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: A following compound or a pharmaceutically acceptable salt thereof is used, where Q represents H or a protecting group, X1 and X2 each represent NH or O, R1 represents C1-C4 alkyl substituted with a halogen-substituted aryl group, R2 and R4 each represents H or C1-C4 alkyl, each R3 represents H, C1-C6 alkyl or C6-C12 aryl, W represents O or S, Y represents, for example, -NH-, L represents a linker, and Ch represents a chelating moiety optionally including one or more non-radiometals or radiometals.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 February 18, 2015, each of which is incorporated herein by reference in its entirety.

[0002] Federally sponsored research or development This invention was made with Government support awarded by the National Institutes of Health (NIH) under Grants K25CA148901-01A1 and U54CA1346751. The Government has certain rights in this invention. [Background technology]

[0003] Prostate-specific membrane antigen (PSMA) is increasingly being recognized as a viable target for imaging and treatment 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 for hormone-resistant types (Ghosh and Heston, 2004; Milowsky et al., 2007). PSMA is also known to be expressed in most solid tumors and tumor neovasculature (Haffner et al., 2012; Haffner et al., 2009). Imaging PSMA can provide insight into androgen signaling (Evans et al., 2011) and response to taxane therapy (Hillier et al., 2011). Previous studies have demonstrated 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 the clinic (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, 64Cu 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) were placed between the large molecule and the target urea to preserve PSMA target binding. Without wishing to be bound by any particular theory, PSMA was deemed suitable as a biomarker for MR molecular imaging due to its extracellular ligand binding site and the estimated high receptor concentration per cell (~3.2 μM / cell volume).

[0004] MR imaging is a clinically relevant non-invasive diagnostic tool for high-resolution anatomical and functional imaging. Molecular MR imaging allows 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 have been widely accepted by clinicians because of their ease of administration and ability to provide T1-weighted positive contrast. Despite advances in the design of high relaxivity contrast agents, 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). With signal amplification techniques, MR may become a sensitive modality for molecular imaging, complementing 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 shift from simple low molecular weight compounds to larger complexes may significantly change the pharmacokinetic profile of the substance (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. dThe sensitivity issue was addressed by creating contrast agents that have a high affinity for MR and thus can minimize the amount of material required for detection with MR (Hanaoka et al., 2008; De Leon-Rodriguez et al., 2010). One solution to enable MR-based receptor imaging was to combine a receptor-specific high affinity ligand with a Gd(III) multimer for detection (Wu et al., 2012).

[0005] An example of such an approach includes molecular imaging of VEGFR2 by preparing multimeric Gd-dendrons with high longitudinal relaxivity (r1) values ​​(De Leon-Rodriguez et al., 2010). Other multimeric materials have reported improved r1 values ​​at higher field strengths, due to the increasing magnetic fields used in MR imaging in both experimental and clinical settings (Mastarone 2011). Optimizing relaxivity at high magnetic fields offers the advantage of greater signal-to-noise and contrast-to-noise ratios (SNR / CNR), with the concomitant benefit of higher spatial resolution and shorter scan times (Rooney 2007). The combination of these concepts, i.e., the use of high-affinity targeting moieties together with sensitive multimeric contrast agents, provides the rationale for investigating targeted MR imaging of cells and tissues expressing prostate-specific membrane antigen (PSMA).

[0006] In addition, it is believed that urea-based agents may also be used in radiotherapy of lesions containing PSMA using radionuclides. 131 I]MIP1095 ((S)-2-(3-((S)-1-carboxy-5-(3-(4-[ 131 I]iodophenyl)ureido)phenyl)ureido)pentanedioic acid) (Zechmann et al., 2014) and 177Clinical studies using that approach with Lu-labeled PSMA-targeting agents (Kulkarni et al., 2014) are underway for the treatment of castration-resistant prostate cancer. This approach would be similar to radioimmunotherapy (RIT), which has proven highly effective in the treatment of lymphoma with two commercially available products that are routinely incorporated into clinical practice. However, due to the use of radiolabeled antibodies for imaging, RIT is fraught with challenges, including long circulation times, unpredictable biological effects, and the possible need for pretargeting methods. Furthermore, antibodies may be less accessible to tumors than pharmacologically engineered low molecular weight agents. Thus, there remains a need for low molecular weight compounds with high binding affinity to PSMA for tumor imaging and radiotherapy.

[0007] Positron-emitting radionuclides 86 Y(half-life [t 1 / 2 ]=14.74 hours, β + =33%, E β+ = 664 keV) is an attractive isotope for molecular imaging (Nayak and Brechbiel, 2011). Yttrium-86 is 86 Sr(p,n) 86 It can be easily prepared in a small medical cyclotron using the Y nuclear reaction (Yoo et al., 2005). - Emitter 90 Y(t 1 / 2 = 64.06 hours, β - =72%, E β- = 2.288 MeV) is widely used in internal radiation therapy (Witzig et al., 2003; Bodei et al., 2004). 86 Y is 90 It is ideal for dosimetric estimation of Y-labeled radiotherapy (Helisch et al., 2004). 86 The antibodies and peptides radiolabeled with Y 90 It has the same properties as its Y-labeled counterpart, making it suitable for use in radiation therapy. 90Accurate absorbed dose estimates can be made for Y (Nayak and Brechbiel, 2011; Palm et al., 2003). 177 Lu 90 Beta particle range shorter than Y (t 1 / 2 =6.7 days, E β- =0.5 MeV) but has similar chelating properties, 90 Not only those radiolabeled with Y, but also potential 177 Suitable imaging surrogates for investigating Lu-based radiotherapy include 86 A similar rationale has been applied to agents for neuroendocrine-targeted peptide receptor radionuclide therapy (Chen et al., 2012). Using a similar approach, potential matched-pair imaging radioisotopes suitable for SPECT imaging have been identified. 203 Pb (half-life, 51.9 hours, E β- = 279-keV gamma rays, 81%) is a therapeutic radionuclide for alpha particle therapy 212 It can be used for Pb (Chappell, et al. 2000; Yong, et al. 2011; Yong, et al. 2012; Yong, et al. 2013). 212 The decay scheme for Pb is 212 It contains Bi, which emits an alpha particle, two beta particles, and some gamma rays upon decay. Alpha particle emitters are particularly attractive for targeted radiotherapy due to their high linear energy deposition properties, such as localized dense ionization (leading to irreparable DNA double-strand breaks and cytotoxicity independent of tissue oxygen content or dose rate) (McDevitt, et al, 1998). 212 Pb and 212 Both Bi and Cr are promising α-particle emitters with well-described radiochemical properties for antibody conjugation, and 224 This can be easily obtained with an Ra generator.

[0008] Radiohalogenated carbamate PSMA inhibitors have also been developed that have shown high binding affinity to PSMA in vitro and, when radiolabeled with the positron emitter F-18, have shown high uptake in PSMA-positive mouse tumor xenografts and fast clearance from normal tissues. The favorable pharmacokinetic profile of this class of compounds (i.e., low nonspecific binding, no metabolism in vivo, and reasonable tumor residence time) has extended imaging studies to molecular radiotherapy. Furthermore, carbamate inhibitors can be coupled to metal chelators using linker functionality similar to urea metal / radiometal agents to maintain high binding affinity to PSMA. Thus, metal or radiometal conjugated carbamate scaffolds can also be used for imaging and treatment of PSMA-expressing cells and tissues. Summary of the Invention

[0009] In some embodiments, the subject matter of the present disclosure has formula (I):

[0010] [ka] (I)

[0011] or a pharma- ceutical acceptable salt thereof, wherein Z is tetrazole or COQ, Q is H or a protecting group, X1 and X2 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, and each R 1 , R 2 and R 4 is independently H or C1-C4 alkyl; each R 3 are independently H, C1-C6 alkyl or C2-C 12 aryl, W is independently O or S, Y is -NH- and may be present or absent, L is a linker, the linker being

[0012] [ka]

[0013] 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 are independently H or C1-C6 alkyl; 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 which may include one or more metals or radiometals.

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

[0015] While certain aspects of the subject matter of the present disclosure, which are addressed in whole or in part by the subject matter of the present disclosure, have been described above, other aspects will become apparent as the description proceeds and in conjunction with the accompanying examples and drawings, which are described in detail below. Having thus generally described the subject matter of this disclosure, reference is now made to the accompanying drawings, which are not necessarily to scale. [Brief description of the drawings]

[0016] [Figure 1A] (A) Structures of Gdl, Gd2 and Gd3 and (B) IC50 curves. [Figure 1B] (A) Structures of Gdl, Gd2 and Gd3 and (B) IC50 curves. [Diagram 2] The concentrations of Gdl–Gd3 in PC3 PSMA-flu (blue) and PC3 PSMA+PIP (red) cell pellets are shown. Data were obtained from ICP-MS analysis. [Diagram 3]The percentage of the incubated dose internalized and cell surface bound (%ID) for Gd1 and Gd2 is shown. Data were obtained from ICP-MS analysis. [Figure 4A] Figure 1 shows T1 contrast enhancement produced by Gd3 in isogenic human PC3 prostate cancer cells versus PSMA+PIP and PSMA-flu cells. (A) Color-coded T1 maps of PIP and flu cells. Relaxivity was determined at 25°C and 9.4T. (B) Quantification of T1 change (ΔΤ1) in PIP and flu cells following treatment with Gd3 (n=4, P<0.05). (C) Cellular uptake of Gd3 in RIP and flu cells. The amount of Gd(III) associated with PIP cell pellets was significantly higher than that of flu cell pellets. Accumulation of Gd3 in PIP cells was blocked by preincubation with ZJ43 (n=4, P<0.05). [Figure 4B] Figure 1 shows T1 contrast enhancement produced by Gd3 in isogenic human PC3 prostate cancer cells versus PSMA+PIP and PSMA-flu cells. (A) Color-coded T1 maps of PIP and flu cells. Relaxivity was determined at 25°C and 9.4T. (B) Quantification of T1 change (ΔΤ1) in PIP and flu cells following treatment with Gd3 (n=4, P<0.05). (C) Cellular uptake of Gd3 in RIP and flu cells. The amount of Gd(III) associated with PIP cell pellets was significantly higher than that of flu cell pellets. Accumulation of Gd3 in PIP cells was blocked by preincubation with ZJ43 (n=4, P<0.05). [Figure 4C]Figure 1 shows T1 contrast enhancement produced by Gd3 in isogenic human PC3 prostate cancer cells versus PSMA+PIP and PSMA-flu cells. (A) Color-coded T1 maps of PIP and flu cells. Relaxivity was determined at 25°C and 9.4T. (B) Quantification of T1 change (ΔΤ1) in PIP and flu cells following treatment with Gd3 (n=4, P<0.05). (C) Cellular uptake of Gd3 in RIP and flu cells. The amount of Gd(III) associated with PIP cell pellets was significantly higher than that of flu cell pellets. Accumulation of Gd3 in PIP cells was blocked by preincubation with ZJ43 (n=4, P<0.05). [Figure 5A] (A) Fluorescence imaging shows cellular uptake and internalization of Gdl-Rh. PSMA+PC3 PIP and PSMA-PC3 flu cells were incubated with serially diluted solutions of Gdl-Rh (4 μM to 4 nM) for 30 min at 37 °C, followed by removal of excess contrast agent with cold PBS. Zoomed-in images of PC3 PIP (B) and PC3 flu (C) at a contrast agent concentration of 4 nM. Rhodamine fluorescence is shown in red, and nuclei counterstained with DAPI are shown in blue. (D) Structure of Gdl-Rh. [Figure 5B] (A) Fluorescence imaging shows cellular uptake and internalization of Gdl-Rh. PSMA+PC3 PIP and PSMA-PC3 flu cells were incubated with serially diluted solutions of Gdl-Rh (4 μM to 4 nM) for 30 min at 37 °C, followed by removal of excess contrast agent with cold PBS. Zoomed-in images of PC3 PIP (B) and PC3 flu (C) at a contrast agent concentration of 4 nM. Rhodamine fluorescence is shown in red, and nuclei counterstained with DAPI are shown in blue. (D) Structure of Gdl-Rh. [Figure 5C](A) Fluorescence imaging shows cellular uptake and internalization of Gdl-Rh. PSMA+PC3 PIP and PSMA-PC3 flu cells were incubated with serially diluted solutions of Gdl-Rh (4 μM to 4 nM) for 30 min at 37 °C, followed by removal of excess contrast agent with cold PBS. Zoomed-in images of PC3 PIP (B) and PC3 flu (C) at a contrast agent concentration of 4 nM. Rhodamine fluorescence is shown in red, and nuclei counterstained with DAPI are shown in blue. (D) Structure of Gdl-Rh. [Figure 5D] (A) Fluorescence imaging shows cellular uptake and internalization of Gdl-Rh. PSMA+PC3 PIP and PSMA-PC3 flu cells were incubated with serially diluted solutions of Gdl-Rh (4 μM to 4 nM) for 30 min at 37 °C, followed by removal of excess contrast agent with cold PBS. Zoomed-in images of PC3 PIP (B) and PC3 flu (C) at a contrast agent concentration of 4 nM. Rhodamine fluorescence is shown in red, and nuclei counterstained with DAPI are shown in blue. (D) Structure of Gdl-Rh. [Figure 6A] The %ID of (A) cell surface bound and (B) internalized Gd3 after 1, 4 and 24 hours is shown. [Figure 6B] The %ID of (A) cell surface bound and (B) internalized Gd3 after 1, 4 and 24 hours is shown. [Figure 7] Viability of PSMA-PC3 flu cells incubated with Gd1, Gd2, Gd3 and Prohance. Contrast agents were incubated with cells at various Gd concentrations for 24 hours at 37°C and viability was measured using the MTS assay. Viability measurements were normalized to cells grown in the absence of contrast agents. [Figure 8] Viability of PSMA+PC3 PIP cells incubated with Gd1, Gd2, Gd3 and Prohance (Gd-DOTA) is shown. Contrast agents were incubated with cells at various Gd concentrations for 24 hours at 37°C and viability was measured using the MTS assay. Viability measurements were normalized to cells grown in the absence of contrast agents. [Figure 9A-B]Gd3 MR imaging of human PC3 prostate cancer PSMA+PIP and PSMA-flu tumor xenografts in male NOD / SCID mice. (A) Enhancement (ΔR1%) maps in PSMA+PC3 PIP and PSMA-PC3 flu tumors were overlaid on T2-weighted images at 40, 80, 120, and 160 min after a single bolus injection of Gd3 into the tail vein. (B) ΔR1% maps in PSMA+ and PSMA- tumors of a trimeric Gd contrast agent without a PSMA-targeting moiety at 40, 80, 120, and 160 min after a single bolus injection of Gd3 into the tail vein. [Figure 10A] (A) T1 time course calculated for the total volume of each tumor between 1 and 1600 min post-injection, (B) zoomed in area of ​​the time course from 0 to 200 min. Highly specific and persistent enhancement in PSMA+PC3 PIP tumors was noted. [Figure 10B] (A) T1 time course calculated for the total volume of each tumor between 1 and 1600 min post-injection, (B) zoomed in area of ​​the time course from 0 to 200 min. Highly specific and persistent enhancement in PSMA+PC3 PIP tumors was noted. [Figure 11] The percent change in relaxivity (%ΔR1) for mice following injection of Gd3 at a dose of 0.05 mmol / Kg (n=3) is shown (p<0.03, PIP:flu). [Figure 12] FIG. 1 shows the in vivo time-dependent change in T1 value of tumor (n=1) before and after injection of 1×PBS (phosphate buffered saline). [Figure 13A-B] (A) Selected MR images shown in FIG. 11, (B) the structure of Gd3. [Figure 14] 1 shows the structure of an 86Y-labeled inhibitor of PSMA. [Figure 15A] Preparative HPLC chromatograms for [86Y]4 are shown. (A) Radioactive HPLC peak. (B) The UV peak at 18.6 min is for unchelated 4 at λ=254 nm. [Figure 15B]Preparative HPLC chromatograms for [86Y]4 are shown. (A) Radioactive HPLC peak. (B) The UV peak at 18.6 min is for unchelated 4 at λ=254 nm. [Figure 16A] Preparative HPLC chromatograms for [86Y]5 are shown. (A) Radioactive HPLC peak. (B) UV peak at 34 min is for unchelated 5 at λ=254 nm. [Figure 16B] Preparative HPLC chromatograms for [86Y]5 are shown. (A) Radioactive HPLC peak. (B) UV peak at 34 min is for unchelated 5 at λ=254 nm. [Figure 17A] Preparative HPLC chromatograms for [86Y]6 are shown. (A) Radioactive HPLC peak, (B) UV peak at 15.8 min is for unchelated 6 at λ=220 nm. (C) HPLC chromatogram for pure [86Y]6. [Figure 17B] Preparative HPLC chromatograms for [86Y]6 are shown. (A) Radioactive HPLC peak, (B) UV peak at 15.8 min is for unchelated 6 at λ=220 nm. (C) HPLC chromatogram for pure [86Y]6. [Figure 17C] Preparative HPLC chromatograms for [86Y]6 are shown. (A) Radioactive HPLC peak, (B) UV peak at 15.8 min is for unchelated 6 at λ=220 nm. (C) HPLC chromatogram for pure [86Y]6. [Figure 18A-C] Whole-body PET-CT imaging of (A) 86Y-4, (B) 86Y-5, and (C) 86Y-6 in mice bearing PSMA+PC3 PIP and PSMA-PC3 flu tumors 2 hours after injection. Mice were injected intravenously (IV) with approximately 3.3 mBq (90 μCi) of radiotracer. PSMA+PC3 PIP (solid arrows), PSMA-PC3 flu (open arrows), K=kidney, GB=gallbladder, GI=gastrointestinal tract, L=left, R=right. Images are decay-corrected and adjusted to the same maximum value. [Figure 19A-C]PET-CT imaging of [86Y]-4 in mice bearing PSMA+PC3 PIP and PSMA-PC3 flu tumors. Images were obtained without (A) and with (B) PSMA blockade using ZJ43, a potent and selective PSMA inhibitor, as a blocker (50 mg / kg). PSMA-specific binding was further checked by the reduced uptake of the radiotracer in both tumor and kidney (another PSMA+ site) upon co-treatment with ZJ43. Mice were intravenously injected with approximately 6.2 MBq (168 μCi) of radiotracer. PSMA+PC3 PIP (solid arrows), PSMA-PC3 flu (open arrows). K=kidney, B=bladder, L=left, R=right. Images are decay-corrected and scaled to the same maximum. Figure 19C shows the structure of ZJ43, a potent and selective PSMA inhibitor. [Figure 20A-C] PET-CT imaging of 86Y-6 in mice bearing PSMA+PC3 PIP and PSMA-PC3 flu tumors (A) 0.5 hours post-injection, (B) 2 hours post-injection, and (C) 12 hours post-injection. Mice were intravenously injected with approximately 6.2 MBq (160 μCi) of radiotracer. PSMA+PC3 PIP (solid arrows), PSMA-PC3 flu (open arrows), K=kidney, L=left, R=right. Images are attenuation corrected and adjusted to the same maximum value. [Figure 21A-B] Screenshots of 3D time course MIP (maximum intensity projection) of 86Y-6PET in a baboon (A) 1-2 hours after injection and (B) 2-3.5 hours after injection are shown. To enhance visualization, radioactivity in the bladder was semi-automatically segmented using thresholding and subsequently removed. MIP 3D rendering was used to overview the distribution of the radiotracer throughout the body. No radiotracer was observed in most normal tissues, except for the bladder (not shown) and kidney (K). Rats were catheterized for this study. Mild uptake in the lacrimal, parotid and salivary glands was noted (short arrows, long arrows and open arrows, respectively). [Figure 22] The structures of 177Lu-SRV171 and related proposed substances for further improving in vivo pharmacokinetics are shown. [Diagram 23] The percentage of ID number (ID2) of 177Lu-SRV171 (0.01–10 μCi / million cells) incubated in PSMA+PIP and PSMA-flu cells after 2 h at 37 °C. Uptake specificity was further checked by co-incubation of 10 μM ZJ43. [Figure 24] 1 shows internalization studies of 177Lu-SRV171 (1 μCi) for up to 24 hours. [Figure 25A-C] SPECT images of male mice bearing PIP and flu tumors using 177Lu-SRV171 (500 μCi) at (A) 2 hours post-injection, (B) 24 hours post-injection, and (C) 96 hours post-injection, show low uptake in the kidney (K), bladder (B), and flu tumor. [Figure 26] Figure 1 shows the tissue biodistribution of 177Lu-SRV171 in different organs 3 h, 24 h, 48 h, 72 h and 96 h after injection. [Figure 27] The structures of 203Pb-SR-IX-ll and 203Pb-SRV171 are shown. [Figure 28A] SPECT-CT images of male mice bearing PIP and flu tumors using 203Pb-SRV171 (left) and 203Pb-SR-IX-ll (right) at (A) 60 min post-injection, (B) 120 min post-injection, and (C) 240 min post-injection. [Figure 28B] SPECT-CT images of male mice bearing PIP and flu tumors using 203Pb-SRV171 (left) and 203Pb-SR-IX-ll (right) at (A) 60 min post-injection, (B) 120 min post-injection, and (C) 240 min post-injection. [Figure 28C] SPECT-CT images of male mice bearing PIP and flu tumors using 203Pb-SRV171 (left) and 203Pb-SR-IX-ll (right) at (A) 60 min post-injection, (B) 120 min post-injection, and (C) 240 min post-injection. [Figure 29]Two lysine-carbamate scaffolds used in the design of compounds of the presently disclosed subject matter are shown: oxypentanedioic acid (OPA), which corresponds to the carbamate scaffold, and amino-pentanedioic acid (NPA), which corresponds to the "reverse" carbamate scaffold. [Diagram 30] HPLC chromatogram of ZCP-01 is shown. [Diagram 31] Electrospray ionization mass spectrometry (ESI-MS) of cryo-[In]ZPC-01. [Diagram 32] HPLC chromatogram of low-temperature [In]ZCP-01 is shown. [Figure 33A] Preparative HPLC chromatograms of [In]ZCP-01 are shown. (A) Radioactive HPLC peak. (B) UV peak after 32 min is for unchelated ZCP-01 at λ=200 nm. [Figure 33B] Preparative HPLC chromatograms of [In]ZCP-01 are shown. (A) Radioactive HPLC peak. (B) UV peak after 32 min is for unchelated ZCP-01 at λ=200 nm. [Fig. 34A-C] 1 shows the uptake of [111In]ZCP-01 in mice bearing PSMA+PC3 Pip and PSMA-PC flu tumor xenografts (A) 2 hours, (B) 4 hours, and (C) 24 hours after injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The subject matter of the present disclosure will now be described in more detail with reference to the accompanying examples and drawings, which illustrate some, but 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 being limited to the embodiments set forth herein. Rather, these embodiments are presented so that the disclosure will satisfy applicable legal requirements. Indeed, one skilled in the art to which the subject matter of the present disclosure pertains will be able to conceive numerous modifications and other embodiments of the subject matter of the present disclosure described herein having the benefit of the teachings presented in the foregoing description and the associated examples and drawings. Therefore, it is to be understood that the subject matter of the present disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

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

[0019] Without wishing to be bound by any particular theory, it was thought that PSMA would be a good target for an MR molecular imaging agent because of the high target concentration per cell (approximately 3 μM / cell volume) and the extracellular location of the ligand binding site. The approach of the present disclosure aims to optimize the binding affinity (minimum K ) of a contrast agent for a particular molecular or cellular target, such that less agent is required for MR detection. d Therefore, the disclosed approach combines high-binding affinity receptor-specific ligands with multimeric Gd(III) materials as one possible solution for MR-based molecular imaging.

[0020] So far, 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 was developed that includes (i) a PSMA targeting moiety, (ii) a linker for pharmacological tuning, and (iii) a chelator that allows for the attachment of a radionuclide. This strategy is suitable for PET imaging and supports the corresponding 90 Serve as a model for radiotherapy using Y-labeled substances 86 Y-labeled DOTA conjugate materials were included. Since DOTA is a strong chelator for many metals, the same DOTA conjugates can be used with other radiotherapeutic radionuclides, such as Lu-177, Ac-225, Bi-213, Bi-212, Pb-212, Cu-67, and Sc-47. In the presently disclosed subject matter, the same urea linker construct was used to systematically explore the potential of PSMA-based MR imaging of PCa by optimizing its relaxometric behavior or MR sensitivity as a high-field contrast agent, as well as its binding affinity, with increasing numbers of Gd chelates (monomeric, dimeric, and trimeric Gd).

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

[0022] [ka] (I)

[0023] or a pharma- ceutical acceptable salt thereof, wherein Z is tetrazole or COQ, Q is H or a protecting group, X1 and X2 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, and each R 1 , R 2 and R 4 is independently H or C1-C4 alkyl; each R3 are independently H, C1-C6 alkyl or C2-C 12 aryl, W is independently O or S, Y is -NH- and may be present or absent, and L is

[0024] [ka]

[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 are independently H or C1-C6 alkyl; 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 which may include one or more metals or radiometals.

[0026] Formula (I) does not include the compounds disclosed in WO 2009 / 002529, WO 2010 / 108125 and WO 2013 / 082338, and in particular the following compounds are specifically excluded from the composition claims herein:

[0027] [ka]

[0028] In a more particular embodiment, the chelating moiety is

[0029] [ka]

[0030] 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] [ka] TIFF0007630149000008.tif146163

[0033] wherein x is selected from the group consisting of 2 and 3, and M is a metal or a radioactive metal, or a pharma- ceutically 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 certain embodiments, 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 more specific 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. Additionally, for SPECT applications, the radiometal is selected from the group consisting of Lu-177 and In-111.

[0035] B. Methods of Using Compounds of Formula (I) for MR Imaging and / or Treatment of PSMA-Expressing Tumors or Cells In some embodiments, the presently disclosed subject matter 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) and forming an image, the compound of formula (I) being:

[0036] [ka] (I)

[0037] wherein Z is tetrazole or COQ, Q is H or a protecting group, X1 and X2 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, and each R 1 , R 2 , R 3 and R 4 is independently H or C1-C4 alkyl, W is independently O or S, Y is -NH- and may be present or absent, L is

[0038] [ka]

[0039] 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 are independently H or C1-C6 alkyl; 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 which may include one or more metals or radiometals, or a pharma- ceutical acceptable salt thereof.

[0040] "Contacting" refers to any action that results in at least one compound, including the imaging agent of the presently disclosed subject matter, coming into physical contact with at least one PSMA-expressing tumor or cell. Contacting may include exposing the cell or tumor to the compound in an amount sufficient to cause contact between the at least one compound and the at least one cell or tumor. The method may 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). The method may be performed in vivo, in which case contacting refers to exposing at least one cell or tumor in a subject to at least one compound of the presently disclosed subject matter, such as administering the compound to the subject by any suitable route. In the presently disclosed subject matter, contacting may include introducing, exposing, etc., the compound at a location remote from the cells to be contacted, and contacting the compound with the cells or tumor by natural (e.g., diffusion) or artificial (e.g., stirring) movement of the subject's bodily functions or bodily fluids. In some embodiments, the tumor or cell is found in vitro, in vivo, or ex vivo.

[0041] "Imaging" means producing an image of a cell, tissue, tumor, body part, etc. using a magnetic resonance (MR) based (magnet that polarizes and excites hydrogen nuclei in water molecules in tissue, thereby producing a detectable signal).

[0042] Formula (I) does not include the compounds disclosed in WO 2009 / 002529, WO 2010 / 108125 and WO 2013 / 082338, and in particular the following compounds are specifically excluded from the imaging claims in this application:

[0043] [ka]

[0044] In a more particular embodiment, the chelating moiety is

[0045] [ka]

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

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

[0048] [ka] TIFF0007630149000014.tif43150 TIFF0007630149000015.tif44150 TIFF0007630149000016.tif70150 TIFF0007630149000017.tif145160

[0049] wherein x is selected from the group consisting of 2 and 3, and M is a metal or a radioactive metal, or a pharma- ceutically 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 more specific 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. Additionally, for SPECT applications, the radiometal 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, 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. 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 cells are found in a subject. In many of its embodiments, the subject treated with the methods of the present disclosure is preferably a human subject. However, it is to be understood that the methods described herein are effective for all vertebrate species encompassed by the term "subject." Thus, a "subject" can include a human subject for medical purposes, e.g., treatment of an existing condition or disease or prophylactic treatment to prevent the onset of a condition or disease, or an animal (non-human) subject for medical, veterinary, or development 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, caprines, such as goats, porcines, such as piglets, grown pigs, equines, such as horses, donkeys, zebras, etc., felines (including wildcats and domestic cats), canines (including dogs), lagomorphs (including rabbits, hares, etc.), rodents (including mice, rats, etc.). The animals can be transgenic animals. In some embodiments, the subject is a human, including but not limited to fetal, neonatal, infant, juvenile, and adult subjects. Additionally, a "subject" may include a patient afflicted or suspected of being afflicted with a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human.

[0054] In some embodiments, a detectably effective amount of the imaging agent of the disclosed method is administered to the subject. In the presently disclosed subject matter, a "detectably effective amount" of the imaging agent is defined as an amount sufficient to obtain an acceptable image using equipment available for clinical use. A detectably effective amount of the imaging agent may be administered in two or more injections. This detectably effective amount of the imaging agent may vary depending on factors such as the individual's degree of sensitivity, the individual's age, sex, and weight, the individual's idiosyncratic response, dosimetry, and other 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 presently disclosed subject matter are rapidly excreted from the body tissues.Typically, the compounds of the presently disclosed subject matter are cleared from the body in less than about 24 hours.More preferably, the compounds of the presently disclosed subject matter are cleared 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 comprising 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 comprising an imaging agent from the kidney occurs more rapidly than clearance from a tumor of a subject.

[0057] In some embodiments, the methods of the disclosure use compounds that are stable in vivo such that substantially all, e.g., greater than about 50%, 60%, 70%, 80%, or more preferably 90%, of the injected compound is not metabolized by the body prior to excretion. In other embodiments, compounds including imaging agents are stable in vivo.

[0058] C.Definition i. Chemical Definition While those of skill in the art will fully understand the following terms as they relate to compounds of formula (I), the following definitions are provided to facilitate explanation of the subject matter of this disclosure and are intended to supplement and illustrate, but not exclude, those definitions that would be apparent to one of skill in the art upon review of this disclosure.

[0059] The terms "substituted" and "substituents" herein, whether preceded by the term "optionally" or not, refer to the ability to change a functional group to another functional group, as long as the valence of all atoms is maintained, as understood by those skilled in the art. When a given structure can be substituted at more than one position with more than one substituent selected from a specified group, 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 nearby, such as another aryl group, which is further substituted at one or more positions, such as with fluorine).

[0060] When substituents or linking groups are designated by their conventional chemical formula written from left to right, these groups are equivalent and include the chemically identical substituents which result from writing the structure from right to left, e.g., CHO- is equivalent to -OCH-, -C(=O)O- is equivalent to -OC(=O)-, -OC(=O)NR- is equivalent to -NRC(=O)O-, etc.

[0061] As used herein, when a bond is placed next to an internal substituent (e.g., -NRC(O)-), the order of the atoms is fixed and the direction of the group is not reversed and is inserted into the structure in the direction shown. In other words, -NRC(O)- is different from -C(O)NR-. As used herein, the term C(O) (e.g., -NRC(O)-) is used to refer to a carbonyl (C=O) group, where the oxygen is double-bonded to the carbon.

[0062] When the phrase "independently selected" is used, the substituents being referenced (e.g., R groups such as groups R1, R2, or variables such as "m" and "n") can be the same or different. For example, both R1 and R2 can be substituted alkyl, or R1 can be hydrogen and R2 can be a substituted alkyl, etc.

[0063] As used herein in referring to a group of substituents, the singular terms (a, an, a(n)) mean at least one. For example, if a compound is substituted with "an" alkyl or aryl, then the compound is optionally substituted with at least one alkyl and / or at least one aryl. Additionally, if a moiety is substituted with R substituents, then the group may be said to be "R-substituted." If a moiety is R-substituted, then 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 the art-recognized structure corresponding to the group bearing that name. For purposes of illustration, certain representative "R" groups listed above 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.Therefore, when a group can be substituted with one or more of a number of substituents, such substitutions are selected to obtain a compound that follows the principles of chemical bonding and is not inherently unstable and / or that would be known to those skilled in the art to be prone to instability 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 according to the principles of chemical bonding known to those skilled in the art to avoid inherently unstable compounds.

[0066] The term "hydrocarbon" as used herein refers to any chemical group containing hydrogen and carbon. Hydrocarbons can be substituted or unsubstituted. As one of ordinary skill in the art will appreciate, any substitution must satisfy all valences. Hydrocarbons can be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrative hydrocarbons are defined in more detail below and 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,” by itself or as part of another substituent, means, unless otherwise specified, a straight-chain (i.e., unbranched) or branched-chain, acyclic or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and which may include divalent and polyvalent radicals, having the specified number of carbon atoms (i.e., C1-C2). 10 means 1 to 10 carbons). In certain embodiments, the term "alkyl" refers to a C alkyl group derived from a hydrocarbon moiety containing 1 to 20 carbon atoms by the removal of one hydrogen atom. 1-20 It refers inclusively to linear (ie, "straight chain"), branched or cyclic, saturated or at least partially, and optionally fully unsaturated (ie, alkenyl and alkynyl) hydrocarbon radicals.

[0068] Representative saturated hydrocarbon radicals 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, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms (i.e., C 1-8 "Higher alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers specifically to C 1-8 In another embodiment, "alkyl" refers to a straight chain alkyl, specifically C 1-8 This refers to a branched alkyl group.

[0070] In certain embodiments, the alkyl group is a C1-C6 alkyl group or a C1-C4 alkyl group. As used herein, the term "C1-C6 alkyl" refers to fully saturated straight chain, branched or cyclic C1-C6 hydrocarbons and hybrids thereof, such as (cycloalkyl)alkyl. Examples of C1-C6 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 butyl (including n-butyl (n-Bu, n Bu), iso-butyl (i-Bu, 1 Bu), sec-butyl (s-Bu, s Bu), tert-Butyl (t-Bu, 1Bu) or cyclobutyl (c-Bu, 0 Bu) and the like.

[0071] Alkyl groups may be optionally substituted with one or more alkyl group substituents, which may be the same or different ("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. Optionally inserted along the alkyl chain is one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, which nitrogen substituents are hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl") or aryl.

[0072] Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, where one or more atoms or functional groups of the alkyl group are replaced 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", by itself or in combination with other terms, means, unless otherwise specified, a stable linear or branched chain or cyclic hydrocarbon radical or combination 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, where the nitrogen, phosphorus and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, P, S and Si may be substituted at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH 25These include -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)- CH3, O-CH3, -O-CH2-CH3 and -CN. Up to two or three heteroatoms may be consecutive, for example -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0074] As noted above, heteroalkyl groups, as used herein, include groups that are attached to the remainder of the molecule via a heteroatom, e.g., -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR and / or -SOR'. When "heteroalkyl" is described and followed by a specific heteroalkyl group, e.g., -NR'R, it is understood that the terms heteroalkyl and -NR'R" are neither redundant nor mutually exclusive; rather, clarity is provided by the recitation of the specific heteroalkyl group. Thus, as used herein, the term "heteroalkyl" should not be construed to exclude specific heteroalkyl groups, e.g., -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 a non-aromatic monocyclic or polycyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Cycloalkyl groups can be optionally partially unsaturated. Cycloalkyl groups can also be optionally substituted with alkyl group substituents, oxo and / or alkylene, as defined herein. Optionally, one or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be inserted along the cyclic alkyl chain, where the nitrogen substituents are hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thereby resulting in a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

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

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

[0079] Cycloheteroalkyl rings may be optionally fused or otherwise attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Heterocycles include those having 1 to 3 heteroatoms independently selected from oxygen, sulfur and nitrogen, where the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. In certain embodiments, the term "heterocycle" 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 fused 6-membered rings having 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where (i) each 5-membered ring has 0-2 double bonds, each 6-membered ring has 0-2 double bonds, and each 7-membered ring has 0-3 double bonds, (ii) the nitrogen and sulfur heteroatom may optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocycles 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, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0080] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder 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 the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

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

[0082] Unsaturated alkyl groups are those that have 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. Alkyl groups that are limited to hydrocarbon groups are referred to as "homoalkyl".

[0083] In particular, the term "alkenyl" as used herein refers to a C alkyl group having at least one carbon-carbon double bond formed by the removal of one hydrogen atom. 1-20Alkenyl refers to a monovalent group derived from a generic straight-chain or branched hydrocarbon moiety. Alkenyl groups include, for example, 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, cycloheptatrienyl, and cyclooctenyl.

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

[0086] The term "alkylene" by itself or as part of another substituent refers to a straight or branched chain divalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight chain, branched, or cyclic. Additionally, the alkylene group can be optionally unsaturated and / or substituted with one or more "alkyl group substituents." One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkylene group (also referred to herein as "alkylaminoalkyl"), with the nitrogen substituent being alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CH2)3-); cyclohexylene (-CH6H6H8 ... 10-);-CH=CH-CH=CH-;-CH=CH-CH2-;-CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, -CH2CH2CH(CH2CH2CH3)CH2-, -(CH2) q -N(R)-(CH2) r - (each of q and r is independently an integer from 0 to about 20, e.g., 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); including methylenedioxyl (-O-CH2-O-) and ethylenedioxyl (-O-(CH2)2-O-). Alkylene groups can have from about 2 to about 3 carbon atoms and can further have 6 to 20 carbons. Typically, alkyl (or alkylene) groups have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being some embodiments of the disclosure. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0087] The term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl, such as, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, etc.). Additionally, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied 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 specified, means an aromatic hydrocarbon substituent which may be a single ring or multiple rings (eg, 1 to 3 rings) that are fused or covalently linked.

[0089] The term "heteroaryl" refers to an aryl group (or ring) having from 1 to 4 heteroatoms selected from N, O, and S (each in a separate ring if polycyclic), where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the remainder of the molecule through 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, 2-isoxazolyl ... The heteroaryl ring systems include aryl, 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 above listed aryl and heteroaryl ring systems 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 brevity, the term "aryl" when used in combination with other terms (e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms "arylalkyl" and "heteroarylalkyl" are intended to include groups in which an aryl or heteroaryl group is bonded to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, etc.) that contains an alkyl group in which a carbon atom (e.g., a methylene group) is replaced, for example, with an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.). However, the term "haloaryl" as used herein is intended to include within its scope only aryl substituted with one or more halogens.

[0091] When a heteroalkyl, heterocycloalkyl, or heteroaryl contains a specific number of members (eg, "3 to 7 members"), the term "member" refers to a carbon or a 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 attached at any position on the alkyl group. The term C4-C 16 Alkylaryl includes alkylaryl groups having a total of 4 to 16 carbon atoms, counting the carbon atoms on the alkyl and aryl groups together. Examples of alkylaryl groups include, but are not limited to, benzyl (phenylmethyl), phenylethyl, and naphthylmethyl. Alkylaryl groups can be substituted or unsubstituted. Substituents are not counted in the total number of atoms in the alkylaryl group unless all atoms in the substituent are larger than the alkylaryl group.

[0093] Further, the formula:

[0094] [ka]

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

[0096] [ka]

[0097] Compound groups, including but not limited to:

[0098] A dashed line representing a bond in a ring structure indicates that the bond can be present or absent in the ring, i.e., the dashed line representing a bond in a 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] Substituents with an broken bond, such as the example shown below, mean that the substituent is directly attached to the molecule at the position shown. No additional methylene (CH2) groups are implied. The symbol

[0100] TIFF0007630149000020.tif8170

[0101] indicates the point of attachment of the moiety to the remainder of the molecule.

[0102] [ka]

[0103] Substituents with two broken bonds, such as the example shown below, mean that the atoms orient as depicted from left to right and should be inserted into the molecule in the direction shown. No additional methylene (CH2) groups are implied unless otherwise specified.

[0104] [ka]

[0105] When a named atom of an aromatic or heteroaromatic ring is defined to be "absent," the named atom is replaced with a direct bond.

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

[0107] Substituents for the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl monovalent and divalent derivative groups (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR', ═O, ═NR', ═N-OR', -NR'R", -SR', -halogen, -SiR'R"R'", and -N(O)R', -C(O)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)R', -S(O)NR'R", -NRSO2R', -CN, and -NO2, where m' is the total number of carbon atoms in such group. R', R", R'", and R"" may each independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. As used herein, an "alkoxy" group is an alkyl attached to the remainder of the molecule through a divalent oxygen. When a compound of the present disclosure includes, for example, more than one R group, each R group is independently selected, as are each R', R", R'" and R"" groups when more than one is present. When R' and R" are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will recognize that the term "alkyl" is intended to include groups that include carbon atoms attached to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0108] Similar to the substituents described above for the alkyl groups, example substituents for the aryl and heteroaryl groups (and their divalent derivatives) vary in number from zero to the total number of open valences in the aromatic ring system, e.g., halogen, -OR', -NR'R", -SR', -halogen, -SiR'R"R'", -OC(O)R', -C(O)R', -COR', -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)R', -S(O) )NR'R", -NRSOR', -CN and -NO, -R', -N, -CH(Ph), fluoro(C-C)alkoxo, and fluoro(C-C)alkyl, where R', R", R'" and R"" can be 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 a compound of the disclosure includes, for example, more than one R group, each R group is independently selected, as are each R', R", R'" and R"" groups when more than one is present.

[0109] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring are optionally of the formula -TC(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 the aryl or heteroaryl ring may optionally be of the formula -A-(CH2) r A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer of 1 to 4.

[0110] One of the single bonds in the thus newly formed ring may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be 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)2NR'-. The substituents R, R', R" and R'" may be independently 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 with another substituent and has the general formula RC(=O)-, where 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 refer to a saturated (i.e., alkyl-O-) or unsaturated (i.e., alkenyl-O- and alkynyl-O-) group attached to the parent molecular moiety through an oxygen atom, where the terms "alkyl", "alkenyl", and "alkynyl" are as defined above. 1-20 Inclusively, it may include linear, branched or cyclic, saturated or unsaturated oxo-hydrocarbon chains, such as methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.

[0113] As used herein, the term "alkoxyalkyl" refers to an alkyl-O-alkyl ether, for example a methoxyethyl or an ethoxymethyl group.

[0114] "Aryloxyl" refers to an aryl-O- group in which the aryl group is as defined above, including substituted aryl. As used herein, the term "aryloxyl" can refer to phenyloxyl or hexyloxyl and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxy.

[0115] "Aralkyl" refers to an aryl-alkyl group in which the aryl and alkyl are as previously described, including substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0116] "Aralkyloxyl" refers to an aralkyl-O- group in which the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl.

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

[0118] "Aryloxycarbonyl" refers to the group aryl-O-CO-. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0119] "Aralkoxycarbonyl" refers to the group aralkyl-O-CO-. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0120] "Carbamoyl" refers to an amide group of the formula -CONH2. "Alkylcarbamoyl" refers to the group R'RN-CO- where one of R and R' is hydrogen and the other of R and R' is alkyl and / or substituted alkyl as defined above. "Dialkylcarbamoyl" refers to the group R'RN-CO- where each of R and R' is independently alkyl and / or substituted alkyl as defined above.

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

[0122] "Acyloxyl" means an acyl-O- group, where acyl is as previously described.

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

[0124] As used herein, "aminoalkyl" refers to an amino group covalently attached to an alkylene linker. In particular, as used herein, the terms alkylamino, dialkylamino, and trialkylamino refer to one, two, or three alkyl groups, respectively, as defined above, attached to the parent molecular moiety through a nitrogen atom. 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. Additionally, R', R" and / or R'" taken together are optionally selected from -(CH2) k-, where 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] An 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 through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0127] "Acylamino" means an acyl-NH- group in which acyl is as previously described. "Aroylamino" means an aroyl-NH- group in which aroyl is as previously described.

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

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

[0130] As used herein, the terms "halo", "halide" or "halogen" refer to fluoro, chloro, bromo and iodo groups. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)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 the -OH group.

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

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

[0134] The term "oxo," as used herein, refers to an oxygen atom that is double bonded to a carbon atom or another element.

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

[0136] The term "thio" refers to the above compounds where a carbon or oxygen atom has been replaced with a sulfur atom.

[0137] The term "sulfate" refers to the -SO4 group.

[0138] The term thiohydroxyl or thiol, as used herein, refers to a group of the formula: --SH.

[0139] The term ureido refers to a urea group of the formula: -NH-CO-NH2.

[0140] Unless expressly defined otherwise, a "substituent" herein includes a functional group selected from one or more of the following moieties, which are defined herein as follows: (A) -OH, -NH2, -SH, -CN, -CF3, -NO2, 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 B are (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl and (ii) is substituted with at least one substituent selected from (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl and the group (b) is substituted with at least one substituent selected from oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0141] As used herein, "lower substituted" or "lower substituent" means a group selected from all of the substituents described above for "substituent", wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5-7 membered heterocycloalkyl.

[0142] As used herein, a "size-limited substitution" or "size-limited substituent" refers to a group selected from all of the substituents listed above for "substituent", where each substituted or unsubstituted alkyl is a substituted or unsubstituted C-C 20 each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4-8 membered heterocycloalkyl.

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

[0144] It will be apparent to one of ordinary skill 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 converted from one isomeric form to another.

[0145] Unless otherwise specified, a structure depicted herein is intended to include all stereochemical forms of that structure, i.e., both R and S configurations for each asymmetric center. Accordingly, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

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

[0147] It is well known in the art how to prepare optically active forms, for example by resolution of racemates (racemic compounds), asymmetric synthesis, or synthesis from optically active starting materials. Resolution of racemic compounds can be accomplished by conventional methods, for example by crystallization in the presence of a resolving agent or by chromatography, for example using a chiral HPLC column. Numerous geometric isomers of olefins, C=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the presently disclosed subject matter. Cis and trans geometric isomers of the compounds of the presently disclosed subject matter are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral (enantiomeric and diastereomeric) and racemic forms, as well as all geometric isomeric forms of a structure, are intended, unless a specific stereochemistry or isomeric form is specifically indicated.

[0148] The compounds described herein may have one or more charged atoms. For example, the compounds may 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 may be associated with a suitable counterion. How to prepare salts or exchange counterions is well known in the art. Generally, such salts may be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (e.g., Na, Ca, Mg, or K hydroxides, carbonates, bicarbonates, etc.) or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or an organic solvent or a mixture of the two. The counterion may be changed, for example, by ion exchange techniques such as ion exchange chromatography. All zwitterions, salts, and counterions are contemplated unless the counterion or salt is specifically indicated. In certain embodiments, the salt or counterion may 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 readily available reagents that do not attack the regenerated functional group or other functional groups in the molecule. Suitable protecting groups are known in the art and continue to be 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, such as those 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 ( tProtecting groups include methyl (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 skill in the art will recognize appropriate situations in which protecting groups are necessary and will be able to select protecting groups appropriate for use in a particular environment.

[0150] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of a hydrogen by deuterium or tritium, or the replacement of a carbon by 13 C- or 14 Compounds having this structure except for the substitution at a 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 at one or more of the atoms that constitute such compounds. For example, the compounds may be radioisotopes, such as tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0152] ii. Pharmaceutical salts The compounds of the present disclosure may exist as pharma- ceutically acceptable salts. The term "pharma-ceutically acceptable salts" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific 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, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolyl arsanilate, hexylresorcinate, hydrabamine, hydrobromide, The salts may include hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate (e.g., (+)-tartrate, (-)-tartrate or mixtures thereof, including racemic mixtures), or teoclate. These salts may be prepared by methods known to those skilled in the art. Other pharma- ceutically acceptable salts may be found, for example, in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0153] Also included are base addition salts, such as sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. 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, either in pure form or in a 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, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and also those derived from 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-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like.

[0154] Also included are salts of amino acids such as arginate and salts of organic acids such as glucuronic or galacturonic acids, see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present disclosure contain both basic and acidic functional groups that allow the compounds to be converted into base or acid addition salts. The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. 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 may exist in unsolvated and solvated forms (including hydrated forms). In general, solvated forms are equivalent to unsolvated forms and are within the scope of the present disclosure. Certain compounds of the present disclosure may exist in various crystalline or amorphous forms. In general, all physical forms are equivalent for the applications envisioned by the present invention and are intended to be within the scope of the present disclosure.

[0156] iii. General Definitions Although specific terms are used herein, they are used in a generic and descriptive sense only and not for purposes of limitation. For the sake of clarity, specific definitions are provided 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 animals refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significantly increased anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cell markers. In some circumstances, cancer cells take the form of a tumor, and such cells may be present locally in the animal's body or circulate in the bloodstream as independent cells.

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

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

[0160] An "effective amount" of a substance is an amount of the substance sufficient to elicit a desired biological response or measurable difference when compared to a control. As one skilled in the art will appreciate, the absolute amount of a particular substance effective to treat a disease, disorder, condition, or injury may vary depending on factors such as the substance delivered, the mode of administration, the age, weight, and general health of the subject, the desired biological endpoint, and the desired therapeutic effect. Ultimately, the attending clinician will determine the appropriate amount and dosing regimen. For example, an "effective amount" of a substance may be an amount sufficient to produce a measurable image when the compound is used for imaging, or an amount sufficient to ameliorate a disease symptom when the compound is used for treatment. One skilled in the art will also appreciate that an effective amount of a substance may be administered in a single dose or may be achieved by multiple separate doses.

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

[0162] Following long-standing patent law convention, in this application, including the claims, the singular indefinite and definite articles refer to "one or more." Thus, for example, reference to "a subject" includes a plurality of subjects unless the context clearly indicates otherwise (e.g., a plurality of subjects), and so forth.

[0163] Throughout this specification and claims, the term "comprise" is used in a non-exclusive sense unless the context clearly indicates otherwise. Similarly, the term "include" and its grammatical variations are intended to be open-ended, such that the listing of items does not exclude other similar items that may be substituted for or in addition to the listed items.

[0164] For purposes of this specification and the appended claims, unless otherwise specified, all numbers expressing amounts, sizes, dimensions, proportions, shapes, ratios, parameters, percentages, parameters, quantities, characteristics, and other numerical values ​​used in the specification and claims are to be understood as being modified in all instances by the term "about," even if the term "about" is not specifically attached to a value, amount, 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, precise but can be approximate and / or larger or smaller as needed, reflecting tolerances, customary factors, rounding, measurement errors, and the like, as well as other factors known to those of ordinary skill in the art. For example, the term "about" when referring to a value can include variations of the specified amount 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%, and in some embodiments, ±0.1%, as such variations are appropriate for carrying out the disclosed methods or using the disclosed compositions.

[0165] Additionally, when the term "about" is used in connection with one or more numbers or numerical ranges, it should be understood to refer to all such numbers inclusive of every number within a range, and the term modifies that range by extending the boundaries above and below the numerical values ​​set forth. By reciting numerical ranges with endpoints, all numbers, e.g., integers (including fractions thereof) contained within that range (e.g., 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. EXAMPLES

[0166] The following examples are included to guide those skilled in the art in practicing representative embodiments of the subject matter of the present disclosure. In light of this disclosure and the general level of skill in the art, those skilled in the art will appreciate that the following examples are merely illustrative, and that numerous changes, modifications, and alterations may be made without departing from the scope of the subject matter of the present disclosure. The synthetic descriptions and specific examples provided below are merely for illustrative purposes, and are not to be construed as limiting in any way to producing 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 provide anatomical, functional and molecular information. MR molecular imaging can combine this established and widespread clinical modality and its high spatial resolution with molecular profiling in vivo. However, the sensitivity of MR is inherently low, so a high local concentration of biological target is required to generate a 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. To this end, three high-affinity low molecular weight gadolinium (Gd)(III)-based PSMA-targeted contrast agents, graded Gd1, Gd2 and Gd3 with 1–3 Gd chelates per molecule, respectively, were synthesized (Figure 1A). The aim of this study was to evaluate the PSMA binding affinity and longitudinal relaxivity (r1) of the synthesized materials. The cellular uptake of the materials in PSMA-expressing (isogenic) and non-expressing control cells was assessed by inductively coupled plasma mass spectrometry (ICP-MS).Finally, the ability of the materials to distinguish PSMA-expressing from control cells was assessed 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-pentaazaheptacosane-21,25,27-tricarboxylic acid, Gd1 Compound Gd1 was prepared according to a recent report. Compound 1 was prepared in three steps as described below. Commercially available N-Boc-1,4 diaminobutane (68 mg, 0.36 mmol, in 0.5 mL 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 DMSO) and DIEA (132 μl, 0.75 mmol) and stirred at 40° C. for 4 h. The solvent was evaporated and the solid residue was purified by reverse phase C chromatography. 18 Purification by flash chromatography (5.5 g, Agilent SF10) using water and acetonitrile (each 0.1% TFA) afforded the Boc-protected 7 after lyophilization. Yield: 146 mg, ca. 55%. ESI-MS 740 [M+H] + The compound obtained in that step was then treated with ice-cold TFA / CH2Cl2 (1 / 1) solution and stirred at ambient temperature for 2 h. The solvent was evaporated, the residue was dried under vacuum and purified by reversed-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 reversed-phase flash chromatography (5.5 g, Agilent SF10) to give 7. Yield approx. 104 mg, 40%. 1 H NMR(DMSO-d6)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 Gd2(CO3)3 (85 mg, 0.17 mmol) and stirred at 60 °C for 14 h. ESI-MS:C 48 H 77 N 10 O 17 Theoretical value for S: 797.5183 [M+H] + , Found: 797.5212. The compound was then 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 in 5 min, 100% A to 80% A, 0% B to 20% B from 5 to 25 min, 80% A to 20% A and 20% B to 80% B from 25 to 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)acetamide)-3,10,16,25,31,33-hexaazahexatriacontane-11,30,34,36-tetracarboxylic acid, digadolinium(III) salt, Gd2 The compound was prepared according to Scheme 2. To a solution of N-Bis-Boc-L-lysine NHS (3 gm, 6.7 mmol, in 10 mL DMF) was added Fmoc-Lys(Boc)-OH (2.49 g, 6.7 mmol) and the solution was sonicated at room temperature for 1 h until a clear solution was obtained. The solution was stirred at room temperature for 4 h and the solvent was removed under vacuum to give 4 in near quantitative yield. Compound 4 was further purified on a silica gel column using 3 / 97 MeOH / CHCl as eluent. 1H NMR(CDCl3)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 / CH2Cl2 solution and stirred at room temperature for 2 h. After solvent evaporation, the solid residue was washed with 3x3 mL of diethyl ether and dried under vacuum to give 5 as the TFA salt. Compound 5 was obtained in quantitative yield and was used without further purification after lyophilization. 1 H NMR (DO) 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) was added 5.2TFA (32 mg, 0.04 mmol) and DIEA (0.78 mmol, 136 μL) in portions over 45 min at room temperature. The solution was then stirred for an additional 2 hours and the reaction completion was monitored using HPLC. After completion of the reaction, the reaction was purified by HPLC to give 6. Compound 6 was treated with 20% piperidine solution for Fmoc group and C 18 Purified by flash chromatography using a 90 / 10 H2O / CH3CN (0.1% TFA each) solution and lyophilized. ESIMS: 1046 [M+H] + The lyophilized compound (50 mg, 0.047 mmol) was dissolved in distilled water (2 mL) and added to a solution of Gd2(CO3)3 (0.26 mmol, in 3 mL of water) and stirred at 60 °C for 12 h. Compound 7 was 18Purified by flash chromatography using a gradient of 90 / 10 to 80 / 20 H2O / CH3CN (each 0.1% TFA) and lyophilized. To a solution of 3 (25 mg, 0.004 mmol) in DMSO was added 7 (40 mg, 0.003 mmol) slowly over 30 min and stirred at room temperature for approximately 2 h until the reaction was complete. Completion of the reaction was monitored by HPLC. Upon completion, the reaction mixture was subsequently purified by HPLC and the product was lyophilized. ESI-MS: 1813.08 [M+H] + , Measured value: 1813.08. The compound was then purified by HPLC. Method 1: C 64 H 103 Gd2N 15 O 26 The theoretical value for is 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. Elution gradient was 100% A and 0% B in 5 min, 100% A to 80% A and 0% B to 20% B in 5-25 min, 80% A to 20% A and 20% B to 80% B in 25-30 min.

[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 a 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 DMF) was added TSTU (440 mg, 1.47 mmol) and TEA (541 μL, 3.39 mmol), and the resulting solution was stirred at room temperature for 4 h until the reaction was complete as monitored by TLC. The solvent was removed under high vacuum, and the residue was dissolved in CHCl and purified on a silica gel column using a 40 / 60-50 / 50 EtOAc / hexane solution as eluent. The product-containing fractions were combined and evaporated to give the desired product as a colorless solid. Yield approximately 310mg. NMR (CDCl3): δ7.56 (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-tetraazahexacosane-1,3,7,22-tetracarboxylic acid 2,2,2-trifluoroacetate, 10 Compound 10 was prepared according to previous reports. Briefly, to a solution of tris-t-Bu protected 3 (100 mg 0.135 mmol, in 1.35 ml DMF) was added H-Lys(Boc)(Ot-Bu) (59.5 mg, 0.175 mmol) followed by DIEA (70.7 μL, 0.135) and the clear solution was stirred overnight at room temperature. The solution was then concentrated under vacuum to a clear oily residue. The residue was dissolved in 2:1 MeCN / water (6 mL) and lyophilized to give the product as a clear foam. 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 an ice-cold solution of TFA / CH2Cl2, followed by dropwise addition of TES (278 μL, 1.7 mmol). The clear solution was stirred for 5 h 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 / CH3CN (0.1% TFA 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-tetraazahexacosane-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 h and concentrated to a tan residue. The residue was dissolved in 1 / 1 water / acetonitrile (3 mL) and concentrated to C. 18 Purification was achieved by reversed-phase flash chromatography using a step gradient consisting of 100% water, 0.1% TFA, followed by 80 / 20, 60 / 40 water / acetonitrile (each 0.1% TFA). Each gradient step consisted of approximately 144 mL of solvent volume. The flow rate was 40 mL / min. Fractions containing the desired product were concentrated to a residue and lyophilized to give (3S,7S)-5,13,20,28-tetraoxo-32-(2,4,6-triethynylphenoxy)-4,6,12,21,27-pentaazadotriacontane-1,3,7,22-tetracarboxylic acid as a white solid. 169 mg, 54% yield. ESI-MS, C 43 H 57 N5O 13 [M+H] + The theoretical value is 852.4 and the measured value is 851.9. 1 H NMR (400MHz, DMSO-d6) 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 C NMR (100MHz, DMSO-d6) δ175.0, 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 To a mixture containing (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) was added 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 hours. 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, 10x250mm column and a gradient consisting of 0-95% acetonitrile:water over 20 minutes. The desired product (003) eluted at 6.1-7.1 minutes. Fractions containing 003 were combined, concentrated, and lyophilized to give a white solid. 13 mg, 34% yield. ESI-MS C 94 H 141 Gd3N 26 O 34 [MH] - Theoretical value is 2650.7, measured value is 2648.9.

[0175] Scheme 1

[0176] [ka]

[0177] Scheme 2

[0178] [ka]

[0179] Scheme 3

[0180] [ka]

[0181] result The structures of representative PSMA contrast agents containing Lys-Glu urea as the targeting moiety: monomeric, dimeric, and trimeric Gd (Gd1, Gd2, and Gd3) are shown in Figure 1A. A multi-step solution-phase synthetic method was developed to prepare the target compounds, which are 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 high relaxivity. The structure of Gd1 is based on a recently reported lead-86Y-labeled imaging agent for positron emission tomography (PET), which has demonstrated high and 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, the yield of the coupling reaction was significantly improved when the reaction was carried out in a sonication bath at room temperature. Gd3 contains a phenolic core to which three Gd(III)-DOTA are attached via rigid triazole bonds using click chemistry as previously reported by Mastarone et al. A PSMA-targeting functional group was conjugated to the core via the phenolic oxygen. Gd3 showed relatively high relaxivity as a result of the more rigid triazole linker moiety. The compounds were purified by reversed-phase HPLC and characterized by LCMS. To confirm whether the Gd(III)-containing moiety of the materials had a potentially negative effect on the binding affinity of the probe, the PSMA inhibition constant (Ki) values ​​were determined for Gd1, Gd2 and Gd3 using a fluorescence-based PSMA inhibition assay and are listed 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 a reference ligand. As expected, all compounds showed high binding affinity, with Ki values ​​highest for Gd1 (0.45 nM), followed by Gd3 (7.19 nM), and lowest for Gd2 (18.18 nM). When imaged at 9.4 T and 25 °C, solution phantoms showed binding affinities ranging from 3.0 to 6.2 mM-1 s -1 (Gd(III)), 3.0-12.5 mM -1 s -1 The results show that the r1 relaxivity in PBS varied between the contrast agents (Table 1). As expected, Gd1 had the lowest relaxivity at 25°C, followed by Gd2 and Gd3. To confirm the selectivity and specificity of the agents, human prostate cancer cells genetically engineered to express high amounts of PSMA (PC3 PIP) and corresponding wild-type, non-PSMA expressing cells (PC3 flu) were selected as negative controls (Banerjee, Angew., 2001). After incubation with Gd1 or Gd2, pelleted PSMA+PC3 PIP and PSMA-PC3 flu cells showed no change in T1-weighted MR contrast or R1. Conversely, T1-weighted images of both cell lines incubated with Gd3 showed significant MR contrast enhancement in PSMA+PC3 PIP cells compared to unlabeled cells and to PSMA-PC3 flu cell pellets, as shown in Figure 4A. Enhancement and T1 measurements by MRI of cell pellets in the presence and absence of 50 μM Gd3 showed, after removal of Gd3 from the cell lines by washing with standard medium, a higher enhancement and a smaller difference in T1 relaxation rate, ΔR, between Gd3-treated PIP cells and control PIP cells compared to Gd3-treated flu and control flu control cells. 1 4B and 4C).

[0185] Selective blocking experiments by performing co-incubation of Gd3 and ZJ43 indeed showed a significant blockade of T1 enhancement. Cells incubated with Gd3 in the presence of ZJ43 showed only minor changes in T1 values ​​in both types of cells, indicating that ZJ43 was able to specifically block Gd3 binding. These results show that Gd3 exhibits receptor-specific cell binding to PSMA+PC3 PIP cells and that PSMA-mediated contrast enhancement is seen, proving the concept of receptor-mediated endocytosis. Indeed, ICP-MS analysis of cells after post-imaging analysis showed that only little Gd(III) was associated with PC3 flu cell pellets, whereas PC3 PIP cell pellets had high Gd amounts (Figures 2 and 3). PSMA+PC3 PIP cell pellets had estimated intracellular Gd(III) concentrations of 22.82 μM for Gd3, followed by 12.5 μM and 7.2 μM for Gd2 and Gd1, respectively (Figure 2). Thus, the difference between ΔR1 of PIP cells and that of flu cells reflects the change due to specific Gd3 binding to PSMA in PIP cells.

[0186] Cellular internalization assays revealed that the percentage of the incubated dose that underwent internalization (%ID) in PSMA+PC3 PIP cells for Gd1 and Gd2 was 9.06 ± 0.31 and 21.63 ± 3.51, respectively, after 4 h of incubation, at which time only 2.42 ± 0.11 and 3.51 ± 1.32%ID were associated with the cell surface (Figure 3). In addition, a slightly higher nonspecific uptake was associated with Gd2 in PSMA-PC3 flu cells, which is consistent with its lower K i To further investigate cellular uptake and internalization, Rhodamine Red was used. TMWe prepared a dual-modality Gd monomer contrast agent labeled with -X to confirm PSMA-mediated internalization of this class of contrast agents (Figure 5D). As expected, this agent showed specific and high accumulation only in PSMA+PC3 PIP cells (Figure 5A-C). These results indicate that cellular receptors expressed at this level can be detected by MRI using these simple targeting agents.

[0187] Time-dependent internalization studies were performed for Gd3 after 1, 4, and 24 h of incubation (Figures 6A and 6B). Cellular uptake after 1 and 4 h was high and specific, with 28.30 ± 0.47 and 39.92 ± 3.59%ID in PSMA+PC3 PIP cells, respectively, and after 24 h of incubation, approximately 89.69 ± 3.90%ID was observed. 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 T1-weighted enhancement of Gd3 in PSMA+PC3 PIP cell pellets and the high and specific accumulation of Gd3 in PSMA+PIP cells.

[0188] Prior to evaluation of Gd3 for imaging in live mice, its biocompatibility was examined using a cell proliferation assay. Various concentrations of Gd3 were incubated with PSMA+PC3 PIP and PSMA-PC3 flu cells for 24 h. Gd3 had no significant effect on PSMA-flu cell viability (i.e., approximately 90% viability) up to Gd(III) concentrations of 1 mM (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 may be due to the high cellular internalization of Gd3 as well as the long incubation time (24 h) used.

[0189] In vivo MR imaging was performed after intravenous injection of Gd3 (0.05 mmol / Kg dose) at 9.4 T in male NOD / SCID mice bearing PSMA+PC3 PIP (right) and PSMA-PC3 flu (left) tumors implanted subcutaneously in the right and left lower flank, respectively. No nonspecific uptake occurred in both PIP and flu tumors during the first 20 min after injection. A steep drop in T1 values ​​was observed in all tissues, with the highest value of 0.63 s for PSMA flu, followed by 0.57 s for PIP tumors and 0.261 s for muscle. Importantly, a fast clearance of the contrast agent from muscle and flu tumors was observed. Contrast enhancement in PIP tumors was highest at 36% 40 min after injection and remained high at 30% until 1.5 h after injection. After 3 h, the T1 values ​​of muscle and flu tumors were confirmed to have returned to their initial values, while the T1 values ​​of PIP tumors showed no significant change.

[0190] In vivo MR imaging of Gd3 was also performed after a single bolus intravenous injection (0.06 mmol / kg) in mice bearing PSMA+PC3 PIP and PSMA-PC3 flu tumor xenografts implanted subcutaneously in the right and left lower flank, respectively. Figure 9A shows quantitative contrast enhancement mapping of 1 mm sections for both tumors 40-160 min after injection (ΔR1). Contrast enhancement remained constant for at least 3 h in PSMA+PC3 PIP tumors, but rapidly decreased in PSMA-PC3 flu tumors and muscle tissue. The change in T1 value for PSMA+PC3 PIP tumors (Figures 10A and 10B) reached a nadir of 1819 ± 76 ms (mean ± SD, mean 36% enhancement in R1 value, n = 4) in the first 40-60 min, remained constant at 29% until 90 min, and slowly decreased to 24% 190 min after injection. For PSMA-PC3 flu tumors, the highest contrast enhancement was approximately 24% 20 min after injection, followed by a rapid decay of contrast enhancement (40 min, ΔR1<20%). These results demonstrate specific contrast enhancement (P≦0.05) for PSMA+PC3 PIP tumors 80 and 120 min after injection. As shown in Figure 9B, these results were directly compared to other mice administered a non-targeting trimeric Gd probe in the same manner (no tumor enhancement was observed) (Mastarone, 2011).

[0191] Under the same experimental conditions, a control study using saline (PBS) showed no change in T1 values ​​for PIP and flu tumors (Figure 12).

[0192] Without wishing to be bound by any particular theory, upon binding to PSMA, the rotational correlation time of Gd3 was increased compared to the unbound state. Binding may also have altered the hydration number and water exchange rate for each agent, altering the relaxivity values ​​from those expected for the free contrast agent (Caravan et al., 2007). In addition, at high magnetic fields, the extended rotational correlation time may be accompanied by a slight decrease in relaxivity due to interactions of the contrast agent with cellular components (Caravan, P., et al. 2009; De Leon-Rodriguez, LM, et al. 2010; Geninatti-Crich, S. 2011). By leveraging sensitive polymeric Gd(III) complexes in combination with established PSMA-targeting small molecules, we have performed PSMA-targeted MR molecular imaging in vitro and in vivo.

[0193] In summary, the Gd-based contrast agent Gd3 was successfully used for PSMA-specific MR imaging in vivo in mouse xenografts. Optimization of the construct for translational use in prostate and other cancers is ongoing.

[0194] Example 2 PSMA receptor concentration Number of receptors per cell (NRC) = 4.9 x 10 6 , r 細胞 = 8.75 μm

[0195]

number

[0196]

number

[0197] Therefore, 0.05 seconds -1 Changes in organization t 0 Relaxivity is required to see approximately 10% enhancement (assuming 1=2 seconds) (assuming receptor:contrast is 1:1).

[0198]

number

[0199] Example 3 Prostate-specific membrane antigen for dosimetric estimation 86 Preclinical evaluation of Y-labeled inhibitors overview 86 Y (half-life = 14.74 hours, 33%β + ) are part of a new class of positron-emitting isotopes with relatively long physical half-lives that allow for long-term imaging of biological processes. 86 Preparation and biodistribution study of Y-labeled PSMA-conjugated urea (Figure 14), and the corresponding 90 Y- and 177 Imaging of the most pharmacokinetically favorable substances in non-human primates for radiation dosimetry in preparation for clinical trials with Lu-labeled substances has been reported.

[0200] [ 86 A multi-step synthesis was used to prepare [Y]-4–6. PSMA inhibition constants were evaluated by competitive binding assays. In vivo characterization was performed using tumor-bearing male mice by PET / CT. 86 Y]-4 to 6, and [ 86 Y]-4 and [ 86 A biodistribution study of [Y]-6 was performed. Quantitative whole-body PET scans were recorded to determine the kinetics of [Y]-6 in 14 organs in male baboons. 86 Y]-6 was used.

[0201] Compound [ 86 Y]-4–6 were obtained in high radiochemical yield and purity with specific activities exceeding 83.92 GBq / μmol. PET imaging and biodistribution studies using PSMA1 / 2-positive PC-3 PIP and PSMA-negative PC-3 flu tumor-bearing mice demonstrated that [ 86We found that compounds [Y]-4–6 showed high site-specific uptake in PSMA-positive PC-3 PIP tumors beginning 20 min after injection and remaining high after 24 h. 86 [Y]-6 showed the highest tumor uptake and retention, with 32.17 ± 7.99 and 15.79 ± 6.44% of dose / g (%ID / g) at 5 and 24 hours, respectively. Low radioactivity concentrations were associated with blood and normal organs, except for the kidney, which is a PSMA-expressing tissue. PET imaging in baboons revealed that all organs had a biphasic (fast and slow) clearance, with the highest uptake in the kidney at 25 minutes (8%ID / g). Individual absolute uptake kinetics were used to calculate radiation doses using OLINDA / EXM software. The highest mean absorbed dose was observed in the renal cortex, with 1.9 mGy / MBq[ 86 Y]-6.

[0202] Materials and Methods Solvents and chemicals obtained from commercial sources were of analytical grade or better 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, IL). Carrier-free [ 86[Y](NO3)3 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, TX). Yttrium(III) nitrate, triethylsilane (Et3SiH), diisopropylethylamine (DIEA) and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were purchased from Thermo Fisher Scientific (Pittsburgh, PA) 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), I2 and 1% ninhydrin in EtOH. Flash chromatography was performed using silica gel MP SiliTech 32-63 D60A purchased from Bodman (Aston, Pa.) All experiments were performed in duplicate or triplicate to ensure reproducibility. 1 H NMR spectra were recorded using a Bruker Ultrashield TM Recorded on a 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm downfield referenced to proton resonances resulting from incomplete deuteration of the NMR solvent. Low-resolution ESI mass spectra were obtained from a Bruker Daltonics Esquire 3000 Plus spectrometer (Billerica, MA). High-resolution mass spectra were obtained from the University of Notre Dame Mass Spectrometry & Proteomics Facility (Notre Dame, IN) using ESI, either by direct infusion on a Bruker micrOTOF-II or by a C coupled to a Bruker micrOTOF-Q II. 18 The elution was obtained by LC elution on an ultra-high pressure Dionex RSLC column.

[0203] 4-6 and [ 89 Purification of 4–6 was carried out using a Phenomenex C column attached to a Waters 600E Delta LC system. 18 Luna 10x250mm 2 The column was run with a Waters 486 variable wavelength UV / Vis detector, both controlled by Empower software (Waters Corporation, Milford, MA) (Figures 15A and 15B, 16A and 16B, 17A, 17B and 17C). HPLC was run using solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) with the following methods: Method 1: Elution gradient was 75% A and 25% B (5 min), 75% A to 60% A and 25% B to 40% B (5 to 25 min), 60% A to 75% A and 40% B to 25% B (25 to 30 min) with a flow rate of 8 mL / min. Method 2: Flow rate was 8 mL / min. The elution gradient was 100% A and 0% B (0-5 min), 100% A-45% A and 0% B-55% B (5-45 min). 86 HPLC purification of 4–6 was performed on a Varian Prostar system (Palo Alto, CA) equipped with a model 490 UV absorbance detector and a Bioscan NaI scintillation detector connected to a Bioscan Flow-count system (Bioscan, Washington, DC, USA). 86 For HPLC purification of 4–6, Waters Novapak C 18 150x3.9mm 2 A column was used. HPLC was performed using solvent A (0.1% TFA in water) and solvent B (0.1% TFA in CH3CN) with a flow rate of 1 mL / min. An isocratic method of 85% A and 15% B for 25 min was used. 86 A gradient method (78% A and 22% B from 0 to 5 min, 78% A to 58% A and 22% B to 42% B from 5 to 25 min) was used to purify [ 86A gradient method (0-5 min: 88% A and 12% B, 5-25 min: 88% A-68% A and 12% B-32% B) was used for [Y]5. 86 [Y]6 was used for purification. Specific activity was calculated as the radioactivity eluting at the retention time of the product during preparative HPLC purification divided by the mass corresponding to the area under the curve of UV absorption. All final compounds were obtained with radiochemical purity of >95% as determined 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 CHCl (3 mL) followed by DMF (3 mL). A solution of 20% piperidine in DMF (3x3 mL) was added to the resin, which was then gently shaken on a mechanical shaker for 30 min at ambient temperature. The resin was washed with DMF (3x3 mL) and CHCl (3x3 mL). The generation of the free amine was confirmed by the Kaiser test (Kaiser et al., 2010). The coupling efficiency was evaluated by the Kaiser test. The sequence above was repeated for two more coupling steps with Fmoc-Phe-OH and DOTA-(t-butyl ester)3-CO2H. The final compound was cleaved from the resin using TFA / CH2Cl2 (1 / 1) and concentrated under vacuum to give 3. The concentrated product was purified by CHCl3 / C. 18 Purification was performed using a SepPak Vac 2g column. The product was eluted with a solution of 70 / 30 water / acetonitrile (0.1% TFA each) and lyophilized. ESI-MS: 974 [M+H] + To a solution of 3 (15 mg, 15.4 μmol, in 1 mL DMSO) was added 1 (15 mg, 26.18 μmol) and TEA (30 μL) and allowed to stand at ambient temperature for 2 h. After removal of the solvent, compound 5 was purified by HPLC (Method 1). 1 H NMR(DMSO-d6)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). 13C NMR(DMSO-d6)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 is 1431.7042 [M+H] + , Measurement 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-pentaazaheptacosane-21,25,27-tricarboxylic acid, 6 Compound 6 was prepared in three steps as described below. Commercially available N-Boc-1,4 diaminobutane (27 mg, 0.15 mmol, in 0.5 mL 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 DMSO) and DIEA (132 μL, 0.75 mmol) and stirred at 40° C. for 4 h. The solvent was evaporated and the solid residue was purified by reverse phase C chromatography. 18 Purification by flash chromatography (5.5 g, Agilent SF10) with water and acetonitrile (0.1% TFA in each) afforded the Boc-protected 7 after lyophilization. Yield: approx. 55%. ESI-MS 740 [M+H] +The compound obtained in this step was then treated with ice-cold TFA / CH2Cl2 (1 / 1) solution and stirred at ambient temperature for 2 h. The solvent was evaporated and the residue was dried under vacuum and purified by reversed-phase flash chromatography (5.5 g, Agilent SF10) to give 7 in moderate yield. 1 H NMR(DMSO-d6)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 DMSO) was added 1 (10 mg, 17.4 μmol, in 200 μL DMSO) and DIEA (27 μL, 170 μmol) and allowed to stand at ambient temperature for 2 h. After solvent evaporation, the residue was dissolved in water and purified by HPLC (Method 2) to give 6. t , 22.5 minutes. 1 H NMR(DMSO-d6)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] + , Measurement 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 YNO3 (0.5 M) and the mixture (pH 6.1) was incubated at 90 °C for 30 min. A solution of EDTA (200 μL, 30 mM, pH 6.0) was added and the reaction mixture was incubated at 40 °C for 10 min to complex unreacted yttrium(III). The resulting compound was purified by HPLC (Method 2, R t, 21 min), concentrated by evaporation and freeze-dried. 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 , 26 min) HPLC purification. ESI-MS: 1517 [M+H] + C 69 H 96 N 12 O 21 Theoretical value for Y, [M+H] + 1516.5793;Measurement 1516.5793

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

[0209] Radiochemistry: 86 Y]4~5 and [ 86 Radiolabeled with [Y] 86 Y] was carried out according to the same general method as described in 6.

[0210] [ 86 Y]6 A freshly prepared solution of ascorbic acid (50 μL, 220 μg) 86Radiolysis was prevented by adding YNO3 (111-148 MBq (3-4 mCi), 0.1 M in 500 μL of nitric acid) to a solution. Approximately 50-70 μg of 6 (in 0.3 M NaOAc) (purged under N2 for 2-3 min) was added to the solution and neutralized by adding 60 μL of 3 M NaOAc to a pH of approximately 5.5-6, followed by brief vortexing of the mixture and then incubation at 95 °C for 20 min. The reaction mixture was diluted with 1 mL of water. Complexation was monitored by injecting 10-15 μL aliquots of the solution into the HPLC. The radiolabeled product [ 86 [Y]6 was obtained in approximately 90-95% radiochemical yield and >98% radiochemical purity as determined by ITLC (Gelman ITLC strips, 10 mM EDTA). t , for the desired product as a mixed isomeric compound in about 13.9-14.8 min, and R for the free ligand. t The elution time was 15.8 min. The specific activity was >83.92 GBq / μmol (n=5). The acidic eluate was neutralized with 20 μL of 1 M sodium carbonate solution and the volume of the eluate was reduced to dryness in 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 eluted peak, only one peak was isolated at around 14.3 min when the tracer was reinjected into the HPLC. 86 To confirm the isomerization of Y6, compound 6 was added to a support. 86 Radiolabeled with Y and the mixture was analyzed by HPLC. Only one peak was isolated at 14.3 min. 86 For [Y]4-5, a single radiolabeled peak was isolated. 86 Y]4R t is 14.0 min, and the R of non-chelated 4 t is 15.5 minutes, and [ 86 Y]5 is R t = 16.9 min, R for non-chelated 5 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 i The values ​​of PSMA expression were calculated 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 were genetically engineered to express high levels of PSMA (PC-3 PIP) or to naturally produce low levels of PSMA (PC-3 flu) (Dr. Warren Heston, Cleveland Clinic, Cleveland, OH).

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

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

[0214] For biodistribution assays, NOD / SCID mice bearing PSMA+PC-3 PIP and PSMA-PC-3 flu xenografts were injected with 0.55 MBq (15 μCi) via the tail vein. 86 Y-4 or 86Y-6. In each case, four mice were sacrificed by cervical dislocation at 1, 2, 5, and 24 hours after injection. Heart, lung, liver, stomach, pancreas, spleen, fat, kidney, muscle, small intestine, large intestine, bladder, and PSMA+PC-3 PIP and PSMA-PC-3 flu tumors were rapidly removed. A 0.1 mL blood sample was also taken. Each organ was weighed and tissue radioactivity was measured in 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 serially diluted samples of 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 with 3% isoflurane and maintained under 1.5% isoflurane (v / v). 86 Y-4 or 86 In Y-6, n=3, 86 Y-5 (n=2) were injected via the tail vein with 3.33–6.21 MBq (90–168 μCi) of radioactive tracer formulated in 100 μL of saline, pH ≈7. For binding specificity studies, mice were injected with 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). 86 30 min before injection of Y-4, and another mouse was injected subcutaneously. 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 anesthetic flow rate 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 latency, and scattered radiation. No partial volume correction (PVC) was performed. After each PET scan, a CT scan was performed for anatomical co-registration. To facilitate the co-registration of PET and CT images, a special bed for mice was used that fits both the PET and CT scanners. Mice were under anesthesia and immobilized during transfer between the scanners and during both scans. The reconstructed PET and CT images were then manually co-registered by passing them through a rigid body transformation and aligning natural landmarks (e.g., mouse limbs and bed contour) using the AMIDE software (sourceforge.net / amide). Data were displayed and analyzed with AMIDE.

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

[0217] 86 Y-6 Anubis baboon (baboon) PET imaging A male Anubis baboon (8 years old, 27.1 kg) was used. 86The biodistribution of Y-6 was studied. Baboons were positioned in supine position during imaging. For attenuation correction, low-dose CT images were taken immediately before the first and last PET images. PET and CT images were registered between time points using a Hermes workstation (Hermes Medical Solutions, Greenville, SC). Contours of 14 source organs were delineated on the CT with the aid of fused PET / CT images. Decay-corrected mean radioactivity concentrations (Bq / g) were extracted for each source organ from the PET images. Contours were delineated on the PET images of kidney, renal cortex, and prostate. Decay-corrected total radioactivity per organ quantified in the PET images showed an almost one-to-one correspondence with the administered radioactivity for the first six time points (within 1 h), confirming that all administered radioactivity was utilized in the PET images, after which the total amount was less than the administered amount due to urination. The total amount of radioactivity quantified in each PET image was used to obtain whole-body retention kinetics.

[0218] Nine static PET images were obtained using a bolus of 80.7MBq (2.2mCi). 86 Images were taken 5, 10, 15, 20, 35 min, 1, 2, 3, 5, and 23 h after intravenous administration of Y-6. Images were taken in 2D mode with 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 organ volume to obtain the total radioactivity per organ per time point. Measurements were then decay corrected and divided by the mass of the baboon organ and the injected radioactivity, determined from the CT density and volume of the contoured organ, to obtain the percentage of initial radioactivity per gram (FIA / g) for each time point and organ. Baboon FIA / g values ​​were then converted to human FIA (per organ) using the following equation (Schwartz et al., 2011; Woodard et al., 1975):

[0220]

number

[0221] In the formula, whole body mass ヒヒ = 27.1kg, total body mass ヒト =73.7kg.

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

[0223]

number

[0224] In the formula, A1, A2, λ1 bio and λ2 bio are the fit parameters. The sum of A1 and A2 gives the back-extrapolated fraction of administered radioactivity in each organ at time zero, and λ1 bio and λ2 biois the biological clearance constant. The equation for the time-integrated activity coefficient (TIAC, formerly known as residence time (Bolch et al., 2009)) for each source organ is, as the name implies, integrated into equation (2) and the physical decay term λ φ was obtained by introducing (depending on the isotope used).

[0225]

number

[0226] TIAC 90 Y, 177 Lu and 86 For Y, the corresponding physical decay constants voided are: 90 Y λφ=0.01083h -1 (T 1 / 2 =64.0 hours); 177 Lu λφ=0.00429h -1 (T 1 / 2 = 161.52 hours) and 86 Y λφ=0.04702h -1 (T 1 / 2 = 14.74 h). Radiation absorbed dose was obtained by converting time-integrated activity to absorbed dose according to the MIRD absorbed fraction method (Bolch et al., 2009) using OLINDA / EXM software (Stabin et al., 2005). TIAC for the bladder was obtained using the MIRD bladder model implemented in OLINDA / EXM. Input to the model required whole-body TIAC, which was fitted to whole-body retention kinetics.

[0227]

number

[0228] The voiding interval was set to 2 hours. The TIAC was then entered into OLINDA / EXM (Stabin et al., 2005) to obtain the absorbed dose per unit activity for the 14 organs. Renal cortex dose values ​​were obtained by using a specific kidney model in OLINDA / EXM. Absorbed doses from organs outside the kidney were 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 proxy for the whole body. The self-dose component of the absorbed dose per unit activity for the salivary glands was obtained using 3D-RD Monte Carlo (EGSnrc) and human CT imaging of the salivary glands. The cross-dose component was obtained by assuming that the cross-dose of an organ of similar size (pancreas) is the same.

[0229] Measured radioactivity concentration (Bq / cm) per organ at each time point 3 The ) values ​​were decay-corrected and divided by the mass of the baboon organ and the injected activity, determined from the CT density and the volume of the contoured area, to obtain the fraction of initial activity per gram (FIA / g) for each time point and each organ. The baboon FIA / g values ​​were then converted using an equation related to human FIA (per organ) (Olszewski et al., 2004; Schwartz et al., 2011). The resulting human FIA values ​​were then plotted as a function of time and fitted to a biexponential expression to calculate the time-integrated activity coefficient (TIAC, previously known as residence time (Woodard et al., 1975)) values ​​for each source organ. The radiation absorbed dose was obtained by converting the time-integrated activity to absorbed dose according to the MIRD absorbed fraction method (Woodard et al., 1975) using OLINDA / EXM software (Bolch et al., 2009).

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

[0231] result Compounds 4 and 5 were prepared using solid- and liquid-phase combinatorial peptide synthesis methods as shown in Schemes 4 and 5. Compounds 1 and 4 were prepared as previously reported (Banerjee, Pullambhatla, Byun, et al., 2011). The synthesis of DOTA-conjugated ligand 5 was carried out using standard fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) starting from Fmoc-Lys(Boc)-Wang resin according to Scheme 5. Three phenylalanine residues were coupled to the resin-bound lysine, followed by conjugation of DOTA, and then the compound was cleaved from the resin with a 1 / 1 mixture of TFA / CH2Cl2 to give 3 in moderate yield (~20%). The free ε-amine of the lysine of 3 was then 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) in the presence of diisopropylethylamine at 40° C. for 4 h, followed by removal of the Boc group to give 7 in moderate yield after purification by HPLC. Compound 7 was then conjugated with 1 to give 6 in good yield. The stable yttrium ( 89 Y) complexes were prepared by incubating conjugates 4–6 with an aqueous solution of YNO3 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 makes the compounds neutral overall, but [ 86 / 89It should be noted that Y]6 has four coordinated carboxylic acids, which makes the overall compound negatively charged. 86 Y] 4 to 6, follow the same general procedure [ 86 Y]NO3 and ligand concentration 10 -6 The solution was prepared by reacting in boiling water at 30 M for 30 min at pH 5-6.

[0232] [ka] a. (i) 20% piperidine / DMF; (ii) Fmoc-Phe-OH, HOBT, HBTU, steps i-ii were repeated twice for X=2 and three times for X=3; (iii) 20% piperidine / DMF; (iv) DOTA-tris(tert-butyl ester)-CO2H, HOBT, HBTU, DIEA; (v) TFA / TES / H2O (98 / 0.5 / 1.5); b. DMSO / TEA, room temperature; c. Y(NO3)3 / NaOAc, pH 5.5, 90℃, 20 min; d. 86Y(NO3)3 / ascorbic acid / NaOAc, pH 5.5, 90℃, 20 min.

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

[0234] [ka]

[0235] Scheme 5:6 and [ 86 / 89 Synthesis of Y6

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

[0237] [Table 2]

[0238] Small Animal PET Imaging Whole-body PET / CT images 86 Y-4, 86 Y-5 and 86 Y-6 (Figures 18A, 18B, 18C, 19A, 20A, 20B and 20C). All three radiotracers allowed visualization 2 hours after injection in PSMA+PC-3 PIP tumors and in the kidney, a known PSMA-expressing organ (Figures 18A, 18B and 18C). Renal uptake of the radiotracers is due in part to excretion pathways for these substances, as well as specific uptake due to expression of PSMA in mouse proximal tubules (Stabin et al., 2005). Substances 86 Y-5 showed nonspecific accumulation in the gastrointestinal tract, presumably due to increased hydrophobicity caused by the three Phe residues in the linker moiety. 86 PET-CT images of Y-4 were taken 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 PSMA+PC-3 PIP tumors and in the kidneys and bladder was observed for up to 4 hours (Figure 19A). Radioactivity in the bladder and kidneys had largely disappeared by 18 hours, although some radioactivity remained in the PSMA+PC-3 PIP tumors. As a further test of in vivo binding specificity, 86Blockade studies of Y-4 were performed by pretreating animals with 50 mg / kg of the potent and selective PSMA inhibitor ZJ43 (Silver et al., 1997). Figure 19B shows that ZJ43 inhibits PSMA not only in tumors but also in another PSMA-expressing tissue, the renal cortex. 86 Figures 20A, 20B, and 20C show that the binding of Y-4 can be blocked by 0.5, 2, and 12 hours after injection. 86 This is a PET-CT image of Y-6. Importantly, 86 Y-6 demonstrated faster radioactivity clearance from normal tissues, and by 12 hours post-injection, radioactivity was largely excreted by the kidneys, resulting in clean tumor / background contrast. Clean imaging of PSMA+PC-3 PIP tumors was achieved as early as 15 minutes. Notably, 86 Y-6 is 86 Y-4 and 86 It does not contain the additional phenylalanine moiety of Y-5, but instead utilizes the p-isothiocyanatobenzyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, which adds an additional carboxylate to provide stronger metal retention and reduced lipid solubility.

[0239] Biodistribution in mice Based on the imaging results, 86 Y-4 and 86 Y-6 was further evaluated in a standard biodistribution 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 post-injection. Both radiotracers showed PSMA-dependent binding in PSMA+PC-3 PIP tumor xenografts, 86Y-4 showed high tumor uptake as early as 1 hour post-injection (29.3±8.7%ID / g) with relatively slow clearance to 15.7±1.7%ID / g at 5 hours and 5.9±0.8%ID / g at 24 hours post-injection. PSMA+PC-3 PIP tumor to PSMA-PC-3 flu tumor uptake ratios ranged from 89 at 1 hour to as high as 229 at 24 hours. Blood and normal tissues, such as heart, liver, stomach, and pancreas, showed no significant uptake (approximately 1%ID / g), decreasing to less than 0.02%ID / g at 24 hours. PSMA+PC-3 PIP tumor to muscle ratios were also high, achieving a maximum of 1046 at 24 hours. Kidney uptake was high as expected, peaking at 244.9±8.8%ID / g at 1 hour, decreasing to 1.5±0.7%ID / g by 24 hours.

[0240] Table 4 shows 86 Organ %ID / g uptake values ​​for Y-6 are shown. 86 Y-6 rapidly accumulated in PSMA+PC-3 PIP tumors within 1 hour of injection with an uptake value of 26.6±1.9%ID / g. Radiotracer concentration continued to increase in PSMA+PC-3 PIP tumors with a peak uptake of 32.2±8.0%ID / g 5 hours after injection. Tumor uptake remained high up to 24 hours after injection. Normal organs, including blood, heart, liver, spleen, stomach, and pancreas, showed low uptake at 1 hour, decreasing to less than 0.4%ID / g by 5 hours. 86 Renal uptake of Y-6 was 86.5±13.6%ID / g and 54.0±9.2%ID / g at 1 and 2 hours, respectively. 86 This was much less than in the case of Y-4.

[0241] [Table 3]

[0242] [Table 4]

[0243] Baboon PET imaging and 86 Pharmacokinetics of Y-6 Figures 21A and 21B show a PET study in which the radiotracer is seen in the liver, salivary glands, kidneys and bladder. Kidneys, renal cortex and whole prostate were outlined in each PET image for quantification. All organs showed a biphasic (rapid and slow) biological clearance. The kidneys showed the highest uptake at approximately 25 minutes post-injection (8%ID / g). Sixty-eight percent of the radioactivity seen in the kidneys was cleared with a biological half-life of approximately 1 hour (0.84 hours), with the remaining radioactivity clearing with a biological half-life of 16.6 hours. The majority of the radioactivity in the renal cortex (66%) was cleared with a biological half-life of 1.1 hours, with the remaining radioactivity clearing with a biological half-life of approximately 19 hours. Significant uptake and retention was seen in the liver and salivary glands, except for 68 Ga-labeled PSMA target substance and 124 / 131 This was mild compared to PET scans of patients imaged with I-MIP-1095 (Zechmann et al., 2014). Table 5 summarizes the biological clearance kinetics for all organs. The TIACs used in dose calculations are shown in Table 6.

[0244] [Table 5]

[0245] [Table 6]

[0246] Organ absorbed dose Table 7 shows the doses in mGy / MBq. 86 Y, 90 Y / 177A detailed list of organ absorbed doses for Lu. For all isotopes, the renal cortex received the highest absorbed dose per unit activity. Therefore, in patient-specific absorbed dose treatment planning, the renal cortex is considered to be the dose-limiting organ for therapeutic radiometals (Baechler et al., 2012; Hobbs et al., 2009), followed by the urinary bladder. Diagnostic isotopes 86 For Y, an effective dose of 0.099 mSv / MBq was also calculated using OLINDA / EXM.

[0247] [Table 7]

[0248] Essay Three methods were used to perform non-human primate dosimetry: 86 Y-labeled PSMA-targeting materials were synthesized and evaluated. These compounds contain DOTA- or DOTA mono-amide chelated radiometals bound to targeting ureas similar to those previously published (Banerjee et al., 2010; Banerjee, Pullambhatla, Byun, et al., 2011). DOTA and its derivatives have been shown to be useful for the detection of PET ( 86 Y) or radiopharmaceutical treatment ( 90 It has been noted that the pharmacokinetics depends on the radiometal chelator used, including for compounds specifically designed to bind to PSMA. Without wishing to be bound by any theory, this is believed to be due primarily to the net charge of the radioligand and the stability of the metal chelate complex. Specifically, 68 In previous reports on Ga-labeled PSMA-linked DOTA conjugates, 68 Ga-4 showed the fastest clearance from normal tissues, including the kidney (Banerjee et al., 2010). However, in the present study, 86 An unexpectedly high renal uptake of Y-4 was observed. 86Evaluation of Y-6 demonstrated the desirable low kidney uptake and high tumor retention required for radiation therapy and was subsequently selected for quantitative PET imaging in baboons for dosimetry.

[0249] Binding specificity studies (Figure 19B) showed that after 1 h 86 Nearly all binding of Y-4 in the kidney was shown to be specific and not due to excretion. Evidence suggests that a more organized and rapid blood flow in the renal parenchyma compared to the tumor may explain the longer retention of many of these substances in the tumor than in the kidney. PSMA binding affinity is one factor that may determine tumor versus kidney uptake, but other factors such as lipid solubility, charge, plasma protein binding, and molecular weight may also play important roles. Estimated renal cortical dose 1.19 mGy / MBq ( 90 Y) and 0.245mGy / MBq( 177 Lu) was calculated to be 1.97 mGy / MBq ( 90 Y) and 0.45mGy / MBq ( 177 Lu) (Baechler et al., 2012) (in which the renal cortex was the dose-limiting organ).

[0250] The commonly used and clinically available chelator DOTA was used for all three radioligands because DOTA and many DOTA derivatives are known to form kinetically and thermodynamically stable complexes. The corresponding Y(III) complexes have been shown in many cases to be stable in vivo, a desirable feature for a chelator. Importantly, DOTA has 86 Y(III) is chemically distinct from the lanthanides, e.g. 177 Lu(III) and actinides, e.g. 225 It has also been reported that PSMA forms stable complexes with a number of trivalent metal ions, including Ac(III). Moreover, PSMA-conjugated urea-based compounds are stable under the radiolabeling conditions used for DOTA.

[0251] recent years,90 Y or 177 Lu-labeled versions of the PSMA-targeting monoclonal antibody J591 have shown promising results in phase I and II clinical trials (Bander et al., 2005; Tagawa, Akhtar, et al., 2013; Tagawa, Milowsky, et al., 2013). 111 In-labeled antibodies were used for dosimetry calculations (Vallabhajosula et al., 2005). Although these radiolabeled monoclonal antibodies have potential for tumor detection and treatment, their limited tumor targeting and relatively high absorbed dose to red bone marrow militate against routine clinical use. As an alternative approach, 131 Early clinical results using I-labeled PSMA-targeted urea-based small molecules showed high dose delivery to malignant 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 kidneys (1.45 mGy / MBq) (Zechmann et al., 2014). The largest contributor to this salivary gland absorbed dose is likely to be the uptake of free iodine, which is also evidenced by the relatively high (0.91 mGy / MBq) thyroid absorbed dose, which did not occur in the present study. Overall, clearance rates from normal organs were similar, with the exception of the kidneys, 86 Y-6 is faster than published results (Zechmann et al., 2014).

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

[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 177 Cellular uptake studies using Lu-SRVI71, Figure 24, show high uptake in PSMA+PIP and negligible uptake in PSMA-flu tumors. In addition, internalization studies revealed that approximately 44% of the total cell-bound radioactivity was internalized. Furthermore, upon co-incubation with 10 μM N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ43), a PSMA-specific inhibitor, approximately 90% blockade in PSMA+ cells was observed, further confirming the excellent specificity of the material (Figure 23). In vivo evaluation was performed in standard PSMA+PIP and PSMA-flu mouse xenografts, as well as SPECT imaging with VECT or an instrument equipped with an ultrasensitive mouse collimator as shown in Figures 25A, 25B, and 25C. The highest accumulation of radioactivity was seen in PSMA+PIP tumors at all time points. Other visible organs were the kidney and bladder. Biodistribution studies at 2 hours (59.1±12.8%ID / g) and 24 hours (40.6±5.8ID / g) (n=4) indeed demonstrated high uptake and retention (~180 PIP:flu at 2 hours) in PSMA+ tumors with high specificity. Early renal uptake was high at 2 hours (89.3±28.9%), 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 encouraging regarding the feasibility of preparing such low molecular weight theranostic agents with desirable pharmacokinetics for radionuclide therapy. Furthermore, the data support that this class of low molecular weight agents can be effectively internalized when conjugated to PSMA.

[0258] Example 5 Overview of the synthesis and use of ZCP-01 and related materials for PSMA-based targeted radionuclide therapy The preparation and use of PSMA-bound ureas conjugated to chelated radiometals via various linking groups for imaging and possible radiotherapy of PSMA-expressing tumors has been described in several patents and publications as well as in this patent application (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). PSMA inhibitors based on the novel lysine-carbamate scaffold oxypentanedioic acid (OPA) and the "reverse" carbamate scaffold amino-pentanedioic acid (NPA), including F-18 labeled analogs, have been recently developed. The F-18 labeled NPA and OPA compounds showed selective uptake in PSMA-positive tumor mouse xenografts.

[0259] ZCP-01, a DOTA-PEG-linked lysine OPA carbamate, has been synthesized as an example for complexing radiometals for imaging and radiotherapy of PSMA-positive tumors and tissues. Conjugation of a wide range of metal chelating ligands and linkers, previously disclosed in WO 2009 / 002529(A2) and WO 2010 / 108125(A2) for use with urea, to the OPA and NPA scaffolds provides novel radiolabeled materials for imaging and / or radiotherapy of prostate cancer.

[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-triazatetracosane-18,22,24-tricarboxylic acid, ZCP-01 Referring to Scheme 7, to a solution of compound 4 (8.5 mg, 0.015 mmol, in 200 μL DMSO) was added diisopropylethylamine (27 μL, 0.255 mmol), followed by slow addition of DOTA-NHS (15.2 mg, 0.023 mmol, in 200 μL DMSO), and the resulting solution was stirred at room temperature for 2 hours. The solution was then diluted with water and purified by HPLC. HPLC Method: Phenomenex C 18 Luna, 10mm * 250 mm, flow rate: 8 ml / min, λ: 200 nm, 220 nm, solvent: H2O and CH3CN (each 0.1% TFA). Gradient method: 0-20 min, 100 / 0 H2O / CH3CN-80 / 20 H2O / CH3CN; 20-30 min, 80 / 20 H2O / CH3CN-0 / 100 H2O / CH3CN; 31 min, 100 / 0 H2O / CH3CN. 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 InNO3 (0.5 M) and the mixture (pH 6) was incubated at 90 °C for 30 min. A solution of EDTA (200 μL, 30 mM, pH 6.0) was added and the reaction mixture was incubated at 40 °C for 10 min to complex the unreacted indium(III). The resulting compound was purified by HPLC (same as ZPC-01), concentrated by evaporation and lyophilized. ESI-MS: 1066 [M+H] + C39 H 64 InNO 20 The theoretical value for is 1065.79.

[0262] 111 Preparation of In-ZPC-01 1.0 μl of 0.1 N HCl 111 InCl3 (1 mCi) was added to 1 mM Ourea-PEG-DOTA in 20 μl of 0.2 M NaOAc. The pH of the mixture was about 4.0. Then, 20 μl of 0.2 M NaOAc was added to adjust the pH to about 6. The mixture was kept at 50° C. for 1 h and purified by radio-HPLC using an isocratic method with a mobile phase of 90% water (containing 0.1% TFA) and 10% CH3CN (0.1% TFA). Flow rate: 1.0 mL / min; λ: 200 nm and C 18 Column (25x4.6mm), Varian microsob-MV 100-5. Radiolabeling 111 In]ZPC-01 eluted at 14.9 min, and the unlabeled chelator eluted at 32 min.

[0263] Scheme 7

[0264] [ka] a. DCC, N-hydroxysuccinimide, CH2Cl2; b. diisopropylethylamine, DMSO; TFA / water; d. DO3A-NHS (commercially available from Macrocylics)

[0265] result ZCP-01 and [In]-ZCP-01 showed high binding affinity, with Ki values ​​ranging from 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 Figures 34A, 34B, and 34C, in vivo SPECT imaging of [In]-ZCP-01 was performed in mice bearing PSMA+PC3 PIP and PSMA-PC3 flu tumor xenografts implanted subcutaneously in the right and left flank, respectively. 111 In]-ZCP-01 intravenous injection. 111 [In]-ZCP-01 allowed visualization of PSMA+PC3 PIP tumors and the kidney, a known PSMA-expressing organ, at 2 and 4 hours post-injection, but resulted in nonspecific uptake in flu tumors. By 24 hours post-injection, radioactivity had largely been cleared from the tumor and kidney.

[0268] These results are highly encouraging regarding the feasibility of preparing such low molecular weight theranostic agents with desirable pharmacokinetics for tumor imaging and radiotherapy.

[0269] References All publications, patent applications, patents, and other references cited herein are indicative of the level of ordinary skill in the art to which the subject matter of this disclosure pertains. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although a number of patent applications, patents, and other references have been mentioned in this specification, it is understood that such mention is not an admission that these documents form part of the general knowledge in the field.

[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-prostatespecific 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)Ytrastuzumab 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, CM, 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] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, one skilled in the art will recognize that certain changes and modifications may be practiced within the scope of the appended claims. Preferred embodiments of the present invention include the following. 1. Formula (I): TIFF0007630149000047.tif44170 (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 TIFF0007630149000048.tif55170 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 Alkyl -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; 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 TIFF0007630149000049.tif218170 wherein q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8. 3.(C4) TIFF0007630149000050.tif197170 TIFF0007630149000051.tif91170 (wherein M is a metal or a radioactive metal). or a pharma- ceutically acceptable salt thereof. 1. A compound of formula (I) selected from the group consisting of: 4. The compound according to 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 according to claim 1, wherein the non-radioactive metal is Gd-157 (stable isotope). 6. The compound according to claim 1, wherein the radioactive metal 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. The method according to claim 1, wherein the radioactive metal is selected from the group consisting of Y-86 and Sc-44. 8. The compound according to claim 1, wherein the radioactive metal 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) TIFF0007630149000052.tif44170 (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 TIFF0007630149000053.tif54170 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 Alkyl -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; 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 TIFF0007630149000054.tif49170 TIFF0007630149000055.tif176170 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 TIFF0007630149000056.tif44170 TIFF0007630149000057.tif250163 TIFF0007630149000058.tif60170 (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 according to claim 9, wherein the compound is selected from the group consisting of: 12. 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. 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. 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. The method of claim 9, wherein said imaging comprises positron emission tomography (PET) imaging and said radiometal is selected from the group consisting of Y-86 and Sc-44. 16. The method of claim 9, wherein the imaging comprises single photon emission computed tomography (SPECT) imaging and the radioactive metal 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. 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 according to claim 9, wherein the one or more PSMA-expressing tumors or cells are present in a subject. 21. The method according to claim 20, wherein the compound comprising the imaging agent is excreted from a tumor or cell of the subject. 22. The compound comprising an imaging agent, TIFF0007630149000059.tif162170 TIFF0007630149000060.tif97170 (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 kidneys of the subject more quickly than the tumor of the subject.

Claims

1. A compound of formula (I) or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 (In the formula, Z is tetrazole or CO 2 Q. Q is H, or benzyl, p-methoxybenzyl (PMB), tertiary butyl ( t a protecting group selected from the group consisting of methoxymethyl (MOM), methoxyethoxymethyl (MEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), benzyloxymethyl (BOM), trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), and triphenylmethyl (trityl, Tr); 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; R 1 is a C substituted with a 4-bromophenyl group 1 -C 4 is alkyl, R 2 and R 4 are each independently H or C 1 -C 4 is alkyl, Each R 3 are independently H, C 1 -C 6 Alkyl or C 6 -C 12 is aryl, W is independently O or S; Y is -NH- and may be present or absent; and 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; and Ch is a chelating moiety that may contain one or more non-radioactive or radioactive metals and is a DOTA derivative.

2. The L is represented by the following formula: 【Chemistry 3】 2. The compound of claim 1, wherein:

3. 3. The compound of claim 1 or 2, or a pharma- ceutically acceptable salt thereof, wherein the non-radioactive metal is selected from the group consisting of Lu, Gd, Ac, Bi, Pb, Cu, In, Sc and Y.

4. 4. The compound of claim 3, or a pharma- ceutically acceptable salt thereof, 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.

5. 5. The compound of claim 4, or a pharma- ceutically acceptable salt thereof, wherein the radiometal is selected from the group consisting of Lu-177 and In-111.

6. 6. The compound according to claim 5, or a pharma- ceutically acceptable salt thereof, wherein the radiometal is Lu-177.

7. 1. A pharmaceutical composition for imaging, diagnosing or treating one or more prostate specific membrane antigen (PSMA) expressing tumors or cells, comprising: A pharmaceutical composition comprising a compound of the following formula (I) or a pharma- ceutically acceptable salt thereof: 【Chemistry 4】 (In the formula, Z is tetrazole or CO 2 Q. Q is H, or benzyl, p-methoxybenzyl (PMB), tertiary butyl ( t a protecting group selected from the group consisting of methoxymethyl (MOM), methoxyethoxymethyl (MEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), benzyloxymethyl (BOM), trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), and triphenylmethyl (trityl, Tr); 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; R 1 is a C substituted with a halogen-substituted aryl group 1 -C 4 is alkyl, R 2 and R 4 are each independently H or C 1 -C 4 is alkyl, Each R 3 are independently H, C 1 -C 6 Alkyl or C 6 -C 12 is aryl, W is independently O or S; Y is -NH- and may be present or absent; and L is, 【Chemistry 5】 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; and Ch is a chelating moiety that may contain one or more non-radioactive or radioactive metals and is a DOTA derivative.

8. R 1 The pharmaceutical composition according to claim 7, wherein the halogen-substituted aryl group in the formula (I) is a 4-bromophenyl group.

9. The L is represented by the following formula: 【Chemistry 6】 The pharmaceutical composition according to claim 7, wherein

10. 10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the non-radioactive metal is selected from the group consisting of Lu, Gd, Ac, Bi, Pb, Cu, In, Sc and Y.

11. 11. The pharmaceutical composition of claim 10, 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.

12. 12. The pharmaceutical composition of claim 11, wherein the radiometal is selected from the group consisting of Lu-177 and In-111.

13. The pharmaceutical composition of claim 12, wherein the radiometal is Lu-177.

14. The pharmaceutical composition of any one of claims 7 to 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.

15. The pharmaceutical composition of any one of claims 7 to 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.

16. The pharmaceutical composition of claim 15, wherein the one or more PSMA-expressing tumors or cells are prostate tumors or cells.

17. The pharmaceutical composition of any one of claims 7 to 9, wherein the one or more PSMA-expressing tumors or cells are in vitro, in vivo or ex vivo.

18. The pharmaceutical composition of any one of claims 7 to 9, wherein the one or more PSMA-expressing tumors or cells are present in a subject.

19. 20. The pharmaceutical composition of claim 18, wherein the compound is excreted from a tumor or cell of the subject.

Images