Psma binding ligand-linker conjugates and methods for use thereof

Abstract

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

Description

PSMA BINDING LIGAND-LINKER CONJUGATES AND METHODS FOR USE THEREOF BACKGROUND OF THE DISCLOSURE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No.63 / 519,469, filed August 14, 2023, and from U.S. Provisional Application No.63 / 520,054, filed August 16, 2023, the disclosure of each of which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The present invention relates to small molecules having high affinity and specificity to prostrate-specific membrane antigen (PSMA) and methods of using them for therapeutic and diagnostic purposes. SUMMARY OF THE RELATED ART

[0003] Prostate cancer (PCa) is the most diagnosed cancer in American men and one of the major causes of cancer-related deaths, second to lung cancers. Generally, patients with localized disease can be treated with radical prostatectomy and / or radiation therapy. Patients with metastatic forms of PCa can be temporarily treated with androgen deprivation strategies, however, most patients with metastatic disease eventually experience disease progression, that can evolve into metastatic castration-resistant prostate cancer (mCRPC), which has a low survival rate. Chemotherapy (e.g., docetaxel, cabazitaxel) is often given following anti-androgen therapy failure, but responses are often marginal, with associated on- and off-target toxicity, especially in elderly men. Better therapeutic approaches are clearly needed for late-stage PCa patients.

[0004] Prostate-specific membrane antigen (PSMA) is a type II transmembrane protein that is highly overexpressed by majority of all prostate cancers. PSMA expression is further upregulated in poorly differentiated, metastatic, hormone-refractory carcinomas and in cancer cells from mCRPC patients. Furthermore, PSMA exhibits robust internalization from the cell surface making it an ideal target for imaging and therapy. Indeed, PSMA-targeted radiopharmaceutical therapies have shown promise in clinical trials, with several radioligand therapy and antibody-drug conjugate (ADCs) demonstrating efficacy in preclinical studies. Problems associated with relapse, off-target toxicity, immunogenicity of antibody-based therapies, as well as the stringent requirements imposed on the facilities that produce and manage radiopharmaceutical production, represent significant challenges for drug development, FDA-approval, and use in the oncology community for agents to treat mCRPC.

[0005] Therefore, there remains a need for effective therapies that accurately localize to the prostate cancer lesions without significant off-target toxicity yet are associated with lower manufacturing costs and avoid regulatory challenges associated with radiopharmaceutical manufacturing and handling. SUMMARY OF THE DISCLOSURE

[0006] Small molecule-drug conjugates (SMDCs) represent an attractive alternative to the more conventional ADC approach for PSMA-targeted chemotherapeutic delivery. Both technologies typically include a PSMA-targeting motif (antibody vs enzyme inhibitor), a spacer, a cleavable linker, and a potent cytotoxic payload. After binding to cell-surface PSMA, these agents are expected to internalize and accumulate in endosomes and lysosomes, which enables efficient release of the cytotoxic payload in the target cells, typically by enzymatic cleavage. Compared to anti-PSMA antibodies, however, small- molecule PSMA inhibitors exhibit similarly accurate localization to the prostate cancer lesions, but their considerably lower molecular weight and simpler molecular characterization is associated with lower manufacturing costs, flexibility in determining the optimal dose regimen, and higher tolerated doses. The toxicity profile of PSMA-SMDCs is expected to be much lower than ADCs, as they have shorter residence time and undergo more rapid and uniform diffusion into the tumor mass compared to normal organs such as kidneys, lacrimal glands, and salivary glands.

[0007] Thus, provided herein are therapeutics and diagnostics for prostate cancer that capitalize on the potency and specific affinity of small-molecule inhibitors to PSMA. Accordingly, in one aspect the present disclosure provides compounds of structural formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety;L is an acid-cleavable linker and is attached to provide a carbamate moiety to formula (I); each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

[0008] The disclosure also provides pharmaceutical compositions comprising the compounds of the disclosure as disclosed herein and a pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer, and / or diluent.

[0009] One aspect of the disclosure provides methods of delivering a therapeutic or diagnostic agent to a subject. Such methods include administering a therapeutically effective amount of an effective amount of a compound of the disclosure as disclosed herein or a pharmaceutical composition of the disclosure as disclosed herein to a subject in need of such agent.

[0010] One aspect of the disclosure provides methods of treating a patient with cancer (for example, prostate cancer). Such methods include administering to the patient an effective amount of a compound of the disclosure as disclosed herein or a pharmaceutical composition of the disclosure as disclosed herein.

[0011] Another aspect of the disclosure provides methods of imaging one or more cancer cells (such as prostate cancer cells) in a patient. Such methods include administering to the patient an effective amount of a compound of the disclosure as disclosed herein or a pharmaceutical composition of the disclosure as disclosed herein.

[0012] Another aspect of the present disclosure provides compounds of formula (II)or a pharmaceutically acceptable salt thereof, whereineach Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4.

[0013] Another aspect of the present disclosure provides compounds of formula (III)or a pharmaceutically acceptable salt thereof, wherein each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

[0014] Another aspect of the present disclosure provides compounds of formula (IV)or a pharmaceutically acceptable salt thereof, whereinD is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety; L is an acid-cleavable linker and is attached to provide a carbamate moiety to formula (IV); each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4.

[0015] In another aspect, the present disclosure provides a method of synthesizing the compounds of formula (I), the method comprises contacting a compound of formula (V)or a pharmaceutically acceptable salt thereof, with a compound of formula (III) as described herein,or a pharmaceutically acceptable salt thereof, wherein each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2m is an integer of from 1 to 5;n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

[0016] In another aspect, the present disclosure provides a method of synthesizing the compounds of formula (IV), the method comprises contacting a compound of formula (V)( ) or a pharmaceutically acceptable salt thereof, with a compound of formula (II) as described herein,or a pharmaceutically acceptable salt thereof, wherein each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4. BRIEF DESCRIPTION OF FIGURES

[0017] The accompanying drawings are included to provide a further understanding of the compositions and methods of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments of the disclosure and, together with the description, serve to explain the principles and operation of the disclosure.

[0018] Figure 1A is a1H NMR spectra of Compound 1 (CTT2274).

[0019] Figure 1B is a31P NMR spectra of Compound 1 (CTT2274).

[0020] Figure 2A is a graph of the IC50curve of Compound 1 (CTT2274) with human PSMA.

[0021] Figure 2B is a graph of the IC50curve of Compound 1 (CTT2274) with mouse PSMA.

[0022] Figure 2C is a graph of the IC50curve of Compound 8 (CTT2101) with human PSMA.

[0023] Figure 3 is a western blot of PSMA expression in PC3, PC3-PIP, and C4-2B cell lines and PDX tumor.

[0024] Figure 4A is a graph of the in vitro efficacy of Compound 8 (CTT2101), Compound 1 (CTT2274), Compound 2 (CTT227X), and free MMAE in PC3 cells.

[0025] Figure 4B is a graph of the in vitro efficacy of Compound 8 (CTT2101), Compound 1 (CTT2274), Compound 2 (CTT227X), and free MMAE in PC3-PIP cells.

[0026] Figure 4C is a graph of the in vitro efficacy of Compound 8 (CTT2101), Compound 1 (CTT2274), Compound 2 (CTT227X), and free MMAE in C4-2B cells.

[0027] Figure 5A is a graph of the change in mouse body weight over time for Compound 8 (CTT2101) versus MMAE.

[0028] Figure 5B is a graph of the fold-change in tumor volume over time for Compound 8 (CTT2101) versus MMAE

[0029] Figure 5C is a spaghetti plot of mouse tumor volumes for Compound 8 (CTT2101) over time. Each line represents one mouse.

[0030] Figure 5D is a spaghetti plot of mouse tumor volumes for MMAE over time. Each line represents one mouse.

[0031] Figure 5E is a spaghetti plot of mouse tumor volumes for PBS over time. Each line represents one mouse.

[0032] Figure 5F is a Kaplan-Meier survival curve for Compound 8 (CTT2101), MMAE, and PBS.

[0033] Figure 5G is a Kaplan-Meier survival sub-analysis of Compound 8 (CTT2101) based on starting tumor volume.

[0034] Figure 6A is a graph of tumor volume versus study day for mice treated with Compound 8 (CTT2101).

[0035] Figure 6B is a graph of tumor volume versus study day for mice treated with Compound 8 (CTT2101).

[0036] Figure 6C is a graph of tumor volume versus study day for mice treated with MMAE.

[0037] Figure 6D is a graph of tumor volume versus study day for mice treated with MMAE.

[0038] Figure 7A is a graph of the change in mouse body weight over time for Compound 8 (CTT2101), Compound 1 (CTT2274), MMAE, and PBS.

[0039] Figure 7B is a graph of the fold-change in tumor volume over time for Compound 8 (CTT2101), Compound 1 (CTT2274), MMAE, and PBS.

[0040] Figure 7C is a spaghetti plot of mouse tumor volumes for Compound 8 (CTT2101) over time.

[0041] Figure 7D is a spaghetti plot of mouse tumor volumes for Compound 1 (CTT2274) over time.

[0042] Figure 7E is a spaghetti plot of mouse tumor volumes for MMAE over time.

[0043] Figure 7F is a spaghetti plot of mouse tumor volumes for PBS over time.

[0044] Figure 7G is a Kaplan-Meier survival curve for Compound 8 (CTT2101), Compound 1 (CTT2274), MMAE, and PBS.

[0045] Figure 8 is a plot of the body weight and analyte concentrations in blood from mice treated with Compound 1 (CTT2274) and MMAE.

[0046] Figure 9A is a spaghetti plot of change in mouse body weight over time for Compound 1 (CTT2274).

[0047] Figure 9B is a spaghetti plot of change in tumor volume over time for Compound 1 (CTT2274).

[0048] Figure 9C is a spaghetti plot of fold-change in tumor volumes over time for Compound 1 (CTT2274).

[0049] Figure 9D is a Kaplan-Meier survival curve for Compound 1 (CTT2274).

[0050] Figure 10 are spaghetti plots of change in tumor volume over time for Compound 1 (CTT2274) for individual mice. DETAILED DESCRIPTION

[0051] Before the disclosed processes and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, and as such can, of course, vary. It is also to be understood that the terminology used herein is forthe purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents, patent applications, and publications referred to herein are incorporated by reference to the extent they are consistent with the present disclosure. Terms and ranges have their generally defined definition unless expressly defined otherwise.

[0053] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms may also be used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g. CH3–CH2–), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., –CH2–CH2–), which is equivalent to the term “alkylene.” Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene. All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S).

[0054] The term “amino” refers to –NH2.

[0055] The term “acetyl” refers to –C(O)CH3.

[0056] As herein employed, the term “acyl” refers to an alkylcarbonyl or arylcarbonyl substituent wherein the alkyl and aryl portions are as defined herein.

[0057] The term “alkyl” as employed herein refers to saturated straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such, “alkyl” encompasses C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, and C12groups. Alkyl groups may be branched or unbranched. Examples of alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0058] The term “alkenyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms. As such, “alkenyl” encompasses C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, and C12groups. Examples of alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0059] The term “alkynyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon triple bonds, having from 2 to 12 carbon atoms. As such, “alkynyl” encompasses C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, and C12groups. Examples of alkynyl groups include, without limitation, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0060] An “alkylene,” “alkenylene,” or “alkynylene” group is an alkyl, alkenyl, or alkynyl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups. Examples of alkylene groups include, without limitation, methylene, ethylene, propylene, and butylene. Examples of alkenylene groups include, without limitation, ethenylene, propenylene, and butenylene. Examples of alkynylene groups include, without limitation, ethynylene, propynylene, and butynylene.

[0061] The term “alkoxy” refers to –O(C1-C6alkyl).

[0062] The term “cycloalkyl” as employed herein is a saturated and partially unsaturated cyclic hydrocarbon group having 3 to 12 carbons. As such, “cycloalkyl” includes C3, C4, C5, C6, C7, C8, C9, C10, C11, and C12cyclic hydrocarbon groups. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. A “cycloalkyl” group also includes fused multicyclic (e.g., bicyclic) ring systems such as decahydronaphthyl and octahydro-1H- indenyl. of the fused rings is non-aromatic, provided that at least one ring is aromatic, such as indenyl

[0063] The term “C3-C6cycloalkyloxy” refers to groups of the formula –O(C3-C6cycloalkyl).

[0064] An “aryl” group is a C6-C14aromatic moiety comprising one to three aromatic rings. As such, “aryl” includes C6, C10, C13, and C14cyclic hydrocarbon groups. A representative aryl group is a C6-C10aryl group. Particular aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An “aryl” group also includes fused multicyclic (e.g., bicyclic) ring systems in which one or more of the fused rings is non- aromatic, provided that at least one ring is aromatic, such as indenyl.

[0065] An “aralkyl” or “arylalkyl” group comprises an aryl group covalently linked to an alkyl group wherein the moiety is linked to another group via the alkyl moiety. An representative aralkyl group is –(C1-C6)alkyl(C6-C10)aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl. For example, an arC1-C3alkyl is an aryl group covalently linked to a C1-C3alkyl.

[0066] As used herein, the term “fused” when used to define rings, e.g., bicyclic fused ring systems, refers to bicyclic, tricyclic, etc. ring systems sharing 2 or more atoms, includingbridged systems. Examples of such fused ring systems are (1S,4R)-2- azabicyclo[2.2.1]heptane; 2-azabicyclo[2.2.2]octane; 2,5-diazabicyclo[2.2.2]octane; 2-oxa-5- azabicyclo[2.2.2]octane; isoindoline; 1,2,3,4-tetrahydro-2,6-naphthyridine; 1,2,3,4- tetrahydroisoquinoline; 1,2,3,4-tetrahydro-1,4-(epiminomethano)naphthalene; and 3- azabicyclo[3.1.0]hexane.

[0067] A “heterocyclyl” or “heterocyclic” or “heterocycloalkyl” group is a mono- or bicyclic (e.g., fused) ring structure having from 3 to 12 atoms, (3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 atoms), for example 4 to 8 atoms, wherein one or more ring atoms are independently N, O, or S, and the remainder of the ring atoms are quaternary or carbonyl carbons. Examples of heterocyclic groups include, without limitation, epoxy, oxiranyl, oxetanyl, azetidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, thiazolidinyl, thiatanyl, dithianyl, trithianyl, azathianyl, oxathianyl, dioxolanyl, oxazolidinyl, oxazolidinonyl, decahydroquinolinyl, piperidonyl, 4-piperidonyl, thiomorpholinyl, dimethyl-morpholinyl, and morpholinyl. Specifically excluded from the scope of this term are compounds having adjacent ring O and / or S atoms. The heterocyclic groups can be attached to a parent group (i.e., the point of attachment) via any ring atom, including one of the heteroatoms or one of the carbon atoms, in the heterocyclic ring group. As chemically required, the heterocyclic ring may be attached to one or more other groups, for instance if operating as a bridging group. The term “heterocyclyl” also includes fused multicyclic (e.g., bicyclic) ring systems in which one or more of the fused rings is aromatic or non-aromatic, provided that at least one ring is non-aromatic contains an N, O, or S ring atom. Examples of such fused multicyclic ring systems are indolinyl, indolin-2-yl, 2,3- dihydrobenzofuran-2-yl, and 2,3,4,5-tetrahydrobenzo[d]oxazol-2-yl. Each of these examples is a 9-membered heterocyclyl.

[0068] As used herein, the term “heteroaryl” refers to a group having 5 to 14 ring atoms, preferably 5, 6, 10, 13, or 14 ring atoms; having 6, 10, or 14 ʌ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to three heteroatoms that are each independently N, O, or S. “Heteroaryl” also includes fused multicyclic (e.g., bicyclic) ring systems in which one or more of the fused rings is non-aromatic, provided that at least one ring is aromatic and at least one ring contains an N, O, or S ring atom. The heteroaryl groups can be attached to a parent group (i.e., the point of attachment) via any ring atom, including one of the heteroatoms or one of the carbon atoms, in the heteroaryl ring group. As chemically required, the heteroaryl may be attached to one or more other groups, for instance if operating as a bridging group.

[0069] Examples of heteroaryl groups include acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzo[d]oxazol-2(3H)-one, 2H-benzo[b][1,4]oxazin-3(4H)-one,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, furanyl, furazanyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3- triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.

[0070] An “arylene,” “heteroarylene,” or “heterocyclylene” group is a bivalent aryl, heteroaryl, or heterocyclyl group, respectively, as defined hereinabove, that is positioned between and serves to connect two other chemical groups.

[0071] As employed herein, when a moiety (e.g., cycloalkyl, aryl, heteroaryl, heterocyclyl, urea, etc.) is described as “optionally substituted” without expressly stating the substituents, it is meant that the group optionally has multiple non-hydrogen substituents, for example, from one to five, or from one to four, or from one to three, or one, or two non-hydrogen substituents.

[0072] The term “halogen” or “halo” as employed herein refers to chlorine, bromine, fluorine, or iodine.

[0073] The term “haloalkyl” refers to an alkyl chain in which one or more hydrogens have been replaced by a halogen. Representative haloalkyls are trifluoromethyl, difluoromethyl, fluorochloromethyl, chloromethyl, and fluoromethyl.

[0074] The term “hydroxyalkyl” refers to –alkylene–OH.

[0075] The term "substituted", as used herein, means that a hydrogen radical of the designated moiety is replaced with the radical of a specified substituent, provided that the substitution results in a stable or chemically feasible compound. The term "substitutable", when used in reference to a designated atom, means that attached to the atom is a hydrogen radical, which can be replaced with the radical of a suitable substituent.

[0076] The phrase "one or more” substituents, as used herein, refers to a number of substituents that equals from one to the maximum number of substituents possible based on the number of available bonding sites, provided that the above conditions of stability and chemical feasibility are met. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and the substituents may be either the same or different. As used herein, the term "independently selected" means that the same or different values may be selected for multiple instances of a given variable in a single compound.

[0077] A “protecting group” as used herein include, but are not limited to, optionally substituted benzyl, t-butyl ester, allyl ester, alkyl esters (e.g., methyl, ethyl), fluorenylmethoxycarbonyl groups (Fmoc), and amino, carboxylic acid and phosphorus acid protecting groups described in Greene's Protective Groups in Organic Synthesis, 4th Edition (which is incorporated by reference). In some embodiments, R1is a carboxylic acid protecting group (e.g., a methyl or t-butyl ester). In some embodiments, R2is a nitrogen protecting group (e.g., Boc, or benzyl). Optionally benzyl groups include, but are not limited to, unsubstituted benzyl, triphenylmethyl (trityl), diphenylmethyl, o-nitrobenzyl, 2,4,6- trimethylbenzyl, p-bromobenzyl, p-nitrobenzyl, p-methoxybenzyl (PMB), 2,6- dimethoxybenzyl, 4-(methylsulfinyl)benzyl, 4-sulfobenzyl, 4-azidomethoxybenzyl, and piperonyl, and benzyl protecting groups for carboxylic and phosphorus acids disclosed in Greene’s Protective Groups in Organic Synthesis (the relevant parts of which are incorporated by reference).

[0078] It is to be understood that each individual atom present in Formula (I) and the compounds within Formula (I), may be present in the form of any of its naturally occurring isotopes, with the most abundant isotope(s) being preferred. Thus, by way of example, each individual hydrogen atom present in Formula (I), or in the formulae depicted hereinafter, may be present as a1H,2H (deuterium; D), or3H (tritium; T) atom, preferably1H. Similarly, by way of example, each individual carbon atom present in formula (I), or in the formulae depicted hereinafter, may be present as a12C,13C, or14C atom, preferably12C.

[0079] As used herein, “an effective amount” of a compound is an amount that is sufficient to negatively modulate or inhibit the activity of PSMA.

[0080] As used herein, a “therapeutically effective amount” of a compound is an amount that is sufficient to ameliorate or in some manner reduce a symptom or stop or reverse progression of a condition, or negatively modulate or inhibit the activity of PSMA. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.

[0081] As used herein, “treatment” means any manner in which the symptoms or pathology of a condition, disorder or disease in a patient are ameliorated or otherwise beneficially altered.

[0082] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need. In general, the disclosed compositions and methods provide improvements in treatment of cancer, particularly prostate cancer, capitalizing on the potency and specific affinity of small- molecule inhibitors of PSMA. COMPOUNDS

[0083] One aspect of the disclosure provides compound having the structural formula (I):or a pharmaceutically acceptable salt thereof, wherein D is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety; L is an acid-cleavable linker and is attached to provide an amide moiety to formula (I); each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

[0084] The compounds of structure formula (I) may have a specific stereochemistry. For example, in certain embodiments, the disclosure provides compounds having a formula:wherein D, L, X, m, n, R1, R2, R3, and R4are as described herein.

[0085] The compounds of the disclosure as described herein include at least one X, wherein each X is independently a natural or unnatural n Į-amino acid. The C-terminus of X forms an amide with the nitrogen carrying R2, as shown in formula (I). Additionally, the N- terminus of X is attached to the adjacent carbonyl to provide an amide moiety.

[0086] In certain embodiments, each X is independently selected from a naturally occurring amino acid (e.g. L-amino acids) or unnaturally occurring amino acid (e.g., D-amino acids). In certain embodiments as described herein, each X is independently selected from a naturally occurring amino acid (e.g. L-amino acids). For example, in certain embodiments, at least one X is independently selected from alanine, glycine, isoleucine, leucine, proline, valine, phenylalanine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, cysteine, methionine, asparagine, or glutamine. In certain embodiments, at least one X is selected from phenylalanine, tryptophan, tyrosine, or histidine. In certain embodiments, at least one X is phenylalanine. In certain embodiments, at least one X is tryptophan. In certain embodiments, at least one X is 4-biphenylalanine. When X is a naturally occurring amino acid, the side chains of such amino acids may be further substituted. For example, the amino acid side chains may be substituted with H, – OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.

[0087] In certain embodiments as described herein, at least one X has a structure

[0088] In certain embodiments, each R6is independently selected from (i) H, (ii) C1-C6alkyl, (iii) hydroxy(C1-C6alkyl), (iv) C1-C6alkoxy(C1-C6alkyl), (v) C2-C6alkenyl, (vi) R8R9N(C1- C6alkyl), (vii) halogen, (viii) halo(C1-C6alkyl), (ix) carboxyl(C1-C6alkyl), (x) thio(C1-C6alkyl), (xi) C1-C6alkylthio(C1-C6alkyl, (xii) R8R9NC(O)(C1-C6alkyl), (xiii) C3-C10cycloalkyl(C1-C6alkyl), (xiv) C3-C8heterocyclyl(C1-C6) alkyl, (xv) aryl(C1-C6alkyl), (xvi) heteroaryl(C1-C6alkyl), (xvii) R17C(O)NR8(C1-C6alkyl), (xviii) C3-C10cycloalkyl, (xix) C3-C8heterocyclyl, (xx) aryl, and (xvi) heteroaryl; wherein R8and R9are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl), and wherein R17is C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, or heteroaryl. In certain embodiments, each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1- C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl); wherein R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl).

[0089] In certain embodiments as described herein, at least one X has a structure

[0090] In certain embodiments, p is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, q is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, each R7is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), and heteroaryl(C1-C6alkyl); wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and each R10and R11is independently selected from hydrogen and C1-C6 alkyl (e.g., C1-C4 alkyl, or C1-C3 alkyl). For example, in certain embodiments, p is 1. In some embodiments as described herein, q is 1. In certain embodiments, p is 1, q is 1, and R7is aryl (e.g., phenyl).

[0091] Each R17is independently selected from C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), and heteroaryl(C1-C6alkyl), wherein R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl).

[0092] In certain embodiments as described herein, at least one X has a structure

[0093] In certain embodiments, r is in an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, s is in an integer from 1 to 7 (e.g., from 1 to 5, or from 1 to 4, or from 1 to 3). In certain embodiments, each R12is independently selected from H, –OH, – SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), and heteroaryl(C1-C6alkyl); wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). For example, in certain embodiments, r is 1. In some embodiments as described herein, s is 1. In certain embodiments, r is 1 and s is 1. In certain embodiments, R12is H.

[0094] In certain embodiments as described herein, at least one X has a structure

[0095] In certain embodiments, a is an integer from 1 to 4 (e.g., from 1 to 3). In certain embodiments, b is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, each of R13and R14is independently selected from H, –OH, –SH, –NO2, – COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). In certain embodiments, R13and R14are H.

[0096] In certain embodiments as described herein, at least one X has a structure

[0097] In certain embodiments, c is an integer of from 1 to 3 (e.g., is 1, or is 2). In certain embodiments, d is an integer of from 1 to 4 (e.g., of from 1 to 3). In certain embodiments, each of R15and R16is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1- C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). In certain embodiments, R15and R16are H.

[0098] In certain embodiments as described herein, at least one X has a structure

[0099] In certain embodiments, t is an integer from 1 to 6 (e.g., from 1 to 5, or from 1 to 4). In certain embodiments, u is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments v is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, each R18is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1- C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). In certain embodiments, each R18is independently selected from C1-C6alkyl.

[0100] In certain embodiments as described herein, at least one X has a structure of

[0101] As described above, the compounds of structural formula (I) are of any of the previous embodiments wherein n is an integer from 1 to 4, or from 1 to 3. In certain embodiments, n is 1 or 2. In other embodiments, n is 1. For example, in some embodiments, n is 1 and X is phenylalanine. In other embodiments, n is 1 and X is In other embodiments as described herein, n is 1 and X is

[0102] In certain embodiments, the compounds of formula (I) are of any of the previous embodiments wherein m is an integer from 1 to 5. For example, in certain embodiments asdescribed herein, m is an integer from 1 to 4, or from 1 to 3. In certain embodiments, m is 1 or 2. In other embodiments of the disclosure as described herein, m is 1.

[0103] In certain embodiments, the compounds are of any of the previous embodiments wherein R1, R2, and R3are independently selected from one of groups (a)-(o): (a) hydrogen, C1-C6alkyl or a protecting group. (b) hydrogen or C1-C6alkyl. (c) C1-C6alkyl or a protecting group. (d) C1-C6alkyl. (e) hydrogen or a protecting group. (f) hydrogen. (g) a protecting group. (h) Any of groups (a)-(d), where C1-C6alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl. (i) Any of groups (a)-(d), where C1-C6 alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, sec-butyl or tert-butyl. (j) Any of groups (a)-(d), where C1-C6alkyl is methyl, ethyl, n-propyl or tert-butyl. (k) Any of groups (a)-(d), where C1-C6alkyl is methyl, ethyl or tert-butyl. (l) Any of groups (a)-(d), where C1-C6alkyl is methyl or ethyl. (m) Any of groups (a)-(d), where C1-C6alkyl is methyl. (n) Any of groups (a)-(d), where C1-C6alkyl is ethyl. (o) Any of groups (a)-(d), where C1-C6alkyl is tert-butyl.

[0104] In certain embodiments of the compounds of the disclosure as described herein, R1, R2, and R3are H.

[0105] In certain embodiments, at least one of R1, R2, and R3is not H, and the remaining R1, R2, and R3are H.

[0106] In certain embodiments, the compounds are of any of the previous embodiments wherein R4is H or methyl. In certain embodiments, R4is H.

[0107] The compounds of the disclosure as described herein include an acid-cleavable linker L (e.g., the linker that decomposes in acidic environment or other desired conditions to release the therapeutic or diagnostic agent). Such linkers when attached to the agent canutilize the lower intracellular pH (for example, pH 5.5.) or extracellular pH (for example, pH 6.5) of target cell types, compared to that of blood or normal tissues, to trigger the controlled release of the agent.

[0108] As described above, L is attached to provide a carbamate moiety to formula (I). In certain embodiments of the disclosure, the linker L comprises a phosphoramidate group. For example, in certain embodiments, L comprises the phosphoramidate of formula: and R1and R3are as provided above with respect to formula (I). Incertain embodiments, R1is hydrogen, and / or R3is hydrogen.

[0109] In certain embodiments of the disclosure, L is selected from: orwherein R5is H, –OH, or C1-C6alkoxyl, and R1, R2, and R3, are as provided above with respect to formula (I).

[0110] In certain embodiments of the disclosure, L is selected from: wherein R5is H, –OH, or C1-C6alkoxyl, and R1, R2, andR3, are as provided above with respect to formula (I).

[0111] In certain embodiments of the disclosure, R5is H, –OH, or C1-C4alkoxyl. In some embodiments, R5is H, –OH, or C1-C2alkoxyl. In some embodiments, R5is H, –OH, or – OCH3. In some embodiments, R5is H or –OCH3. In some embodiments, R5is H. In some embodiments, R5is –OCH3.

[0112] In certain embodiments of the disclosure, L is

[0113] As used herein, the term “PhosAm-2” is used to denote the compounds of formula (I) having such a linker L. “PhosAm-2”-containing compounds can be derived from “PhosAm-A”-containing azides of formula (V), as described below.

[0114] In certain embodiments of the disclosure, L is selected from: wherein R1, R2, and R3, are as provided above with respect to formula()

[0115] In certain embodiments of the disclosure, L is

[0116] As used herein, the term “PhosAm-1” is used to denote the compounds of formula (I) having such a linker L. “PhosAm-1”-containing compounds can be derived from “PhosAm-B”-containing azides of formula (V), as described below.

[0117] As provided above, the compounds of formula (I) as described herein include a therapeutic or diagnostic agent moiety, D. In general, D is attached to the linker L through – NR2–, –S–, or –O–. It is to be understood that the agents may actually be derivatives, with modifications at the linking site. For example, D can be modified to comprise –NR2–, –S–, or –O– for attaching to L.

[0118] As described above, in some embodiments, D is a therapeutic agent. A therapeutic agent is a molecule that is useful in the treatment of a disease. Examples of therapeutic agents include chemotherapeutic agents, antibodies, antibody fragments, toxins, enzymes, nucleases such as a ribonuclease (RNase) or DNase I, hormones, cytokines,chemokines, angiogenesis inhibitors, antisense oligonucleotides, small interfering RNA (siRNA), chelators, boron compounds, photoactive agents, small molecules, antibiotics, and radioisotopes. For example, in some embodiments as described herein, D is an anti- angiogenic agent, a cytotoxic agent, a cytokine, a chemokine, an apoptotic agent, a prodrug, a toxin, an enzyme, a radioisotope, an immunomodulator, an antibiotic, an agent active in the CNS or a hormone.

[0119] A chemotherapeutic agent includes, for example, an anticancer agent, an antineoplastic agent, and a cytotoxic agent. Examples of anti-cancer chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecins, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, methotrexate, mitomycin, navelbine, nitrosurea, plicamycin, procarbazine, raloxifene, tamoxifen, TAXOL, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing. Chemotherapeutic agents of use against infectious organisms include, but are not limited to, acyclovir, albendazole, amantadine, amikacin, amoxicillin, amphotericin B, ampicillin, aztreonam, azithromycin, bacitracin, BACTRIM, BATRAFEN, bifonazole, carbenicillin, caspofungin, cefaclor, cefazolin, cephalosporins, cefepime, ceftriaxone, cefotaxime, chloramphenicol, cidofovir, Cipro®, clarithromycin, clavulanic acid, clotrimazole, cloxacillin, doxycycline, econazole, erythrocycline, erythromycin, FLAGYL®, fluconazole, flucytosine, FOSCARNET®, furazolidone, ganciclovir, gentamycin, imipenem, isoniazid, itraconazole, kanamycin, ketoconazole, lincomycin, linezolid, meropenem, miconazole, minocycline, naftifine, nalidixic acid, neomycin, netilmicin, nitrofurantoin, nystatin, oseltamivir, oxacillin, paromomycin, penicillin, pentamidine, piperacillin-tazobactam, rifabutin, rifampin, rimantadine, streptomycin, sulfamethoxazole, sulfasalazine, tetracycline, tioconazole, tobramycin, tolciclate, tolnaftate, trimethoprim sulfamethoxazole, valacyclovir, vancomycin, zanamir, and zithromycin.

[0120] Hormones can be used as a therapeutic agent themselves or in combination with other chemotherapeutic agents. Progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate, have been used in cancers of the endometrium and breast. Estrogens such as diethylstilbestrol and ethinyl estradiol have been used in cancers such as prostate cancer. Antiestrogens such as tamoxifen have been used in cancers such as breast cancer. Androgens such as testosterone propionate and fluoxymesterone have also been used in treating breast cancer. Corticosteroid hormones such as prednisone and dexamethasone can improve the effective of other chemotherapeutic agents. Cytokines that are used as therapeutic agents include, but are notlimited to, lymphokines, monokines, growth factors, and polypeptide hormones. Examples of cytokines include but are not limited to human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, tumor necrosis factor-Į, tumor necrosis factor-ȕ, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), NGF-ȕ, platelet-growth factor, TGF-Į, TGF-ȕ, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factor, interferon-Į, interferon-ȕ, interferon-Ȗ, macrophage-CSF (M- CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), IL-1, IL-1Į, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, and lymphotoxin. Examples of angiogenesis inhibitors that are used as therapeutic agents include, but are not limited to, angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PIGF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Flt- 1 antibodies and peptides, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-ȕ, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP- 470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, and minocycline. Examples of small molecules for use as therapeutic agents include, but are not limited to, abrin, amantadine, amoxicillin, amphotericin B, ampicillin, aplidin, azaribine, anastrozole, azacytidine, aztreonam, azithromycin, bacitracin, trimethoprim / sulfamethoxazole, Batrafen, bifonazole, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carbenicillin, caspofungin, carmustine, cefaclor, cefazolin, cephalosporins, cefepime, ceftriaxone, cefotaxime, celecoxib, chlorambucil, chloramphenicol, ciprofloxacin, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, diphtheria toxin, DNase I, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), doxycycline, cyano-morpholino doxorubicin, doxorubicin glucuronide, duocarmycin (DUBA), epirubicin glucuronide, ethinyl estradiol, 7-ethyl-10-hydroxycamptothecin (SN-38), estramustine, estrogen receptor binding agents, etoposide, etoposide glucuronide, etoposide phosphate, erythrocycline, erythromycin, flagyl, farnesyl-protein transferase inhibitors, floxuridine (FUdR), 3ƍ,5ƍ-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, ganciclovir, gentamycin, gelonin, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, imiquimod, isoniazid, itraconazole, kanamycin, ketoconazole, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, minocycline, naftifine, nalidixic acid, neomycin, navelbine, nitrosurea, nystatin, ranpirnase, oxacillin, paromomycin, penicillin, pentamidine, piperacillin-tazobactam, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, pokeweed antiviral protein, PSI-341, seco-duocarmycin (seco-DUBA), semustine, rifabutin, rifampin, rimantadine, streptomycin, sulfamethoxazole, sulfasalazine, streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, tetracycline, thalidomide, thioguanine, thiotepa, teniposide, topotecan, transplatinum, trimethoprim sulfamethoxazole, uracil mustard, valacyclovir, vancomycin, vinblastine, vinorelbine, vincristine, zanamir, zithromycin, and (R)-5-chloro-N2-[4-(4-methylpiperazin-1-yl)phenyl]-N4-[(tetrahydrofuran-2- yl)methyl]pyrimidine-2,4-diamine ((R)-9b).

[0121] In embodiments, D disclosed herein include therapeutic agents for the treatment of cancer and non-cancer therapeutic agents. These therapeutic agents include organic small molecules: including all hydroxyl and amine-containing therapeutic agents for the treatment of cancer, for example, molecules that inhibit the replication of DNA (e.g., doxorubicin, epirubicin, calecheamicin, camptothecin), molecules that stabilize or disrupt microtubules (e.g., paclitaxel, docetaxel, epothilone), molecules that affect the Na+ / K+pump (e.g., strophanthidin), molecules that affect the function of the Golgi apparatus (e.g., norrisolide and active derivatives of norrisolide). These therapeutic agents also include inorganic small molecules, such as all hydroxyl and amine containing therapeutic agents for the treatment of cancer, for example, cisplatin or oxoplatin. Examples of linked anti-tumor agents include, for example, CO-doxorubicin, and CO-strophanthidin.

[0122] In other embodiments, D disclosed herein include but are not limited to proteins: including proteins of human and non-human origin, for example, antibodies (e.g. trastuzumab), hormones (e.g. leutinizing hormone, follicle stimulating hormone), cytokines (e.g. IL-6), growth factors (e.g. G-CSF), bacterial or plant toxins (e.g., Pseudomanas toxin, gelonin, ricin, abrin) and tumor-targeting soluble proteins of any type; peptides including engineered and natural peptides that are toxic to tumor cells, that alter the architecture or function of such cells, or target other molecules to tumor cells or cells in the tumor that serve to support tumor cells, for example, lysins, TAT-related proteins that enhance cell penetration; nucleic acids such as RNA, for example, anti-sense RNA, silencing RNA, toxin aptamers, DNA such as naturally-occurring and synthetic oligonucleotides and higher molecular weight structures, for example, plasmid and viral vectors that express RNAs orproteins that are toxic to tumor cells; particles such as polymer-derived, protein-derived, metal-derived and inorganic-based particles of any size, for example, nanoparticles loaded with therapeutic agents, detectable labels or imaging agents such as fluorescent dyes or radionuclides; small molecules such as both inorganic and organic small molecules that target cell surface receptors or otherwise bind to the surface or other accessible intracellular or extracellular components of tumor cells.

[0123] Therapeutic agents also include drugs that are active in the CNS, for example, L- Dopa, Ritalin, Cymbalta, Namenda, and Gleevec.

[0124] In other embodiments, D is an anticancer agent, an antineoplastic agent, or a cytoxic molecule. In one embodiment, D is selected from the group consisting of an amine group containing antineoplastic agent say, for example, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and doxorubicin.

[0125] In one embodiment, D is selected from MMAE, MMAF, doxorubicin, cabazitaxel, docetaxel, paclitaxel, gemcitabine, imiquimod, SN-38, DUBA, seco-DUBA, (R)-9b, and gemcitabine.

[0126] In one embodiment, D is selected from monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), exatecan, N-Me-L-Ala-maytansinol, gemcitabine, seco- duocarmycin (seco-DUBA), gemcitabine-monophosphate, doxorubicin, cabazitaxel, docetaxel, paclitaxel, imiquimod, 7-ethyl-10-hydroxycamptothecin (SN-38), duocarmycin (DUBA), or (R)-5-Chloro-N2-[4-(4-methylpiperazin-1-yl)phenyl]-N4-[(tetrahydrofuran-2- yl)methyl]pyrimidine-2,4-diamine ((R)-9b).

[0127] As described above, in some embodiments, D is a diagnostic agent. A diagnostic agent is a molecule that may be used in imaging studies such as magnetic resonance imaging (MRI), magnetic resonance tomography (MRT), positron emission tomography (PET), computer tomography (CT), single-photon emission computed tomography (SPECT) and optical imaging, such as x-ray. Diagnostic agents are detectable or traceable labels. Examples of diagnostic agents used in these studies include, but are not limited to, radioisotopes, dyes (including those using a biotin-streptavidin complex), enzymes, contrast agents, fluorescent compounds or molecules such as a fluorescent dye, paramagnetic ions (for MRI), and small molecules including both inorganic and organic small molecules that target cell surface receptors or otherwise bind to the surface or other accessible intracellular or extracellular components of tumor cells.

[0128] In certain embodiments, D is a radioisotope, an imaging agent, a fluorescent dye, a near-IR dye, an enzyme, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an ultrasound label, or a radioacoustic label.

[0129] As described above, D is attached to L through an –NR2–, –S–, or –O– moiety. For example, in some embodiments as described herein, D is attached to L through –NR2–. In another embodiment, D is attached to L through –O–.

[0130] In certain embodiments, the disclosure provides the following example compounds represented by the formula:. In certain embodiments, D is MMAE.

[0131] In certain embodiments, the disclosure provides the following example compounds of the formula:

[0132] In one aspect, the present disclosure provides pharmaceutical compositions comprising a compound of the disclosure as described herein and a pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer, and / or diluent. Pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer, diluent, etc. to be included are determined by the composition being administered and by the method of administering the composition. There are a wide variety of suitable formulations of pharmaceutical composition including optional pharmaceutically acceptable carriers, excipients, stabilizers, etc. Acceptablecarriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990).

[0133] Pharmaceutical compositions suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.

[0134] As used herein, the term “pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the above-identified compounds and exhibit minimal or no undesired toxicological effects. Examples of such salts include, but are not limited to acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid. Non-toxic pharmaceutical base addition salts include salts of bases such as sodium, potassium, calcium, ammonium, and the like. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt. In certain embodiments, the pharmaceutically acceptable salt is a potassium salt. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable addition salts.

[0135] The active compound (e.g., the compounds of formula (I)) is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount without causing serious toxic effects in the patient treated. A dose of the active compound for all of the above-mentioned conditions is in the range from about 0.01 to 300 mg / kg, preferably 0.1 to 100 mg / kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient per day. A typical topical dosage will range from 0.01-3% wt / wt in a suitable carrier. The effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.

[0136] In certain embodiments, the dose of the active compound as described herein is administered based on the mg per kilogram body weight of the recipient. In certain embodiments, the dose of the active compound as described herein is administered once per every 7 days (e.g., once per week).

[0137] In certain embodiments, the dose of the active compound for all of the above- mentioned conditions may be in the range of about 0.1 to 20 mg / kg, or about 0.1 to 15 mg / kg, or about 0.1 to 10 mg / kg, or about 0.1 to 5 mg / kg, or about 0.5 to 20 mg / kg, or about 0.5 to 15 mg / kg, or about 0.5 to 10 mg / kg, or about 0.5 to 5 mg / kg, or about 1 to 20 mg / kg, or about 1 to 15 mg / kg, or about 1 to 10 mg / kg, or about 1 to 5 mg / kg, or about 2 to 20 mg / kg, or about 2 to 15 mg / kg, or about 2 to 10 mg / kg, or about 2 to 5 mg / kg. In certain embodiments as described herein, the dose of active compound is from about 0.254 to about 0.45 mg / kg, about 0.6 to about 0.75 mg / kgm about 1.3 to about 1.6 mg / kg, about 3.4 to about 3.8 mg / kg, about 8 to about 10 mg / kg, or about 15 to about 20 mg / kg. In certain embodiments, doses of the active compound of formula (I) are about 0.36 mg / kg, abot 0.72 mg / kg, about 1.44 mg / kg, about, about 9 mg / kg or about 18 mg / kg. In certain embodiments as described herein, the dose of active compound is at least 0.36 mg / kg per every 7 days, or at least 0.72 mg / kg per every 7 days, or at least 1.44 mg / kg per every 7 days, or at least 3.6 mg / kg per every 7 days, or at least 9 mg / kg per every 7 days, or at least 18 mg / kg per every 7 days.

[0138] The pharmaceutical compositions disclosed herein may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. The pharmaceutical compositions can be prepared as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to use, or as emulsions.

[0139] In certain aspects, the disclosure also provides methods of using the disclosed compounds for therapeutic and diagnostic purposes. For example, the disclosure provides methods of treating or ameliorating a disease or condition that can include administering an effective amount of one or more of the compounds as described herein or one or more of the pharmaceutical compositions as described herein to a subject in need thereof.

[0140] In embodiments, the compounds of the disclosure deliver an anticancer drug to a selected tissue. Cancer can be lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, and melanoma.

[0141] Thus, in certain embodiments, the disclosure provides methods of treating a patient with prostate cancer by administering an effective amount of compound as describedherein or the pharmaceutical composition as described herein to the patient. The amount of the compound and regiment can be routinely determined using art-recognized techniques.

[0142] In certain embodiments, the disclosure provides methods for imaging one or more cancer cells (such as prostate cancer cell) in a patient by administering to the patient an effective amount of compound as described herein or the pharmaceutical composition as described herein. The method may further include imaging the compound in vivo. The imaging can be performed with any imaging techniques known in the art.

[0143] Another aspect of the disclosure provides compounds having the structural formula (II):or a pharmaceutically acceptable salt thereof, wherein each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4.

[0144] In certain embodiments, each Y is independently selected from a naturally occurring amino acid (e.g. L-amino acids) or unnaturally occurring amino acid (e.g., D-amino acids). In certain embodiments as described herein, each Y is independently selected from a naturally occurring amino acid (e.g. L-amino acids). For example, in certain embodiments, at least one Y is independently selected from alanine, glycine, isoleucine, leucine, proline, valine, phenylalanine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, cysteine, methionine, asparagine, or glutamine. In certain embodiments, at least one Y is selected from phenylalanine, tryptophan, tyrosine, or histidine. In certain embodiments, at least one Y is phenylalanine. In certain embodiments, at least one Y is tryptophan. In certain embodiments, at least on Y is 4-biphenylalanine. When Y is a naturally occurring amino acid, the side chains of such amino acids may be further substituted. For example, the amino acid side chains may be substituted with –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, – NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl); wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.

[0145] In certain embodiments as described herein, at least one Y has a structure

[0146] In certain embodiments, each R6is independently selected from (i) H, (ii) C1-C6alkyl, (iii) hydroxy(C1-C6alkyl), (iv) C1-C6alkoxy(C1-C6alkyl), (v) C2-C6alkenyl, (vi) R8R9N(C1- C6alkyl), (vii) halogen, (viii) halo(C1-C6alkyl), (ix) carboxyl(C1-C6alkyl), (x) thio(C1-C6alkyl), (xi) C1-C6alkylthio(C1-C6alkyl), (xii) R8R9NC(O)(C1-C6alkyl), (xiii) C3-C10cycloalkyl(C1-C6alkyl), (xiv) C3-C8 heterocyclyl(C1-C6) alkyl, (xv) aryl(C1-C6 alkyl), (xvi) heteroaryl(C1-C6 alkyl), (xvii) R17C(O)NR8(C1-C6alkyl), (xviii) C3-C10cycloalkyl, (xix) C3-C8heterocyclyl, (xx) aryl, and (xvi) heteroaryl; wherein R8and R9are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl), and wherein R17is C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, or heteroaryl. In certain embodiments, each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1- C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl); wherein R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl).

[0147] In certain embodiments as described herein, at least one Y has a structure

[0148] In certain embodiments, p is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, q is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). Incertain embodiments, each R7is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl); wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and each R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). For example, in certain embodiments, p is 1. In some embodiments as described herein, q is 1. In certain embodiments, p is 1, q is 1, and R7is aryl (e.g., phenyl).

[0149] Each R17is independently selected from C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, and heteroaryl, wherein R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl).

[0150] In certain embodiments as described herein, at least one Y has a structure

[0151] In certain embodiments, r is in an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, s is in an integer from 1 to 7 (e.g., from 1 to 5, or from 1 to 4, or from 1 to 3). In certain embodiments, each R12is independently selected from H, –OH, – SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl); wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and each R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). For example, in certain embodiments, r is 1. In someembodiments as described herein, s is 1. In certain embodiments, r is 1 and s is 1. In certain embodiments, R12is H.

[0152] In certain embodiments as described herein, at least one X has a structure

[0153] In certain embodiments, a is an integer from 1 to 4 (e.g., from 1 to 3). In certain embodiments, b is an integer from 1 to 5 (e.g., from 1 to 4, or from 1 to 3). In certain embodiments, each of R13and R14is independently selected from H, –OH, –SH, –NO2, – COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). In certain embodiments, R13and R14are H.

[0154] In certain embodiments as described herein, at least one Y has a structure

[0155] In certain embodiments, c is an integer of from 1 to 3 (e.g., is 1, or is 2). In certain embodiments, d is an integer of from 1 to 4 (e.g., of from 1 to 3). In certain embodiments, each of R15and R16is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and -C1- C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl). In certain embodiments, R15and R16are H.

[0156] In certain embodiments as described herein, at least one X has a structure of

[0157] In certain embodiments, the compounds of structural formula (II) are of any of the previous embodiments wherein n is an integer of from 1 to 4 or from 1 to 3. In certain embodiments, n is 1 or 2. In other embodiments, n is 1. For example, in some embodiments, n is 1 and Y is phenylalanine. In other embodiments, n is 1 and Y is In other embodiments as described herein, n is 1 and Y is

[0158] In certain embodiments, the compounds of formula (II) are of any of the previous embodiments wherein m is an integer from 1 to 5. For example, in certain embodiments asdescribed herein, m is an integer of from 1 to 4 or from 1 to 3. In certain embodiments, m is 1 or 2. In other embodiments of the disclosure as described herein, m is 1.

[0159] Another aspect of the disclosure provides compounds having the structural formula (III):or a pharmaceutically acceptable salt thereof, wherein each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

[0160] The compounds of structure formula (III) may have a specific stereochemistry. For example, in certain embodiments, the disclosure provides compounds having a formula:wherein X, m, n, R1, R2, R3, and R4are as described with respect to formula (III).

[0161] In certain embodiments as described herein, the compounds of formula (III) include X as defined in any of the previous embodiments with respect to formula (I). In certain embodiments as described herein, the compounds of formula (III) include n as defined in any of the previous embodiments with respect to formula (I). In certain embodiments as described herein, the compounds of formula (III) include m as defined in any of the previous embodiments with respect to formula (I). In certain embodiments as described herein, the compounds of formula (III) include R1, R2, R3, and R4as defined in any of the previous embodiments with respect to formula (I).

[0162] Another aspect of the disclosure provides compounds having the structural formula (IV):or a pharmaceutically acceptable salt thereof, wherein D is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety; L is an acid-cleavable linker and is attached to provide a carbamate moiety to formula (IV); each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer of from 1 to 4.

[0163] In certain embodiments as described herein, the compounds of formula (IV) include D as defined in any of the previous embodiments with respect to formula (I). Incertain embodiments as described herein, the compounds of formula (IV) include L as defined in any of the previous embodiments with respect to formula (I). In certain embodiments as described herein, the compounds of formula (IV) include Y as defined in any of the previous embodiments with respect to formula (II). In certain embodiments as described herein, the compounds of formula (IV) include m as defined in any of the previous embodiments with respect to formula (II). In certain embodiments as described herein, the compounds of formula (IV) include n as defined in any of the previous embodiments with respect to formula (II). In certain embodiments as described herein, the compounds of formula (IV) include R1, R2, R3, and R4as defined in any of the previous embodiments with respect to formula (I).

[0164] Another aspect of the disclosure provides a method for synthesizing a compound of formula (I), as described above. The method includes contacting a compound of formula (V)or a pharmaceutically acceptable salt thereof, with a compound of formula (III), as described herein,or a pharmaceutically acceptable salt thereof, wherein each X is independently a natural or unnatural n Į-amino acid, wherein the C-terminus of X forms an amide with the nitrogen carrying R2; m is an integer of from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

[0165] Another aspect of the disclosure provides a method for synthesizing a compound of formula (IV), as described above. The method includes contacting a compound of formula (V)or a pharmaceutically acceptable salt thereof, with a compound of formula (II), as described herein,or a pharmaceutically acceptable salt thereof, wherein each Y is independently a natural or unnatural n Į-amino acid, wherein the N-terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4. DEFINITIONS

[0166] As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

[0167] As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” PSMA with a compound includes the administration of a compound described herein to a subject or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing PSMA.

[0168] As used herein, the term “subject” or “patient,” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

[0169] As used herein, the phrase “pharmaceutically acceptable salt” refers to both pharmaceutically acceptable acid and base addition salts and solvates. Such pharmaceutically acceptable salts include salts of acids such as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic, formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric, tartaric, maleic, hydroiodic, alkanoic such as acetic, HOOC-(CH2)n-COOH where n is 0-4, and the like. Non-toxic pharmaceutical base addition salts include salts of bases such as sodium, potassium, calcium, ammonium, and the like. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable addition salts. EXAMPLES

[0170] The compounds disclosed herein can be made using procedures familiar to the person of ordinary skill in the art and as described herein. For example, compounds of structural formula (I) can be prepared according to general procedures (below), and / or analogous synthetic procedures. One of skill in the art can adapt the reaction sequences of Examples 1 and 2, and general procedures to fit the desired target molecule. Of course, in certain situations one of skill in the art will use different reagents to affect one or more of the individual steps or to use protected versions of certain of the substituents. Additionally, one skilled in the art would recognize that compounds of the disclosure can be synthesized using different routes altogether.

[0171] General Information: For the compounds described in the examples below,1H and31P NMR spectra were recorded on a Brucker Avance Neo 500 MHz spectrometer. High- resolution mass spectrometry (HRMS) spectra were obtained on an Applied Biosystems 4800 MALDI-TOF / TOF mass spectrometer. The scavenging resins, azide agarose and DBCO agarose, were purchased from Click Chemistry Tools. DBCO-NHS was purchased from AmBeed. PSMA-617 (vipivotide tetraxetan) was purchased from TargetMol (#T12573). All other chemical reagents used for synthesis and purification were purchased from AmBeed, Fisher, or Sigma Aldrich (unless otherwise noted) and used as supplied. Monomethyl auristatin E (MMAE, KP-20340) was purchased from eNovation Chemicals. Penicillin / streptomycin (P / S) was purchased from Corning. Fetal bovine serum albumin (FBS), HyClone™ RPMI 1640 with L-glutamine and DPBS without calcium and magnesium were purchased from Cytiva. Trypan Blue was purchased from VWR. CELLSTAR® μCLEAR tissue culture treated, clear bottom, 96-well plates (#655098) were purchased from Greiner Bio-One. Trypsin 0.25% - EDTA 1 mm (1X) and recombinant mousePSMA / FOLH1 protein (#4946-ZN) were purchased from R&D Systems. Collagenase Type I (0.25%) was purchased from STEMCELL Technologies. CellTiter-Glo® Luminescent Cell Viability Assay (G7572) reagents were purchased from Promega.

[0172] Example 1. Preparation and characterization of Compound 1 (CTT2274)

[0173] Compound 3 (DBCO-Bip): H-p-Phenyl-L-phenylalanine (272.92 mg, 1.13 mmol) was dissolved with 8 mL of anhydrous DMF in a 10 mL glass conical vial. DIPEA (656 μL, 486.6 umol) was added dropwise followed by the addition of the DBCO-NHS (303.25 mg, 753.6 umol) powder. The reaction was stirred at 1100 Mot / min at RT for 27 h. The reaction was diluted with DCM (4 mL) and transferred to a 50 mL recovery flask to rotovap at 60°C. Crude product was dissolved in EtOAc (20 mL) and washed sequentially with 1N HCl (10 mL, 3x), ddH2O (10 mL, 2x), and Brine (20 mL, 2x). Organic layer was dried on Na2SO4, volatiles were removed under reduced pressure, and resulting residue was dried under high vacuum. The crude product was dissolved in DCM and purified by silica (20 g) column chromatography (95:5 DCM:MeOH with 1% acetic acid) to give a solid in 87% yield.

[0174] Compound 6 (CTT1298): CTT1298 was synthesized as described in Ganguly, T. et al. A high-affinity [18 F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer. Nucl Med Biol 42, 780–787 (2015) and International Patent Application WO2014143736A1, each of which is incorporated by reference.

[0175] Compound 4 (CTT2270): Dissolved 1 eq DBCO-Bip (78.78 mg, 149.04 umol) with anhydrous DMF (600 μL) and transferred to a 2 mL glass conical vial with 5 molecular sieves.1.5 eq of dry DIPEA (39 μL, 223.6 umol) was added to the vial, followed by 1.3 eq TSTU (58.3 mg, 193.8 umol). The reaction was stirred for 12 min at RT, 1100 Mot / min, and monitored by silica TLC (90:10 EtoAc:ACN) to identify DBCO-BIP-NHS.2 eq CTT1298 (230.86 mg, 297.9 umol) was dissolved in ddH2O (1.1 mL) with 2 eq NaHCO3(25 mg, 297.6 umol). Reaction mixture was frozen and lyophilized overnight. Crude material was purified by C18 (1 g, 6 cc, WAT036905) and C8 (1 g, 6 cc, WAT054570) Waters Sep-Pak, using 40% ACN ddH2O and 30% ACN in ddH2O, respectively, to elute the product. Isolated yield was 18%.

[0176] Compound 5 (MMAE-PhosAm-azide): MMAE-PhosAm-Azide was synthesized as described in Olatunji, F. P. et al. Modular Smart Molecules for PSMA-Targeted Chemotherapy. Mol Cancer Ther 21, 1701 (2022), Olatunji, F. P., Herman, J. W., Kesic, B. N., Olabode, D. & Berkman, C. E. A click-ready pH-triggered phosphoramidate-based linker for controlled release of monomethyl auristatin E. Tetrahedron Lett 61, (2020., and Olatunji, F. P., Herman, J. W., Kesic, B. N., Olabode, D. & Berkman, C. E. Corrigendum to “A click- ready pH-triggered phosphoramidate-based linker for controlled release of monomethyl auristatin E” [Tetrahedron Lett. Volume 61, Issue 41, 8 October 2020, 152398]. Tetrahedron Lett 71, (2021), each of which is incorporated by reference. MMAE was purchased from eNovation Chemicals (Cat #KP-20340).

[0177] Compound 1 (CTT2274): In a 1.5 mL polypropylene microcentrifuge tube, 1.1 eq MMAE-PhosAm-azide (28.83 mg, 20.6 umol) was dissolved, assisted with pulse centrifugation for 10 s at 14.5 rcf, in ddH2O (60 μL) with 1.5 eq of NaHCO3(27.4 umol). In a separate 1.5 mL microcentrifuge tube, 1 eq of CTT2270 (22.11 mg, 18.3 umol) was dissolved in ddH2O (160 μL) with 1.5 eq of NaHCO3(27.4 umol). CTT2270 was added dropwise to MMAE-PhosAm-azide and stirred at RT, 500 Mot / min, for 1 h. Any unreacted DBCO-Bip and MMAE-PhosAm-azide was scavenged with azide agarose and DBCO agarose (Click Chemistry Tools), respectively, as previously described for CTT227X. Final scavenged material was desalted with a C8 Waters Sep-Pak (1 g, 6 cc, WAT054570) conditioned with ACN, equilibrated with ddH2O, and eluted with 35% ACN. ACN was removed by evaporation before freezing the pooled fractions and lyophilizing the product overnight. Isolated yield was 71%. The1HNMR and31PNMR for CTT2274 are shown in Figures 1A and 1B.

[0178] Example 2. Preparation and characterization of Compound 2 (CTT227X)

[0179] Compound 2 (CTT227X): Dissolved 1.1 eq DBCO-Bip (16.86 mg, 12 umol) with MeOH (120 μL) in a 1.5 mL polypropylene microcentrifuge tube. In a separate 1.5 mL microcentrifuge tube, 1 eq MMAE-PhosAm-azide (15.11 mg, 10.8 umol) was dissolved, assisted with pulse centrifugation for 10 s at 14.5 rcf, in ddH2O (120 μL) with 3.5 eq of NaHCO3(38.3 umol). MMAE-PhosAm-azide was added dropwise to DBCO-Bip and stirred at RT, 400 Mot / min, for 4 h 5 min. MeOH (additional 300 μL) was added to the reaction to help dissolve the material since a cloudy white precipitate began to develop shortly after adding MMAE-PhosAm-azide to DBCO-Bip. The final % MeOH in the reaction was 78%. Any unreacted DBCO-Bip and MMAE-PhosAm-azide was scavenged with azide agarose and DBCO agarose (Click Chemistry Tools), respectively. Briefly, 1 mL scavenging resin (i.e., 2 mL of 50% agarose slurry) was rinsed 2x with 1 mL of a 10% DMSO, 30% ethanol, 100 mM NaHCO3solution. The entire reaction solution was added to the rinsed resin and placed on a rotisserie for 35 min at RT. Filtrate of the resin was collected, and the resin was rinsed with ddH2O (1 mL, 3x), collecting each wash with the original filtrate. The sample was frozen and lyophilized overnight. Final scavenged material was desalted with a C18 Waters Sep-Pak (1 g, 6 cc, WAT036905) conditioned with ACN, equilibrated with ddH2O, and eluted with 40% ACN. ACN was removed by evaporation before freezing the pooled fractions and lyophilizing the product overnight. Isolated yield was 58%.

[0180] Example 3. Preparation and characterization of Compound 8 (CTT2101)

[0181] Compound 7 (CTT1400): CTT1400 was synthesized as described in Olatunji, F. P. et al. Modular Smart Molecules for PSMA-Targeted Chemotherapy. Mol Cancer Ther 21, 1701 (2022), Olatunji, F. P., Herman, J. W., Kesic, B. N., Olabode, D. & Berkman, C. E. A click-ready pH-triggered phosphoramidate-based linker for controlled release of monomethyl auristatin E. Tetrahedron Lett 61, (2020., and Olatunji, F. P., Herman, J. W., Kesic, B. N., Olabode, D. & Berkman, C. E. Corrigendum to “A click-ready pH-triggered phosphoramidate- based linker for controlled release of monomethyl auristatin E” [Tetrahedron Lett. Volume 61, Issue 41, 8 October 2020, 152398]. Tetrahedron Lett 71, (2021). MMAE was purchased from eNovation Chemicals (Cat #KP-20340), each of which is incorporated by reference. Accordingly, CTT1298 was dissolved in ddH2O to make a 0.43M solution.125 ^L of this solution was added to a 1 mL conical vial.1M TEA-Bicarb buffer was added to the 1mL conical vial containing the CTT1298 solution.1.8 equivalents of DBCO-PEG4-NHS was dissolved in DMSO (to make a 0.26M solution) and added to the vial dropwise. This reaction stirred vigorously overnight at 4°C. The reaction was then purified via prep HPLC and dried down through lyophilization. Before lyophilization, 1.2 equivalents of NaHCO3were added to neutralize the pH.

[0182] Compound 8 (CTT2101): In a 1.5 mL polypropylene microcentrifuge tube, 1.1 eq MMAE-PhosAm-azide (21.51 mg, 15.4 umol) was dissolved, assisted with pulse centrifugation for 10 s at 14.5 rcf, in ddH2O (60 μL) with 1.5 eq of KHCO3(20.85 umol). In aseparate 1.5 mL microcentrifuge tube, 1 eq of CTT1400 (18.21 mg, 13.9 umol) was dissolved in ddH2O (110 μL) with 1.5 eq of KHCO3(20.85 umol). CTT1400 was added dropwise to MMAE-PhosAm-azide and stirred at RT, 500 Mot / min, for 1 h. Any unreacted DBCO-Bip and MMAE-PhosAm-azide was scavenged with azide agarose and DBCO agarose (Click Chemistry Tools), respectively, as previously described for CTT227X, except 100 mM KHCO3was used instead of 100 mM NaHCO3for rinsing the scavenging resin. Final scavenged material was desalted with a C8 Waters Sep-Pak (1 g, 6 cc, WAT054570) conditioned with ACN, equilibrated with ddH2O, and eluted with 20% ACN. ACN was removed by evaporation before freezing the pooled fractions and lyophilizing the product overnight. Isolated yield was 71%. This reaction is shown in Scheme 5.

[0183] Example 4. IC50Determination and Mode of Inhibition Studies

[0184] IC50determination and mode of inhibition studies with human PSMA are routinely performed as previously described [4-6]. With recombinant mouse PSMA, finalized reaction conditions were based on monitoring product formation over time at 37°C by HPLC analysis to generate a progress curve and identify a linear range. In the IC50studies, 0.5 μL of recombinant mouse PSMA (0.22 μg) was diluted 1:1999 in 50 mM tris pH 7.4 with 1% triton X-100. Reactions were prepared with 175 μL of 50 mM tris pH 7.4, 25 μL of 10 μM substrate (N-[4-(phenylazo)-benzoyl]-glutamyl-g-glutamic acid, PABGgG), and 25 μL inhibitor or tris buffer. Reactions were initiated with 25 μL of 1:1999 diluted recombinant mouse PSMA for a total reaction volume of 250 μL and incubated for 15 min at 37°C. Final concentration range of inhibitor was 0.1 nM – 10 μM. in the mode of inhibition studies, the mouse PSMA concentration is 100-fold greater than the IC50 studies and pre-incubated at 37°C for 10 min with an inhibitor concentration that is 10-fold greater than the determined IC50or 50 mM tris buffer pH 7.4 (40 μL total sample volume). After pre-incubation, the enzyme-inhibitor / buffer sample was rapidly diluted with 1 mM substrate in 50 mM tris pH 7.4 with 1% triton buffer (3960 μL). A 200 μL sample was removed every 5 min over the course of 1 h to monitor product formation. All reactions were quenched with 25 μL of 2.5% trifluoroacetic acid in methanol and samples were prepared for HPLC analysis as previously described [6]. The IC50of the compounds were compared using both human PSMA (hPSMA) and mouse PSMA (mPSMA). For hPSMA, the IC50values for CTT2101 and CTT2274 were found to be 3.97 ± 0.126 nM and 3.97 ± 0.134 nM, respectively. For mPSMA, the IC50of CTT2274 was found to be 105 ± 4.79 nM. MMAE showed <10 μM IC50value for hPSMA. CTT227X and MMAE showed no appreciable inhibition at 10 μM to mPSMA. Binding of CTT2101 and CTT2274 was determined to be irreversible. Figures 2A and 2B depicts the IC50curves of CTT2274 against hPSMA and mPSMA, respectively, and Figure 2C depicts the IC50curve of CTT2101 against hPSMA.

[0185] Example 5. Western Blot Analysis

[0186] PSMA protein expression levels in the JAX Laboratories PDX prostate adenocarcinoma model (TM00298) as well as the PC3, C4-2B, and PC3-PIP prostate cancer cell lines were verified by western blot analysis. C4-2B cells were kindly gifted from Eva Corey at the University of Washington. PC3, C4-2B, and PC3-PIP cells were cultured in RPMI 1640 with L-glutamine and DPBS without calcium and magnesium (HyClone™) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) at 37 °C, 5.0% CO2until 70-80% confluency.

[0187] An approximately 6 mm cube, frozen tumor sample from the PDX prostate adenocarcinoma model was thawed for 5 min at 37°C in 1 mL of RPMI-1640 media (1% P / S 2% FBS). The tissue in media was centrifugation for 3 min at 65 rcf and the media was replaced with 2 mL of collagenase Type 1 (0.25%, STEMCELL Technologies), and incubated for 1 h at 37°C. Cells were disrupted by passing the cells three times through a 20 gauge, 1- inch needle on a 3 cc syringe. Collagenase Type 1 was neutralized with 1 mL of 5% FBS RPMI-1640 (1% P / S). The digested cell sample was centrifuged for 5 min at 65 rcf and the supernatant was discarded. Digested cells were resuspended in PBS by pipette and centrifuges for 3 min at 65 rcf, discarding the supernatant. Cells were lysed with 1 mL of ice- cold lysis buffer (150 mM NaCl, 1% NP-40, 50 mM tris-HCl, pH 7.4) supplemented with 1X APExBIO protease inhibitor cocktail in DMSO with EDTA (#K1019) and incubated at 4°C for 20 min. Lysed cells were centrifuged for 10 min at 4°C, 14500 rcf, and the supernatant was collected for protein quantification.

[0188] PC3, C4-2B, and PC3-PIP cells were cultured in complete media (RPMI-1640, 10% FBS, 1% penicillin / streptomycin) in T-75 flasks at 37 °C, 5.0% CO2until 70-80% confluency. Cells were washed with cold (4°C) PBS and incubated with 0.5 mL of ice-cold lysis buffer (150 mM NaCl, 1% NP-40, 50 mM tris-HCl, pH 7.4) supplemented with 1X APExBIO protease inhibitor cocktail in DMSO with EDTA. Cells were gently scraped from the flask and transferred to a microcentrifuge tube to incubate at 4°C for 20 min. Cells were centrifuged for 10 min at 4°C, 14,500 rcf. Supernatant was collected for protein quantification with the Bio-Rad Protein Assay, following the manufacturer’s standard procedure for microtiter plates and using bovine serum albumin to prepare a protein standard curve. Absorbance at 595 nm as measured with a Fluostar Omega Microplate reader with the Omega software version 1.02 and Mars Data Analysis Software Program version 1.10 (BMG Labtech).

[0189] Cell lysates were prepared in sample buffer (50 mM Tris-HCl pH 6.5, 2% SDS, 8% glycerol, 0.1% bromophenol blue, 5.3% ȕ-mercaptoethanol) and boiled at 100°C for3 min. Proteins were resolved by SDS-PAGE on 10% polyacrylamide gels in tris-glycine SDS buffer (25 mM Tris pH 8.3, 192 mM glycine, 0.1% SDS) using 200V for 35 min. Proteins were transferred 0.2 μm nitrocellulose membranes in Towbin Buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) for 1 h at 100V. Membranes were rinsed with tris buffered saline (TBS) (20 mM tris pH 7.6, 150 mM NaCl) and blocked for 30 min at RT with 3% gelatin in TBS. Membranes were washed with tris buffered saline-Tween 20 (TBS-T) (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.05% Tween 20). Membranes were probed with 1:1000 PSMA (D4S1F) rabbit mAb (#12702, Cell Signaling Technology) and 1:1000 GAPDH (D16H11) XP rabbit mAb (#5174, Cell Signaling Technology), each diluted in 1% gelatin in TBS-T. Proteins were detected using the colorimetric BioRad Immun-Blot Goat Anti-Rabbit IgG (H + L)-AP Assay Kit (#1706460), following the manufacturer’s instructions.

[0190] PSMA expression in PC3, PC3-PIP, and C4-2B cell lines and PDX tumor was determined via western blot. The western blot is shown in Figure 3. The PC3 cell line did not express PSMA while the PC3-PIP and C4-2B cell lines and PDX tumor sample did express PSMA.

[0191] Example 6. In vitro cytotoxicity

[0192] The Cell-Titer-Glo Luminescent Cell Viability assay (Promega) was used to measure the effectiveness of each CTT compound at reducing cell viability in prostate cancer cell lines with different PSMA expression levels. PC3 is PSMA negative, C4-2B is PSMA positive, and PC3-PIP is PSMA positive, which was confirmed by western blot analysis. All cell lines were cultured in complete media (RPMI-1640, 10% FBS, 1% penicillin / streptomycin). Cells were uplifted with trypsin / EDTA, counted with trypan blue, and serially diluted with complete media to prepare working concentrations between 100,000 cells / mL and 1,560 cells / mL.100 μL of cells were plated in triplicate for each drug or control well in clear, flat bottom, 96-well tissue culture plates (Grenier Bio-One #). One 96-well plate was used per cell line. Each cell viability assay was repeated three times (n = 3) with a different cell passage. Experimental wells had a final cell number of 2500 cells / well, and each cell line had a cell standard curve prepared with a final cell number range of 156 – 10,000 cells / well to verify the linear relationship of the chemiluminescent signal with the increasing cell number. Cells were allowed to adhere overnight at 37 °C, 5.0% CO2before treating the cells with drugs. MMAE was dissolved in DMSO and all CTT compounds were dissolved in 100 mM HEPES pH 7.4.100 μL of 0.2 nM – 20 nM drug in complete media, vehicle (0.2% DMSO or 2% 100 mM HEPES pH 7.4 in media), or complete media was added to each well to achieve a total volume of 200 μL. Empty wells were filled with 200 μL of phosphate buffered saline (PBS). The final drug concentrations were 0.1 nM, 1 nM, and 10 nM for MMAE, CTT2101, CTT2274, and CTT227X. The final percentages of DMSO and 100mM HEPES pH 7.4 buffer in media were 0.1% and 2% in complete media, respectively, which were maintained during the assay. Only the cells plated to generate the cell standard curve were treated with complete media alone. During the 72 h drug treatment period, 150 μL was removed from each well every 24 h to replace with fresh drug, vehicle, or complete media. Given that the media pH can begin to drop as the cells grow, replacing the media every 24 h was expected to reduce the likelihood of pre-mature drug release during the assay. After the 72 h drug treatment period, 100 μL was removed from all wells and the 96- plates were allowed to equilibrate to room temperature (RT) for 30 min.100 μL of the CellTiter-Glo reagent was added to each well and the chemiluminescence was measured according to the manufacturer’s instructions with a Fluostar Omega Microplate reader with the Omega software version 1.02 and Mars Data Analysis Software Program version 1.10 (BMG Labtech). Viability (% control) was calculated as follows:

[0193] AVG LUM is the calculated average of luminescence measured.

[0194] The in vitro cytotoxicity of CTT2101, CTT2274, CTT227X, and MMAE were tested against three prostate cancer cell lines, PC3, PC3-PIP, and C4-2B. In the PC3 cell line, MMAE was the only compound to show a dose-responsive effect; CTT2101, CTT2274, and CTT227X showed non-specific killing (Figure 4A). In the PC3-PIP cell line, CTT2101, CTT2274, and MMAE showed a dose-responsive effect. At 1 nM drug concentration, MMAE showed significantly more killing than any other drug and resulted in 30.33% cell viability. At 10 nM drug concentration, MMAE resulted in the lowest cell viability (25.33%) with CTT227X having no appreciable effect, resulting in 89.67% cell viability. CTT2274 produced the second smallest cell viability (47.33%) while treatment with CTT2101 resulted in 63.67% cell viability (p=0.03) (Figure 4B). Lastly, in the C4-2B cell line, MMAE exhibited a near complete killing of cells at 1 and 10 nM drug concentrations while CTT2101 and CTT2274 exhibit no appreciable effect until 10 nM drug concentrations were reached (Figure 4C). At 10 nM, CTT2101, CTT2274, and CTT227X resulted in 78%, 38.67%, and 91.33% cell viability, respectively. CTT2274 resulted in significantly more cell death than CTT2101 at this concentration (p=0.003).

[0195] Example 7. Evaluation of the Efficacy of CTT2101 in Comparison to MMAE

[0196] The study was conducted at The Jackson Laboratory in Sacramento, California. Male NSG mice (Stock #005557) ages 6-8 weeks were subcutaneously inoculated with 40 μL of the TM00298 patient-derived xenograft prostate cancer tumor in the right flank.29Mice were randomized to CTT2101, MMAE, or PBS treatment groups based on tumor volume,and t-tests were performed to ensure that no statistically significance differences between groups were observed. Thirty animals (n=10 / group) were administered 0.8 mg / kg of CTT2101 (equivalent to 0.2 mg / kg MMAE) in 100 μL 1X PBS, 0.2 mg / kg MMAE in 100 μL 1X PBS, or 100 μL 1X PBS via intravenous tail vein injection. The dose 0.2 mg / kg of MMAE was selected based on a previously published study and the known LD50of MMAE when intravenously administered to mice, 1-2 mg / kg. Animals were dosed once per week for a total of six doses starting on Day 0. Beginning on Day 39, animals were dosed every three days for a total of six doses. Throughout the study, animals were observed daily for signs of morbidity and mortality. Body weights were collected twice per week. Tumor volume measurements as measured by calipers (width2× 0.5 × length) were performed twice per week. Animals were provided supportive saline and Nutra Gel as needed directed by veterinary staff. Animals were euthanized when tumor volume exceeded 2,000 mm3, body condition score ^ 2, or if tumor was found to be ulcerated or necrotic. This study was conducted in a blinded manner.

[0197] The efficacy of CTT2101 was evaluated in comparison to MMAE using a PDX model of prostate cancer in mice. All tumor volumes were between 49.87-308.42 mm3at the start of the study. Prior to receiving the first dose, average tumor volumes were 175.11 ± 69.24 mm3, 175.94 ± 77.01 mm3, and 175.94 ± 73.78 mm3for the CTT2101, MMAE, and PBS groups, respectively. Throughout the course of the study, animals maintained steady body weights (Figure 5A). Looking at changes in tumor growth over time, it is evident that MMAE results in a more pronounced and sustained tumor suppression over time compared to CTT2101, even when dosing frequency is increased to once every three days (Figure 5B). Figures 5C-E show the tumor growth spaghetti plots for CTT2101, MMAE, and PBS, respectively. Based on this data, tumor burden at the start of treatment drives time to reach the exponential growth phase (Figure 5C).

[0198] Median survival was found to be 56, 70, and 48 days for the CTT2101, MMAE, and PBS groups, respectively. No statistically significant difference in survival was noted between the CTT2101 and PBS. Statistical significance in survival was observed when comparing the CTT2101 and MMAE groups (p = 0.0017) and the MMAE and PBS groups (p < 0.0001) (Figure 5F). However, when mice in the CTT2101 group are analyzed by starting tumor volume, median survival in mice with tumors less than 200 mm3is 63 days while median survival in mice with tumors greater than 200 mm3is only 48 days; this difference is statistically significant (p = 0.0052) (Figure 5G). In the MMAE group, there is no statistically significant difference in survival based on initial tumor volume (data not shown). Moreover, mice in the CTT2101 group with tumors less than 200 mm3at the start of dosing showed delayed tumor regrowth after treatment; mice with small initial tumors did not show markedincrease in tumor volume until approximately Day 35 while animals with larger tumors are the start of dosing showed an increased in tumor volume at approximately Day 21. The same trend is seen in the MMAE group, wherein animals with the smaller tumors showed tumor regrowth starting at approximately Day 42 while the animals with larger tumors showed tumor regrowth starting at approximately Day 28 (Figure 6A-D).

[0199] Example 8. Evaluation of the Efficacy of CT2274 in Comparison to CTT2101 and MMAE

[0200] This study was conducted at The Jackson Laboratory in Sacramento, California. Male NSG mice (Stock #005557) ages 6-8 weeks were subcutaneously inoculated with 40 μL of the TM00298 patient-derived xenograft prostate cancer tumor in the right flank. Mice were randomized to CTT2101, CTT2274, MMAE, or PBS treatment groups based on tumor volume, and t-tests were performed to ensure that no statistically significance differences between groups were observed. Forty animals (n=10 / group) were administered 3.8 mg / kg of CTT2101 (equivalent to 1 mg.kg MMAE) in 100 μL 1X PBS, 3.6 mg / kg of CTT2274 (equivalent to 1 mg.kg MMAE) in 100 μL 1X PBS, 1 mg / kg MMAE in 100 μL 1X PBS, or 100 μL 1X PBS via intravenous tail vein injection. Animals were dosed once per week for a total of six doses beginning on Day 0. Throughout the study, animals were observed daily for signs of morbidity and mortality. Body weights were collected twice per week. Tumor volume measurements as measured by calipers (width2× 0.5 × length) were performed twice per week. Starting at Day 86, body weight and tumor volume measurements were switched to once per week. Animals were provided supportive saline and Nutra Gel as needed directed by veterinary staff. Animals were euthanized when tumor volume exceeded 2,000 mm3, body condition score ^ 2, or if tumor was found to be ulcerated or necrotic. This study was conducted in a blinded manner.

[0201] The efficacy of CTT2274 to CTT2101 and MMAE was evaluated using the same patient-derived xenograft prostate cancer model as used in the first efficacy study. For this study, the dose of MMAE was increased five-fold to 1 mg / kg, and the doses of CTT2101 and CTT2274 were equivalently. Throughout the study, mice receiving CTT2101, CTT2274, and PBS showed no fluctuations in body weight; however, the mice receiving MMAE showed a sawtooth pattern in body weight. The transient decreases in body weight were consistently noted two days after dose administration. Following cessation of treatment on Day 35, mice in the MMAE group exhibited a steady decrease in body weight followed by a subsequent increase (Figure 7A).

[0202] All tumor volumes were between 62-172 mm3at the start of the study. Prior to receiving the first dose, average tumor volumes were 98.90 ± 33.41 mm3, 98.95 ± 29.25mm3, 98.60 ± 32.05 mm3, and 98.83 ± 32.99 mm3for the CTT2101, CTT2274, MMAE, and PBS groups, respectively. As observed in the first study, CTT2101 showed modest tumor suppression compared to PBS, although there was no statistically significant difference between the two groups. In contrast, CTT2274 and MMAE showed statistically significant tumor suppression compared to PBS, and importantly, no statistically significant difference was noted between CTT2274 and MMAE throughout the study (Figure 7B). Figures 7C-F show the tumor growth spaghetti plots for the CTT2101, CTT2274, MMAE, and PBS groups, respectively.

[0203] Average survival was found to be 53 days for the CTT2101 group and 48 days for the PBS group. In the CTT2274 group, no mice met euthanasia criteria or succumbed to tumor burden. In the MMAE group, two mice were found dead (attributed to drug toxicity) and one mouse succumbed to tumor burden. No statistically significant difference in survival was found between the CTT2101 and PBS groups (p=0.09). However, mice treated with CTT2274 survived significantly longer than CTT2101 (p<0.0001) and PBS (p<0.0001). (Figure 7G). From this study, it was concluded: 1) 3.6 m / kg of CTT2274 produced prolonged tumor suppression, even after cessation of treatment, on par with MMAE, 2) 3.6 mg / kg of CTT2274 does not exhibit obvious signs of toxicity (changes in body weight or other physical body signs), and 3) CTT2101 is inferior to CTT2274 efficacy for both tumor suppression and overall survival.

[0204] Example 9. Safety / Toxicity of CTT2274 Compared to MMAE

[0205] The study was conducted at The Jackson Laboratory in Sacramento, California. Male C56BL / 6J mice (Stock #000664) ages 6-8 weeks were used for this study (n=3 / group / time point). Two weeks prior to dosing, mice were bled via retroorbital route and 200 μL of blood was collected; 150 μL of blood was processed to serum and 50 μL of whole blood was collected in a tube with K2EDTA. Two days prior to dosing, mice were grouped based on body weight. On Day 0, mice were administered a single dose of 3.6 mg / kg of CTT2274 (equivalent to 1 mg / kg MMAE), 36 mg / kg of CTT2274 (equivalent to 10 mg / kg MMAE), 1 mg / kg of MMAE, or 10 mg / kg of MMAE in 100 μL of 1X PBS via intravenous tail vein injection. At 8, 24, and 168 hours post-dose administration blood was collected via retroorbital bleed and processed as described. At 336 hours post-dose administration, terminal blood collection was performed via cardiocentesis. All samples were sent to the Comparative Pathology Laboratory at the University of California-Davis for analysis. Complete blood counts and comprehensive chemistry panels were performed on all samples. Body weight measurements and clinical observations were recorded daily. This study was conducted in a blinded manner. Figure 8 highlights key blood chemistry and hematology findings, respectively. The black, dotted lines represent 95% confidence intervalreference ranges. It is well understood that reference ranges vary by sex, strain, age, and bleed route; therefore, this data and reference ranges are to be used as a guide in future study design. The high doses of both MMAE and CTT2274 were found to be toxic and result in death. Mice in the 3.6 mg / kg CTT2274 group maintained a steady body weight throughout the study while the remaining groups all exhibited decreases. The 1 mg / kg MMAE group was trending towards recovering by Day 14 in the study. No mice in the 1 mg / kg MMAE or 3.6 mg / kg CTT2274 were found dead or needed to be euthanized during the study.

[0206] Several important changes were observed in CBC and blood chemistry parameters as well. Both 1 mg / kg MMAE and 3.6 mg / kg CTT2274 showed hyperglycemia. While the exact mechanism is not known, hyperglycemia is also observed in patients treated with Padcev® and Adcetris®, and these findings indicate that glucose will need to be closely monitored in subsequent studies. Both 1 mg / kg MMAE and 3.6 mg / kg CTT2274 also showed increases in potassium and phosphorus levels by Day 2; interestingly, these values did not return to baseline by the end of the study. Increases in potassium and phosphorus could be elevated due to acute kidney injury, but serum creatinine and BUN values were normal, indicating AKI was unlikely. Insulin resistance or hemolysis could also cause increased potassium and phosphorus. Importantly, the 1 mg / kg MMAE group showed while the 3.6 mg / kg CTT2274 group showed inconsequential fluctuations. Padcev®, Adcetris®, and Polivy® all note thrombocytopenia as a potential adverse reaction. Moderate lymphopenia was observed in both the 1 mg / kg MMAE and 3.6 mg / kg CTT2274 groups, although it was more pronounced in animals treated with MMAE. Lymphopenia is also seen in patient treated with Padcev®, Tivdak®, and Polivy®, and this further indicates bone marrow suppression. The 1 mg / kg MMAE group also showed a decrease in red blood cell count on Day 7, indicating potential bone marrow suppression, while the 3.6 mg / kg CTT2274 group’s values remained unchanged (hemoglobin, and hematocrit trends mimicked that of RBC). Alkaline phosphatase (ALP) was notably elevated in the 1 mg / kg MMAE treated mice at 8 hrs but returned to levels comparable to that of the 3.6 mg / kg CTT2274 group by 24 hrs. Lastly, the absolute neutrophil count in the 1 mg / kg MMAE group experience dramatic fluctuations while the 3.6 mg / kg CTT2274 group experienced clinically insignificant changes throughout the study. All other CBC and chemistry parameters showed insignificant fluctuations.

[0207] While this safety / toxicity study has its limitations, several important pieces of information were gleaned: 1) 36 mg / kg CTT2274 and 10 mg / kg MMAE are acutely toxic and result in animal death; 2) 3.6 mg / kg CTT2274 demonstrates minimal safety issues as compared to a comparable dose of 1 mg / kg MMAE; 3) fluctuations in hematology and clinical chemistry seen in the 3.6 mg / kg dose of CTT2274 are in line with previously published data for MMAE ADC comparators.

[0208] To summarize, it has been demonstrated that 3.6 mg / kg of CTT2274 is efficacious and has a favorable safety profile for continued drug development. Table 1 outlines how CTT2274 compares to free MMAE, competing ADCs, and similar MMAE SMDCs based on key drug commercialization aspects and desirability for future compound development.

[0209] Table 1.

[0210] Discussion

[0211] MMAE is a promising treatment option for patients with PCa; however, safe and effective delivery of MMAE remains a challenge. ADCs utilizing MMAE rely on unstable linkers, cause considerable adverse events, and are expensive to manufacture. Here, the efficacy and safety of CTT2274 was demonstrated.

[0212] In in vitro cytotoxicity studies, as expected, only MMAE induced cell death in the PSMA- PC3 cell line. CTT2101 and CTT2274 had an effect in both PSMA+ cell lines at 10 nM concentrations, but CTT2274 resulted in less cell viability. Importantly, it is demonstrated that removal of the PSMA-binding moiety, as seen in CTT227X, does not cause cell death, indicating that selective uptake is observed.

[0213] In the first efficacy study, a PDX model was utilized of prostate cancer to evaluate CTT2101 in comparison to MMAE. CTT2101 showed modest tumor suppression at a dose of 0.8 mg / kg (equivalent to 0.2 mg / kg of MMAE). Initially, animals were dosed once per week for six weeks. At the conclusion of that dosing cycle, increasing the dosing frequency to once every three days for six doses was evaluated to determine if the dosing further suppress tumor growth. At this point in time, the average tumor volumes were 943.74 ± 462.06, 416.92 ± 260.60, and 1175.68 ± 570.90 mm3for the CTT2101, MMAE, and PBS groups, respectively. Increasing the dosing frequency to once every three days proved to be ineffective with tumors this large. It was evident that tumor volume at the start of treatment had a profound impact on treatment effectiveness and overall survival. Keeping clinical translatability in mind, CTT2101 was modified to include the biphenyl motif, with the hopes of increasing circulation time so that a once per week dosing regimen could be maintained.

[0214] In the second efficacy study, CTT2101, CTT2274, and MMAE were compared in the same PDX model of prostate cancer. The doses administered were increased to be equivalent to 1 mg / kg MMAE based on the modest therapeutic effect of CTT2101 seen in the first efficacy study. Furthermore, the tumor volume at the start of treatment did not exceed 200 mm3based on the information gleaned from the first study. This combination of changes (biphenyl motif, increased dose, and control of tumor volume at start of treatment) proved to be highly effective. While MMAE caused saw-tooth pattern changes in body weight, with drops consistently occurring two days after dosing and eventually steadily decreasing, the mice treated with CTT2274 did not show any appreciable changes in weight throughout the duration of the study. Additionally, the first mouse to die in the MMAE group was likely due MMAE toxicity, as the animal only had a 218.02 mm3tumor. Additionally, it should be noted that the PDX tumors exhibited variable growth kinetics, with several tumors growing much more slowly than others. However, all animals were included in analyses, and statistically significant differences were still observed.

[0215] The safety of CTT2274 was compared to equivalent doses of MMAE in mice. As previously discussed, MMAE ADCs have numerous known associated toxicities. Moreover, due to MMAE’s mechanism of action, it is expected that organs and tissues with high cell turnover rates (e.g. bone marrow, gastrointestinal tract, spleen, thymus) are more likely to be negatively impacted by MMAE-based therapies. Additionally, PSMA is expressed on healthy proximal tubule epithelial cells in the kidney, albeit at lower levels than tumor cells. While PSMA is expressed in proximal tubule cells, these cells also express MDR1, a known MMAE efflux transporter. Knowing this, it is hypothesized that CTT2274 may cause kidney injury.

[0216] While CTT2274 showed promising efficacy, survival, and safety results, this study is not without its limitations. As with any animal model of cancer, the subcutaneous PDX PCa model used is not fully indicative of the human condition. Humans diagnosed with PCa have localized disease that often metastasizes to the bone, lungs, liver, and other tissues, which this model does not recapitulate. Future studies will seek to determine the pharmacokinetics and complete safety / toxicity profile of CTT2274.

[0217] In this study, it was demonstrated that CTT2274 is a safe and efficacious treatment for PCa. The addition of the biphenyl motif on CTT2274 significantly improves treatment durability and overall survival compared to CTT2101, which does not possess the biphenyl motif. Moreover, it was demonstrated that CTT2274 is at least as efficacious, if not more so, than MMAE. Furthermore, treatment with CTT2274 results in at least equivalent or improved overall survival compared to MMAE. Lastly, it was demonstrated that CTT2274 has a more favorable safety / toxicity profile than MMAE. These results support the need for additional in vivo studies to evaluate the clinical translatability of CTT2274.

[0218] Example 10. Dose Ranging Efficacy Study

[0219] The study was conducted at The Jackson Laboratory in Sacramento, California using the TM00298 patient-derived xenograft (PDX) prostate cancer tumor model. Male NSG mice (Stock #005557) ages 6-8 weeks were subcutaneously inoculated with 40 μL of tumor in the right flank. Mice were randomized to CTT2274 or PBS treatment groups based on tumor volume; t-tests were performed to ensure that no statistically significant differences were observed between groups. Fifty-six (n=8 / group) mice were administered 0.36 mg / kg, 0.72 mg / kg, 1.44 mg / kg, 3.6 mg / kg, 9 mg / kg, or 18 mg / kg of CTT2274 in 100 μL 1X PBS or 100 μL of 1X PBS via intravenous tail vein injection. Mice were dosed once per week for six weeks starting on Day 0. Throughout the study, animals were observed daily for signs of morbidity and mortality. Body weights were collected twice per week. Tumor volume measurements as measured by calipers (width2× 0.5 × length) were performed twice per week. Animals were euthanized when tumor volume exceeded 2,000 mm3, body condition score (BCS) ^ 2, or if tumor was found to be ulcerated or necrotic. This study was conducted in a blinded manner.

[0220] Results

[0221] At the start of the study, mouse body weights were 31.26 ± 1.92, 30.94 ± 2.55, 31.19 ± 1.74, 29.98 ± 2.27, 31.16 ± 1.80, 31.49 ± 2.03, and 30.91 ± 1.22 g for the 0.36 mg / kg, 0.72 mg / kg, 1.44 mg / kg, 3.6 mg / kg, 9 mg / kg, 18 mg / kg, and PBS groups, respectively (Figure 9A). The 18 mg / kg group experienced an initial decrease in weight on Study Day 17 and further weight loss was observed on Study Day 21. The 9 mg / kg group saw a decrease in weight beginning on Study Day 52 with animals’ body weights fluctuating for the remainder of the study. The 0.36 mg / kg, 0.72 mg / kg, 1.44 mg / kg, and 3.6 mg / kg groups did not experience such decreases or fluctuations.

[0222] All tumor volumes were between 54.47-234.54 mm3at the start of the study. Prior to receiving the first dose, average tumor volumes were 111.94 ± 42.45 mm3, 112.14 ± 46.54 mm3, 111.76 ± 44.36 mm3, 111.53 ± 50.88 mm3, 112.05 ± 51.58 mm3, 112.24 ± 61.48 mm3, and 111.88 ± 55.24 mm3for the 0.36 mg / kg, 0.72 mg / kg, 1.44 mg / kg, 3.6 mg / kg, 9 mg / kg, 18 mg / kg, and PBS groups, respectively (Figures 9B and 9C). A dose-responsive trend in tumor suppression was observed. Figure 10 shows tumor growth trends of each mouse across groups. This data shows that the 0.36 mg / kg and 0.72 mg / kg doses, when dosed once per week for six weeks, provide little tumor growth suppression. The 1.44 mg / kg dose provides for improved tumor suppression at the same regimen. Further, the 3.6 mg / kg and 9 mg / kg doses at the same regimen are very effective at suppressing tumor growth, as tumors do not appear to reach the exponential growth phase until Study Day 84, which represents asustained 7 week-long period of tumor suppression following the cessation of dosing. Interestingly, mice #07 and #22 in the 9 mg / kg group had no measurable tumor starting on Study Day 28 and Study Day 80, respectively. Mouse #79 in the 9 mg / kg group had no measurable tumor between Study Days 45-84; however, the tumor eventually became measurable on Study Day 87.

[0223] Median survival was determined to be 38, 47, 70, 21, and 40 days for the 0.36 mg / kg, 0.72 mg / kg, 1.44 mg / kg, 18 mg / kg, and PBS groups respectively (Figure 9D). All mice in the 18 mg / kg group were euthanized on Study Day 21 at the recommendation of the study veterinary due to abnormal ambulation. All mice in the 0.36 mg / kg, 0.72 mg / kg, and PBS groups were euthanized due to meeting tumor volume euthanasia criteria. The mice in the 1.44 mg / kg group that did not reach the conclusion of the study were euthanized due to meeting tumor volume euthanasia criteria. The mice in the 9 mg / kg group that did not reach the conclusion of the study were euthanized due to a BCS of 2; one mouse was also noted to have abnormal breathing / gasping due to a mass of unknown origin above the heart pressing on the trachea. At the conclusion of the study (Study Day 90), all mice in the 3.6 mg / kg group, six mice in the 9 mg / kg group, and two mice in the 1.44 mg / kg group were alive. Survival statistical comparisons between groups is shown in Table 2.

[0224] Table 2.

[0225] Given the ambulation abnormalities observed in the 18 mg / kg group, two mice were selected at random to be euthanized and formalin-fixed for gross necropsy with histopathology. In both mice, clinical signs and abnormal gait were attributed to axonal degeneration and neuronal degeneration affecting the dorsal root ganglia and the white matter of the ventral spinal cord.

[0226] Based on the above example dosing study in mice, it is expected that safe and efficacious dose ranges for compounds in accordance with the disclosure including, e.g., CTT2274, can be achieved within ranges that are physiologically safe, effective, and / or tolerable (e.g., as suggested by the efficacy in the mice receiving dosages within the 1.44 to 9 mg / kg range).

[0227] Examples 11-34. Additional SMDCs according to the disclosure

[0228] Additional example SMDCs are prepared essentially according to the procedures noted above and shown in Table 3. In particular, an azide is reacted with a targeting molecule to result in the corresponding reaction product, i.e., a triazole-containing SMDC. Thus the example compounds in Table 3 are the product of a reaction between the azide and the alkyne. For each of the examples in Table 3, the targeting molecule (i.e., alkyne) is CTT2270, CTT23002, CTT23012, CTT23022, CTT23032, CTT23042, or CTT23052, the structures of which are:

[0229] Further example SMDCs precursors are prepared essentially according to the procedures noted above and are also shown in Table 3. In particular, an azide is reacted with an alkyne containing a carboxylic acid to result in the corresponding reaction product, i.e., a triazole-containing SMDC precursor. Each of the triazole-containing SMDC precursor examples in Table 3 can be further derivatized with a targeting moiety using the carboxylic acid moiety (e.g., through various amide bond forming reactions). For each of the examples in Table 3, the targeting molecule (i.e., alkyne) is DBCO-Bip, the structure of which is:

[0230] Table 3.

[0231] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be incorporated within the spirit andpurview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated herein by reference for all purposes.

Claims

We claim:

1. A compound of structural formula (I)or a pharmaceutically acceptable salt thereof, wherein D is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety; L is an acid-cleavable linker and is attached to provide a carbamate moiety to formula (I); each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

2. The compound for claim 1 having a formula:wherein D is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety; L is an acid-cleavable linker and is attached to provide a carbmate moiety to formula (I);each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

3. The compound according to claim 1 or claim 2, wherein D is a therapeutic agent.

4. The compound according to any of claims 1 to 3, wherein D is an anti-angiogenic agent, a cytotoxic agent, a cytokine, a chemokine, an apoptotic agent, a prodrug, a toxin, an enzyme, a radioisotope, an immunomodulator, an antibiotic, an agent active in the CNS or a hormone.

5. The compound according to any of claims 1 to 3, wherein D is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), exatecan, N-Me-L-Ala-maytansinol, gemcitabine, seco-duocarmycin (seco-DUBA), gemcitabine-monophosphate, doxorubicin, cabazitaxel, docetaxel, paclitaxel, imiquimod, 7-ethyl-10- hydroxycamptothecin (SN-38), duocarmycin (DUBA), or (R)-5-Chloro-N2-[4-(4- methylpiperazin-1-yl)phenyl]-N4-[(tetrahydrofuran-2-yl)methyl]pyrimidine-2,4-diamine ((R)-9b).^ 6. The compound according to claim 1 or claim 2, wherein D is a diagnostic agent.

7. The compound according to claim 1 or claim 2, wherein D is a radioisotope, an imaging agent, a fluorescent dye, a near-IR dye, an enzyme, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an ultrasound label, or a radioacoustic label.

8. The compound according to any of claims 1 to 7, wherein D is attached to L through – NR2–.

9. The compound according to any of claims 1 to 7, wherein D is attached to L through – O–.

10. The compound according to any of claims 1 to 7, wherein L is selected from:wherein R5is H, –OH, or C1-C6alkoxyl.

11. The compound according to any of claims 1 to 7, wherein L is selected from: wherein R5is H, –OH, or C1-C6alkoxyl.

12. The compound of claim 10 or 11, wherein R5is H.

13. The compound of claim 10 or 11, wherein R5is –OCH3.

14. The compound according to any of claims 1 to 7, wherein L is15. The compound according to any of claims 1 to 14, wherein the N-terminus of X is attached to the adjacent carbonyl to provide an amide moiety; 16. The compound according to any of claims 1 to 15, wherein each X is independently selected from a naturally occurring amino acid (e.g. L-amino acids) or unnaturally occurring amino acid (e.g., D-amino acids).

17. The compound according to any of claims 1 to 16, wherein at least one X has a structurewherein each R6is independently selected from (i) H, (ii) C1-C6alkyl, (iii) hydroxy(C1- C6alkyl), (iv) C1-C6alkoxy(C1-C6alkyl), (v) C2-C6alkenyl, (vi) R8R9N(C1-C6alkyl), (vii) halogen, (viii) halo(C1-C6alkyl), (ix) carboxyl(C1-C6alkyl), (x) thio(C1-C6alkyl), (xi) C1-C6alkylthio(C1-C6alkyl), (xii) R8R9NC(O)(C1-C6alkyl), (xiii) C3-C10cycloalkyl(C1-C6alkyl), (xiv) C3-C8heterocyclyl(C1-C6) alkyl, (xv) aryl(C1-C6alkyl), (xvi) heteroaryl(C1-C6alkyl), and (xvii) R17C(O)NR8(C1-C6alkyl); wherein R8and R9are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl), and wherein R17is C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1- C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, or heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and R10and R11are independently selected from hydrogen and C1-C6alkyl.

18. The compound according to any of claims 1 to 16, wherein at least one X has a structurewherein p is an integer from 1 to 5;q is an integer from 1 to 5; each R7is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1- C6alkyl), or heteroaryl(C1-C6alkyl); and each R17is independently selected from C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, and heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and each R10and R11are independently selected from hydrogen and C1-C6alkyl.

19. The compound according to claim 18, wherein p is 1.

20. The compound according to claim 18 or claim 19, wherein q is 1.

21. The compound according to claim 18, wherein p is 1, q is 1 and R7is aryl (e.g. phenyl).

22. The compound according to any of claims 1 to 16, wherein at least one X has a structurewherein r is in an integer from 1 to 5; s is in an integer from 1 to 7; andeach R12is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1- C6alkyl), or heteroaryl(C1-C6alkyl); wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and each R10and R11are independently selected from hydrogen and C1-C6alkyl.

23. The compound according to claim 22, wherein r is 1.

24. The compound according to claim 22 or claim 23, wherein s is 1.

25. The compound according to claim 18, wherein at least one X has a structure whereina is an integer from 1 to 4; b is an integer from 1 to 5; and each of R13and R14is independently selected from H, –OH, –SH, –NO2, –COOH, C1- C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.

26. The compound according to claim 22, wherein at least one X has a structurewherein c is an integer from 1 to 3; d is an integer from 1 to 4; and each of R15and R16is independently selected from H, –OH, –SH, –NO2, –COOH, C1- C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.

27. The compound according to any of claims 1 to 26, wherein n is an integer from 1 to 3.

28. The compound according to any of claims 1 to 26, wherein n is 1 or 2.

29. The compound according to any of claims 1 to 26, wherein n is 1.

30. The compound according to any of claims 1 to 26, wherein n is 1 and X is phenylalanine.

31. The compound according to any of claims 1 to 26, wherein n is 1 and X is32. The compound according to any of claims 1 to 26, wherein n is 1 and X is33. The compound according to any of claims 1 to 32, wherein m an integer from 1 to 4 (e.g., an integer of from 1 to 3).

34. The compound according any of claims 1 to 32, wherein m is 1 or 2.

35. The compound according to any of claims 1 to 32, wherein m is 1.

36. The compound according to any of claims 1 to 35, wherein R1, R2, and R3are independently H or C1-C4alkyl.

37. The compound according to any of claims 1 to 35, wherein R1, R2, and R3are independently H or methyl.

38. The compound according to any of claims 1 to 35, wherein R1, R2, and R3are independently H.

39. The compound according to any of claims 1 to 35, wherein R4is H or methyl..

40. The compound according to any of claims 1 to 35, wherein R4is H.

41. The compound according to any of claims 1 to 40 of formula:

42. The compound of claim 41, wherein D is MMAE.

43. The compound of any of claims 1 to 40 of formula:.

44. A pharmaceutical composition comprising a compound of any one of claims 1 to 43 and a pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer, and / or diluent.

45. A method of delivering a therapeutic or diagnostic agent to a subject, wherein the method comprises administering a therapeutically effective amount of a compound according to any one of claims 1 to 43 or a pharmaceutical composition according to claim 44 to a subject in need of such agent, wherein D is a therapeutic or diagnostic agent.

46. A method of treating a patient with prostate cancer, the method comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 43 or a pharmaceutical composition according to claim 44.

47. A method for imaging one or more prostate cancer cells in a patient comprising administering to the patient a compound according to any one of claims 1 to 43 or a pharmaceutical composition according to claim 44.

48. A compound of structural formula (II)or a pharmaceutically acceptable salt thereof, wherein each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4.

49. The compound according to claim 48, wherein each Y is independently selected from a naturally occurring amino acid (e.g. L-amino acids) or unnaturally occurring amino acid (e.g., D-amino acid).

50. The compound according to claim 48 or claim 49, wherein at least one Y has a structurewherein each R6is independently selected from (i) H, (ii) C1-C6alkyl, (iii) hydroxy(C1- C6alkyl), (iv) C1-C6alkoxy(C1-C6alkyl), (v) C2-C6alkenyl, (vi) R8R9N(C1-C6alkyl), (vii) halogen, (viii) halo(C1-C6alkyl), (ix) carboxyl(C1-C6alkyl), (x) thio(C1-C6alkyl), (xi) C1-C6alkylthio(C1-C6alkyl), (xii) R8R9NC(O)(C1-C6alkyl), (xiii) C3-C10cycloalkyl(C1-C6 alkyl), (xiv) C3-C8 heterocyclyl(C1-C6) alkyl, (xv) aryl(C1-C6 alkyl), (xvi) heteroaryl(C1-C6alkyl), (xvii) R17C(O)NR8(C1-C6alkyl),^(xviii) C3-C10cycloalkyl, (xix) C3-C8heterocyclyl, (xx) aryl, and (xvi) heteroaryl; wherein R8and R9are independently selected from hydrogen and C1-C6alkyl (e.g., C1-C4alkyl, or C1-C3alkyl), and wherein R17is C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1- C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, or heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.

51. The compound according to claim 48 or claim 49, wherein at least one Y has a structurewherein p is an integer from 1 to 5; q is an integer from 1 to 5; and each R7is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1- C6alkyl), or heteroaryl(C1-C6alkyl); each R17is independently selected from C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), heteroaryl(C1-C6alkyl), C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, and heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6 alkyl, hydroxy(C1-C6 alkyl), C1-C6 alkoxy(C1-C6 alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and each R10and R11is independently selected from hydrogen and C1-C6alkyl.

52. The compound according to claim 51, wherein p is 1.

53. The compound according to claim 51 or claim 52, wherein q is 1.

54. The compound according to claim 51, wherein p is 1, q is 1 and R7is aryl (e.g. phenyl).

55. The compound according to claim 48 or claim 49 wherein at least one Y iswherein r is in an integer from 1 to 5; s is in an integer from 1 to 7; and each R12is independently selected from H, –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl, C3-C8heterocyclyl, aryl, heteroaryl, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1- C6 alkyl), or heteroaryl(C1-C6 alkyl); wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with –OH, –SH, –NO2, –COOH, C1-C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), halogen, C3-C10cycloalkyl(C1-C6alkyl), C3-C8heterocyclyl(C1-C6alkyl), aryl(C1-C6alkyl), or heteroaryl(C1-C6alkyl), and R10and R11are independently selected from hydrogen and C1-C6alkyl.

56. The compound according to claim 55, wherein r is 1.

57. The compound according to claim 55 or claim 56, wherein s is 1.

58. The compound according to claim 51, wherein at least one Y has a structurewherein a is an integer from 1 to 4; b is an integer from 1 to 5; and each of R13and R14is independently selected from H, –OH, –SH, –NO2, –COOH, C1- C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.^ 59. The compound according to claim 55, wherein at least one Y is whereinc is an integer from 1 to 3; d is an integer from 1 to 4; and each of R15and R16is independently selected from H, –OH, –SH, –NO2, –COOH, C1- C6alkyl, hydroxy(C1-C6alkyl), C1-C6alkoxy(C1-C6alkyl), C2-C6alkenyl, –NR10R11, R10R11N(C1-C6alkyl), and halogen; wherein R10and R11are independently selected from hydrogen and C1-C6alkyl.

60. The compound according to any of claims 48 to 59, wherein n is in the range of 1 to 3.

61. The compound according to any of claims 48 to 59, wherein n is 1 or 2.

62. The compound according to any of claims 48 to 59, wherein n is 1.

63. The compound according to any of claims 48 to 59, wherein n is 1 and Y is phenylalanine.

64. The compound according to any of claims 48 to 59, wherein n is 1 and Y is65. The compound according to any of claims 48 to 59, wherein n is 1 and Y is66. The compound according to any of claims 48 to 65, wherein m is in the range of 1 to 4 (e.g., in the range of 1 to 3).

67. The compound according any of claims 48 to 65, wherein m is 1 or 2.

68. The compound according to any of claims 48 to 65, wherein m is 1.

69. A compound of formula (III)or a pharmaceutically acceptable salt thereof, whereineach X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

70. The compound of claim 68 having a formula:wherein each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

71. The compound of claim 69 or claim 70, wherein X is as defined as in any of claims 15 to 26.

72. The compound of any of claims 69 to 71, wherein n is defined as in any of claims 27 to 32.

73. The compound of any of claims 69 to 72, wherein m is defined as in any of claims 33 to 35.

74. The compound of any of claims 69 to 73, wherein R1, R2, R3, and R4is defined as in any of claims 36 to 40.

75. A compound of formula (IV)or a pharmaceutically acceptable salt thereof, wherein D is a therapeutic or diagnostic agent attached to L through an –NR2–, –S–, or –O– moiety; L is an acid-cleavable linker and is attached to provide a carbamate moiety to formula (IV); each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4.

76. The compound of claim 75, wherein D is as defined in any of claims 3 to 9.

77. The compound of claim 75 or claim 76, wherein L is as defined in any of claims 10 to 14.

78. The compound of any of claims 75 to 77, where Y is as defined in any of claims 48 to 59.

79. The compound of any of claims 75 to 78, wherein n is as defined in any of claims 60 to 65.

80. The compound of any of claims 75 to 79, wherein m is as defined in any of claims 66 to 68.

81. The compound of any of claims 75 to 80, wherein R1, R2, R3, and R4is defined as in any of claims 36 to 40.

82. A method of synthesizing a compound of formula (I) as described in any one of claims 1 to 43, the method comprising contacting a compound of formula (V)or a pharmaceutically acceptable salt thereof, with a compound of formula (III) (e.g., as described in any one of claims 69 to 74)or a pharmaceutically acceptable salt thereof, wherein each X is independently a natural or unnatural n Į-amino acid, wherein the C- terminus of X forms an amide with the nitrogen carrying R2; m is an integer from 1 to 5; n is an integer from 1 to 4; and R1, R2, R3, and R4are independently H, C1-C6alkyl, or a protecting group.

83. A method of synthesizing a compound of formula (IV) as described in any one of claims 75 to 81, the method comprising contacting a compound of formula (V) (V) or a pharmaceutically acceptable salt thereof, with a compound of formula (II) (e.g., as described in any one of claims 48 to 68)or a pharmaceutically acceptable salt thereof, wherein each Y is independently a natural or unnatural n Į-amino acid, wherein the N- terminus of Y is attached to the adjacent carbonyl to provide an amide moiety; m is an integer from 1 to 5; and n is an integer from 1 to 4.

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