PSMA binding ligand-linker conjugates and methods for using the same

Abstract

To provide compositions for treating diseases involving pathogenic cell populations that express PSMA.SOLUTION: A composition comprises a ligand of PSMA (B), a linker (L), and a drug (D), wherein the linker is covalently bound to the drug and the linker is covalently bound to the ligand, where the linker comprises a chain of at least seven atoms, provided that the ligand is not an antibody or an antigen-binding fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The invention described herein relates to compounds and methods for treating diseases of the prostate and related diseases, such as prostate cancer. More particularly, embodiments of the invention described herein relate to conjugates of a biologically active agent linked to a PSMA-binding ligand. [Background technology]

[0002] The prostate is one of the male reproductive organs found in the pelvis, below the bladder. It functions to produce and store semen, which provides nutrients and fluids that are important for the survival of sperm introduced into the vagina during reproduction. Like many other tissues, the prostate is prone to developing malignant (cancerous) or benign (non-cancerous) tumors. The American Cancer Society estimated that in 2005, over 230,000 men would be diagnosed with prostate cancer, and over 30,000 men would die from the disease. In fact, prostate cancer is one of the most common male cancers in Western society, and the second most common form of malignancy in American men. Current methods of treating prostate cancer include hormone therapy, radiation therapy, surgery, chemotherapy, photodynamic therapy, and combination therapy. The choice of treatment generally depends on the stage of the cancer. However, many of these treatments affect the quality of life of patients, especially those over the age of 50 who are diagnosed with prostate cancer. For example, the use of hormone drugs is often accompanied by side effects such as osteoporosis and liver damage. Such side effects can be mitigated by using treatments that are more selective or specific for tissues involved in the disease state, and avoid non-target tissues such as bone or liver. As described herein, prostate-specific membrane antigen (PSMA) represents a target for such selective or specific treatments.

[0003] PSMA is named primarily for its high level of expression in prostate cancer cells, but its specific function on prostate cancer cells remains unresolved. PSMA is overexpressed in malignant prostate tissue when compared to other organs of the human body such as the kidney, proximal small intestine, and salivary glands. PSMA is expressed in the brain, but its expression is minimal, and most of PSMA's ligands are polar and cannot penetrate the blood-brain barrier. PSMA is a type II cell surface membrane-bound glycoprotein with a molecular weight of approximately 110 kD, containing an intracellular segment (amino acids 1-18), a transmembrane domain (amino acids 19-43), and a large extracellular domain (amino acids 44-750). The functions of the intracellular segment and the transmembrane domain are currently not believed to be significant, but the extracellular domain is involved in several different activities. PSMA plays a role in the central nervous system, where it metabolizes N-acetyl-aspartylglutamate (NAAG) to glutamate and N-acetylaspartate. Thus, it is sometimes called N-acetylglucosamine (NAALADase).PSMA is also sometimes called folate hydrolase I (FOLH I) or glutamate carboxypeptidase (GCP II) due to its role in the proximal small intestine in removing gamma-linked glutamate from poly-gamma-glutamylated folates and alpha-linked glutamate from peptides and small molecules.

[0004] PSMA also shares similarities with the human transferrin receptor (TfR), since both PSMA and TfR are type II glycoproteins. More specifically, PSMA shows 54% and 60% homology to TrR1 and TfR2, respectively. However, while TfR exists only in a dimeric form due to the formation of its interchain sulfhydryl bridges, PSMA can exist in either a dimeric or monomeric form.

[0005] Unlike many other membrane-bound proteins, PSMA is rapidly internalized into cells in a manner similar to cell surface-bound receptors such as vitamin receptors. PSMA can be internalized through clathrin-coated pores and subsequently recycled to the cell surface or go to lysosomes. The dimeric and monomeric forms of PSMA have been suggested to be interconvertible, but direct evidence of the interconversion is under debate. Even so, only dimeric PSMA has enzymatic activity, not the monomeric form.

[0006] Although the activity of PSMA on the cell surface of prostate cells is still under investigation, it has been recognized by the inventors herein that PSMA is a target that may make it feasible to selectively and / or specifically deliver biologically active agents, including diagnostic, imaging and therapeutic agents, to such prostate cells. Summary of the Invention

[0007] It has been discovered that biologically active agents linked to a ligand capable of binding to prostate specific membrane antigen (PSMA) via a linker can be useful in imaging, diagnosing and / or treating prostate cancer and related diseases involving pathogenic cell populations that express or overexpress PSMA. PSMA is a cell surface protein that is internalized by a process similar to the endocytosis observed for cell surface receptors such as vitamin receptors. It has therefore been discovered that certain conjugates comprising a linker with a defined length and / or a defined diameter and / or preselected functional groups along its length can be used to treat, image and / or diagnose such diseases.

[0008] In one exemplary embodiment of the present invention, a compound of the formula: BLD Conjugates having the formula: are described, where B is a ligand that binds to or targets prostate specific membrane antigen (PSMA), L is a linker, and D is a drug. As used herein, the term drug D collectively includes therapeutic agents, cytotoxic agents, imaging agents, diagnostic agents, and the like, unless otherwise indicated or context dictates. For example, in one exemplary configuration, the conjugates described herein are used to eliminate a pathogenic cell population, and thus drug D is a therapeutic agent, cytotoxic agent, and the like. In another exemplary configuration, the conjugates described herein are used to image and / or diagnose a disease or disease state, and thus drug D is an imaging agent, diagnostic agent, and the like. Other configurations are also contemplated and described herein. Analogs and derivatives of each of the foregoing B, L, and D are also contemplated and described herein, and as used herein, the terms B, L, and D should be understood to collectively refer to such analogs and derivatives.

[0009] In one exemplary embodiment, the linker L can be a releasable or non-releasable linker. In one embodiment, the linker L is at least about 7 atoms long. In one variation, the linker L is at least about 10 atoms long. In one variation, the linker L is at least about 14 atoms long. In another variation, the linker L is about 7 to about 31, about 7 to about 24, or about 7 to about 20 atoms long. In another variation, the linker L is about 14 to about 31, about 14 to about 24, or about 14 to about 20 atoms long.

[0010] In an alternative embodiment, linker L is at least about 10 angstroms (Å) in length. In one variation, linker L is at least about 15 Å in length. In another variation, linker L is at least about 20 Å in length. In another variation, linker L is in the range of about 10 Å to about 30 Å in length.

[0011] In an alternative embodiment, at least a portion of the length of the linker L is about 5 Å or less in diameter at the end connected to the binding ligand B. In one variation, at least a portion of the length of the linker L is about 4 Å or less in diameter or about 3 Å or less at the end connected to the binding ligand B. It is understood that the exemplary embodiment including a diameter requirement of about 5 Å or less, about 4 Å or less, or about 3 Å or less includes a requirement for a linker of a given length, thereby defining a cylindrical-like portion of the linker. Illustratively, in another variation, the linker includes a cylindrical portion of at least about 7 Å in length and about 5 Å or less, about 4 Å or less, or about 3 Å or less in diameter at the end connected to the binding ligand.

[0012] In another embodiment, the linker L comprises one or more hydrophilic linkers capable of interacting with one or more residues of PSMA that include amino acids with hydrophobic side chains, such as residues of Ser, Thr, Cys, Arg, Orn, Lys, Asp, Glu, Gln, etc. In another embodiment, the linker L comprises one or more hydrophobic linkers capable of interacting with one or more residues of PSMA that include amino acids with hydrophilic side chains, such as residues of VaI, Leu, Ile, Phe, Tyr, Met, etc. It should be understood that the above-mentioned embodiments and aspects may be included in the linker L alone or in combination with each other. For example, a linker L that is at least about 7 atoms long and has a diameter of about 5 Å or less, about 4 Å or less, or about 3 Å or less is contemplated and described herein, and a linker L that comprises one or more hydrophilic linkers capable of interacting with one or more residues of PSMA that include residues of VaI, Leu, Ile, Phe, Tyr, Met, etc. is also contemplated and described herein.

[0013] In another embodiment, one end of the linker is unbranched and comprises a chain of carbon, oxygen, nitrogen and sulfur atoms. In one embodiment, the chain of carbon, oxygen, nitrogen and sulfur atoms is at least 5 atoms long. In one variation, the linker chain is at least 7 atoms long or at least 10 atoms long. In another embodiment, the chain of carbon, oxygen, nitrogen and sulfur atoms is unsubstituted. In one variation, a portion of the chain of carbon, oxygen, nitrogen and sulfur atoms is cyclized with a divalent fragment. For example, a linker (L) comprising a dipeptide Phe-Phe can comprise a piperazine-1,4-diyl structure in which the two nitrogens are cyclized with an ethylene fragment, or a substituted variant thereof.

[0014] In another embodiment, a pharmaceutical composition is described herein, the pharmaceutical composition comprising a conjugate described herein in an amount effective for treating diseases and disease states, diagnosing diseases and disease states, and / or imaging tissues and / or cells associated with pathogenic cell populations that express or overexpress PSMA. Illustratively, such pharmaceutical compositions also include one or more carriers, diluents and / or excipients.

[0015] In another embodiment, methods of treating diseases and disease states, diagnosing diseases and disease states, and / or imaging tissues and / or cells associated with pathogenic cell populations that express or overexpress PSMA are described herein. Such methods include administering a conjugate described herein and / or a pharmaceutical composition containing a conjugate described herein in an amount effective to treat diseases and disease states, diagnose diseases and disease states, and / or image tissues and / or cells associated with pathogenic cell populations that express or overexpress PSMA.

[0016] <CROSS REFERENCE TO RELATED APPLICATIONS> This application claims priority to U.S. Provisional Application No. 60 / 956,489, filed Aug. 17, 2007, and U.S. Provisional Application No. 61 / 074,358, filed Jun. 20, 2008, the disclosures of which are incorporated herein by reference in their entireties. [Brief description of the drawings]

[0017] [Figure 1A] Cell-bound radioactivity versus concentration of SK28-99mTc (Kd=18.12 nM) in the presence (▲) or absence (■) of excess PMPA. [Figure 1B] In vitro binding studies using LNCaP cells and SK33 (14 atom linker). LNCaP cells containing increasing concentrations of DUPA-99mTc in the presence (▲) or absence (■) of excess PMPA. [Diagram 2] (■) Cell-bound radioactivity versus concentration of SK28-99mTc at 4°C and (▲) 37°C. [Figure 3A] Plot of cell-bound radioactivity versus concentration of DUPA-linker-99mTc imaging agent: (■) linker with 0 atoms (Kd=171 nM); (▲) linker with 7 atoms (Kd=68 nM); (▼) linker with 14 atoms (Kd=15 nM); (◆) linker with 16 atoms (Kd=40 nM). [Figure 3B] 13 is a KD value of the DUPA-linker-99mTc compound binding to LNCaP cells. [Figure 4] Plot of tumor volume versus days post-injection for LNCaP tumors: (a) 25 million + Matrigel; (b) 25 million + HC Matrigel; (c) 5 million + Matrigel; (d) 5 million + HC Matrigel. [Figure 5A] Plots of tumor volume versus days post-injection for (a) LNCaP tumors (27 mice) and (b) KB cells (5 mice) and A549 cells (5 mice). [Figure 5B] Plot of tumor volume versus days after injection for KB cells (5 mice) and A549 cells (5 mice). [Figure 6A]Mice pre-injected with LNCaP tumors (set 1) treated with 1 ng / kg SK28-99mTc (14 atom linker), the image on the left shows a white light image, which shows an overlay of the radiographic and white light images. In each panel, the mouse on the right was treated with 50 mg / kg PMPA (to block PSMA binding) and the mouse on the left was treated without the addition of PMPA. [Figure 6B] Mice pre-injected with LNCaP tumors treated with 1 ng / kg SK28-99mTc (14 atom linker) (set 2), the image on the left shows a white light image, which shows an overlay of the radiographic and white light images. In each panel, the mouse on the right was treated with 50 mg / kg PMPA (to block PSMA binding) and the mouse on the left was treated without the addition of PMPA. [Figure 6C] Mice pre-injected with LNCaP tumors treated with 1 ng / kg SK28-99mTc (14 atom linker) (set 3), the image on the left shows a white light image, which shows an overlay of the radiographic and white light images. In each panel, the mouse on the right was treated with 50 mg / kg PMPA (to block PSMA binding) and the mouse on the left was treated without the addition of PMPA. [Figure 6D] Shown is a single mouse study of LNCaP tumors imaged using a Kodak imaging device 4 hours after subcutaneous injection (administered via intraperitoneal) of 1 ng / kg SK28-99mTc, with the image on the left showing a kidney-shielded radiographic image overlaid with an unshielded white light image, and the image on the right showing a kidney-shielded radiographic image overlaid with an unshielded X-ray image. [Figure 7A] Mice pre-injected with LNCaP tumors treated with SK60-99mTc (0 atom linker). The image on the left shows a white light image, the image in the middle shows a radiographic overlay of the white light image, and the image on the right shows a radiographic overlay of the white light image with the mouse kidneys masked. [Figure 7B]Mice pre-injected with KB cells treated with SK28-99mTc (14 atom linker). The image on the left shows a white light image, the image in the middle shows a superposition of the radiographic image and the white light image, and the image on the right shows a superposition of the radiographic image and the white light image with the mouse kidneys masked. [Figure 7C] A mouse pre-injected with A549 cells treated with SK28-99mTc (14 atom linker). The image on the left shows a white light image, the image in the middle shows a superposition of the radiographic image and the white light image, and the image on the right shows a superposition of the radiographic image and the white light image with the mouse kidneys masked. [Figure 7D] Whole-body images of solid tumor xenografts in nu / nu mice taken 4 hours after injection of 150 μCi of DUPA-99mTc. Whole-body radiographic and white-light image overlays of LNCaP tumor-bearing mice treated with DUPA-99mTc in the absence (a, c) or presence (b, d) of 100-fold molar excess PMPA. Whole-body radiographic and white-light image overlays of A549 tumor-bearing (e) or KB tumor-bearing (f) mice similarly treated with DUPA-99mTc. [Figure 8A] Biodistribution of tumors implanted in the trunk of male nude mice, measured as cpm counted directly on tissue, of SK28-99mTc with or without PMPA (competitor) is shown for LNCaP tumors (a) with and (b) without PMPA, (c) A549 tumors, and (d) KB tumors. [Figure 8B] Tumor biodistribution data for tumors implanted in the trunk of male nude mice are shown for LNCaP tumors, tumors and kidneys only for SK28-99mTc with or without PMPA (competitor) (b) (a). [Figure 8C] Biodistribution study of DUPA-99mTc in nu / nu mice bearing LNCaP, A549 or KB tumors. [Figure 9A]Acute MTD (single dose) showing percent body weight change following administration of saline alone, single doses of 1.1 μmol / kg, 2.3 μmol / kg, 4.5 μmol / kg and 9 μmol / kg SK71. [Figure 9B] Chronic MTD represents the percent change in body weight after 5 doses of saline alone, 2 μmol / kg, and 4 μmol / kg given on alternate days (M, W, F, M, W). [Figure 10A] FIG. 1 is an efficacy study showing tumor volumes in animals treated with 1 μmol / kg of conjugate SK71 given in 5 doses on alternate days (M, W, F, M, W). [Figure 10B] Efficacy study showing tumor volumes in animals treated with saline alone on alternate days (M,W,F,M,W) for 5 doses (control group). [Figure 10C] Efficacy study showing tumor volume in animals treated with excess PSMA and 1 μmol / kg of conjugate SK71 given in 5 doses on alternate days (M, W, F, M, W) (competition). [Figure 11] This was an efficacy study (1 μmol / kg every other day for 10 days, i.e., 5 doses). [Figure 12A] [3H]-thymidine incorporation into LNCaP cells following treatment with SK71 (IC50 approx. 2 nM). [Figure 12B] [3H]-thymidine incorporation into LNCaP cells following treatment with SK77 (IC50 approximately 3 nM). [Figure 12C] [3H]-thymidine incorporation into LNCaP cells following treatment with SK37 (IC50 approx. 33 nM). [Figure 12D] [3H]-thymidine incorporation into LNCaP cells following treatment with SK45 (IC50 approx. 29 nM). [Figure 13A] Effect of SK71 (1.5 μmol / kg) treatment on tumor volume in nu / nu mice pre-treated with LNCaP cells in HC Matrigel: (■) treated mice, (●) untreated mice, (▲) treated mice pre-injected with 100-fold molar excess of PMPA. [Figure 13B]Effect of SK71 (1.5 μmol / kg) treatment on the rate of body weight change in nu / nu mice pretreated with LNCaP cells in HC Matrigel: (■) treated mice, (●) untreated mice, (▲) treated mice pre-injected with 100-fold molar excess of PMPA. [Figure 13C] Effect of SK71 (2.0 μmol / kg) treatment on tumor volume in nu / nu mice pre-treated with LNCaP cells in HC Matrigel. (■) Treated mice, (●) Untreated mice, (▼) Treated mice pre-injected with 30-fold molar excess of PMPA. [Figure 13D] Effect of SK71 (2.0 μmol / kg) treatment on the rate of body weight change in nu / nu mice pretreated with LNCaP cells in HC Matrigel: (■) treated mice, (●) untreated mice, (▲) treated mice pre-injected with a 30-fold molar excess of PMPA. [Figure 14A] Effect of SK77 (2.0 μmol / kg) treatment on tumor volume in nu / nu mice pretreated with LNCaP cells in HC Matrigel. (■) Untreated mice, (▼) Treated mice. [Figure 14B] Effect of SK77 (2.0 μmol / kg) treatment on the rate of body weight change in nu / nu mice pretreated with LNCaP cells in HC Matrigel. (■) Untreated mice, (▼) Treated mice. [Figure 15A] Energy-minimized computer model of the MUPA 99mcTc imaging agent conjugate (9 atom linker). [Figure 15B] Energy-minimized computer model of MUPA 99mcTc imaging agent conjugate (syn-SK33, 14 atom linker). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Drug delivery conjugates are described herein in which a PSMA-binding ligand is attached to a releasable or non-releasable linker that is attached to a drug, therapeutic, diagnostic or imaging agent.

[0019] Illustratively, the bivalent linkers described herein have the following formula: BL-TA BL-IA BL-DA wherein B is a PSMA-binding moiety, including analogs or derivatives thereof, L is a linker, TA is a therapeutic agent, including analogs or derivatives thereof, IA is an imaging agent, including analogs or derivatives thereof, and DA is a diagnostic agent, including analogs or derivatives thereof. The linker L can include multiple bivalent linkers, including the bivalent linkers described herein. It should also be understood that, as used herein, TA collectively refers to therapeutic agents and their analogs and derivatives, IA collectively refers to imaging agents and their analogs and derivatives, and DA collectively refers to diagnostic agents and their analogs and derivatives.

[0020] The linker may also include one or more spacer linkers, and optionally additional releasable linkers. The spacer and releasable linkers may be attached to each other in any order or combination. Similarly, the PSMA binding ligand may be attached to a spacer linker or a releasable linker. Similarly, the drug, therapeutic agent, diagnostic agent, or imaging agent may be attached to a spacer linker or a releasable linker. These components of the conjugate may be linked via existing or additional heteroatoms on the targeting ligand, drug, therapeutic agent, diagnostic agent, imaging agent, releasable linker, or spacer linker, respectively. Exemplary heteroatoms include nitrogen, oxygen, sulfur, and heteroatoms of the formula: -(NHR 1 NHR 2 )-, -SO-, -(SO2)- and -N(R 3 )O-, where R 1 , R 2 and R 3are independently selected from hydrogen, alkyl, heteroalkyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, and the like, each of which can be optionally substituted.

[0021] In one exemplary embodiment, compounds comprising linkers having length and diameter of a given dimension are described herein. In one embodiment, linkers that meet one or more minimum length requirements or length requirements that fall within a given range are described herein. In another embodiment, it can be understood that meeting a minimum length requirement is determined by computer modeling of an extended conformation of the linker. In another embodiment, it should be understood that meeting a minimum length requirement is determined by having a certain number of atoms (with or without substitution) that form a backbone of atoms that connects the binding ligand (B) with the drug (D). In another embodiment, the backbone of atoms is cyclized by another bivalent fragment. In another embodiment, linkers that meet one or more maximum or minimum diameter requirements are described herein. In another embodiment, it is understood that meeting a maximum or minimum diameter requirement is determined by computer modeling of various conformations of the linker created as a configuration such as a space-filling model, a CPK model, etc. In another embodiment, it is understood that meeting a maximum or minimum diameter requirement can apply to one or more selected portions of the linker, such as the portion of the linker proximal to the binding ligand (B) or the portion of the linker distal to the drug (D). In another embodiment, linkers are described herein that meet one or more chemical composition requirements, such as linkers that include one or more polar groups that can positively interact with one or more Arg or Lys side chain nitrogens and / or Asp or Glu side chain oxygens found in the funnel portion of PSMA. In one embodiment, linkers are described herein that meet one or more chemical composition requirements, such as linkers that include one or more non-polar groups that can positively interact with one or more Tyr or Phe side chain carbons found in the funnel portion of PSMA.

[0022] In one embodiment, the atom length of the linker is defined by the number of atoms separating the binding or targeting ligand B, or its analog or derivative, and the drug D, or its analog or derivative. Thus, in a configuration in which the binding ligand or its analog or derivative is directly attached to the drug D, or its analog or derivative, the linkage is also referred to herein as a "zero atom" linker. Such a zero atom linker is understood to include a configuration in which B and D are directly attached by removing a hydrogen atom from the attachment point of B and D, respectively. Such a zero atom linker is also understood to include a configuration in which B and D are attached via an overlapping heteroatom by removing a hydrogen atom from one of B or D and a heteroatom functional group such as OH, SH, NH2, etc. from the other of B or D. It is also understood that such zero atom linkers include configurations in which B and D are linked through a double bond that can be formed by removing two hydrogen atoms from the attachment points of B and D, respectively, or B and D are linked through one or more overlapping heteroatoms by removing two hydrogen atoms, one hydrogen atom and one heteroatom functional group such as OH, SH, NH2, etc., or two heteroatom functional groups from B or D, respectively. In addition, B and D can be linked through a double bond that is formed by removing a double-bonded heteroatom functional group such as O, S, NH, etc., from either or both of B or D. It is also to be understood that such heteroatom functional groups include those that are bonded to saturated carbon atoms, unsaturated carbon atoms (including carbonyl groups), and other heteroatoms. Similarly, linker lengths greater than zero atoms can be modified in a similar manner. It is defined by:

[0023] Thus, in another exemplary embodiment, linkers (L) are described that have a chain length of at least 7 atoms. In one variation, linkers (L) are described that have a chain length of at least 14 atoms. In another variation, linkers (L) are described that have a chain length ranging from about 7 to about 20 atoms. In another variation, linkers (L) are described that have a chain length ranging from about 14 to about 24 atoms.

[0024] In another embodiment, the length of the linker (L) is defined by measuring the length of the extended conformation of the linker. Such extended conformation can be measured by art-recognized computer modeling programs such as PC Model 7 (MMX). Thus, in another exemplary embodiment, linkers having chain lengths of at least 15 Å, at least 20 Å, or at least 25 Å are described.

[0025] In another embodiment, a linker is described that has at least one hydrophobic side group, such as an alkyl, cycloalkyl, aryl, arylalkyl, etc. group, each of which may be optionally substituted. In one embodiment, the hydrophobic group is included in the linker by incorporating one or more Phe or Tyr groups (including substituted variants thereof, as well as analogs and derivatives thereof) into the linker chain. It is understood that such Phe and / or Tyr side groups can form positive pi-pi (π-π) interactions with the Tyr and Phe residues found in the funnel of PSMA. In addition, it is understood that the presence of large side chain branches, such as the arylalkyl groups found on Phe and Tyr, can provide a level of conformational rigidity to the linker, thus restricting the degrees of freedom, reducing winding, and promoting an extended conformation of the linker. Without being bound by theory, such entropy restriction can increase the overall binding energy of the conjugates conjugated as described herein. In addition, it is understood that the increased rigidity that can be provided by sterically hindered side chains such as Phe and Tyr described herein can reduce or prevent wrapping and interaction between the ligand and the imaging agent. For example, energy minimization calculations of representative 9-atom and 14-atom linkers (see, for example, Figures 15A and 15B) show that there are no intramolecular interactions between the ligand and the imaging agent. Furthermore, the presence of two Phe side chains in the side chains appears to promote a more extended conformation in syn-SK33 (Figure 15B) than in the aminohexanoic acid-containing conjugate (Figure 15A).

[0026] It has been discovered herein that the funnel-like tunnel leading to the catalytic or active site of PSMA imposes length, shape and / or chemical composition requirements on the linker portion of conjugates of PSMA-binding ligands and therapeutic, diagnostic and imaging agents that positively and negatively affect the interaction of these conjugates with PSMA. Described herein are exemplary embodiments of these conjugates that include such length, shape and / or chemical composition requirements for the linker. Such length, shape and / or chemical composition requirements were evaluated using molecular modeling. For example, space-filling and surface models of PSMA complexed with (s)-2-(4-iodobenzenesylphosphonomethyl)-pentanedioic acid [2-PMPA derivative] PDB ID code 2C6P were generated using PROTEIN EXPLORER. The PROTEIN EXPLORER model confirmed a 20 Å deep funnel and also showed diameter features at various positions along the funnel that can be used to define linkers with favorable structural properties. In addition, the model showed that there are a number of hydrophobic residues in close proximity to the active site of PSMA that could provide additional binding interactions if corresponding functional groups were included in the linker. Finally, the model showed that there are three hydrophobic pockets that could provide additional binding interactions if corresponding functional groups were included in the linker.

[0027] In another exemplary embodiment, the following tripeptide is linked to MUPA by a 9 atom linker: 99m Molecular models were generated for the Tc conjugate (shown in FIG. 15A) and for the synSK33 conjugate (shown in FIG. 15B) which contains a branched, 14 atom linker. The models were generated using energy-minimized PC Model 7 (MMX) with the following bond length parameters: CC(sp 3 -sp 3 ) = 1.53 Å, CC(sp 3 -sp 2 ) = 1.51 Å, CN(sp 3 -N) = 1.47 Å, CN(sp 2-N)=1.38 Å. Such a model can be used to calculate the length of the linker connecting the binding ligand (B) and the drug (D). In addition, such a model can be modified to generate extended conformations, which can then be used to calculate the length of the linker connecting the binding ligand (B) and the drug (D).

[0028] The first human PSMA gene was cloned from LNCaP cells and reported to be located on chromosome 11p11-12. In addition, a PSMA-like gene is located at locus 11q14.3. Crystal structures of PSMA have been reported at different resolutions by two different groups, each showing that the active site contains two zinc atoms, confirming that PSMA is also considered a zinc metalloprotease. Davis et al, PNAS, 102:5981-86, (2005) reported a low resolution (3.5 Å) crystal structure, while Mesters et al, The EMBO Journal, 1-10 (2006) reported a higher resolution (2-2.2 Å) crystal structure (these disclosures are incorporated herein by reference). The crystal structure shows that PSMA is a homodimer containing a protease domain, an apical domain, a helical domain, and a CPG2 dimerization domain. The protease domain of PSMA contains a binuclear zinc site, a catalytic residue, and a substrate-binding region (also called the substrate-binding arginine patch) that includes three arginine residues. In the crystal structure, the two zinc ions in the active site are each linked to a phosphate oxygen or, in the co-crystal structure, to the phosphinic acid moiety of the inhibitor GPI 1843. In the high-resolution crystal structure of the extracellular domain, PSMA co-crystallized with both a strong inhibitor, a weak inhibitor, and glutamic acid at 2.0, 2.4, and 2.2 Å, respectively. The high-resolution crystal structure shows a 20 Å deep funnel-shaped tunnel that leads to the catalytic or active site of PSMA. The funnel is lined with the side chains of numerous Arg and Lys residues, Asp and Glu residues, and Tyr and Phe residues.

[0029] In another embodiment, the linker (L) is a chain of atoms selected from C, N, O, S, Si and P. The linker can have a wide variety of lengths, such as ranging from about 7 to about 100. The atoms used to form the linker can be combined in any chemically relevant manner, such as a chain of carbon atoms forming an alkylene group, a chain of carbon and oxygen atoms forming a polyoxyalkylene group, a chain of carbon and nitrogen atoms forming a polyamine, and the like. In addition, it should be understood that the bond linking atoms in the chain can be saturated or unsaturated, such as, for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, and the like can be divalent radicals included in the linker. In addition, it should be understood that the atoms forming the linker can also cyclize with each other to form a divalent cyclic radical in the linker. In each of the above and other linkers described herein, the chain forming the linker can be substituted with a wide variety of groups.

[0030] In another embodiment, a linker (L) is described that includes at least one releasable linker. In one variation, a linker (L) is described that includes at least two releasable linkers. In another variation, a linker (L) is described that includes at least one self-immolative linker. In another variation, a linker (L) is described that includes at least one releasable linker that is not a disulfide. In another embodiment, a linker (L) that does not include a releasable linker is described.

[0031] It is understood that a releasable linker can be used when the drug to be delivered is advantageously released from the binding ligand-linker conjugate and the free drug has the same or nearly the same effect on the target as when administered without the targeting provided by the conjugates described herein. In another embodiment, the linker L comprises a non-releasable linker. It is understood that a non-releasable linker can be used when the drug is advantageously retained by the binding ligand-linker conjugate in imaging, diagnosis, and the use of the conjugates is described herein. It should be understood that the selection of a releasable or non-releasable linker can be performed regardless of the application or configuration of the respective conjugate without limiting the invention described herein. It should be further understood that the linker L described herein includes a variety of atoms, chains of atoms, functional groups, and combinations of functional groups. Where appropriate, the linker L can be referred to in the present disclosure by the presence of spacer linkers, releasable linkers, and heteroatoms. However, such options should not be considered as limiting the definition of the linker L described herein.

[0032] The linker (L), including the spacer linker and / or releasable linker (i.e., cleavable linker), can be any biocompatible linker. The releasable or cleavable linker can be, for example, a linker susceptible to cleavage under reducing or oxidizing conditions present in a cell, a pH-sensitive linker, which can be an acid-labile or base-labile linker, or a linker that can be cleaved by a biochemical or metabolic process, such as an enzyme-labile linker. In one embodiment, the spacer and / or releasable linker comprises about 1 to about 30, or about 2 to about 20 atoms. Low molecular weight linkers (i.e., those having an approximate molecular weight of about 30 to about 300) are also described. Precursors of such linkers can be selected to have either nucleophilic or electrophilic functional groups, or both, optionally in protected form with easily cleavable protecting groups to facilitate their use in the synthesis of intermediate species.

[0033] As used herein, the term "releasable linker" refers to a linker that includes at least one bond that can be cleaved under physiological conditions (e.g., a pH-labile, acid-labile, oxidation-labile or enzyme-labile bond). The cleavable bond can be located within the cleavable linker and / or at one or both ends of the cleavable linker. It should be understood that physiological conditions that result in bond cleavage include, for example, standard chemical hydrolysis reactions that occur at physiological pH or as a result of compartmentalization into a cellular organelle, such as an endosome, that has a lower pH than the pH of the cellular matrix. Illustratively, the bivalent linkers described herein can be cleaved under other physiological or metabolic conditions, such as the action of a glutathione-mediated mechanism. It is understood that the lability of the cleavable bond can be adjusted by including functional groups or fragments in the bivalent linker L that can assist or promote such bond cleavage, also referred to as adjacent group assistance. The lability of the scissile bond can also be tailored by substitution changes at or near the scissile bond, such as, for example, including an alpha branch adjacent to the scissile disulfide bond, increasing the hydrophobicity of the silicon substituent of the moiety having a hydrolyzable silicon oxygen bond, homologating an alkoxy group that forms part of a hydrolyzable ketal or acetal, etc. In addition, it is understood that the bivalent linker L can include additional functional groups or fragments that can assist or facilitate additional fragmentation of the PSMA binding drug linker conjugate following bond scission of the releasable linker.

[0034] In another embodiment, the linker has specific length, diameter and / or functional group requirements and includes one or more spacer linkers and / or radicals that form a releasable linker that together form a linker as described herein.

[0035] Another exemplary embodiment of the linkers described herein includes releasable linkers that cleave under the conditions described herein by a chemical mechanism involving beta-elimination. In one embodiment, such releasable linkers include beta-thio, beta-hydroxy, and beta-amino substituted carboxylic acids and their derivatives, such as esters, amides, carbonates, carbamates, and urea compounds. In another embodiment, such releasable linkers include 2- and 4-thioaryl esters, carbamates, and carbonates.

[0036] It should also be understood that releasable linkers can be referred to by the groups they contain, such as, for example, disulfide groups, ketal groups, etc., illustratively as described herein. Thus, it is understood that a cleavable bond may link two adjacent atoms within a releasable linker and / or may link another linker, or a binding ligand B or a therapeutic, diagnostic, or imaging agent D, as described herein, at either or both ends of the releasable linker. If a cleavable bond links two adjacent atoms within a releasable linker, after cleavage of the bond, the releasable linker is cleaved into two or more fragments. Alternatively, if the cleavable bond is between the releasable linker and another moiety, such as an additional heteroatom, a spacer linker, another releasable linker, a drug D or an analog or derivative thereof, or a binding ligand B or an analog or derivative thereof, after cleavage of the bond, the releasable linker is separated from the other moiety.

[0037] In another embodiment, the releasable linkers and spacer linkers can be arranged such that after cleavage of a bond of the bivalent linker, the released functional group assists adjacent groups in severing or cleaving additional bonds, as described above. Exemplary embodiments of such bivalent linkers or portions thereof include those having the formula: [ka] wherein X is a heteroatom such as nitrogen, oxygen or sulfur, n is an integer selected from 0, 1, 2 and 3, R is hydrogen or a substituent, including a substituent capable of stabilizing a positive charge inductively or by resonance of an aryl ring such as alkoxy, and * ) indicates the point of attachment of a heteroatom forming an additional spacer or releasable linker or a bivalent linker, or the point of attachment of a drug or an analog or derivative thereof, or a vitamin or an analog or derivative thereof. It is understood that other substituents can be present on the aryl ring, the benzylic carbon, the alkanoic acid, or the methylene bridge, including, but not limited to, hydroxy, alkyl, alkoxy, alkylthio, halo, and the like. Assisted cleavage can include mechanisms involving benzylinium intermediates, benzyne intermediates, lactone cyclization, oxonium intermediates, beta elimination, and the like. It is further understood that in addition to fragmentation following cleavage of the releasable linker, the initial cleavage of the releasable linker can be facilitated by an anchimeric assisted mechanism.

[0038] In this embodiment, the cyclizable hydroxyalkanoic acid can promote cleavage of the methylene bridge, for example by oxonium ion, to promote bond scission or promote subsequent fragmentation following bond cleavage of the releasable linker. Alternatively, acid-catalyzed oxonium ion-assisted cleavage of the methylene bridge can initiate a fragmentation cascade of this exemplary bivalent linker or fragment thereof. Alternatively, acid-catalyzed hydrolysis of the carbamate can promote beta-elimination of the cyclizable hydroxyalkanoic acid, for example by oxonium ion, to promote cleavage of the methylene bridge. It is understood that other chemical mechanisms of bond scission or cleavage under metabolic, physiological, or cellular conditions described herein can initiate such a fragmentation cascade. It is understood that other chemical mechanisms of bond scission or cleavage under metabolic, physiological, or cellular conditions described herein can initiate such a fragmentation cascade.

[0039] Exemplary mechanisms for cleavage of the bivalent linkers described herein include the following 1,4 and 1,6 fragmentation mechanisms: [ka] where X is an exogenous or endogenous nucleophile, such as glutathione or a bioreductive agent, and either Z or Z' is a PSMA-binding ligand or a drug, therapeutic, diagnostic or imaging agent, or either Z or Z' is a PSMA-binding ligand or a drug, therapeutic, diagnostic or imaging agent linked via another portion of the bivalent linker. Although the above fragmentation mechanism is depicted as a concerted mechanism, it should be understood that any number of separate steps can occur to effect the final fragmentation of the bivalent linker to the final product shown. For example, it is understood that bond cleavage can also occur by acid-catalyzed elimination of a carbamate moiety, which may be adjacent-group assisted by stabilization provided by either the aryl group of the beta sulfur or disulfide illustrated in the examples above. In these variations of this embodiment, the releasable linker is a carbamate moiety. Alternatively, fragmentation can be initiated by nucleophilic attack on the disulfide group, resulting in cleavage to form a thiolate. Thiolates can intermolecularly displace carbonate or carbamate moieties to form the corresponding thiacyclopropanes. In the case of benzyl-containing bivalent linkers, after exemplary disulfide bond cleavage, the resulting phenylthiolates can be further fragmented to release carbonate or carbamate moieties by forming a resonance stabilized intermediate. In any of these cases, the releasable properties of the exemplary bivalent linkers described herein can be achieved by any mechanism that may be related to existing chemical, metabolic, physiological, or biological conditions.

[0040] Other exemplary mechanisms of releasable linker bond cleavage include oxonium assisted cleavage as follows: [ka] wherein Z is a vitamin or an analog or derivative thereof, or a drug or an analog or derivative thereof, or a vitamin or drug moiety together with other moieties of a polyvalent linker, such as a drug or vitamin moiety comprising one or more spacer linkers and / or other releasable linkers, respectively. In this embodiment, acid catalyzed elimination of the carbamate results in release of CO and the nitrogen-containing moiety attached to Z, and results in the formation of a benzyl cation that can be scavenged by water or any other Lewis acid.

[0041] In one embodiment, the releasable linker comprises a disulfide.

[0042] In another embodiment, the releasable linker can be a divalent radical comprising alkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, or alkylenesulfonylaziridin-1-yl, each of which is optionally coupled to a substituent X, as defined below. 2 has been replaced with.

[0043] Exemplary additional releasable linkers include methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, carbonylcycloalkylideneiminyl, alkylenethio, alkylenearylthio, and carbonylalkylthio, each of which optionally contains a substituent X as defined below. 2 has been replaced with.

[0044] In the foregoing embodiments, the releasable linker can include oxygen, and the releasable linker can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and 1-alkoxycycloalkylenecarbonyl, each of which optionally includes a substituent X, as defined below. 2 where the releasable linker is substituted with an optionally substituted aryl and the releasable linker is bonded to the oxygen to form an acetal or ketal. Alternatively, the releasable linker can include oxygen and the releasable linker can be methylene, where the methylene is substituted with an optionally substituted aryl and the releasable linker is bonded to the oxygen to form an acetal or ketal. Further, the releasable linker can include oxygen and the releasable linker can be a sulfonylalkyl, where the releasable linker is bonded to the oxygen to form an alkylsulfonate.

[0045] In another embodiment of the above releasable linker embodiments, the releasable linker can include nitrogen and can be iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, and carbonylcycloalkylideneiminyl, each of which optionally contains a substituent X, as defined below. 2 and the releasable linker is attached to the nitrogen to form a hydrazone. In an alternative embodiment, the hydrazone can be acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form a variety of acylhydrazone releasable linkers.

[0046] Alternatively, the releasable linker can include oxygen and can be an alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl, each of which optionally contains a substituent X as defined below. 2 and the releasable linker bonds with oxygen to form a silanol.

[0047] In the above releasable linker embodiments, the drug can include a nitrogen atom, the releasable linker can include nitrogen, the releasable linker can be carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, the releasable linker can be bonded to the nitrogen of the heteroatom to form an amide, and can also be bonded to the nitrogen of the drug to form an amide.

[0048] In the above releasable linker embodiments, the drug can include an oxygen atom, the releasable linker can include a nitrogen, the releasable linker can be a carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, and the releasable linker can form an amide or can be bonded to the drug nitrogen to form an ester.

[0049] Substituent X 2 is alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl, alkylcarboxylate, alkylalkanoate, guanidinoalkyl, R 4 -Carbonyl, R 5 -carbonylalkyl, R 6 -Acylamino and R 7 -acylaminoalkyl, where R 4 and R 5 are each independently selected from an amino acid, an amino acid derivative, and a peptide; and R 6 and R 7 are each independently selected from an amino acid, an amino acid derivative, and a peptide. In this embodiment, the releasable linker may contain nitrogen and the substituent X 2 and the releasable linker can form a heterocycle.

[0050] The heterocycle can be pyrrolidine, piperidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, pyrrolidinone, piperidinone, oxazolidinone, isoxazolidinone, thiazolidinone, isothiazolidinone and succinimide.

[0051] In one embodiment, the polyvalent linker described herein has the following formula: [ka] [wherein n is an integer selected from 1 to about 4; R a and R b are each independently selected from the group consisting of hydrogen and alkyl, including lower alkyl, such as C1-C4 alkyl, which is optionally branched; or R a and R b together with the carbon atom to which it is attached form a carbocyclic ring; R is an optionally substituted alkyl group, an optionally substituted acyl group or an appropriately selected nitrogen protecting group; and * ) represents a point of attachment for a drug, vitamin, imaging agent, diagnostic agent, other polyvalent linker, or other moiety of the conjugate.

[0052] In another embodiment, the polyvalent linker described herein has the following formula: [ka] wherein m is an integer selected from 1 to about 4; R is an optionally substituted alkyl group, an optionally substituted acyl group, or an appropriately selected nitrogen protecting group; and * ) represents a point of attachment for a drug, vitamin, imaging agent, diagnostic agent, other polyvalent linker, or other moiety of the conjugate.

[0053] In another embodiment, the polyvalent linker described herein has the following formula: [ka] wherein m is an integer selected from 1 to about 4; R is an optionally substituted alkyl group, an optionally substituted acyl group, or an appropriately selected nitrogen protecting group; and *) represents a point of attachment for a drug, vitamin, imaging agent, diagnostic agent, other polyvalent linker, or other moiety of the conjugate.

[0054] In another embodiment, the linker L comprises one or more spacer linkers. Such spacer linkers may optionally be substituted with a substituent X, as defined below. 1 and the releasable linker can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-aroxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, each of which is optionally substituted with a substituent X as defined below. 2 and the spacer linker and releasable linker are each attached to the spacer linker to form a succinimid-1-yl alkyl acetal or ketal.

[0055] The spacer linker can be carbonyl, thionocarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimid-3-yl, each of which is optionally represented by a substituent X as defined below. 1In this embodiment, the spacer linker can include an additional nitrogen and the spacer linker can be alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, each of which is optionally substituted with a substituent X, as defined below. 1 and the spacer linker is bonded to the nitrogen to form an amide. Alternatively, the spacer linker can include an additional sulfur, and the spacer linker can be alkylene and cycloalkylene, where the spacer linker is optionally substituted with carboxy, and the spacer linker is bonded to the sulfur to form a thiol. In another embodiment, the spacer linker can include sulfur, and the spacer linker can be 1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, where the spacer linker is bonded to the sulfur to form a succinimid-3-yl thiol.

[0056] As an alternative to the above described embodiments, the spacer linker can contain nitrogen and the releasable linker can be a divalent radical comprising an alkyleneaziridin-1-yl, a carbonylalkylaziridin-1-yl, a sulfoxylalkylaziridin-1-yl or a sulfonylalkylaziridin-1-yl, each of which optionally contains a substituent X as defined below. 2 In this alternative embodiment, the spacer linker can be carbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, each of which is optionally substituted with a substituent X, as defined below. 1 and the spacer linker is attached to a releasable linker to form an aziridine amide.

[0057] Substituent X 1is alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl, alkylcarboxylate, alkylalkanoate, guanidinoalkyl, R 4 -Carbonyl, R 5 -carbonylalkyl, R 6 -Acylamino and R 7 -acylaminoalkyl, where R 4 and R 5 are each independently selected from an amino acid, an amino acid derivative, and a peptide; and R 6 and R 7 are each independently selected from an amino acid, an amino acid derivative, and a peptide. In this embodiment, the spacer linker may contain nitrogen and the substituent X 1 and the spacer linker is attached thereto to form a heterocycle.

[0058] Additional examples of spacer linkers include alkylene-amino-alkylenecarbonyl, alkylene-thio-(carbonylalkylsuccinimide-3-yl), and the like, and are represented by the formula: [ka] wherein integers x and y are 1, 2, 3, 4, or 5.

[0059] In another embodiment, linkers comprising hydrophilic regions are also described. In one embodiment, the hydrophilic region of the linker forms all or part of a spacer linker included in the conjugates described herein. Exemplary hydrophilic spacer linkers are described in PCT International Application PCT / US2008 / 068093 (filed June 25, 2008), the disclosure of which is incorporated herein by reference.

[0060] As used herein, the term "cycloalkyl" includes molecular fragments or radicals that include a divalent chain of carbon atoms, part of which forms a ring. As used herein, the term cycloalkyl should be understood to include fragments and radicals that are attached to a ring atom or a non-ring atom, such as, for example, cyclopropyl, cyclohexyl, 3-ethylcyclopent-1-yl, cyclopropylethyl, cyclohexylmethyl, and the like.

[0061] As used herein, the term "cycloalkylene" includes molecular fragments or radicals that include a divalent chain of carbon atoms, part of which forms a ring. As used herein, the term cycloalkyl should be understood to include fragments and radicals that are attached to ring atoms or non-ring atoms, such as, for example, cyclopropa-1,1-diyl, cyclopropa-1,2-diyl, cyclohexa-1,4-diyl, 3-ethylcyclopenta-1,2-diyl, 1-methylenecyclohex-4-yl, and the like.

[0062] As used herein, the terms "heteroalkyl" and "heteroalkylene" include molecular fragments or radicals containing monovalent and divalent groups, respectively, formed from linear or branched chains of carbon atoms and heteroatoms (wherein heteroatoms are selected from nitrogen, oxygen, and sulfur), such as, for example, alkoxyalkyl, alkyleneoxyalkyl, aminoalkyl, alkylaminoalkyl, alkyleneaminoalkyl, alkylthioalkyl, alkylenethioalkyl, alkoxyalkylaminoalkyl, alkylaminoalkoxyalkyl, alkyleneoxyalkylaminoalkyl, and the like.

[0063] As used herein, the term "heterocyclyl" includes molecular fragments or radicals that include a monovalent chain of carbon atoms and heteroatoms (wherein the heteroatoms are selected from nitrogen, oxygen, and sulfur) of which a portion forms a ring, including at least one heteroatom, such as aziridine, pyrrolidine, oxazolidine, 3-methoxypyrrolidine, 3-methylpiperazine, etc. Thus, as used herein, heterocyclyl includes alkylheterocyclyl, heteroalkylheterocyclyl, heterocyclylalkyl, heterocyclylheteroalkyl, and the like. As used herein, the term heterocyclyl should be understood to include fragments and radicals attached to a ring atom or a non-ring atom, such as, for example, tetrahydrofuran-2-yl, piperidin-1-yl, piperidin-4-yl, piperazin-1-yl, morpholin-1-yl, tetrahydrofuran-2-ylmethyl, piperidin-1-ylethyl, piperidin-4-ylmethyl, piperazin-1-ylpropyl, morpholin-1-ylethyl, and the like.

[0064] As used herein, the term "aryl" includes fragments or radicals that contain aromatic monocyclic or polycyclic rings of carbon atoms, for example, phenyl, naphthyl, and the like.

[0065] As used herein, the term "heteroaryl" includes molecular fragments or radicals containing aromatic monocyclic or polycyclic rings of carbon atoms and at least one heteroatom selected from nitrogen, oxygen, sulfur, e.g., pyridinyl, pyrimidinyl, indolyl, benzoxazolyl, and the like.

[0066] As used herein, the term "substituted aryl" or "substituted heteroaryl" includes molecular fragments or radicals that contain an aryl or heteroaryl that is substituted with one or more substituents, such as, for example, alkyl, heteroalkyl, halo, hydroxy, amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, aminosulfonyl, carboxylate, alkoxycarbonyl, aminocarbonyl, cyano, nitro, etc. It is to be understood that the alkyl group of such substituents can be optionally substituted with halo.

[0067] As used herein, the term "iminoalkylidenyl" includes molecular fragments or radicals which contain an alkylene, as defined herein, and a divalent radical containing a nitrogen atom, where the terminal carbon of the alkylene is double bonded to the nitrogen atom, such as, for example, -(CH)=N-, -(CH)2(CH)=N-, -CH2C(Me)=N-, and the like.

[0068] As used herein, the term "amino acid" refers to aminoalkylcarboxylates containing a group corresponding to naturally occurring amino acids such as serine, cysteine, methionine, aspartic acid, glutamic acid, and the like, where the alkyl radical is optionally substituted with alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like.

[0069] For example, in one embodiment, the amino acid has the general formula: [ka] wherein R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting group; R' and R" are hydrogen or a substituent, each occurrence of which is independently selected; and q is an integer, such as 1, 2, 3, 4, or 5. Illustratively, R' and / or R" independently correspond to hydrogen or the side chains present in naturally occurring amino acids, such as, but not limited to, methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, and derivatives and protected derivatives thereof. The formula set forth above includes all stereochemical variations. For example, the amino acids can be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, and the like. In one variation, the amino acids can be selected from phenylalanine, tyrosine, and the like, and derivatives and substituted variations thereof.

[0070] As used herein, the terms "arylalkyl" and "heteroarylalkyl" include molecular fragments or radicals containing aryl and heteroaryl, respectively, as defined herein, substituted with a straight-chain or branched-chain alkylene group, e.g., benzyl, phenylethyl, α-methylbenzyl, picolinyl, pyrimidinylethyl, and the like.

[0071] It is to be understood that the above-described terms may be combined to form chemically related groups, such as, for example, "haloalkoxyalkyl," which refers to trifluoromethyloxyethyl, 1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like.

[0072] As used herein, the term "amino acid derivative" generally refers to aminoalkylcarboxylates containing groups corresponding to the side chains found in naturally occurring amino acids such as those found in serine, cysteine, methionine, aspartic acid, glutamic acid, and the like, where the amino or carboxylate radical, respectively, is optionally substituted with alkyl, carboxylalkyl, alkylamino, and the like, or optionally protected, and the intervening divalent alkyl fragment is optionally substituted with alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like.

[0073] As used herein, the term "peptide" includes fragments or radicals that include a series of amino acids covalently bonded to one another through amide bonds and analogs and derivatives of amino acids.

[0074] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form 3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is optionally substituted with alkyl or substituted aryl.

[0075] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form a 3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl, together with the drug, forms an acylaziridine, or an analog or derivative thereof.

[0076] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form a 1-alkoxycycloalkyleneoxy.

[0077] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form an alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.

[0078] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker that together form a dithioalkylcarbonyl hydrazide, where the hydrazide forms a hydrazone, or an analog or derivative thereof, with the drug.

[0079] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form a 3-thiosuccinimid-1-ylalkylcarbonyl hydrazide, where the hydrazide forms a hydrazone, or an analog or derivative thereof, with the drug.

[0080] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form a 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the disubstituted silyl is substituted with an alkyl or an optionally substituted aryl.

[0081] In another embodiment, the bivalent linker comprises a plurality of spacer linkers selected from the group consisting of naturally occurring amino acids and their stereoisomers.

[0082] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker that together form a 3-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate, or an analog or derivative thereof, with the drug.

[0083] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker and a releasable linker that together form a 3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate, or an analog or derivative thereof, with the drug, and the aryl is optionally substituted.

[0084] In another embodiment, the bivalent linker comprises a spacer linker and a releasable linker that together form a 3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the alkylidene forms a hydrazone or an analog or derivative thereof with the drug, each alkyl is independently selected, and the oxyalkyloxy is optionally substituted with alkyl or optionally substituted aryl.

[0085] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker that together form a 3-dithioalkyloxycarbonyl hydrazide.

[0086] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker and a releasable linker that together form a 3-dithioalkylamino, where the amino forms a vinylic amide or an analog or derivative thereof with the drug.

[0087] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker and a releasable linker that together form a 3-dithioalkylamino, where the amino forms a vinylic amide or an analog or derivative thereof with the drug and the alkyl is ethyl.

[0088] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker that together form a 3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate, or an analog or derivative thereof, with the drug.

[0089] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker and a releasable linker that together form a 3-dithioalkylaminocarbonyl, where the carbonyl, with the drug, forms a carbamate, or an analog or derivative thereof, and the alkyl is ethyl.

[0090] In another embodiment, the bivalent linker comprises a releasable linker, a spacer linker and a releasable linker that together form a 3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or carbamoylaziridine, or an analog or derivative thereof, with the drug.

[0091] In another embodiment, the polyvalent linker comprises linked spacer linkers and releasable linkers that form a polyvalent 3-thiosuccinimid-1-ylalkyloxymethyloxy group and has the formula: [ka] where n is an integer from 1 to 6, the alkyl group is optionally substituted, and the methyl is optionally substituted with an additional alkyl or an optionally substituted aryl group, each of which is represented as an independently selected group R. * ) indicates the point of attachment of the multivalent linker fragment to other moieties of the conjugates described herein.

[0092] In another embodiment, the polyvalent linker comprises a spacer linker and a releasable linker that link to form a polyvalent 3-thiosuccinimid-1-ylalkylcarbonyl group and has the formula: [ka] where n is an integer from 1 to 6, and the alkyl group is optionally substituted. * ) indicates the point of attachment of the polyvalent linker fragment to other moieties of the conjugates described herein. In another embodiment, the polyvalent linker comprises a spacer linker and a releasable linker that link to form a polyvalent 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy group, where the disubstituted silyl is substituted with alkyl and / or optionally substituted aryl groups.

[0093] In another embodiment, the polyvalent linker comprises a spacer linker and a releasable linker that link to form a polyvalent dithioalkylcarbonyl hydrazide group or a polyvalent 3-thiosuccinimid-1-ylalkylcarbonyl hydrazide group and has the formula: [ka] where n is an integer from 1 to 6, the alkyl group is optionally substituted, and the hydrazide forms a hydrazone with (B), (D) or another portion of the polyvalent linker (L). * ) indicates the point of attachment of the multivalent linker fragment to other moieties of the conjugates described herein.

[0094] In another embodiment, the polyvalent linker comprises a spacer linker and a releasable linker that link to form a polyvalent 3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene group and has the formula: [ka] where each n is an integer independently selected from 1 to 6, the alkyl groups are each independently selected and optionally substituted, for example with alkyl or optionally substituted aryl, and the alkylidene forms a hydrazone with (B), (D) or another portion of the polyvalent linker (L). * ) indicates the point of attachment of the multivalent linker fragment to other moieties of the conjugates described herein.

[0095] Additional exemplary linkers are described in WO2006 / 012527, the disclosure of which is incorporated herein by reference. Additional linkers are described in the table below, * ) atoms are points of attachment for additional spacers or releasable linkers, drugs and / or binding ligands.

[0096] Exemplary Releasable Linkers [Table 1]

[0097] The spacer and releasable linkers described herein are each divalent. In addition, the linkage between the spacer linker, the releasable linker, the drug D, and the ligand B can occur at any atom found in the various spacer linkers, the releasable linkers, the drug D, and the ligand B.

[0098] The drug may contain a nitrogen atom and the releasable linker may optionally contain a substituent X 2 The releasable linker can be a haloalkylenecarbonyl substituted with, where the releasable linker is attached to the nitrogen of the drug to form an amide.

[0099] The drug may contain an oxygen atom and the releasable linker may optionally contain a substituent X 2 The releasable linker can be a haloalkylenecarbonyl substituted with an aryl group, which is bonded to an oxygen of the drug to form an ester.

[0100] The drug can include a double-bonded nitrogen atom, and in this embodiment the releasable linker can be alkylenecarbonylamino and 1-(alkylenecarbonylamino)succinimid-3-yl, which can be bonded to the nitrogen of the drug to form a hydrazone.

[0101] The drug can include a sulfur atom, and in this embodiment the releasable linker can be alkylenethio and carbonylalkylthio, and the releasable linker can bond with the sulfur of the drug to form a disulfide.

[0102] In another embodiment, the binding or targeting ligand that can bind or target PSMA is a phosphoric acid, a phosphonic acid, or a phosphinic acid, or a derivative thereof.In one embodiment, the phosphoric acid, the phosphonic acid, or a phosphinic acid, or a derivative thereof, comprises one or more carboxylic acid groups.In another embodiment, the phosphoric acid, the phosphonic acid, or a phosphinic acid, or a derivative thereof, comprises one or more thiol groups or a derivative thereof.In another embodiment, the phosphoric acid, the phosphonic acid, or a phosphinic acid, or a derivative thereof, comprises one or more carboxylic acid bioequivalents, such as, for example, an optionally substituted tetrazole.

[0103] In another embodiment, the PSMA ligand is a derivative of pentanedioic acid. Illustratively, the derivative of pentanedioic acid has the following formula: [ka] wherein X is RP(O)(OH)CH- (see, e.g., U.S. Pat. No. 5,968,915, incorporated herein by reference); RP(O)(OH)N(R 1 )- (see, e.g., U.S. Pat. No. 5,863,536, incorporated herein by reference); RP(O)(OH)O- (see, e.g., U.S. Pat. No. 5,795,877, incorporated herein by reference); RN(OH)C(O)Y- or RC(O)NH(OH)Y (wherein Y is -CR1R2-, -NR3- or -O-) (see, e.g., U.S. Pat. No. 5,962,521, incorporated herein by reference); RS(O)Y, RSO2Y or RS(O)(NH)Y (wherein Y is -CR1R2-, -NR3- or -O-) (see, e.g., U.S. Pat. No. 5,902,817, incorporated herein by reference); or RS-alkyl (wherein R is, for example, hydrogen, alkyl, aryl or arylalkyl, each optionally substituted) (see, e.g., J. Med. Chem. 1999, 144, 147, incorporated herein by reference). 46:1989-1996 (2003)).

[0104] In each of the above formulas, R, R1, R2, and R3 are each independently selected from hydrogen, C1-C9 linear or branched alkyl, C2-C9 linear or branched alkenyl, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, and aryl. In addition, R, R1, R2, and R3 can each be optionally substituted with one or more groups selected from, for example, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, halo, hydroxy, nitro, trifluoromethyl, C1-C6 linear or branched alkyl, C2-C6 linear or branched alkenyl, C1-C4 alkoxy, C2-C4 alkenyloxy, phenoxy, benzyloxy, amino, and aryl. In one embodiment, aryl is selected from 1-naphthyl, 2-naphthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, benzyl, and phenyl, in each case aryl can be optionally substituted with one or more groups, illustratively 1 to 3 groups, selected from halo, hydroxy, nitro, trifluoromethyl, C1 to C6 straight or branched alkyl, C2 to C6 straight or branched alkenyl, C1 to C4 alkoxy, C2 to C4 alkenyloxy, phenoxy, benzyloxy, and amino. In one variation of each of the above formulas, R is not hydrogen.

[0105] Exemplary PSMA ligands described in U.S. Pat. No. 5,968,915 include 2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid; 2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid; 2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid; 2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid; 2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid; 2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid; 2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid. 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid; 2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic acid; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid.

[0106] Exemplary PSMA ligands described in U.S. Pat. No. 5,863,536 include N-[methylhydroxyphosphinyl]glutamic acid; N-[ethylhydroxyphosphinyl]glutamic acid; N-[propylhydroxyphosphinyl]glutamic acid; N-[butylhydroxyphosphinyl]glutamic acid; N-[phenylhydroxyphosphinyl]glutamic acid; N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid; N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid; and N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid.

[0107] Exemplary PSMA ligands described in U.S. Pat. No. 5,795,877 include 2-[[methylhydroxyphosphinyl]oxy]pentanedioic acid; 2-[[ethylhydroxyphosphinyl]oxy]pentanedioic acid; 2-[[propylhydroxyphosphinyl]oxy]pentanedioic acid; 2-[[butylhydroxyphosphinyl]oxy]pentanedioic acid; 2-[[phenylhydroxyphosphinyl]oxy]pentanedioic acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid; 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid; 2-[[(phenylmethyl)hydroxyphosphinyl]oxy]pentanedioic acid; and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid.

[0108] Exemplary PSMA ligands described in U.S. Pat. No. 5,962,521 include 2-[[(N-hydroxy)carbamoyl]methyl]pentanedioic acid; 2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]pentanedioic acid; 2-[[(N-butyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid; 2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid; 2-[[(N-hydroxy-N-phenyl)carbamoyl]methyl]pentanedioic acid; 2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]pentanedioic acid; 2-[[(N-ethyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid; 2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]pentanedioic acid; 2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]pentanedioic acid. 2-[[(N-hydroxy-N-4-pyridyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(methyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(benzyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(phenyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydrox [[N-hydroxy(2-phenylethyl)carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy(ethyl)carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy(propyl)carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy(3-phenylpropyl)carboxamido]methyl]pentanedioic acid; and 2-[[N-hydroxy(4-pyridyl)carboxamido]methyl]pentanedioic acid.

[0109] Exemplary PSMA ligands described in U.S. Pat. No. 5,902,817 include 2-[(sulfinyl)methyl]pentanedioic acid; 2-[(methylsulfinyl)methyl]pentanedioic acid; 2-[(ethylsulfinyl)methyl]pentanedioic acid; 2-[(propylsulfinyl)methyl]pentanedioic acid; 2-[(butylsulfinyl)methyl]pentanedioic acid; 2-[(phenylsulfinyl]methyl]pentanedioic acid; 2-[[(2-phenylethyl)sulfinyl]methyl]pentanedioic acid. ;2-[[(3-phenylpropyl)sulfinyl]methyl]pentanedioic acid;2-[[(4-pyridyl)sulfinyl]methyl]pentanedioic acid;2-[(benzylsulfinyl)methyl]pentanedioic acid;2-[(sulfonyl)methyl]pentanedioic acid;2-[(methylsulfonyl)methyl]pentanedioic acid;2-[(ethylsulfonyl)methyl]pentanedioic acid;2-[(propylsulfonyl)methyl]pentanedioic acid;2-[(butylsulfonyl)methyl]pentanedioic acid;2-[(phenylsulfonyl)methyl]pentanedioic acid 2-[[(2-phenylethyl)sulfonyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl)sulfonyl]methyl]pentanedioic acid;2-[[(4-pyridyl)sulfonyl]methyl]pentanedioic acid;2-[(benzylsulfonyl)methyl]pentanedioic acid;2-[(sulfoximinyl)methyl]pentanedioic acid;2-[(methylsulfoximinyl)methyl]pentanedioic acid;2-[(ethylsulfoximinyl)methyl]pentanedioic acid;2-[(propanyl)sulfonyl]methyl]pentanedioic acid 2-[(phenylsulfoximinyl)methyl]pentanedioic acid; 2-[(butylsulfoximinyl)methyl]pentanedioic acid; 2-[(phenylsulfoximinyl)methyl]pentanedioic acid; 2-[[(2-phenylethyl)sulfoximinyl]methyl]pentanedioic acid; 2-[[(3-phenylpropyl)sulfoximinyl]methyl]pentanedioic acid; 2-[[(4-pyridyl)sulfoximinyl]methyl]pentanedioic acid; and 2-[(benzylsulfoximinyl)methyl]pentanedioic acid. do.

[0110] Derivatives of pentanedioic acid described herein have been reported to have high binding affinity to PSMA and include, but are not limited to, the following phosphonic and phosphinic acid derivatives: [ka] where the dissociation constant of the EI complex (K i (Current Medicinal Chem. 8:949-.957(2001); Silverman, “The Organic Chemistry of Drug Design and Drug Action,” Elsevier Academic Press (2 nd Ed. 2003), the disclosure of which is incorporated herein by reference.

[0111] In another exemplary embodiment, the derivative of pentanedioic acid contains a thiol group, for example, the following formula: [ka] wherein the inhibition constant of the EI complex (IC 50 values) are shown.

[0112] In another embodiment, the PSMA ligand is a urea compound of two amino acids. In one embodiment, the amino acid contains one or more additional carboxylic acids. In another embodiment, the amino acid contains one or more phosphoric acids, phosphonic acids, phosphinic acids, sulfinic acids, sulfonic acids, or boronic acids. In another embodiment, the amino acid contains one or more thiol groups or derivatives thereof. In another embodiment, the amino acid contains one or more bioequivalents of carboxylic acids, such as tetrazoles.

[0113] In another embodiment, the PSMA ligand is an aminocarbonyl derivative of pentanedioic acid. Illustratively, the aminocarbonyl pentanedioic acid derivative has the following formula: [ka] [In the formula, R 1 and R 2 are each selected from hydrogen and optionally substituted carboxylic acids such as thiolacetic acid, thiolpropionic acid, etc., malonic acid, succinic acid, glutamic acid, adipic acid, etc. Exemplary aminocarbonylpentanedioic acid derivatives are described in J. Med. Chem. 44:298-301 (2001) and J. Med. Chem. 47: 1729-38 (2004), the disclosures of which are incorporated herein by reference.

[0114] In another embodiment, the PSMA ligand is a compound having the formula: [ka] [Table 2]

[0115] It is understood that the urea compounds described herein may also be advantageous for the preparation of the ligands described herein due to the sub-nanomolar potency, water solubility and / or long-term stability of these compounds. The urea compounds described herein can generally be prepared as described herein from commercially available starting materials.

[0116] In each of the above exemplary pentanedioic acid and urea compounds, there is at least one asymmetric carbon atom. Thus, the above exemplary formulas are intended to refer to all stereoisomers, individually and collectively, as pure enantiomers or mixtures of enantiomers and / or diastereomers, including, but not limited to, racemic mixtures and mixtures that contain one epimer at the first asymmetric carbon atom but also allow mixtures of other asymmetric carbons, including racemic mixtures.

[0117] In another exemplary embodiment, the conjugate is a urea compound of an aminodicarboxylic acid, such as aspartic acid, glutamic acid, and the like, with another aminodicarboxylic acid or an analog thereof, such as, for example, a compound of the formula: [ka] wherein Q is an aminodicarboxylic acid or analog thereof, such as aspartic acid, glutamic acid, and the like, n and m are each selected from an integer from 1 to about 6, and * ) represents the attachment point of the linker L.

[0118] In another embodiment, the PSMA ligand comprises at least four carboxylic acid groups, or comprises at least three free carboxylic acid groups after the PSMA ligand is attached to an agent or linker. As described herein, the carboxylic acid groups of the PSMA ligand are understood to include biological equivalents of the carboxylic acid.

[0119] Illustratively, the PSMA ligand is a compound having the formula: [ka]

[0120] In another embodiment, the PSMA ligand is 2-[3-(1-carboxy-2-mercapto-ethyl)-ureido]-pentanedioic acid (MUPA) or 2-[3-(1,3-dicarboxy-propyl)-ureido]-pentanedioic acid (DUPA).

[0121] Other illustrative examples of PSMA ligands include quisqualate, aspartic acid glutamic acid (Asp-Glu), Glu-Glu, Gly-Glu, gamma-Glu-Glu, beta-N-acetyl-L-aspartic acid-L-glutamic acid (β-NAAG), and the like.

[0122] In another exemplary embodiment, the linker is a urea compound of an amino dicarboxylic acid, such as aspartic acid, glutamic acid, etc., with another amino dicarboxylic acid or its analog, and the linker is a peptide of amino acids, including naturally occurring and non-naturally occurring amino acids. In one embodiment, the linker is a peptide of amino acids selected from Glu, Asp, Phe, Cys, beta amino Ala, and amino alkyl carboxylic acids, such as Gly, beta Ala, amino valeric acid, amino caproic acid, etc. In another embodiment, the linker is a peptide of amino acids selected from Glu, Asp, Phe, Cys, beta amino Ala, and amino alkyl carboxylic acids, such as Gly, beta Ala, amino valeric acid, amino caproic acid, etc. In another embodiment, the linker is a peptide of at least one Phe. In a variant, the linker is a peptide of at least two Phe residues, or at least three Phe residues. In another embodiment, the linker is a peptide of Glu-Phe, or a dipeptide of amino alkyl carboxylic acid and Phe. In another embodiment, the linker is a peptide comprising Glu-Phe-Phe, or a tripeptide of an aminoalkyl carboxylic acid and two Phe residues. In another embodiment, the linker is a peptide comprising one or more Phe residues, where at least one Phe is about 7 to about 11 or about 7 to about 14 atoms away from the binding ligand B. In another embodiment, the linker is a peptide comprising Phe-Phe, where at least one of the Phe atoms is about 7 to about 11 or about 7 to about 14 atoms away from the binding ligand B. In each of the above embodiments and variations, one or more of the Phe residues can be replaced with Tyr or another substitution variant. It should be understood that the above may be interchanged in a similar manner.

[0123] In another exemplary embodiment, the linker is a urea compound of an aminodicarboxylic acid such as aspartic acid, glutamic acid, etc., with another aminodicarboxylic acid or analog thereof, and the linker comprises one or more aryl or arylalkyl groups, each optionally substituted, attached to the backbone of the linker. In another embodiment, the linker comprises one or more aryl or arylalkyl groups, each optionally substituted, attached to the backbone of the linker, separated from the binding ligand B by about 7 to about 11 atoms. In another embodiment, the linker comprises two aryl or arylalkyl groups, each optionally substituted, attached to the backbone of the linker, where one aryl or arylalkyl group is separated from the binding ligand B by about 7 to about 11 atoms, or about 7 to about 14 atoms, and the other alkyl or arylalkyl group is separated from the binding ligand B by about 10 to about 14 atoms, or about 10 to about 17 atoms.

[0124] As described herein, the conjugates use PSMA-binding ligands to target cells expressing or overexpressing PSMA. Once delivered, the conjugates bind to PSMA. It is understood that in certain embodiments, the conjugates remain on the surface of the cell for a sufficient time for imaging and / or diagnosis. In another embodiment, the conjugates are internalized into cells expressing or overexpressing PSMA by endogenous cellular mechanisms such as endocytosis for subsequent imaging and / or diagnosis, or treatment. Once internalized, the conjugates may remain intact or may degrade, disassemble, or otherwise be altered to allow for the release of the agent forming the conjugate. It is understood that in imaging and / or diagnostic configurations, the agent may remain intact as a conjugate or may be released once internalized into the target cell. It is further understood that in therapeutic configurations, it is advantageous for the agent to be released from the conjugate once internalized into the target cell.

[0125] In one exemplary embodiment, the agent is an imaging agent. In another exemplary variation, the agent is a diagnostic agent. In another exemplary variation, the agent is a chemotherapeutic agent.

[0126] In one embodiment, the imaging agent is a radioisotope covalently attached to a linker. In another embodiment, the imaging agent is a radioisotope, such as a radioactive metal isotope coordinated to a chelating group. Exemplary radioactive metal isotopes include: 111 In, 99m Tc, 64 Cu, 67 Cu, 67 Ga, 68 These include technetium, rhenium, gallium, gadolinium, indium, copper, and the like, including isotopes such as Ga. Additional exemplary radionuclide imaging agents are described in U.S. Pat. No. 7,128,893, the disclosure of which is incorporated herein by reference. Additional exemplary chelating groups are tripeptides or tetrapeptides. , the following formula: [ka] wherein R is independently selected at each occurrence from H, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, etc., each of which is optionally substituted. It should be understood that one R contains a heteroatom such as nitro, oxygen, or sulfur and is the attachment point for the linker L. Illustratively, the following chelating groups are depicted: [ka] wherein X is oxygen, nitrogen, or sulfur, X is attached to a linker, L, and n is an integer from 1 to about 5.

[0127] In another embodiment, the imaging agent is a fluorescent agent. Fluorescent agents include, but are not limited to, Oregon Green fluorescent agents, including but not limited to, Oregon Green 488, Oregon Green 514, etc.; AlexaFluor fluorescent agents, including but not limited to, AlexaFluor 488, AlexaFluor 647, etc.; BODIPY fluorescent agents, including but not limited to, fluorescein and related analogues, BODIPY Fl, BODIPY 505, etc.; rhodamine fluorescent agents, including but not limited to, tetramethylrhodamine, etc.; DyLight fluorescent agents, including but not limited to, DyLight 680, DyLight 800, etc.; CW800, Texas Red, phycoerythrin, etc. Exemplary fluorescent agents are illustrated by the following exemplary general structure: [ka] where X is oxygen, nitrogen or sulfur, X is attached to a linker L; and Y is OR a , N.R. a 2 or NR a 3 + and Y′ is O, NR a or NR a 2 + wherein R is independently selected at each occurrence from H, fluoro, sulfonic acid, sulfonate, and salts thereof, and the like; R a is hydrogen or alkyl.

[0128] [ka] wherein X is oxygen, nitrogen, or sulfur, X is attached to a linker, L; R is independently selected at each occurrence from H, alkyl, heteroalkyl, and the like; and n is an integer from 0 to about 4.

[0129] In another embodiment, the imaging agent is a PET imaging agent or a FRET imaging agent. PET imaging agents include: 18 F, 11 C.64 Cu, 65 Cu. FRET imaging agents include: 64 Cu, 65 Cu etc. 18 F, 11 It is understood that in the case of C, the imaging isotope may be present in any part of the linker or in a structure attached to the linker. For example, 18 In the case of F, fluoroaryl groups such as fluorophenyl, difluorophenyl, fluoronitrophenyl, and the like are described. For example, 11 In the case of C, alkyl and alkylaryl are mentioned.

[0130] In another embodiment, the chemotherapeutic agent is a cytotoxic compound. The cytotoxic compounds described herein operate by any of a number of mechanisms of action. In general, cytotoxic compounds interfere with cellular mechanisms important for cell survival and / or cell proliferation and / or cause apoptosis.

[0131] A drug can be any molecule capable of regulating or otherwise modifying a cellular function, including pharma- ceutically active compounds. Suitable molecules include peptides, oligopeptides, retro-inverse oligopeptides, proteins, protein analogs in which a peptide bridge is replaced by at least one non-peptide bridge, apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins; antigens and their antibodies; heptenes and their antibodies; hormones, lipids, phospholipids; liposomes; toxins; antibiotics; sedatives; bronchodilators; beta-blockers; antibacterial agents; antihypertensive agents; antiarrhythmic agents, cardiac glycosides. The therapeutic agents may include, but are not limited to, cardiovascular agents including antianginal agents, antianginal agents, and vasodilators; central nervous agents including stimulants, psychotropic agents, antimanic agents, and depressants; antiviral agents; antihistamines; anticancer agents including chemotherapeutic agents; tranquilizers; antidepressants; H-2 antagonists; anticonvulsants; antiemetics; prostaglandins and prostaglandin analogs; muscle relaxants; anti-inflammatory substances; stimulants; decongestants; antiemetics; diuretics; antispasmodics; antiasthmatics; antiparkinsonian agents; expectorants; antitussives; mucolytics; and mineral and nutritional additives.

[0132] Furthermore, the drug can be any drug known in the art to be cytotoxic, to enhance tumor penetrance, to inhibit tumor cell proliferation, to promote apoptosis, to reduce anti-apoptotic activity of target cells, to be used in the treatment of diseases caused by infectious agents, to enhance endogenous immune responses directed against pathogenic cells, or to be useful in the treatment of disease states caused by any type of pathogenic cell. Suitable drugs for use in the present invention include adrenocorticoids and corticosteroids, alkylating agents, antiandrogens, antiestrogens, androgens, aclamycin and aclamycin derivatives, estrogens, antimetabolites such as cytosine arabinoside, purine analogs, pyrimidine analogs and methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, taxanes such as tamoxifen, taxol, paclitaxel, paclitaxel derivatives, Taxotere®, maytansines and their analogs and derivatives, cyclophosphamide, daunomycin, doxorubicin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, Teniposide, mitomycin, discodermolide, microtubule inhibitors, epothilones, tubulysin, cyclopropylbenz[e]indolone, seco-cyclopropylbenz[e]indolone, O-Ac-seco-cyclopropylbenz[e]indolone, bleomycin and any other antibiotics, nitrogen mustards, nitrosoureas, vincristine, vinblastine, analogs and derivatives thereof, such as deacetylvinblastine monohydrazide, colchicine, colchicine derivatives, Included are allocolchicine, thiocolchicine, trityl cysteine, halichondrin B, dolastatins such as dolastatin 10, amanitins such as alpha-amanitin, camptothecin, irinotecan and other camptothecin derivatives, geldanamycin and geldanamycin derivatives, estramustine, nocodazole, MAP4, colcemid, inflammatory and pro-inflammatory agents, peptide and peptidomimetic signal transduction inhibitors, and any other art-recognized drug or toxin.Other drugs that can be used in the present invention include penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and any other art-recognized antibacterial compound.

[0133] Exemplary drugs and other therapeutic agents are described in U.S. Patent Application Nos. 2005-0002942-A1, 2001-0031252-A1, and 2003-0086900-A1. Exemplary imaging agents and diagnostic agents are described in U.S. Patent Application No. 2004-0033195-A1 and International Patent Publication No. WO03 / 097647. The disclosures of each of the foregoing patent application publications are incorporated herein by reference.

[0134] The invention described herein also includes pharmaceutical compositions comprising a binding ligand (B) drug delivery conjugate in an amount effective to eliminate a population of pathogenic cells in a host animal when administered in one or more doses. The binding ligand drug delivery conjugate is preferably administered to the host animal parenterally, for example, intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously or intrathecally. Alternatively, the binding ligand drug delivery conjugate can be administered to the host animal by other medically useful methods, such as oral administration, and any effective dose and suitable therapeutic dosage form, including sustained release dosage forms, can be used.

[0135] Examples of parenteral dosage forms include aqueous solutions of the active agent dissolved in isotonic saline, 5% glucose, or other well-known pharma- ceutically acceptable liquid carriers, such as liquid alcohols, glycols, esters, and amides. The parenteral dosage form of the present invention can be in the form of a reconstitutable lyophilizate containing a dose of the drug delivery conjugate. In one aspect of the embodiment of the present invention, any of the numerous sustained release dosage forms known in the art may be administered, such as biodegradable carbohydrate matrices described in U.S. Pat. Nos. 4,713,249, 5,266,333, and 417,982, the disclosures of which are incorporated herein by reference, or a slow-release pump (osmotic pump) may be used.

[0136] In one exemplary embodiment, at least one additional composition comprising a therapeutic factor can be administered to the host in combination with or as an adjuvant to the methodology detailed above to enhance the elimination of pathogenic cell populations mediated by the binding ligand drug delivery conjugate, and two or more additional therapeutic factors can also be administered. The therapeutic factor can be selected from chemotherapeutic agents or other therapeutic factors capable of complementing the efficacy of the administered binding ligand drug delivery conjugate.

[0137] In one exemplary embodiment, a therapeutically effective combination of these agents can be used. In one embodiment, for example, a therapeutically effective amount of a therapeutic agent, for example, about 0.1 MIU / m in a multiple dose daily regimen, is used. 2 / dose / day approximately 15 MIU / m 2 / Dose / Day Range or, for example, about 0.1 MIU / m in a multiple dose daily regimen. 2 / dose / day to 7.5 MIU / m 2 Amounts ranging from 0.1 mg / dose / day can be used with the binding ligand drug delivery conjugates to eliminate, reduce or neutralize pathogenic cells in a host animal that has the pathogenic cells (MIU = milli-international units; m 2 = Approximate body surface area of ​​an average human).

[0138] In another embodiment, chemotherapeutic agents, for example, which are themselves cytotoxic or which can act to enhance tumor permeability, are also suitable for use in the methods of the invention in combination with binding ligand drug delivery conjugates. Such chemotherapeutic agents include adrenocorticoids and corticosteroids, alkylating agents, antiandrogens, antiestrogens, androgens, aclamycin and aclamycin derivatives, estrogens, antimetabolites such as cytosine arabinoside, purine analogs, pyrimidine analogs, and methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, taxol, paclitaxel, paclitaxel derivatives, Taxotere®, cyclophosphamide, daunomycin, doxorubicin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycin, discodermolide, microtubule inhibitors, epothilones, tubulysin, cyclopropylbenzo[e]indolone, seco-cyclopropylbenz[e]indolone, ... benzo[e]indolone, O-Ac-seco-cyclopropylbenzo[e]indolone, bleomycin and any other antibiotics, nitrogen mustards, nitrosoureas, vincristine, vinblastine, analogs and derivatives thereof, such as deacetylvinblastine monohydrazide, colchicine, colchicine derivatives, allocolchicine, thiocolchicine, trityl cysteine, halichondrin B, dolastatins such as dolastatin 10, amanitins such as alpha-amanitin, camptothecin, irinotecan and other camptothecin derivatives, geldanamycin and geldanamycin derivatives, estramustine, nocodazole, MAP4, colcemid, inflammatory and pro-inflammatory agents, peptide and peptidomimetic signal transduction inhibitors, and any other art recognized drug or toxin.Other drugs that can be used in the present invention include penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, maytansine and its analogs and derivatives, gemcitabine, and any other art-recognized antibacterial compound.

[0139] The therapeutic factor may be administered to the host animal before, after, or simultaneously with administration of the binding ligand drug delivery conjugate. The therapeutic agent may be administered to a subject as part of the same composition containing the binding ligand drug delivery conjugate or as part of a different composition than the binding ligand drug delivery conjugate. Any such therapeutic composition containing a therapeutically effective dose of the therapeutic agent may be used in the present invention.

[0140] In addition, two or more types of binding ligand drug delivery conjugates can be used. Illustratively, for example, the host animal can be treated with a co-administration protocol of conjugates with different vitamins but the same drug. In other embodiments, the host animal can be treated with conjugates that include the same binding ligand bound to different drugs or that include multiple binding ligands bound to multiple drugs. In another exemplary embodiment, binding ligand drug delivery conjugates can be used with the same or different drugs, including multiple vitamins and multiple drugs as part of the same drug delivery conjugate.

[0141] In another exemplary embodiment, any effective regimen for administering the binding ligand drug delivery conjugate can be used. For example, the binding ligand drug delivery conjugate can be administered as a single dose or can be divided into multiple dose daily regimens. In another embodiment, a staggered regimen, for example, 1-3 days per week, can be used as an alternative to daily treatment, and for purposes of defining the present invention, such intermittent or staggered daily regimens are considered equivalent to daily treatment and within the scope of the methods of the present invention. In one embodiment, the host is treated with multiple injections of the binding ligand drug delivery conjugate to eliminate the population of pathogenic cells. In another embodiment, the host is injected multiple times (preferably about 2 to up to about 50 times) with the binding ligand drug delivery conjugate, for example, at 12-72 hour intervals or 48-72 hour intervals. In other embodiments, additional injections of the binding ligand drug delivery conjugate can be administered to the patient at intervals of days or months after the first injection, and the additional injections prevent recurrence of the disease state caused by the pathogenic cells.

[0142] Illustratively, the binding ligand drug delivery conjugates can be administered parenterally, for example, intradermally, subcutaneously, intramuscularly, intraperitoneally or intravenously, to an animal or patient suffering from a disease state in combination with a pharma- ceutically acceptable carrier. In another embodiment, the binding ligand drug delivery conjugates can be administered to an animal or patient by other procedures that are medically useful, and an effective dose can be administered in a standard or sustained release dosage form. In another aspect, the method of treatment can be used alone or in combination with other methods of treatment approved for treating disease conditions mediated by activated macrophages.

[0143] Described herein are methods for imaging pathogenic cell populations that express or overexpress PSMA.

[0144] Described herein is a method for diagnosing diseases and disease states associated with pathogenic cell populations that express or overexpress PSMA. The compounds described herein selectively and / or specifically bind to cells that express or overexpress PSMA. In addition, they show selectivity not only between pathogenic cells and normal cells, but also between pathogenic cell populations (see FIG. 8, where LnCAP cells expressing PSMA are preferentially visualized compared to non-expressing A549 or KB tumors). In addition, the reaction is specific for PSMA binding, as shown by competition studies performed with the conjugates described herein, where binding is determined using the conjugates alone or in the presence of excess amounts of PMPA, a known binding ligand for PSMA. Binding in both kidney and tumor is blocked by the presence of excess amounts of PMPA (see, for example, the method examples described herein).

[0145] In another embodiment, the conjugate has a binding constant K of about 100 nM or less. d In another embodiment, the conjugate has a binding constant K of about 75 nM or less. d In another embodiment, the conjugate has a binding constant K of about 50 nM or less. d In another embodiment, the conjugate has a binding of about 25 nM or less. number K d has.

[0146] In another embodiment, the conjugates described herein exhibit selectivity for cells or tissues that express or overexpress PSMA with at least 3-fold selectivity or at least 5-fold selectivity over normal tissues, such as blood, heart, lung, liver, spleen, duodenum, skin, muscle, bladder, and prostate. In one variation, the conjugates described herein exhibit selectivity for cells or tissues that express or overexpress PSMA with at least 10-fold selectivity over normal tissues. It is understood that the selectivity observed in imaging is indicative of the selectivity that may be observed in the treatment of disease conditions that respond to the selective or specific elimination of cells or cell populations that express or overexpress PSMA.

[0147] The daily dosage unit of the drug delivery conjugate can vary significantly depending on the condition of the host, the condition of the disease being treated, the molecular weight of the conjugate, its route of administration and tissue distribution, and the possibility of concomitant use of other therapeutic treatments such as radiation therapy. The effective amount administered to the patient is based on the body surface area, the patient's weight, and the physician's assessment of the patient's condition. The effective dose can range from about 1 ng / kg to about 1 mg / kg, about 1 μg / kg to about 500 μg / kg, about 1 μg / kg to about 100 μg / kg, and about 1 μg / kg to about 10 μg / kg.

[0148] In general, any method can be utilized in accordance with the present invention to form conjugates between the bivalent linker (L) and the binding ligand (B), or an analog or derivative thereof, and between the bivalent linker (L) and the drug, or an analog or derivative thereof (including any intervening heteroatoms). Also, any art-recognized method can be used to form conjugates between the spacer linker, the releasable linker, and one or more heteroatoms forming the bivalent linker (L). Conjugates can be formed by direct bonding between any of these molecules, such as through hydrogen, ionic, or covalent bonds. Covalent bonds can occur, for example, through the formation of amide, ester, disulfide, or imino bonds between acid, aldehyde, hydroxy, amino, sulfhydryl, or hydrazo groups.

[0149] The synthetic method is selected depending on the selection of the optional heteroatom or heteroatoms already present in the spacer linker, the releasable linker, the drug and / or the binding ligand. In general, the relevant bond-forming reactions are described in Richard C. Larock, "Comprehensive Organic Transformations, a guide to functional group preparations," VCH Publishers, Inc. New York (1989) and Theodora E. Greene & Peter GM Wuts, "Protective Groups ion Organic Synthesis," 2d edition, John Wiley & Sons, Inc. New York (1991), the disclosures of which are incorporated herein by reference.

[0150] More specifically, disulfide groups can generally be formed by reacting alkyl or aryl sulfonylthioalkyl derivatives or corresponding heteroaryl dithioalkyl derivatives, such as pyridin-2-yl dithioalkyl derivatives, with alkylene thiol derivatives. For example, the required alkyl or aryl sulfonylthioalkyl derivatives can be prepared according to the method of Ranasinghe and Fuchs, Synth. Commun. 18(3), 227-32 (1988), the disclosure of which is incorporated herein by reference. Another method for preparing asymmetric dialkyl disulfides is based on the thiol exchange of asymmetric heteroaryl-alkyl disulfides, such as 2-thiopyridinyl, 3-nitro-2-thiopyridinyl, etc., with alkyl thiols, as described in WO88 / 01622, European Patent Application No. 0116208A1, and U.S. Patent No. 4,691,024, the disclosures of which are incorporated herein by reference. Additionally, carbonates, thiocarbonates and carbamates can generally be formed by reacting hydroxy-, thio- or amine-substituted compounds, respectively, with activated alkoxycarbonyl derivatives bearing an appropriate leaving group. EXAMPLES

[0151] The compounds described herein can be prepared by conventional organic synthesis methods. In addition, the compounds described herein can be prepared as shown below. Unless otherwise indicated, all starting materials and reagents are commercially available. All amino acid starting materials were purchased from Chem-Impex Int (Chicago, IL). 1 1 H NMR spectra were obtained using a Bruker 500 MHz cryoprobe unless otherwise indicated.

[0152] Example 1A. General synthesis of PSMA inhibitor intermediates of conjugates. Specific synthesis of DUPA derivative 2-[3-(1,3-bis-tert-butoxycarbonyl-propyl)-ureido]-pentanedioic acid 1-tert-butyl ester (I) is illustrated.

[0153] [ka]

[0154] SK09. To a mixture of L-glutamic acid di-tert-butyl ester HCl (1.0 g, 3.39 mmol) and triphosgene (329.8 mg, 1.12 mmol) in CHCl (25.0 mL) cooled to -78 °C was added triethylamine (1.0 mL, 8.19 mmol). After stirring at -78 °C under nitrogen for 2 h, a mixture of L-Glu(OBn)-O-tert-Bu (1.2 g, 3.72 mmol) and triethylamine (600 μL, 4.91 mmol) in CHCl (5.0 mL) was added. The reaction mixture was allowed to reach room temperature over 1 h and stirring was continued at room temperature overnight. The reaction mixture was washed with 1N HCl and brine and dried over NaSO. The crude product was purified by flash chromatography (hexane:EtOAc = 1:1, R t =0.67) to give SK09 (1.76 g, 90.2%). 30 H 46N2O9;W=578.69g / mol;colorless oil; 1 H NMR(CDCl3) δ1.43(s,9H,CH3- t Bu); 1.44(s,9H,CH3- t Bu);1.46(s,9H,CH3- t Bu);1.85(m,1H,Glu-H);1.87(m,1H,Glu-H);2.06(m,1H,Glu-H);2.07(m,1H,Glu-H);2.30(m,2H,Glu-H);2.44(m,2H,Glu- H);4.34[s(broad),1H,αH];4.38[s(broad),1H,α-H];5.10(s,2H,CH2-Ar);5.22[s(broad),2H,urea-H);7.34(m,5H,Ar-H). 13 C NMR(CDCl3)δ28.16;28.25;28.54;2.60;30.52;31.73;53.13;53.22;66.58;80.71;82.25; 82.35;128.39;128.71;136.03;156.96;172.01;172.16;172.65;173.13:CI-MS=579(M+H) + , ESI-MS=579(M+H) + , 601 (M+Na adduct).

[0155] SK23. To a solution of compound SK09 (250 mg, 432 mmol) in CH2Cl2 was added 30% Pd / C (50 mg). The reaction mixture was hydrogenated at room temperature at 1 atm for 24 h. The Pd / C was filtered through a pad of Celite and washed with CH2Cl2. The crude product was purified by flash chromatography (hexane:EtOAc=40:60, R t =0.58) to give SK23 (169 mg, 80.2%). 23 H 40 N2O9; MW=488.57 g / mol; colorless oil; 1 H NMR(CDCl3) δ1.46(m,27H,CH3- tBu);1.91(m,2H,Glu-H);2.07(m,1H,Glu-H);2.18(m,1H,Glu-H);2.33(m,2H,Glu-H);2.46(m,2H ,Glu-H);4.31(s(broad),1H,αH);4.35(s(broad),1H,α-H);5.05(t,2H,urea-H);CI-MS=489(M+H) + , ESI-MS=489(M+H) + , 511 (M+Na adduct), 487 (MH) - .

[0156] Example 1B. General synthesis of PSMA inhibitor intermediates of conjugates. Specific synthesis of tertiary butyl protected MUPA derivative 2-[3-(1-tert-butoxycarbonyl-2-mercapto-ethyl)-ureido]-pentanedioic acid di-tert-butyl ester (II) is illustrated.

[0157] [ka]

[0158] SK15. To a mixture of L-glutamic acid di-tert-butyl ester HCl (200 mg, 0.676 mmol) and triphosgene (67 mg, 0.228 mmol) in CHCl (5.0 mL) cooled to -78 °C, triethylamine (50 μL, 0.410 mmol) was added. After stirring at -78 °C for 2 h under nitrogen, D-Cys(Fm) in CHCl (1.0 mL) was added. -O t A mixture of Bu (291.4 mg, 0.774 mmol) and triethylamine (30 μL, 240 mmol) was added. The reaction mixture was allowed to warm to room temperature over 1 h and was left stirring at room temperature overnight. The reaction mixture was washed with 1N HCl and brine and dried over Na2SO4. The crude product was purified by flash chromatography (hexane:EtOAc=50:50, R t =0.6) to give SK15 (374 mg, 86.4%). 35 H 48 N2O7S; MW=640.83g / mol; pale yellow oil;1 H NMR(CDCl3) δ1.45(s,27H,CH3- t Bu);1.88(m,1H,Glu-H);2.10(m,1H,Glu-H);2.32(m,2H,Glu-H);2.97(m,2H,Fm-CH2);3.13(m,2H,Cys-H);4.09(t,1H,Fm-H);4 .38(m,1H,αH);4.66(m,1H,α-H);5.55(d,1H,urea-H);5.67(d,1H,urea-H);7.30(q,2H,Ar-H);7.36(q,2H,Ar-H);7.73(m,4H,Ar-H). 13 C-NMR (CDCl3)δ28.05;28.14;28.42;31.64;36.27;37.25;53.07;53.73;80.51;81.98;82.42;119.85;1 24.95;125.09;127.09;127.51;141.09;145.99;156.76;170.80;172.15;172.43;CI-MS=641(M+H) + ,ESI-MS=641(M+H) + .

[0159] Example 2A. General synthesis of PSMA imaging agent conjugates. The synthesis of the 14 atom linker compound SK28 is illustrated. [ka]

[0160] SK28 was synthesized using standard fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) starting with Fmoc-Cys(Trt)-Wang resin (Novabiochem; catalog no. 04-12-2050). SK28 was purified by reversed-phase preparative HPLC (Waters, xTerra C 18 10 μm; 19 × 250 mm; A = 0.1 TFA, B = acetonitrile (ACN); λ = 257 nm; solvent gradient: 5% B to 80% B in 25 min, wash with 80% B for 30 min) was used to purify (61%). The purified compound was purified by reversed-phase analytical HPLC (Waters, X-Bridge C 188 μm; 3.0 × 15 mm; A = 0.1 TFA, B = ACN; λ = 257 nm, 5% B to 80% B in 10 min, wash in 80% B for 15 min). 47 H 65 N2O 17 S; MW=1060.13g / mol; white solid; R t = 7.7 minutes; 1 H NMR(DMSO-d6 / D2O)δ0.93(m,2H);1.0(m,5H);1.27(m,5H);1.69(m,2H);1.90(m,2H); 1.94(m,2H);2.10(m,2H);2.24(q,2H);2.62(m,2H);2.78(m,4H);2.88(dd,1H);2.96( t,2H);3.01(dd,1H);3.31(dd,1H);3.62(dd,1H);3.80(q,1H,αH);4.07(m,1H,αH);4. 37(m,1H,αH);4.42(m,2H,αH);4.66(m,1H,αH);7.18(m,10H,Ar-H):LC-MS=1061(M+H) + ;ESI-MS=1061(M+H) + .

[0161] Example 2AA The following example compounds were synthesized in a similar manner. [ka]

[0162] Examples 2B-2E. The following compounds were synthesized according to the methods described herein using Fmoc SPPS starting with Fmoc-Cys(Trt)-Wang resin (Novabiochem; Cat. No. 04-12-2050) and purified by reversed-phase preparative HPLC (Waters, xTerra C 18 10 μm; 19 × 250 mm) and purified using reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 15 mm).

[0163] [ka]

[0164] SK60 (0 atom linker): Solvent gradient A=0.1 TFA, B=ACN; λ=220 nm; Solvent gradient: 1% B to 50% B in 25 min, wash with 80% B for 30 min, (75.3%). 21 H 32 NO 14 S; MW=624.58g / mol; white solid; R t = 6.3 minutes; 1 H NMR(DMSO-d6 / D2O)δ1.70(m,2H);1.92(m,2H);2.17(m,2H);2.23(m,2H);2.57(m,1H);2.77(m,4H);3.45(dd ,1H);3.54(dd,1H);3.83(t,1H,αH);4.06(m,1H,αH);4.38(m,1H,α-H);4.63(m,1H,α-H);ESI-MS=625(M+H) + .

[0165] [ka]

[0166] SK62 (7 atom linker): Solvent gradient A=0.1 TFA, TFA, B=ACN; λ=220, 257 nm; Solvent gradient: 1% B to 50% B in 25 min, wash with 80% B for 30 min, (72%). 35 H 48 N8O 18 S; MW=900.86g / mol; white solid; R t =8.2 minutes; 1H NMR(DMOS-d6 / D2O)δ1.62(m,1H);1.70(m,2H);1.79(m,1H);1.90(m,2H);2.09 (t,2H);2.16(m,2H);2.24(m,2H);2.60(m,1H);2.75(m,4H);2.81(m,1H);2.97 (m,1H);3.33(dd,1H);3.60(dd,1H);3.81(t,1H,αH);4.07(m,2H,αH);4.33[m, 1H,α-H];4.39(t,α-H);4.65(m,1H,α-H);7.20(m,5H,Ar-H);ESI-MS=901(M+H) + .

[0167]

change

[0168] SK38 (atomic 16 のリンカー): solvent blending A=10mM NH4OAc, B=ACN; λ=257nm; solvent blending: 25 minutes of 1% B, 80% B, 80% B, 30 minutes of washing, (63%). C 43 H 63 N9O 19 S,MW=1042.07g / mol; white solid; R t = compartmentalization; 1 H NMR(DMSO-d6 / D2O)δ0.94(m,2H);1.08(m,5H);1.27(m,5H);1.66(m,2H);1.70(m,2H);1.79(m ,1H);1.90(m,2H);2.09(t,2H);2.74(m,2H);2.84(m,1H);2.95(t,3H);3.07(d,2H);3.23(m, 1H);3.43(dd,1H);3.52(dt,1H);3.78(m,1H,αH);3.81(m,1H,αH);3.88(m,1H,αH);4.11(m,1 H,αH);4.39[m,2H,α-H];4.65(m,1H,α-H);7.14(m,1H,Ar-H);7.21(m,4H,Ar-H):ESI-MS=1043 (M+H) + .

[0169] [ka]

[0170] SK57 (24 atom linker): Solvent gradient A=0.1 TFA, B=ACN; λ=257 nm; Solvent gradient: 1% B to 50% B in 25 min, wash with 80% B for 30 min, (56%). 45 H 70 N8O 22 S, MW = 1107.14 g / mol; colorless solid; 1 H NMR(DMSO-d6 / D2O)δ1.66(m,2H);2.07(m,4H);2.31(t,1H);2.43(m,1H);2.77(m,2H);2.98(dd,1H);3.14(t,2H);3.24(d,1H);3.40(m,4H,PEG- H);3.46(s,24H,PEG-H);3.78(t,1H);3.81(t,1H);4.03(m,1H,αH);4.40(m,2H,α-H);7.16(m,1H,Ar-H);7.22(m,4H,Ar-H):ESI-MS=1108(M+H) + .

[0171] Example 2F. The following compounds can be synthesized according to the methods described herein. [ka]

[0172] Example 3A. General method for attaching radionuclides to chelating groups. 99mc Radiolabeling of SK28 with Tc to prepare SK33 is exemplified. [ka]

[0173] Preparation of SK28 Formulation Kit. HPLC grade Millipore filtered water (50 mL) was added to a 100 mL bottle and purged with argon for at least 10 minutes. α-D-Sodium glucoheptonate dihydrate (800 mg) was dissolved in argon purged water (5 mL). Stannous chloride dihydrate (10 mg) was dissolved in 0.02 M HCl (10 mL) while bubbling with argon. Stannous chloride (0.8 mL) was added to the sodium glucoheptonate solution under argon. SK28 (1.4 mL) was added to the sodium glucoheptonate / stannous chloride solution under argon. The pH of the reaction mixture was adjusted to 6.8±0.2 using 0.1 N NaOH. Argon purged water (5.2 mL) was added to the reaction mixture to bring the total volume to 10 mL. 1.0 mL of the reaction mixture was dispensed into each vial (10 vials) under argon atmosphere and lyophilized for 36-48 hours. The vials were sealed with rubber stoppers and aluminum seals under argon atmosphere to prepare SK28 formulation kits. The formulation kit vials were stored at -20°C until use.

[0174] 99m Labeling of SK28 with Tc. 99m Radiolabeling of SK28 with Tc can be performed according to published procedures. The compounding vial was warmed to room temperature for 10 min and heated in a boiling water bath for 3 min. Then, 15 mCi of sodium pertechnetate was added. 99m Tc (1.0 mL) was injected and an equal volume of gas was withdrawn from the vial to normalize the pressure. The vial was heated in a boiling water bath for 15-20 min and then cooled to room temperature before use in experiments. Radiochemical purity was analyzed by radio-TLC (>98%), which showed that the syn- and anti-isomers of the radiolabeled compound (SK33 / SK28- 99m Tc) was shown.

[0175] Examples 3B-3E. The following examples were prepared according to the methods described herein (both syn and anti isomers were obtained, but only the syn isomers are shown): [ka] [ka] [ka] [ka]

[0176] Example 3F. The following compounds can be synthesized according to the methods described herein. [ka]

[0177] Example 4. General synthesis of PSMA imaging agent conjugate, exemplified with SK59 using universal PSMA (DUPA) resin, a two atom linker and FITC. This conjugate can also be used to detect circulating tumor cells in prostate cancer patients.

[0178] [ka]

[0179] Synthesis of PSMA Universal Resin and SK59. Universal PSMA Ligand (DUPA) resin was synthesized using Universal NovaTag™ resin (Novabiochem; Cat. No. 04-2-3910). After swelling the resin with DCM (CH2Cl2) and DMF, the Fmoc group was deprotected using 20% ​​piperidine / DMF (N,N-dimethylformamide). tert-Butyl protected DUPA was coupled using HATU [Hexafluorophosphate uronium 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl] and DIPEA (N,N-diisopropylethylamine) in DMF. The side group Mmt (4-methoxytrityl) was removed with 1M HOBT (1-hydroxybenzotriazole) in DCM / TFE (trifluoroethanol). The resin intermediate can be washed with DMF and used immediately in the subsequent synthetic step or it can be washed with DCM / DMF then MeOH and dried for later use.

[0180] The universal PSMA resin can be reacted with commercially available FITC (1.25 equiv.) in the presence of DIPEA (4 equiv.) in DMF to yield the SK59 (2 atom linker) construct. The final compound was cleaved from the resin using a mixture of TFA (trifluoroacetic acid), TIPS (triisopropylsilane) and water. Purification was performed using reversed-phase preparative HPLC (Waters, xTerra C 18 5 μm; 19 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm; solvent gradient: 1% B to 50% B in 25 min, wash at 80% B for 40 min) was used (63%). SK59 was purified by reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 15 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 34 H 33 NO 13 S; MW=751.72g / mol; orange solid, R t =7.2 minutes;ESI-MS=752(M+H) +;774(M+Na) + ;750(MH) - .

[0181] Example 5A. General synthesis of PSMA imaging agent conjugates, exemplified with SK64 using universal PSMA (DUPA) resin, a 16 atom linker, and FITC.

[0182] [ka]

[0183] Universal PSMA resin was synthesized using standard Fmoc SPPS with Fmoc-Glu-(O t The SK64 (16 atom linker) compound was cleaved from the resin using TFA / TIPS / HO. Purification was performed by reversed-phase preparative HPLC (Waters, xTerra C). 18 5 μm; 19 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm; solvent gradient: 1% B to 50% B in 25 min, wash at 80% B for 40 min) (63%). SK64 was purified using reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 47 H 55 N7O 17 S; MW=1022.04g / mol; orange solid, R t =7.8 minutes;ESI-MS= 022(M+H) + ;1020(MH) - .

[0184] Examples 5B-5C. The following compounds were prepared using the synthetic methods described herein: [ka]

[0185] SK63 (7 atom linker, C 39 H 40 NO 17 , molecular weight: 864.76) was synthesized using Universal PSMA resin and Fmoc-Glu-(O t The compound was prepared using standard Fmoc SPPS linked to FITC (Bu)-OH. After coupling with FITC, the compound was cleaved from the resin using a TFA / TIPS / HO cocktail and analyzed by reversed-phase preparative HPLC (Waters, xTerra C 18 5 μm; 19 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm; solvent gradient: 1% B to 50% B in 25 min, wash with 80% B for 40 min) (63%) and purified by reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). SK63:C 39 H 40 NO 16 S; MW=880.83g / mol; orange solid, R t =6.8 minutes;ESI-MS=881(M+H) + ;903(M+Na) + ;863(MH) - .

[0186] [ka]

[0187] SK58 (24 atom linker, C 49 H 62 NO 20S, molecular weight: 1087.11) was prepared using universal PSMA resin and standard Fmoc SPPS coupled to Fmoc-(PEG)6-OH, purified by HPLC (1% B to 60% B in 25 min, washed with 80% B for 40 min) (65%) and reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm, 1% B to 60% B in 10 min, followed by a 15 min wash at 80% B). 49 H6ON6O 20 S; MW=1087.11g / mol; orange solid, R t =7.3 minutes;ESI-MS=1087(M+H) + ;1109(M+Na) + ;1085(MH) - .

[0188] Example 6A. General synthesis of Cys-maleimide PSMA imaging agent conjugates, exemplified with SK56 using Wang PSMA (DUPA) resin, a 28 atom linker and Oregon Green 488 (n=3).

[0189] [ka] Related analogs, where n is an integer from 4 to about 30, can also be prepared according to the methods described herein.

[0190] SK54 was prepared using standard Fmoc SPPS starting from Fmoc-Cys(Trt)-Wang resin (Novabiochem; catalog no. 04-12-2050) and purified using reversed-phase HPLC (Waters, xTerra C 18 The column was purified (63%) using reversed-phase analytical HPLC (Waters, X-Bridge C) with a 10 μm column; 19 × 250 mm; A = 0.1 TFA; B = ACN; λ = 257 nm; solvent gradient: 1% B to 60% B in 25 min, washed with 80% B for 40 min). 185 μm; 3.0 × 50 mm; A = 10 mM NH4OAc, B = ACN; λ = 257 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 38 H 59 N5O 18 S, MW=905.96g / mol; colorless solid; R t =9.2 min, LC-MS=906.3g / mol;ESI-MS=906(M+H) + ;904(MH) - .

[0191] SK56 (24 atom linker). HPLC grade Milli-Q water and saturated NaHCO3 were purged with argon for 10 min. SK54 was dissolved in 1.0 mL of argon purged water while bubbling with argon. The pH of the solution was raised to 6.8 and Oregon Green 488 maleimide dissolved in 1.0 mL of THF was added to the reaction mixture. The reaction was monitored by analytical HPLC (10 mM NH4OAc, pH=7.0; 1% B to 50% B in 10 min, 80% B wash for 15 min) and was complete within 10 min. THF was evaporated and the reaction mixture was diluted with 5.0 mL of 7 mM phosphate buffer. Purification was performed by reversed phase preparative HPLC (Waters, xTerra C 18 10 μm; 19 × 250 mm; A = 7 mM phosphate buffer, pH = 7.2, B = ACN; λ = 488 nm; solvent gradient: 1% B to 50% B in 25 min, wash at 80% B for 40 min) (89%) and analyzed by reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 150 mm; A = 10 mM NH4OAc, B = ACN; λ = 488 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 62 H 70 F2N6O 25 S; MW=1369.31g / mol; orange solid, R t =7.0 min;LC-MS=1370.2;ESI-MS=1391(M+Na) +The following 24 atom linker compounds were prepared in a similar manner to those described herein using the general synthesis described herein.

[0192] Example 6B. The following AlexaFluor 488 conjugate compounds were prepared according to the methods described herein starting with SK55 (n=3).

[0193] [ka] Related analogs, where n is an integer from 4 to about 30, can also be prepared according to the methods described herein.

[0194] Examples 7A-7C The following DUPA imaging agent conjugate compounds SK51, SK45 and SK49 were prepared according to the methods described herein (n=5).

[0195] [ka] SK51 (25 atom linker and AlexaFluor 647, MW approx. 2300 (commercially available from Invitrogen)) Related analogs, where n is an integer from 0 to about 12, can also be prepared according to the methods described herein.

[0196] [ka] SK45 (25 atom linker BODIPY 505, C 67 H 87 BF2N 13 O 20 S, molecular weight: 1475.35) Related analogs, where n is an integer from 0 to about 12, can also be prepared according to the methods described herein.

[0197] [ka] SK49 (25 atom linker - Oregon Green 488, C 71 H 76 F2N 10 O 24 , molecular weight: 1523.48) Related analogs, where n is an integer from 0 to about 12, can also be prepared according to the methods described herein.

[0198] Synthesis of the linker. In each of the preceding examples, the linker was synthesized using standard Fmoc SPPS starting from Fmoc-Cys(Trt)-Wang resin (Novabiochem; catalog number 04-12-2050). 47 H 65 N2O 17 S; MW=1060.13g / mol; white solid; R t = 7.7 minutes; 1 H NMR(DMSO-d6 / D2O)δ0.93(m,2H);1.08(m,5H);1.27(m,5H);1.69(m,2H);1.90(m,2H); 1.94(m,2H);2.10(m,2H);2.24(q,2H);2.62(m,2H);2.78(m,4H);2.88(dd,1H);2.96( t,2H);3.01(dd,1H);3.31(dd,1H);3.62(dd,1H);3.80(q,1H,αH);4.07(m,1H,αH);4. 37(m,1H,αH);4.42(m,2H,αH);4.66(m,1H,αH);7.18(m,10H,Ar-H):LC-MS=1061(M+H) + ;ESI-MS=1061(M+H) + .

[0199] Synthesis of SK51 (AlexaFluor 647 conjugate), SK45 (BODIPY conjugate) and SK49 (Oregon Green 488 conjugate). HPLC grade Milli-Q water and saturated NaHCO3 were purged with argon for 10 min. The linker was dissolved in 1.0 mL of argon-purged water while bubbling with argon. The pH of the solution was raised to 6.8 and AlexaFluor maleimide, BODIPY maleimide or Oregon Green 488 maleimide was dissolved in 1.0 mL of tetrahydrofuran (THF) respectively and added to the reaction mixture. The progress of the reaction was monitored by analytical HPLC (10 mM NH4OAc, pH = 7.0; 1% B to 50% B in 10 min, 80% B wash for 15 min), and the reaction was complete within 10 min. THF was evaporated and the reaction mixture was diluted with 5.0 mL of 1 mM phosphate buffer (pH = 7.2).

[0200] Each compound was analyzed by reversed-phase preparative HPLC (Waters, xTerra C 18 5 μm; 18 × 150 mm; A = 1 mM phosphate buffer, pH = 7.2, B = ACN; λ = 647 or 488 nm; solvent gradient: 1% B to 50% B in 25 min, wash with 80% B for 40 min) and purified using reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 50 mm; A = 10 mM NH4OAc, B = ACN; λ = 588 or 488 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). SK51: MW approx. 2360.13g / mol; blue solid, R t = 6.7 min; (AlexaFluor 647 structure unknown); SK45:C 67 H 87 BF2N 13 O 20 S; MW=1475.35g / mol; orange solid, R t =7.6 minutes;LC-MS=1475.3(M+H) + ; SK49:C 71 H 76 F2N 10 O 24S; MW=1523.48g / mol; orange solid, R t =6.7 min;LC-MS=1524(M+H) + .

[0201] Example 8A. General synthesis of PSMA disulfite linker intermediates for releasable drug conjugates, exemplified by SK68. [ka]

[0202] SK68 was synthesized using standard Fmoc SPPS starting from Fmoc-Cys(Trt)-Wang resin (Novabiochem; catalog no. 04-12-2050) and purified by reversed-phase preparative HPLC (Waters, xTerra C 18 The column was purified (68%) using reversed-phase analytical HPLC (Waters, X-Bridge C) with 10 μm; 19 × 250 mm; A = 0.1 TFA; B = ACN; λ = 257 nm; solvent gradient: 1% B to 50% B in 30 min, washed with 80% B for 40 min). 18 5 μm; 3.0 × 15 mm; A = 0.1 TFA, B = ACN; λ = 257 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 32 H 42 NO 17 S; MW=814.77g / mol; white solid; R t =8.2 minutes; 1 H NMR(DMOS-d6 / D2O)δ1.70(m,3H);1.90(m,3H);2.10(m,2H);2.17(m,2H);2.23(m,2H);2.36(m,1H);2.59(dd,1H);2.79(m,3H);3.04(dd,1H);4 .07(m,2H,αH);4.13(m,1H,αH);4.37[m,1H,α-H];4.47(m,2H,α-H);7.19(m,5H,Ar-H);7.87(d,urea-NH);8.20(d,1H,urea-NH);LC-MS=815.3(M+H) + .

[0203] Example 8B. General synthesis of PSMA disulfite linker intermediates for releasable drug conjugates, exemplified by SK28L. [ka]

[0204] SK28 was synthesized using standard Fmoc SPPS starting from Fmoc-Cys(Trt)-Wang resin (Novabiochem; catalog no. 04-12-2050) and purified using reversed-phase preparative HPLC (Waters, xTerra C 18 The column was purified (61%) using reversed-phase analytical HPLC (Waters, X-Bridge C) with 10 μm; 19 × 250 mm; A = 0.1 TFA; B = ACN; λ = 257 nm; solvent gradient: 5% B to 80% B in 25 min, wash with 80% B for 30 min). 18 5 μm; 3.0 × 15 mm; A = 0.1 TFA, B = ACN; λ = 257 nm, 5% B to 80% B in 10 min, wash in 80% B for 15 min). 47 H 65 N2O 17 S; MW=1060.13g / mol; white solid; R t = 7.7 minutes; 1 H NMR(DMSO-d6 / D2O)δ0.93(m,2H);1.08(m,5H);1.27(m,5H);1.69(m,2H);1.90(m,2H); 1.94(m,2H);2.10(m,2H);2.24(q,2H);2.62(m,2H);2.78(m,4H);2.88(dd,1H);2.96( t,2H);3.01(dd,1H);3.31(dd,1H);3.62(dd,1H);3.80(q,1H,αH);4.07(m,1H,αH);4. 37(m,1H,αH);4.42(m,2H,αH);4.66(m,1H,αH);7.18(m,10H,Ar-H):LC-MS=1061(M+H) + ;ESI-MS=1061(M+H) + .

[0205] Example 9A. General synthesis for preparing disulfide-linked conjugates, exemplified by tubulysin B conjugate SK71 (20 atom linker). [ka]

[0206] Synthesis of EC0312. Tubulysin B (30 mg, 0.036 mmol) was dissolved in ethyl acetate (600 μL) at −15° C. under argon. Isobutyl chloroformate (4.7 μL, 0.054 mmol) and diisopropylethylamine (13.2 μL, 0.076 mmol) were added to the reaction mixture and the reaction was stirred at −15° C. under argon for 45 min. EC0311 (13.4 mg, 0.054 mmol) dissolved in ethyl acetate (500 μL) was added. The reaction mixture was stirred at −15° C. for an additional 15 min and then at room temperature for 45 min. The solvent was evaporated and the residue was purified using a short column (2% to 8% methanol in CHCl) to give EC0312 (34.4 mg, 90.5%). EC0312 was determined by NMR (Varian 300 MHz, CDCl3) and LC-MS = 1058.3 (M+H). + was used to characterize it.

[0207] Synthesis of SK71. HPLC grade Milli-Q water and saturated NaHCO3 were purged with argon for 10 min. SK68 was dissolved in 1.0 mL of argon-purged water while bubbling argon through the solution. The pH of the solution was raised to 6.8 using argon-purged NaHCO3, and EC0312 dissolved in THF (2.0 mL) was added to the reaction mixture. The progress of the reaction was monitored by analytical HPLC (10 mM NH4OAc, pH = 7.0; λ = 254; 1% B to 50% B in 10 min, 80% B wash for 15 min), and the reaction was complete within 10 min. THF was evaporated and the reaction mixture was diluted with 5.0 mL of 2 mM phosphate buffer. SK71 (61.3%) was purified by reversed-phase preparative HPLC (Waters, xTerra C 1810 μm; 19 × 250 mm; A = 2 mM phosphate buffer, B = ACN; λ = 254 nm; 5% B to 80% B in 25 min, wash with 80% B for 40 min) and purified using reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 15 mm; A = 10 mM NH4OAc, B = ACN; λ = 254 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 77 H 109 N 13 O 28 S3: MW=1760.95g / mol; white solid, R t = 7.6 minutes; 1 H NMR (DMSO-d6 / D2O) was consistent with the SK71 structure; HRMS (MALDI) (m / z): (MH) - C 77 H 110 N 13 O 28 S3 calculated 1758.6594; found 1758.7033; LRMS (LCMS) (m / z): (M+H) + Calculated 1761.9; Found 1761.8; UV / Vis: λmax=254nm.

[0208] Example 9B Similarly, the D-Cys analog of SK71 was prepared as described herein. [ka]

[0209] Example 9C. General synthesis for preparing disulfide-linked conjugates, exemplified by tubulysin B conjugate SK77 (31 atom linker). [ka]

[0210] HPLC grade Milli-Q water and saturated NaHCO3 were purged with argon for 10 min. SK68 was dissolved in 1.0 mL of argon-purged water while bubbling with argon. The pH of the solution was raised to 6.8 using argon-purged NaHCO3, and EC0312 dissolved in THF (2.0 mL) was added to the reaction mixture. The progress of the reaction was monitored by analytical HPLC (10 mM NH4OAc, pH = 7.0; λ = 254; 1% B to 50% B in 10 min, 80% B wash for 15 min), and the reaction was complete within 10 min. THF was evaporated and the reaction mixture was diluted with 5.0 mL of 2 mM phosphate buffer. SK77 (61%) was purified by reversed-phase preparative HPLC (Waters, xTerra C 18 10 μm; 19 × 250 mm; A = 2 mM phosphate buffer, B = ACN; λ = 254 nm; 5% B to 80% B in 25 min, wash with 80% B for 40 min) and purified using reversed-phase analytical HPLC (Waters, X-Bridge C 18 5 μm; 3.0 × 15 mm; A = 10 mM NH4OAc, B = ACN; λ = 254 nm, 1% B to 50% B in 10 min, washed with 80% B for 15 min). 93 H 133 N 16 O 28 S3: MW=2006.32 g / mol; white solid, Rt=7.7 min; 1 H NMR(DMSO-d6 / D2O);LC-MS=2007.0(M+H) + .

[0211] Example 9D. Similarly, the D-Cys analog of SK77 was prepared as described herein. was prepared. [ka]

[0212] Example 9E Similarly, the D-Cys propanoic acid analog of SK77 was prepared as described herein. [ka]

[0213] Examples 9F-9G The following DUPA vinblastine and DUPA camptothecin compounds, SK37 and SK45, respectively, were prepared according to the methods described herein. [ka]

[0214] SK37 (vinblastine conjugate, C 93 H 123 N 15 O 26 S2, molecular weight: 1931.19), prepared in 63.1% yield. 93 H 123 N 15 O 26 S2: MW = 1931.19 g / mol; white solid, R t = 7.7 minutes; 1 H NMR(DMSO-d6 / D2O);LC-MS=1932.6(M+H) + .

[0215] [ka]

[0216] SK45 (camptothecin conjugate, C 70 H 83 N 11 O 23 S2, molecular weight: 1510.60), prepared in 66% yield. 70 H 83 N 11 O 23 S2: MW=1510.60g / mol; white solid, R t = 7.5 minutes; 1 H NMR(DMSO-d6 / D2O);LC-MS=1511.1(M+H) + .

[0217] Example 9H Similarly, the Glu-Asp-Phe analog of SK37 was prepared as described herein.

[0218] [ka]

[0219] Example 10. The following compounds were prepared using the synthetic methods described herein: SK125 (FITC conjugate) [ka] SK131 (rhodamine conjugate) [ka] SK179 (FITC conjugate) [ka] FITC conjugate [ka] DyLight 680 conjugate [ka] DyLight 800 conjugate [ka] PET Agent Conjugate [ka] PET Agent Conjugate [ka] PET Agent Conjugate [ka] for example 64 Cu, 65 DOTA conjugates capable of chelating Cu etc. [ka] for example 64 Cu, 65 DOTA conjugates capable of chelating Cu etc. [ka] DTPA conjugates capable of chelating, for example, In, Ga, Ir, Yr, etc. [ka] Tripeptide conjugates capable of chelating, for example, Tc, Tc oxide, etc. [ka]

[0220] The foregoing exemplary embodiments are intended to be illustrative of the present invention and should not be read or interpreted in any way as limiting the invention described herein.

[0221] <Method Example> == Example 1A == In vitro binding studies using LNCaP cells and SK28 (14 atom linker). LNCaP cells (a human prostate cancer cell line overexpressing PSMA, purchased from American Type Culture Collection (ATCC)) were seeded (120,000 cells / well) into two 24-well Falcon plates and grown to adherent monolayers in RPMI (Gibco RPMI medium 1640, Cat. No. 22400) with glutamine (2 mM) + 10% FBS (fetal bovine serum), 1% sodium pyruvate (100 mM) and 1% PS (penicillin streptomycin) for 48 hours at 37°C in a 5% CO2 atmosphere. Cells in the first 24-well plate were incubated with increasing concentrations of K28- 99mThe cells in a second 24-well plate were incubated with 50 μM PMPA for 30 min at 37° C. in a 5% CO2 atmosphere, followed by incubation with increasing concentrations of K28- 99m The cells were incubated with Tc (each concentration in triplicate) for 1 h at 37°C under a 5% CO2 atmosphere (competition studies). The cells were rinsed three times with 1.0 mL of RPMI. The cells were lysed in Tris buffer, transferred to individual gamma scintigraphy vials, and the radioactivity was counted. A plot of cell-bound radioactivity versus radiolabeled compound was used to determine the K d Values ​​were calculated. Competition studies were used to determine the binding specificity of the ligand (DUPA) to PSMA (Figure 1A).

[0222] == Example 1B == In vitro binding studies using LNCaP cells and SK33 (14 atom spacer). LNCaP cells were seeded into 24-well Falcon plates (150,000 cells / well) and allowed to form confluent monolayers for 48 hours. Spent medium from each well was diluted with increasing concentrations of DUPA- 99m The medium was replaced with fresh medium (0.5 mL) containing Tc. After 1 h incubation at 37° C., the cells were rinsed with culture medium (2×1.0 mL) and Tris buffer (1×1.0 mL) to remove any unbound radioactivity. After suspension in Tris buffer (0.5 mL), the cell-bound radioactivity was counted using a γ-counter (Packard, Packard Instrument Company). The dissociation constant (K D ) was calculated using a plot of cell-bound radioactivity versus concentration of radiotracer using nonlinear regression with GraphPad Prism 4. Error bars represent 1 standard deviation (n=3). Experiments were performed three times with similar results (Figure 1B).

[0223] == Example 2 == Quantification of PSMMA molecules on LNCaP cells. LNCaP cells were seeded into 24-well Falcon plates and grown in RPMI (Gibco RPMI medium 1640, Cat. No. 22400) + 10% FBS (fetal bovine serum), 1% glutaric acid and 1% PS (penicillin streptomycin) at 37°C in a 5% CO2 atmosphere for 48 hours to form an adherent monolayer. The cells were then incubated with increasing concentrations of SK28- 99m The cells were incubated with Tc (each concentration in triplicate) for 1 hour at 4°C or 37°C under a 5% CO2 atmosphere. The cells were rinsed three times with 1.0 mL of RPMI. The cells were lysed with Tris buffer, transferred to individual gamma scintillation vials, and counted for radioactivity. A plot of cell-bound radioactivity versus radiolabeled compound was used to calculate the number of PSMA / LNCaP cells. SK28- 99m The radioactivity of a 30 nM sample of Tc (20 uL) was counted at 4°C (to prevent endocytosis of PSMA). The number of moles of a 30 nM sample was 30 nM x 20 uL = (30 x 10 -9 mol / L)×(20×10 -6 L) = 6 × 10 -13 mol. The number of atoms in a 30 nM sample is (6 × 10 -13 mol) × (6.023 × 10 23 atoms / mol)=3.6×10 11 The radioactivity count in 20 uL of a 30 nM sample was 20477 cpm (cpm / atom = 3.6 x 10 11 / 20477=1.76×10 7 The cell-bound radioactivity at saturation point at 4°C was 12,000 cpm. The number of atoms at saturation point was (1.76 × 10 7 atoms) × (12,000 cpm). The number of cells / well is 245,000. The number of PSMA / cell at 4°C is (2.12 × 10 11 ) / 2.45×10 5 =864,396.4=approx. 0.9×10 6 PSMA / LNCaP cells.

[0224] The cell-bound radioactivity at saturation point at 37° C. is 33,000 cpm (approximately 3-fold higher than at 4° C.), indicating that PSMA undergoes endocytosis similar to that by cell surface receptors, releasing and recycling the drug. See FIG. 2.

[0225] == Example 3 == Spacer-dependent binding studies. LNCaP cells were seeded (120,000 cells / well) in 24-well Falcon plates (10 plates) and grown to adherent monolayers in RPMI (Gibco RPMI medium 1640, Cat. No. 22400) + 10% FBS (fetal bovine serum), 1% sodium pyruvate and 1% PS (penicillin streptomycin) at 37°C in a 5% CO2 atmosphere for 48 hours. The cells were then incubated with increasing concentrations of SK60- 99m Tc (0 atom spacer), SK62 -99m Tc (7 atom spacer), SK28 -99m Tc (14 atom spacer), SK38 -99m Tc (16 atom spacer) and SK57- 99m In a separate plate, 50 μM PMPA was incubated for 30 min at 37° C. under 5% CO2, followed by incubation with increasing concentrations of SK60-Tc (24 atom spacer) (each concentration in triplicate) at 37° C. under 5% CO2 for 1 h. 99m Tc (0 atom spacer), SK62 -99m Tc (7 atom spacer), SK28 -99m Tc (14 atom spacer), SK38 -99m Tc (16 atom spacer) and SK57- 99mThe cells were incubated with Tc (24 atom spacer) (each concentration in triplicate) for 1 h at 37 °C under 5% CO2 atmosphere (competition study, data not shown). The cells were rinsed three times with 1.0 mL of RPMI. The cells were lysed with Tris buffer, transferred to individual gamma scintigraphy vials, and the radioactivity was counted. A plot of cell-bound radioactivity versus concentration of radiolabeled compound was used to determine the K d The K values ​​were calculated by plotting the saturation percentage against the concentration of radiolabeled compound and the K value against the spacer length. d Plots of are shown (Figures 3A and B).

[0226] == Example 4 == In vivo growth of human LNCaP tumor cells in nude mice. LNCaP cells were maintained in RPMI 1640 (Gibco RPMI medium 1640, Cat. No. 22400) with glutamine (2 mM), 10% FBS (fetal bovine serum), 1% sodium pyruvate (100 mM) and 1% PS (penicillin streptomycin) at 37°C in a 5% CO2 atmosphere. Athymic male nude mice (nu / nu) 4-5 weeks old were obtained from NCI Charles River and maintained in a sterile environment. Mice were housed in polycarbonate shoebox cages with wire top lids and maintained on a normal diet. Mice were allowed to acclimate for 1 week before inoculation with LNCaP cells. Matrigel and high concentration (HC) Matrigel were purchased from BD Biosciences. Nude mice received 2.5 × 10 in vitro propagated LNCaP cells in 50% Matrigel (100 uL PPMI medium + 100 uL Matrigel) or 50% high-concentration Matrigel (100 uL PPMI medium + 100 uL HC Matrigel). 6 pcs or 5.0 x 10 6 The optimal conditions, including the number of cells, vehicle, etc., were determined by inoculating the cells into each trunk and each flank of nude mice subcutaneously to determine the optimal site. The volume of each tumor was measured vertically twice a week using a caliper, and the body weight was measured once a week (data not shown).

[0227] The volume of each tumor was calculated as 0.5×L×W2, where L is the measurement of the longest axis (mm) and W is the measurement of the axis perpendicular to L (mm). 6 LNCaP cells can grow to 600 mm within 30 days. 3 See Figure 4.

[0228] == Example 5 == Comparison of tumor growth of LNCaP, KB and A549 cells in mice. LNCaP, KB and A549 cells were maintained in RPMI 1640 (Gibco RPMI medium 1640, Cat. No. 22400) with glutamine (2 mM), 10% FBS (fetal bovine serum), 1% sodium pyruvate (100 mM) and 1% PS (penicillin streptomycin) at 37°C in a 5% CO2 atmosphere. Male nude mice (nu / nu) aged 4-5 weeks were obtained from NCI Charles River and maintained in a sterile environment. Mice were housed in polycarbonate shoebox cages with wire top lids and maintained on a normal diet. Mice were allowed to acclimate for 1 week before inoculation with cells.

[0229] For tumor cell inoculation, 5.0 × 10 cells were inoculated in 50% highly concentrated Matrigel. 6 LNCaP cells, 1.0 x 10 in RPMI medium 6 KB cells or 1.0 x 10 in RPMI medium 6 A549 cells were subcutaneously injected into the right side of the trunk of nude mice (some animals were injected bilaterally). The volume of each tumor was measured twice a week in two perpendicular directions using calipers (see Figures 5A and 5B), and the body weight was measured once a week (data not shown). The tumor volume was calculated as 0.5 x L x W2, where L is the measurement of the longest axis (mm) and W is the measurement of the axis perpendicular to L (mm).

[0230] == Example 6A == PSMA- 99m In vivo imaging of tumors in mice using Tc. 3When the volume reached 100 ml, the solution was prepared as described. 99m Tc-labeled compounds (e.g., SK28- 99m Tc, SK60- 99m PMPA was administered by intraperitoneal (subcutaneous) injection. After 4 hours, animals were euthanized and blood was collected by cardiac puncture and transferred to individual gamma scintigraphy vials for each animal. Imaging experiments were performed using a Kodak or gamma scintigraphy camera imager (Figures 6A, 6B, 6C, 7A, 7B, and 7C). [Note: PMPA was administered using SK28- 99m The injection was performed 30 min before Tc injection. 99m The distribution of Tc was restricted to the kidney (Figs. 6A, 6B, and 6C). 99m Tc was injected, but distribution was largely restricted to the kidneys (there was no tumor uptake even after shielding of both kidneys).

[0231] 6A, 6B and 6C show SK28- 99m 6A-6C show images of mice bearing human LNCaP tumors using Tc (radiolabeled 14 atom spacer). 99m LNCaP tumor-bearing mice were imaged using a Kodak camera imager 4 hours after subcutaneous (via intraperitoneal) injection of Tc without (mouse on the left of each set of images) and with 50 mg / kg PMPA (competitor) to block PSMA. The image on the left shows a white light image, and the image on the right shows an overlay of the radiographic and white light images. Figure 6D shows the results of a 1 ng / kg SK28- 99m 1 shows a single mouse study of LNCaP tumors imaged using a Kodak imaging system 4 hours after subcutaneous injection of Tc (administered via intraperitoneal), with the image on the left showing a kidney-shielded radiographic image overlaid with an unshielded white light image, and the image on the right showing a kidney-shielded radiographic image overlaid with an unshielded X-ray image.

[0232] == Example 6B == DUPA- 99m In vivo imaging of tumors in mice using Tc. To further establish the specificity of the DUPA conjugates to prostate cancer cells, DUPA- 99m Tc was injected intraperitoneally into athymic nude mice bearing LNCaP tumors in the shoulders. After 4 hours to allow clearance of unbound conjugates, the retained DUPA- 99m The distribution of Tc was imaged by gamma scintigraphy. As can be seen in Figures 7D(a) and 7D(c), the target 99m The Tc radiotracer accumulated primarily in PSMA-positive LNCaP tumors, with little or no radioactivity in other tissues except the kidney. Importantly, the kidney uptake may be specific to the mouse, since immunohistochemical and RT-PCR analyses suggest that PSMA expression is high in the murine kidney and minimal in the human kidney. The in vivo specificity of the PSMA-targeted imaging agent was confirmed by the DUPA- 99m This was further tested by pre-treating LNCaP tumors with excess PMPA, which blocks all PSMA sites, prior to Tc administration. As shown in Figures 7D(b) and 7D(d), blocked LNCaP tumors were able to upregulate DUPA-9 9m 100% Tc uptake, confirming the specificity of the DUPA conjugate to PSMA in vivo. To further demonstrate this specificity, the radiotracer was also administered to two PSMA-negative mouse xenograft models, A549 (human lung cancer cell line) and KB (human nasopharyngeal carcinoma cell line), again with whole-body imaging. As expected, renal shielding was performed to reveal low levels of DUPA- in other tissues. 99m No radioactivity was observed in either KB or A549 tumors, even after allowing for Tc detection (Figures 7D(e) and 7D(f)). Thus, these studies demonstrate that DUPA- 99m It is confirmed that almost no Tc binding occurs.

[0233] Figure 7D shows that 150 μCi of DUPA- 99m7D(a,c) and 7D(b,d) show whole-body images of solid tumor xenografts in nu / nu mice taken 4 hours after injection of Tc. 99m This is a whole-body radiographic and white light image overlay of a Tc-treated LNCaP tumor-bearing mouse. 99m Overlays of radiographic and white light images were also obtained from mice bearing A549 tumors [7D(e)] or KB tumors [7D(f)] similarly treated with Tc. The kidneys were shielded with lead pads, except for images 7D(a) and 7D(b). All images were taken with DUPA- 99m Images were taken using a Kodak Imaging Station 4 hours after intraperitoneal injection of Tc. Arrows indicate solid tumor xenografts. Similar images were obtained from all five mice in each treatment group.

[0234] == Example 7A == Biodistribution studies. After imaging, all animals were 99m Tc or SK60- 99m Approximately 6-7 hours after administration of Tc [or other radiolabeled compounds (data not shown)], animals were dissected and organs (blood, tumor, heart, liver, kidneys, spleen, skin, muscle, etc.) were transferred to individual gamma scintigraphy vials for each animal and counted for radioactivity. NOTE: Blood samples were collected (using cardiac puncture) immediately after sacrificing the animals and immediately prior to imaging the animals. Plots of tumor vs. tissue cpm / g ratios were used to determine the biodistribution of imaging agents (Figures 8A and 8B).

[0235] == Example 7B == DUPA- in nu / nu mice bearing LNCaP, A549 or KB tumors 99m Tc biodistribution study. Tumor-bearing mice were treated with DUPA- 99mThe animals were euthanized 4 hours after intraperitoneal injection of Tc (50 μmol / kg, 150 μCi) and tissue-accumulated radioactivity was counted using a gamma counter. The injected dose rate per gram of wet tissue was calculated as described in the method example. Data are from one experiment and error bars represent standard deviation (n=5). LNCaP tumors (solid bars), LNCaP tumors in mice pre-injected with 100-fold molar excess of PMPA (hollow bars), A549 tumors (cross-hatched bars), KB tumors (horizontal-hatched bars) (FIG. 8C).

[0236] == Example 8 == Single dose toxicity in surviving mice. SK71 was administered at a single dose as indicated. The data show that the MTD of the conjugate is approximately 4.5 μmol / kg at a single dose (see FIG. 9A).

[0237] == Example 9 == Multiple dose toxicity in surviving mice. SK71 was administered in five doses every other day (M, W, F, M, W). The data show that the MTD of SK71 is 2 μmol / kg in multiple doses and that the conjugate is effective against LNCaP tumors (the mice were administered the MTD before treatment began, 2 weeks prior to implantation of LNCaP cells). All four mice in the saline control group had large tumors, while none of the mice in the two treatment groups had visible tumors 18 days after treatment (see FIG. 9B).

[0238] == Example 10 == Efficacy study comparing control and competitor groups. Animals were treated with (a) 5 doses of 1 μmol / kg of conjugate SK71 administered every other day (M, W, F, M, W) and compared to (b) vehicle-treated animals (FIG. 10B) and (c) animals treated with conjugate together with PMPA. Treatment with 1 μmol / kg reduced tumor size (starting tumor size was approximately 250 mm) over the course of treatment. 3) in the 10-μmol / kg dose. At the low dose of 1 μmol / kg shown in FIG. 10A, tumor volume rebounded upon cessation of treatment. At higher doses of 2 μmol / kg and above, complete disappearance of tumors was observed over the study period. Competition experiments (see FIG. 10C) indicate that successful treatment of transplanted tumors is related to selective or specific targeting of PSMA-mediated delivery.

[0239] == Example 11 == Efficacy study (1 μmol / kg every other day for 10 days, i.e. 5 doses). The data (see FIG. 11) show that tumors in treated animals decreased in size over the treatment period.

[0240] == Example 12 == In vitro evaluation of PSMA targeted therapeutics. Analysis of toxicity of SK71 (FIG. 12A), SK77 (FIG. 12B), SK37 (FIG. 12C) and SK45 (FIG. 12D) towards LNCaP cells in culture. LNCaP cells were pulsed with increasing concentrations of SK71 or SK77 for 2 hours in the presence (▲) or absence (■) of 100-fold molar excess of PMPA. After washing twice, cells were incubated in fresh medium for an additional 66 hours at 37° C. Cell viability was then assessed using the ELISA kit described herein. 3 H]-thymidine incorporation assay. Data are from one experiment and error bars represent standard deviation (n=3 wells per concentration).

[0241] == Example 13 == In vivo efficacy. The effect of SK71 on subcutaneous tumor growth (Figures 13A and 13C) and body weight (Figures 13B and 13D) of treated mice. LNCaP cells in HC Matrigel were implanted subcutaneously into the shoulders of n / n male mice. Once tumors reached 100 mm 3 (13A, 13B) or 330 m 3Upon reaching a volume of 13C, 13D, animals were treated with SK71 at 1.5 μmol / kg (a, b) or 2.0 μmol / kg (c, d). (■) Treated mice, (●) Untreated mice, (▲) Treated mice pre-injected with a 100-fold (13A, 13B) or (▼) 30-fold (13C, 13D) molar excess of PMPA. Data are from a single experiment and error bars represent standard deviation (n=4 (13A, 13B) or 3 (13C, 13D)). Figure 10 shows the in vivo efficacy of SK71.

[0242] In vivo efficacy. Effect of SK77 on subcutaneous tumor growth (FIG. 14A) and body weight of treated mice (FIG. 14B). LNCaP cells in HC Matrigel were implanted subcutaneously into the shoulders of n / n male mice. Once tumors reached 100 mm 3 Once the volume reached 10 μmol / kg, the animals were treated with SK77 (2 μmol / kg). (■) Untreated mice, (▼) Treated mice. Data are from one experiment and error bars represent standard deviation (n=4 mice / group).

Claims

1. A compound of the formula: B-L-D or a salt thereof, B is a urea of ​​two amino acids; L comprises (i) a divalent alkylenecarbonyl and (ii) at least one nitrogen atom, the divalent alkylenecarbonyl being selected from the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, arylalkyl, heteroaryl, carboxy, carboxyalkyl, and alkylcarboxylate. １ where L ranges from about 7 atoms to about 9 atoms in length, and D is a radioisotope of a metal coordinated to a chelating group; A compound or a salt thereof.

2. B is of the formula: 【Chemistry 1】 (In the formula, R 1 is hydrogen, R 2 is a substituted carboxylic acid, the substituted carboxylic acid being covalently bonded to L; 2. The compound of claim 1, which is a compound of the formula:

3. 2. The compound of claim 1, or a salt thereof, wherein L is covalently bonded to B via an amide bond.

4. 2. The compound of claim 1, or a salt thereof, wherein L is covalently bonded to D via an amide bond.

5. Radioactive isotopes of metals ６８ The compound of claim 1 or a salt thereof, wherein

6. Radioactive isotopes of metals ６８ The compound of claim 3 or a salt thereof, wherein

7. Radioactive isotopes of metals ６８ The compound of claim 4 or a salt thereof, wherein

8. The compound of claim 2 or a salt thereof, wherein L is covalently bonded to B via an amide bond.

9. 3. The compound of claim 2 or a salt thereof, wherein L is covalently bonded to D via an amide bond.

10. Radioactive isotopes of metals ６８ The compound of claim 2 or a salt thereof, wherein

11. Radioactive isotopes of metals ６８ The compound of claim 8 or a salt thereof, wherein

12. Radioactive isotopes of metals ６８ The compound of claim 9 or a salt thereof, wherein

13. A pharmaceutical composition comprising a compound of formula B-L-D or a salt thereof, and one or more carriers, diluents or excipients, B is a urea of ​​two amino acids; L comprises (i) a divalent alkylenecarbonyl and (ii) at least one nitrogen atom, the divalent alkylenecarbonyl being selected from the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, arylalkyl, heteroaryl, carboxy, carboxyalkyl, and alkylcarboxylate. １ where L ranges from about 7 atoms to about 9 atoms in length, and D is a radioisotope of a metal coordinated to a chelating group; Pharmaceutical compositions.

14. 14. The pharmaceutical composition of claim 13, wherein the carrier is an aqueous solution.

15. B is of the formula: 【Chemistry 2】 (In the formula, R 1 is hydrogen, R 2 is a substituted carboxylic acid, the substituted carboxylic acid being covalently bonded to L; 14. The pharmaceutical composition of claim 13, which is a compound of the formula:

16. 16. The pharmaceutical composition of claim 15, wherein the carrier is an aqueous solution.

17. Radioactive isotopes of metals ６８ The pharmaceutical composition of claim 13, wherein said compound is Ga.

18. Radioactive isotopes of metals ６８ The pharmaceutical composition of claim 14, wherein said compound is Ga.

19. Radioactive isotopes of metals ６８ The pharmaceutical composition of claim 15, wherein said compound is Ga.

20. Radioactive isotopes of metals ６８ The pharmaceutical composition of claim 16, wherein said compound is Ga.

21. L is covalently bonded to B by an amide bond, and a radioisotope of a metal ６８ The pharmaceutical composition of claim 13, wherein said compound is Ga.

22. L is covalently bonded to D by an amide bond, and a radioisotope of a metal ６８ The pharmaceutical composition of claim 13, wherein said compound is Ga.

23. L is covalently bonded to B by an amide bond, and a radioisotope of a metal ６８ The pharmaceutical composition of claim 14, wherein said compound is Ga.

24. L is covalently bonded to D by an amide bond, and a radioisotope of a metal ６８ The pharmaceutical composition of claim 14, wherein said compound is Ga.

25. A method of imaging comprising administering to a subject a compound of claim 1 or a salt thereof in an amount effective to image tissues or cells associated with a pathogenic cell population that expresses or overexpresses prostate specific membrane antigen.

26. Radioactive isotopes of metals ６８ The method of claim 25 , wherein the metal is Ga.

27. A method of imaging comprising administering to a subject the pharmaceutical composition of claim 13 in an amount effective to image tissues or cells associated with a pathogenic cell population that expresses or overexpresses prostate specific membrane antigen.

28. Radioactive isotopes of metals ６８ The method of claim 27, wherein said first metal is Ga.

29. B is of the formula: 【Chemistry 3】 (In the formula, R 1 is hydrogen, R 2 is a substituted carboxylic acid, the substituted carboxylic acid being covalently bonded to L; The method of claim 27, wherein the compound is

30. Radioactive isotopes of metals ６８ The method of claim 29 , wherein the metal is Ga.

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