMA).Cancer Biol.Therapy.2008;7:974-982. Chappell, L.L.et al.Synthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb, Nucl Med Biol, 27 (2000) 93-100. Chen, Z., Penet, et al.PSMA-targeted theranostic nanoplex for prostate cancer therapy.ACS Nano.2012;6:7752-7762. Cheng, Y.and Prusoff, W.H.Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.Biochem.Pharmacol.1973;22:3099-3108. Cho, S.Y.et al.Biodistribution, tumor detection, and radiation dosimetry of 18 F-DCFBC, a low-molecular-weight inhibitor of prostate-specific membrane antigen, in patients with metastatic prostate cancer.J.Nucl.Med.2012;53:1883-1891. Davis, S.L., et al. Bacterial thymidine kinase as a non-invasive imaging reporter for Mycobacterium tuberculosis in live animals. PloS one 2009; 4:e6297. De Leon-Rodriguez, L.M., et al. MRI detection of VEGFR2 in vivo using a low molecular weight peptoid-(Gd)8-dendron for targeting. J.Am.Chem.Soc. Sep 22 2010;132(37):12829-12831. Evans, M.J., et al. Proc Natl Acad Sci U S A 2011, 108, 9578-9582. Frenzel, T., et al. Invest Radiol 2008, 43, 817-828. Geninatti-Crich, S. Contrast Media Mol Imaging 2011, 6, 421-425. Ghosh, A. and Heston, W.D. Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer. J.Cell.Biochem. 2004;91:528-539. Haffner, M.C., et al. Hum Pathol 2009, 40, 1754-1761 Haffner, M.C., et al. Mod Pathol 2012, 25, 1079-1085. Hanaoka, K., et al. Magn Reson Imaging 2008, 26, 608-617. Helisch, A., et al. Pre-therapeutic dosimetry and biodistribution of86 Y-DOTAPhe1-Tyr3-octreotide versus 111In-pentetreotide in patients with advanced neuroendocrine tumours.Eur.J.Nucl.Med.Mol.Imaging.2004;31:1386-1392. Hillier, S.M., et al.J Nucl Med 2011, 52, 1087-1093. Hobbs, R.F., et al. 124 I PET-based 3D-RD dosimetry for a pediatric thyroid cancer patient: real-time treatment planning and methodologic comparison.J.Nucl.Med.2009;50:1844-1847. Huang, C.H.and Tsourkas, A.Curr Top Med Chem 2013, 13, 411-421. Kaiser, E., et al.Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.Anal Biochem 1970;34:595-598. Kam, B.L., et al.Lutetium-labelled peptides for therapy of neuroendocrine tumours.Eur.J.Nucl.Med.Mol.Imaging.2012;39 Suppl 1:S103-112. Konda, et al.Magma 2001, 12, 104-113. Kulkarni, H.M.W., et al. First clinical results with Lu-177 PSMA-TUM1 for the treatment of castrate-resistant metastatic prostate cancer. J. NUCL. Med. Meeting Abstract Vol 55; 2014. Lanza, M., et al. J Nucl Cardiol 2004, 11, 733-743. Low, P.S., Chelvam, V., Kim, Y. PSMA binding linker conjugates and methods for using WO2011 / 106639, WO 2010 / 045598 A2, WO 2009 / 026177A1. Ma, D., et al. Radioimmunotherapy for model B cell malignancies using 90 Y-labeled anti-CD19 and anti-CD20 monoclonal antibodies. Leukemia. 2002;16:60-66. Major, J.L. and Meade, T.J. Acc Chem Res 2009, 42, 893-903. Mastarone, D.J., et al. A modular system for the synthesis of multiplexed magnetic resonance probes. J Am Chem Soc. Apr 13 2011;133(14):5329-5337. McDevitt, M.R. Radioimmunotherapy with alpha-emitting nuclides, Eur J Nucl Med, 25 (1998)1341-1351. Mease, R.C., et al. PET imaging in prostate cancer: focus on prostate-specific membrane antigen. Curr. Top. Med. Chem. 2013;13:951-962. Milowsky, M.I., et al. Vascular targeted therapy with anti-prostate specific membrane antigen monoclonal antibody J591 in advanced solid tumors. J. Clin. Oncol. 2007;25:540-547. Nayak, T.K. and Brechbiel, M.W. 86 Y based PET radiopharmaceuticals: radiochemistry and biological applications. Med. Chem. 2011;7:380-388. Olson, W.C., et al. Clinical trials of cancer therapies targeting prostate specific membrane antigen. Rev. Recent Clin. Trials. 2007;2:182-190. Olszewski, R.T., et al. NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR. J. Neurochem. 2004;89:876-885. Palm, S., et al. Pharmacokinetics and biodistribution of (86)Y trastuzumab for (90)Y dosimetry in an ovarian carcinoma model: correlative MicroPET and MRI. J. Nucl. Med. 2003;44:1148-1155. Pomper, M.G., Ray, S., Mease, R.C., Foss, C. Labeled inhibitors of prostate specific membrane antigen (PSMA), biological evaluation, and use as imaging agents. WO 2009 / 002529 A2. Rooney, W.D., et al. Magn Reson Med 2007, 57, 308-318. Schulke, N., et al. The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy. Proc. Natl. Acad. Sci. U S A. 2003;100:12590-12595. Schwartz, J., et al. Renal uptake of bismuth-213 and its contribution to kidney radiation dose following administration of actinium-225-labeled antibody. Phys. Med. Biol. 2011;56:721-733. Silver, D.A., et al. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin. Cancer Res. 1997;3:81-85. Song, Y., et al. J Am Chem Soc 2008, 130, 6662 - 6663. Stabin, M.G., et al. OLINDA / EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine. J. Nucl. Med. 2005;46:1023 - 1027. Tagawa, S.T., et al. Bone marrow recovery and subsequent chemotherapy following radiolabeled anti-prostate-specific membrane antigen monoclonal antibody j591 in men with metastatic castration-resistant prostate cancer. Front. Oncol. 2013;3:1 - 6. Tagawa, S.T., et al. Phase II study of Lutetium-177-labeled anti-prostate specific membrane antigen monoclonal antibody J591 for metastatic castration-resistant prostate cancer. Clin. Cancer Res. 2013;19:5182 - 5191. Tse, B.W., et al. Nanomedicine 2015, 10, 375 - 386. Vallabhajosula, S., et al. Pharmacokinetics and biodistribution of 111 In- and 177 Lu-labeled J591 antibody specific for prostate-specific membrane antigen: prediction of 90 YJ591 radiation dosimetry based on111 In or 177 Lu? J.Nucl.Med.2005;46:634-641. Witzig, T.E., et al.Safety of yttrium-90 ibritumomab tiuxetan radioimmunotherapy for relapsed low-grade, follicular, or transformed non-Hodgkin’s lymphoma.J.Clin.Oncol.2003; 21:1263-1270. Woodard, H.Q., et al.Letter: Expression of tissue isotope distribution.J.Nucl.Med.1975; 16:958-959. Wu, X.et al.Bioconjug Chem 2012; 23, 1548-1556. Yong,K.and Brechbiel, M.W.Towards translation of 212Pb as a clinical therapeutic; getting the lead in!, Dalton Trans, 40 (2011)6068-6076. Yong, K.J.et al.(212)Pb-radioimmunotherapy induces G(2)cell-cycle arrest and delays DNA damage repair in tumor xenografts in a model for disseminated intraperitoneal disease, Mol.Cancer Ther.,11(2012)639-648 Yong, K.J.et al.212Pb-radioimmunotherapy potentiates paclitaxel-induced cell killing efficacy by perturbing the mitotic spindle checkpoint, Br.J.Cancer, 108 (2013)2013-2020. Yoo, J., et al. Preparation of high specific activity (86)Y using a small biomedical cyclotron. Nucl. Med. Biol. 2005; 32:891-897. Zechmann, C.M., et al. Radiation dosimetry and first therapy results with a (124)I / (131)I-labeled small molecule (MIP-1095) targeting PSMA for prostate cancer therapy. Eur. J. Nucl. Med. Mol. Imaging. 2014; 41:1280-292.

[0271] For the purpose of a clear understanding, the above subject matter has been described in some detail using examples and instances. However, it will be understood by those skilled in the art that certain changes and modifications can be made within the scope of the appended claims.

Claims

1. Formula (I): 【Chemistry 1】 (I) (In the formula, Z is tetrazole or CO 2 Q. Q is H or a protecting group; X 1 and X 2 are each independently NH or O; a is an integer selected from the group consisting of 1, 2, 3, and 4; c is an integer selected from the group consisting of 0, 1, 2, 3, and 4; Each R 1 , R 2 and R 4 are independently H or C 1 -C 4 is alkyl, Each R 3 are independently H, C 1 -C 6 Alkyl or C 2 -C 12 is aryl, W is independently O or S; Y is -NH- and may be present or absent; L is, 【Chemistry 2】 is a linker selected from the group consisting of During the ceremony, m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; Each R 5 is independently H or each R 6 are independently H or C 1 -C 6 -COOR is an alkyl group 6 and n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; p is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; Ch is a chelating moiety that may contain one or more metals or radiometals. or a pharma- ceutically acceptable salt thereof.

2. The chelating moiety is 【Chemistry 3】 2. The compound of claim 1, wherein q is selected from the group consisting of: 【Request 3】 【Chemical 4】 【change】 where M is a metal or a radioactive metal. or a pharma- ceutically acceptable salt thereof.

2. A compound of formula (I) selected from the group consisting of:

4. 2. The compound of claim 1, wherein the metal is selected from the group consisting of Gd, Lu, Ac, Bi, Pb, Cu, In, Sc and Y.

5. The compound of claim 1, wherein the non-radioactive metal is Gd-157 (stable isotope).

6. 2. The compound of claim 1, wherein the radiometal is selected from the group consisting of Lu-177, Ac-225, Bi-213, Bi-212, Pb-212, Cu-67, In-111, Sc-47, and Y-90.

7. 2. The method of claim 1, wherein the radiometal is selected from the group consisting of Y-86 and Sc-44.

8. 2. The compound of claim 1, wherein the radiometal is selected from the group consisting of Lu-177 and In-111.

9. contacting one or more tumors or cells with an effective amount of a compound of formula (I); forming an image; The compound of formula (I) is 【Chemistry 5】 (I) (In the formula, Z is tetrazole or CO 2 Q. Q is H or a protecting group; X 1 and X 2 are each independently NH or O; a is an integer selected from the group consisting of 1, 2, 3, and 4; c is an integer selected from the group consisting of 0, 1, 2, 3, and 4; Each R 1 , R 2 , and R 4 are independently H or C 1 -C 4 is alkyl, Each R 3 are independently H, C 1 -C 6 Alkyl or C 2 -C 12 is aryl, W is independently O or S; Y is -NH- and may be present or absent; L is a linker, said linker being 【Chemistry 6】 is selected from the group consisting of During the ceremony, m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; Each R 5 is independently H or each R 6 are independently H or C 1 -C 6 -COOR is an alkyl group 6 and n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; p is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; Ch is a chelating moiety that may contain one or more metals or radiometals. or a pharma- ceutical acceptable salt thereof for imaging or treating one or more prostate specific membrane antigen (PSMA) tumors or cells.

10. The chelating moiety is 【Chemistry 7】 【change】 wherein:

10. The method of claim 9, wherein q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8.

11. The compound is 【Chemistry 8】 【change】 【change】 (In the formula, x is selected from the group consisting of 2 and 3; M is a metal or a radioactive metal.

10. The method of claim 9, wherein the compound is selected from the group consisting of:

12. 10. The method of claim 9, wherein the metal is selected from the group consisting of Gd, Lu, Ac, Bi, Pb, Cu, In, Sc and Y.

13. 10. The method of claim 9, wherein the imaging comprises magnetic resonance imaging (MRI) and the non-radioactive metal is Gd-157 (a stable isotope).

14. 10. The method of claim 9, comprising treating one or more prostate specific membrane antigen (PSMA) tumors or cells, wherein the radiometal is selected from the group consisting of Lu-177, Ac-225, Bi-212, Bi-213, Pb-212, Cu-67, In-111, Sc-47, and Y-90.

15. 10. The method of claim 9, wherein the imaging comprises positron emission tomography (PET) imaging and the radiometal is selected from the group consisting of Y-86 and Sc-44.

16. 10. The method of claim 9, wherein the imaging comprises single photon emission computed tomography (SPECT) imaging and the radiometal is selected from the group consisting of Lu-177 and In-111.

17. The method of claim 9, wherein the one or more PSMA-expressing tumors or cells are selected from the group consisting of prostate tumors or cells, metastatic prostate tumors or cells, lung tumors or cells, renal tumors or cells, glioblastoma, pancreatic tumors or cells, bladder tumors or cells, sarcoma, melanoma, breast tumors or cells, colon tumors or cells, germ cells, pheochromocytoma, esophageal tumors or cells, gastric tumors or cells, and combinations thereof.

18. The method of claim 9, wherein the one or more PSMA-expressing tumors or cells are prostate tumors or cells.

19. 10. The method of claim 9, wherein the one or more PSMA-expressing tumors or cells are in vitro, in vivo, or ex vivo.

20. The method of claim 9, wherein the one or more PSMA-expressing tumors or cells are present in a subject.

21. 21. The method of claim 20, wherein the compound comprising an imaging agent is excreted from a tumor or cell of the subject.

22. The compound comprising an imaging agent, 【Chemistry 9】 【change】 and; (In the formula, M is a metal or a radioactive metal. or a pharma- ceutical acceptable salt thereof; 21. The method of claim 20, wherein the tumor is cleared from the subject's kidneys faster than the subject's tumor.

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