BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The present invention relates to small molecules having high affinity and specificity to prostrate-specific membrane antigen (PSMA) and methods of using them for diagnostic and therapeutic purposes.

SUMMARY OF THE RELATED ART

[0002] Prostate cancer is the most commonly diagnosed cancer and second leading cause of cancer death in American men. Hormone ablation or androgen deprivation therapy are widely used in the treatment of prostate cancer, but all prostate cancer eventually becomes hormone resistant, rendering hormone ablation therapy ineffective. Prostate cancer is radiosensitive and, when localized, can be treated with external beam radiation and brachytherapy. To date, only one systemic radionuclide therapy has been approved to treat disseminated bone metastases from prostate cancer. Radium-223 dichloride (Xofigo®) targets bone metabolism/turnover in prostate cancer and was FDA approved in 2013 for palliative treatment of bone metastases. It is not indicated for soft tissue metastases associated with metastatic prostate cancer. Xofigo® modestly increases survival by 3-4 months attributed primarily to a lack of specific targeting to prostate cancer and inability to target soft tissue metastases. Specific targeting of the radiation payload to metastatic sites in advanced and widely disseminated prostate cancer is expected to result in improved efficacy, while minimizing side effects to non-target tissues.

[0003] Prostate-specific membrane antigen (PSMA) is a well characterized biomarker for prostate cancer and can be targeted for diagnostic and therapeutic approaches. PSMA is expressed at low levels on certain normal tissues including the proximal tubules of the kidney and salivary glands but is highly expressed on >90% of prostate cancers. Increased PSMA expression correlates with progression to metastatic and castration resistant disease. Treatment with abiraterone and enzalutamide has been shown to increase PSMA expression and may exacerbate castration resistant prostate cancer (CRPC) through clonal selection of PSMA-expressing androgen independent tumor cells. Various chemical scaffolds have been pursued as PSMA targeting molecules for imaging and therapeutic uses. Effectively addressing prostate cancer and potentially achieving a long term therapeutic benefitcould be achieved by harnessing delivery and affinity of these small- molecule PSMA inhibitors to deliver radiotherapeutic payloads. Indeed, promising therapeutic efficacy results have emerged for two urea-based PSMA inhibitors: PSMA Imaging and Therapy (l&T) and ¹⁷⁷ Lu-PSMA-617, both containing a DOTA chelate complexed with ¹⁷⁷ Lu. ¹⁷⁷ Lu-PSMA-617 treatment results in 40-60% of recipients experiencing a biochemical decline of at least 50% in circulating prostate specific antigen (PSA ₅ O), and a Response Evaluation Criteria in Solid Tumors (RECIST) response rate in soft tissue disease of between 40% and 50%. Hofman et al. demonstrated in a prospective Phase II trial that treatment with ¹⁷⁷ Lu-PSMA-617 yielded 53% partial and 29% complete response rates, minimal side effects, and reduction of pain in men with metastatic CRPC (mCRPC) whose disease progressed after conventional treatments. ¹⁷⁷ Lu-PSMA-617 is currently being evaluated in the Phase 3 VISION trial. Considerable clinical data support the use of PSMA-directed agents for the successful diagnosis and durable treatment of mCRPC and the use of these agents is expected to impart meaningful survival advantages.

[0004] Until now, all small molecule PSMA-targeted agents have been based on a urea scaffold. Up to 30% of men do not respond to treatment, which may be due to reversible binding to PSMA and low overall cellular internalization of the urea scaffold. Salivary glands, and potentially the bone marrow (with long term use) are considered to be the dose limiting organ in many studies using ¹⁷⁷ Lu-PSMA-617 and other radiolabeled urea compounds. Cancer Targeted Technology (CTT) has developed a unique high affinity PSMA-targeted scaffold that, unlike the urea-based scaffolds, binds irreversibly to PSMA. This irreversible binding leads to rapid and extensive internalization by PSMA-expressing tumor cells. This backbone was used to develop an ¹⁸ F-labeled PET imaging agent, CTT1057, that targets PSMA in prostate cancer. CTT completed a Phase 1 trial in September 2017 using CTT 1057, and PET imaging of patients with advanced stage prostate cancer. The trial demonstrated safety and that an intravenous (IV) micro-dose of 370 MBq (10 mCi) detected PSMA positive tumors with greater sensitivity than standard-of-care whole body scans and conventional cross-sectional imaging. Additionally, CTT1057 biodistribution showed lower exposure to kidneys and salivary glands compared to urea agents, which may have considerable safety significance for therapeutic trials.

[0005] The CTT PSMA-targeting backbone was modified to allow delivery of a radiotherapeutic payload, ¹⁷⁷ Lu. ¹⁷⁷ Lu has short tissue penetration of ¹⁷⁷ Lu (2 mm) that allows treatment of small distal metastatic sites, a ~6.6 day half-life to maximize tumor exposure, and commercial clinical grade availability. To allow the PSMA targeting molecule to carry the ¹⁷⁷ Lu to the tumor, a DOTA chelator was joined to the precursor molecule using click chemistry. This radiotherapeutic, CTT1403,

(disclosed in WO 2018/031809) also includes an albumin binding motif that reduces renal clearance and increases tumor bioavailability.

[0006] The CTT1403 drug product is assembled in a 2-step labeling process: 1) chelation of ¹⁷⁷ Lu to DOTA-N ₃ , and 2) click chemistry uniting ¹⁷⁷ Lu-DOTA-N ₃ with the pre assembled CTT1402 precursor:

The 2-step radiolabeling process is required because the phosphoramidate scaffold is incompatible with the high heat (80 - 95°C), low pH (pH 2-4) conditions required to drive ¹⁷⁷ Lu into the DOTA chelator. To retain stability of the CTT1402 precursor, click chemistry of the ¹⁷⁷ Lu-DOTA is required. [0007] Prostate-specific membrane antigen (PSMA) is uniquely overexpressed on the surface of prostate cancer cells as well as in the neovasculature of a variety of solid tumors. As a result, PSMA has attracted attention as a clinical biomarker for detection and management of prostate cancer. Generally, these approaches utilize an antibody specifically targeted at PSMA to direct imaging or therapeutic agents. For example, ProstaScint (Cytogen, Philadelphia, PA), which has been approved by the FDA for the detection and imaging of prostate cancer, utilizes an antibody to deliver a chelated radioisotope (Indium-111). However, it is now recognized that the ProstaScint technology is limited to the detection of dead cells and therefore its clinical relevance is questionable.

[0008] The success of cancer diagnosis and therapy using antibodies is limited by challenges such as immunogenicity and poor vascular permeability. In addition, large antibodies bound to cell-surface targets present a barrier for subsequent binding of additional antibodies at neighboring cell-surface sites resulting in a decreased cell-surface labeling.

[0009] In addition to serving as a cell-surface target for antibodies delivering diagnostic or therapeutic agents, a largely overlooked and unique property of PSMA is its enzymatic activity. That is, PSMA is capable of recognizing and processing molecules as small as dipeptides. Despite the existence of this property, it has been largely unexplored in terms of the development of novel diagnostic and therapeutic strategies. There are a few recent examples in the literature that have described results in detecting prostate cancer cells using labeled small-molecule inhibitors of PSMA.

[0010] Certain phosphoramidate and phosphate PSMA inhibitors have been described in U.S. Patent Nos. 7,696,185, 8,293,725, RE42.275, and in U.S. Patent Application Publication Nos. US-2014-0241985-A1 and US-2016-0030605-A1. Chelated PSMA inhibitors with an albumin binding moiety are disclosed in WO 2018/031809.

SUMMARY OF THE INVENTION

[0011] Provided herein are imaging diagnostics and therapeutics for prostate cancer that capitalize on the potency and specific affinity of small-molecule inhibitors to PSMA. The diagnostic agents can be used to monitor and stratify patients for treatment with appropriate therapeutic agents.

[0012] Accordingly, in one aspect the present disclosure provides compounds of Formula (I)

and pharmaceutically acceptable salts thereof, wherein R ¹ -R ⁴ are independently H, -C1-C6 alkyl, or a protecting group. In a particular embodiment R ⁴ is H or -CH _3.

[0013] In another aspect the present disclosure provides methods for imaging one or more prostate cancer cells or tumor-associated vasculature in a patient comprising administering to the patient a compound or a pharmaceutical composition of either of the preceding aspects.

[0014] In another aspect, the present disclosure provides a method of synthesizing the compounds of the disclosure. The 2-step radiolabeling process used to produce CTT1403 has been found to be time consuming and costly. Radiomanufacturing time and cost are greatly reduced by a single step radiolabeling process according to the disclosure for a fully assembled, label-ready molecule that could be formulated in a kit and rapidly labeled in a “shake and shoot” format. The method disclosed herein eliminates the 2-step cGMP radiolabeling process. The method eliminates use of the expensive click chemistry reagents, DBCO and DOTA-N3.

[0015] All publicly available documents recited in this application are hereby incorporated by reference in their entirety to the extent their teachings are not inconsistent with the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION [0016] In one aspect, the present disclosure provides compounds useful as PET imaging diagnostics and radiotherapeutic agents for prostate cancer that capitalize on the potency and specific affinity of small-molecule inhibitors to PSMA.

[0017] In a first aspect, the compounds have structural Formula (I)

or a pharmaceutically acceptable salt thereof, wherein R ¹ -R ⁴ are independently H, -C1-C6 alkyl, or a protecting group.

[0018] In a particular embodiment of Formula (I), Embodiment 1A, R ⁴ is H or -CH _3.

[0019] The chelator in Formula (I) and Embodiment 1A is Lumi804™, a novel chelator developed by Lumiphore Inc. that can be rapidly and stably radiolabeled at neutral pH, room temperature and within 5 minutes. Incorporation of Lumi804™ allows development of a molecule that can be radiolabeled in a single step, increasing manufacturing efficiency and reducing time and cost of radiomanufacture.

[0020] In embodiment h, a compound of Formula (I) or Embodiment 1A is chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope selected from ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, i ⁸⁸ n ⁸⁸ Re, 9og, 177|_ _U 1533^ 2133^ 225/^ _anc| 223^3

[0021] In embodiment ha, the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ⁸⁹ Zr.

[0022] In embodiment h _b , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ⁶⁴ Cu.

[0023] In embodiment h _e , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is with ⁶⁸ Ga.

[0024] In embodiment h _d , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ^186/188 Re.

[0025] In embodiment he, the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ⁹⁰ Y. [0026] In embodiment h _f , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ¹⁷⁷ Lu.

[0027] In embodiment h _g , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ¹⁵³ Sm.

[0028] In embodiment h _h , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ²¹³ Bi.

[0029] In embodiment In, the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ²²⁵ Ac.

[0030] In embodiment h _j , the compounds of each of the preceding embodiments are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope that is ²²³ Ra.

[0031] In embodiment I2, the compounds are of any of the previous embodiments wherein R ¹ , R ² , and R ³ are independently selected from one of groups (5a)-(5o):

(5a) hydrogen, C1-C6 alkyl or a protecting group.

(5b) hydrogen or C1-C6 alkyl.

(5c) C1-C6 alkyl or a protecting group.

(5d) CrCe alkyl

(5e) hydrogen or a protecting group.

(5f) hydrogen.

(5g) a protecting group

(5h) Any of groups (5a)-(5d), where CrCealkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl.

(5i) Any of groups (5a)-(5d), where C Cealkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, sec-butyl or tert-butyl.

(5j) Any of groups (5a)-(5d), where CrCealkyl is methyl, ethyl, n-propyl or tert-butyl. (5k) Any of groups (5a)-(5d), where CrCealkyl is methyl, ethyl or tert-butyl.

(5I) Any of groups (5a)-(5d), where CrCealkyl is methyl or ethyl.

(5m) Any of groups (5a)-(5d), where CrCealkyl is methyl.

(5n) Any of groups (5a)-(5d), where CrCealkyl is ethyl.

(5o) Any of groups (5a)-(5g), where CrCealkyl is tert-butyl.

[0032] In embodiment l ₃ , the compounds are according to embodiment l ₂ , wherein R ¹ ,

R ² , and R ³ are H. [0033] In embodiment U, the compounds according to embodiment or I3 are chelated with a therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope selected f

[0034] In embodiment I5, the radioisotope is ¹⁷⁷ Lu.

[0035] In embodiment l ₆ , this disclosure provides compounds represented by structural Formula (II), and pharmaceutically acceptable salts thereof, wherein R ⁴ is H or -CH3 (i.e. , compounds of structural formula (I) wherein R ¹ -R ³ are H, Embodiment 1A).

[0036] In embodiment l ₇ , this disclosure provides a compound of structural Formula (II*), and pharmaceutically acceptable salts thereof, wherein R ⁴ is H or -CH3.

[0037] Formulae (I), (II), and (II*) include an albumin-binding moiety that reduces renal clearance resulting in increased bioavailability and therapeutic effect (Dumelin, C.E., et al. , A portable albumin binder from a DNA-encoded chemical library. Angew Chem Int Ed Engl, 2008. 47(17): p. 3196-201). A major obstacle in the development of PSMA-targeted small molecule drugs is the renal accumulation and fast blood clearance imparted by their low- molecular weight. Incorporating an albumin-binding motif addresses this issue. Albumin binding is a well-known drug delivery platform for increasing half-life. Drugs like Levemir®, ^99m Tc-Albures, and ^99m Tc-Nanocoll were specifically designed to take advantage of albumin’s systemic circulation to increase bioavailability (37, 38). Addition of an albumin binding motif (34) to the PSMA-targeting therapeutic agent extends circulation (tumor uptake up to 72 hours), prolongs tumor exposure and increases the overall percent injected dose per gram (%l D/g) in PSMA+ tumor tissue compared to a similar molecule lacking the albumin-binding moiety (Choy, C.J., et al. , 177Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor- Bearing Mice. Theranostics, 2017. 7(7): p. 1928-1939.5479279, and Ling, X., et al., Preclinical Dosimetry, Imaging, and Targeted Radionuclide Therapy Studies of Lu-177- Labeied Albumin-Binding, PSMA-Targeted CTT1403. Mol Imaging Biol, 2019). This is in sharp contrast to the urea-based ¹⁷⁷ Lu-PSMA-617 agent in clinical trials which is largely undetectable in blood after just 24 hours (Delker, A., et al., Dosimetry for 177Lu-DKFZ- PSMA-617: a new radiopharmaceutical for the treatment of metastatic prostate cancer. European Journal of Nuclear Medicine and Molecular Imaging, 2016. 43: p. 42-51). The increased tumor uptake conferred by addition of the albumin-binding moiety translates to a reduction in tumor growth and markedly increased survival in animals treated with CTT1403 (disclosed in WO 2018/031809). This is an innovative breakthrough in the field of PSMA- targeted agents, as the successful translation and commercial viability of small molecule drugs depends on their ability to overcome pharmacokinetic challenges. Several labs have recently incorporated this albumin binding strategy into other radiotherapeutics in development (40-42).

[0038] Formula (II*) herein differs from CTT1403 of WO 2008/031809 in the absence of the of the click chemistry motifs present in the CTT1403 structure as a result of its method of synthesis. Because the molecular weight of Formula (II*) is only -10% greater, the clinical performance of Formula (II*) is substantially the same as that of CTT1403, as binding of this class of PSMA ligands is completely dominated by the interactions between the first 3 residues of the C-terminus of the PSMA-targeting motif (the phosphoramidate end of the molecule) and the active site of PSMA.

[0039] A “protecting group” as used herein include, but are not limited to, optionally substituted benzyl, t-butyl ester, allyl ester, alkyl esters (e.g., methyl, ethyl), fluorenylmethoxycarbonyl groups (Fmoc), and amino, carboxylic acid and phosphorus acid protecting groups described in Greene's Protective Groups in Organic Synthesis, 4th Edition (which is incorporated by reference). In some embodiments, R ¹ is a carboxylic acid protecting group (e.g., a methyl or t-butyl ester). In some embodiments, R ² is a nitrogen protecting group (e.g., Boc, or benzyl).

[0040] Optionally benzyl groups include, but are not limited to, unsubstituted benzyl, triphenylmethyl (trityl), diphenylmethyl, o-nitrobenzyl, 2,4,6-trimethylbenzyl, p-bromobenzyl, p-nitrobenzyl, p-methoxybenzyl (PMB), 2,6-dimethoxybenzyl, 4-(methylsulfinyl)benzyl, 4- sulfobenzyl, 4-azidomethoxybenzyl, and piperonyl, and benzyl protecting groups for carboxylic and phosphorus acids disclosed in Greene’s Protective Groups in Organic Synthesis (the relevant parts of which are incorporated by reference).

[0041] In embodiment Is, the present disclosure provides pharmaceutical compositions comprising a compound of Formula (II*) and a pharmaceutically acceptable carrier.

[0042] In embodiment Ig, the present disclosure provides a method for imaging one or more prostate cancer cells in a patient comprising administering to the patient a compound of Formula (II*) or a pharmaceutical composition thereof. The method may further include imaging the compound of Formula (II*) in vivo. The imaging can be performed with any PET- imaging techniques known in the art.

[0043] WO 2018/031809 discloses assembly of CTT1403 drug product in a 2-step labeling process: 1) chelation of ¹⁷⁷ Lu to DOTA-N ₃ , then 2) click chemistry uniting ¹⁷⁷ LU-DOTA-N3 with the pre-assembled CTT1402 precursor

[0044] Current radiolabeling conditions for the DOTA chelator are very harsh. Indeed, Breeman et al have demonstrated that at pH 4, 80°C yields optimal DOTA-chelator radiolabeling, and incorporation of the radionuclide is compromised as temperature is lowered or pH is increased (43). The current DOTA-N3 labeling conditions require a 15- minute reaction with ¹⁷⁷ Lu at 95°C and pH 4.83 in the presence of radiostabilizing excipients. CTT1402, the precursor to CTT1403, has been shown to be sensitive to low pH (<5) and high temperature (>60°C) and is unlikely to be stable at those conditions. In order to maintain viability of a CTT phosphoramidate molecule, the chelator labeling conditions must become more favorable to the precursor. By contrast, compounds of this disclosure which have the Lumi804™ chelator (see U.S. Patent No. 10,239,878) are rapidly labeled at room temperature and neutral pH. Eliminating the click chemistry reagents DBCO and DOTA-N3 by incorporating the Lumi804™ chelator obviates the foregoing issues.

[0045] Furthermore, the 2-step radiolabeling process is time consuming and costly. Radiomanufacturing time and cost is greatly reduced by using a single step radiolabeling process to produce a fully assembled, label-ready molecule for ready formulation in a kit and rapidly labeled in a “shake and shoot” format. Using a PSMA-targeted, irreversible binding scaffold, this disclosure provides a method that eliminates the 2-step cGMP radiolabeling process.

[0046] Accordingly, in another aspect, the disclosure provides a method of synthesizing a compound of the disclosure wherein a radioisotope is introduced to and chelated by the corresponding non-radiolabeled precursor, as illustrated in the following for the compound of Formula (II*) from its non-radiolabeled precursor, formula (II):

[0047] In embodiment Mi, the method comprises contacting a radioisotope with a compound described herein not chelated to a radioisotope.

[0048] In embodiment M2, the method is according to embodiment Mi wherein the radioisotope is therapeutic radioisotope or a PET-active, SPECT-active, or MRI-active radioisotope.

[0049] In embodiment M ₃ , the method is according to embodiment M ₂ wherein the radioisotope is selected from ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, ^186/188 Re, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵³ Sm, ²¹³ Bi, ²²⁵ Ac, and ²²³ Ra.

[0050] In embodiment M ₄ , the method is according to any of embodiment M1-M3 wherein the radioisotope is ¹⁷⁷ Lu.

[0051] The method eliminates use of the expensive click chemistry reagents, DBCO and DOTA-N ₃ , reducing cost for manufacturing and cost of radiolabeling. [0052] Not only will replacement of click chemistry and expensive click chemistry reagents DBCO and DOTA-N3 with the single step method of this disclosure reduce the cost of production compared to the method of WO 2008/031809, it will also significantly reduce time and personnel required for radiolabeling the molecule prior to administration. This one- step process will be amenable to a “shake and shoot” formulation at the clinical site and could eventually be formatted as a kit for automatic processing by a Neptis or Trasis synthesizer.

[0053] Radiomanufacturing costs of formula (II*) are further diminished by the consequential increase in radiochemical yield when ¹⁷⁷ Lu becomes the sole limiting reagent to the 1-step process (Table 1):

Table 1 Estimated Cost for Radiomanufacturing

A “shake-and-shoot” automated labeling process further reduces manufacturing time and eliminate additional expenditure of ¹⁷⁷ Lu half-lives during shipping to/from the radiomanufacturer’s site.

[0054] An automated “shake and shoot” kit according to this disclosure eliminates the need for a central radiomanufacturing facility and associated shipping challenges in favor of on-site radiolabeling. A kit-based approach to radiolabeling allows same day administration of the final Formula (II*) therapeutic, eliminates overnight shipping costs and loss of radioactivity that occurs during the shipping process, and eliminates potential radiolysis during transit time.

[0055] In another aspect, the disclosure provides for methods of treating a patient with prostate cancer by administering an effective amount of Formula (II*) to the patient. The amount of the therapeutic radioisotope-chelated compound and regiment can be routinely determined using art-recognized techniques.

Definitions

[0056] As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

[0057] As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” PSMA with a compound includes the administration of a compound described herein to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing PSMA.

[0058] As used herein, the term “individual” or “patient,” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

[0059] As used herein, the phrase “pharmaceutically acceptable salt” refers to both pharmaceutically acceptable acid and base addition salts and solvates. Such pharmaceutically acceptable salts include salts of acids such as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic, formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric, tartaric, maleic, hydroiodic, alkanoic such as acetic, HOOC-(CH ₂ ) _n -COOH where n is 0-4, and the like. Non-toxic pharmaceutical base addition salts include salts of bases such as sodium, potassium, calcium, ammonium, and the like. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable addition salts.

[0060] Pharmaceutical compositions suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.

[0061] The term “alkyl” as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, unless otherwise specified. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethyl pentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an “alkyl” group is a linking group between two other moieties, then it may also be a straight or branched chain; examples include, but are not limited to -CH2-, -CH2CH2-, -CH ₂ CH ₂ CHC(CH ₃ )-, -CH ₂ CH(CH ₂ CH3)CH2-.

[0062] The term “aryl” or “Ar” refers to an aromatic ring or an aromatic ring system of 6- 16 members having at least one carbocyclic aromatic ring (e.g., phenyl) optionally fused to one or more aromatic or non-aromatic rings. “Aryl” includes (a) single aromatic rings and (b) fused ring systems having 6-16 annular atoms in which at least one ring is a carbocyclic aromatic ring.

[0063] The term "heteroaryl" or “Het” refers to an aromatic ring or an aromatic ring system containing 5-15 members and at least one heteroatom selected from nitrogen, oxygen and sulfur in an aromatic ring, optionally fused to one or more aromatic or non-aromatic rings. Heteroaryl groups will have 1-4 O, S, or N atoms, provided no O or S is adjacent to another O or S. Most commonly, the heteroaryl groups will have 1 , 2, 3, or 4 heteroatoms. “Heteroaryl” includes (a) single heteroaryl rings and (b) fused ring systems having 5-16 annular atoms in which at least one ring is heteroaryl.

[0064] The term “heterocyclyl” as used herein, means a monocyclic heterocycle or a bicyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O,

N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S. The 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. The monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl. The bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system. Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. Heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.

[0065] The term “oxo” as used herein means a =0 group.

[0066] The term “saturated” as used herein means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like.

[0067] The term “thia” as used herein means a =S group.

[0068] The term “unsaturated” as used herein means the referenced chemical structure contains at least one multiple carbon-carbon bond, but is not aromatic. For example, a unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.

EXAMPLES

Example 1

Formula (II*) Synthesis

[0069] Formula (II) is the radiolabeling precursor to Formula (II*). Nearly all synthetic steps are required for manufacture of Formula (II) are based on the previously established synthesis and manufacture of CTT1402, and are straightforward, involving routine amino acid protection/deprotection steps as well as peptide coupling chemistry. However, the synthesis scheme for Formula (II) eliminates inefficient aqueous chemistry required for CTT1402 manufacture by using the globally protected synthetic intermediate CTT1298- (OMe^OFm rather than CTT1298 itself.

CTT1298 [0070] CTT1298 is a non-water-soluble precursor of CTT1057 and a component of

CTT1402.

CTT1057

Example 2

Formula (ID radiolabelinq stability

[0071] The pH and temperature range in which Formula (II) is stable are identified to define to its stability for ¹⁷⁷ Lu labeling conditions. Solutions of Formula (II) are incubated up to 11 hour at increasing pH (5-8) and temperature (25 - 37°C) for evaluation using HPLC to quantify intact and fragmented Formula (II). At 15-minute intervals, aliquots are removed and analyzed by HPLC to quantify intact and degraded Formula (II). Conditions at which Formula (II) is stable are applied to radiolabeling studies.

Example 3

Formula (II*) radiolabelinq studies

[0072] ¹⁷⁷ Lu radiolabeling conditions of Formula (II) to produce Formula (II*) are optimized within the temperature and pH ranges identified in the stability study described above. However, Lumi804™ can be labeled with ¹⁷⁷ Lu within 5 minutes at physiological pH. Radiolabeling is conducted with and without stabilizing excipients present at increasing concentrations. The cocktail of radiostabilizing excipients for CTT1403 is 4mM Genticic, 6mM Sodium Ascorbate and 4mM L-methionine, which are adjusted to accommodate the new Lumi804 chelating agent. Lumiphore Inc. reports that metalation of Lumi804™ occurs within 1 min exposure to ¹⁷⁷ Lu. Chelation is assessed over a time period of 1, 5, 10 and 15 minutes over a range of 25-37°C and pH 5-8 as dictated by the Formula (II) stability studies. Radiochemical yield, purity and presence of impurities in the reaction solutions are monitored by RP-HPLC.

[0073] Radiolabeled Formula (II*) is purified following the process modified from the CTT1403 purification procedure. Briefly, the radiolabeled solution is loaded onto a C18 Sep- Pak cartridge preconditioned with 0.5 M Ammonium Acetate (pH 4.8). The cartridge is first flushed with wash solution (combination of excipients as discussed for radiolabeling plus 5% ethanol). The radiolabeled product is selectively eluted from the cartridge with a solution of 50% ethanol plus excipients. Radiochemical purity of Formula (II*) is determined by RP- HPLC. The final Formula (II*) drug product should have a radiochemical purity to < 95%. The drug product ia adjusted to 8 mL in 0.9% saline plus excipients to maintain starting concentration for stability studies (see Formula (ll)/Formula (II*) Stability Studies below).

Example 4

Formula (II) Precursor Storage Stability

[0074] Aliquots of lyophilized Formula (II*) precursor (~5 pg each) stored at -20°C and 4°C (+/- 5°C) are assessed by RP-HPLC to demonstrate storage stability at 1 , 2, 3, and 6 months post-manufacture.

Example 5

Formula (II*) Radiotherapeutic Stability

[0075] Aliquots (~5 pg) of purified radiolabeled Formula (II*) are stored at -20°C (+/- 5°C) and 4°C for RP-HPLC assessment of purity and radiolysis 4, 8, 24, 48 and 72 hours after radiolabeling to determine its stability at 4°C for same-day patient administration or (4°C) or storage and shipment from a central manufacturer (-20°C).

[0076] Cold ¹⁷⁶ Lu-Formula (II*). Up to 250 mg ¹⁷⁶ Lu-Formula (II*), will be made as a “cold” reference marker for use as an analytical HPLC standard to confirm the identity, purity, and stability of Formula (II*). The preparation of this “Cold” standard will be achieved by complexing ¹⁷⁶ Lu with the Formula (II) precursor.

Example 6

In vitro and in vivo characterization of Formula (II*).

[0077] The in vitro characteristics of Formula (II*) and ¹⁷⁶ Lu-Formula (II) as a non radioactive surrogate for ¹⁷⁷ Lu-labeled Formula (II) (i.e. , Formula (II*) are determined.

[0078] Serum stability studies. Stability of ¹⁷⁶ Lu-Formula (II) (1 pg) in mouse and human serum (1 ml_) is determined over 24 hours (1 , 2, 4, 6, 24 hours). After the incubation period, 50 pl_ methanol is added to samples to precipitate proteins, and samples are centrifuged. The recovery supernatant (40 mI_) is analyzed by LC-MS/MS on a Sciex 6500 QTrap coupled to a Shimadzu LC system with CTC PAL auto-sampler. Serum samples with Formula (II*) are also analyzed by direct infusion on a Thermo Fisher Scientific Q Extractive high- resolution accurate mass spectrometer to monitor intact Formula (II*).

[0079] PSMA Binding: IC50 is determined for Formula (II*) as previously described for various inhibitor-conjugates (44, 46, 47). To determine the mode of inhibition (irreversible, slowly reversible or rapidly reversible). The same activity recovery experiments as previously described is employed (46). [0080] In vivo Biodistribution: Formula (II*) is evaluated in a biodistribution study in PSMA+ (PC3-PIP) tumor-bearing mice as previously described (Choy, C.J., et al. , 177Lu- Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice. Theranostics,

2017. 7(7): p. 1928-1939.5479279). Forty NCr nude mice are injected with saline and Matrigel containing ¹ 10 ⁶ PC3-PIP cells subcutaneously in the right shoulder and grown to approximately 0.8 cm across (longest axis of measurement) (n=5/group). A single injection of 1.85 MBq Formula (II*) is administered to each animal. Organs and tissues including blood, lung, liver, spleen, kidney, bladder, muscle, fat, heart, brain, pituitary, prostate, testes, bone, marrow, adrenals, pancreas, thymus, tumor, salivary glands, stomach, small intestine, upper large intestine and lower large intestine are harvested and counted on a gamma counter for 3 minutes each at 4, 48 and 168 hours post-injection. Results from Formula (II*) are compared to historical data collected for CTT1403 in normal mouse tissues and PSMA+ tumor to demonstrate similar localization between the two compounds.

[0081] Evaluation of Formula anti-tumor efficacy: Efficacy of Formula (II*) is assessed in PSMA+ tumor-bearing mice (n=6/group) prepared as described for biodistribution studies. Mice bearing PSMZ+ tumors are injected with 29 MBq Formula (II*) (the initial single injection dose demonstrated efficacious for CTT1403 (20) or saline as a control. Therapeutic efficacy of Formula (II*) is assessed by monitoring tumor volume, and survival to either of the following endpoints: tumor growth to 1.5 cm at longest axis or 120 days post-therapeutic injection.

[0082] Like CTT1403 and CTT1057, Formula (II*) localizes to tumor and only at very low levels in non-PSMA expressing tissues. Thus, with this profile, Formula (II*) demonstrates similar efficacy to CTT1403.