WO2011008985A2

WO2011008985A2 - Methods and compositions for improved delivery of therapeutic and diagnostic agents

Info

Publication number: WO2011008985A2
Application number: PCT/US2010/042175
Authority: WO
Inventors: Niren Murthy; Michael E. Davis; Dongin Kim; Madhuri Dasari; Sungmun Lee; Seungjun Lee; Jay Sy; Mark Goodman
Original assignee: Georgia Tech Research Corporation; Emory University
Priority date: 2009-07-15
Filing date: 2010-07-15
Publication date: 2011-01-20
Also published as: WO2011008985A3

Abstract

Among other things, this disclosure provides for compounds having a targeting moiety linked to a therapeutic moiety and/or a diagnostic moiety; the synthetic methods for making the compounds; pharmaceutical compositions comprising the compounds; and, methods for treating or diagnosing disorders. Some of the compounds include a linker which can be cleaved under physiological conditions, including linkers comprising an amide or disulfide bond. Examples of targeting moieties are derivatives of DNA binding agents, which may comprise derivatives of Hoechst 33258. Examples of therapeutic moieties are compounds useful for treating a cancer, which may comprise derivatives of nucleotides and nucleosides such as gemcitabine. This abstract is intended to be used as a scanning tool for the purposes of searching in the particular art and is not intended to limiting of the present disclosure.

Description

METHODS AND COMPOSITIONS FOR IMPROVED DELIVERY OF THERAPEUTIC AND DIAGNOSTIC AGENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of United States Application No. 61/225,649, filed July 15, 2009, which is hereby incorporated by reference in its entirety.

GOVERNMENT INTEREST

[002] This invention was made with government support under agreement numbers EEC-9731643 and BES-0546962 awarded by the National Science Foundation and agreement number 1 R21EB006418-01 awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0031 This disclosure generally relates to chemical compositions comprising a targeting moiety linked via a chemical linker to either a therapeutic moiety or a diagnostic moiety, and methods of using the same.

BACKGROUND OF THE INVENTION

[004] In diseased states, intrinsic cellular pathways and extrinsic events cause cell death and result in the exposure of the intracellular contents to extracellular spaces. As one result, genomic DNA becomes exposed, and the concentration of extracellular DNA (E- DNA) increases. In this context, E-DNA comprises both extracellular DNA and accessible DNA. Accessible DNA may be found in both dead cells and non-intact cells (i.e. cells with non-intact cell membranes). Consequently, E-DNA provides a molecular target for the diagnosis and treatment of multiple diseases associated with necrosis such as cancer, cardiac dysfunction, sepsis, atherosclerosis, stroke and dementia. An ideal and novel clinical reagent could selectively bind E-DNA, and thereby create a means to image or deliver therapeutic agents. Described here are a series of membrane-impermeable, bi- functional small molecules containing a DNA-binding agent that selectively targets E- DNA coupled to therapeutic compounds or imaging agents.

[005] In cancers, multiple events lead to cell death and the accumulations of E-DNA. As tumors enlarge, the nutritional demands of the growing and dividing cells exceed vascularization and blood flow. As result, many cells within a tumor die of oxygen deprivation and create zones of dead tissue referred to as necrotic cores. Such necrotic cores contain high concentrations of E-DNA. Cancer treatments increase and enhance the levels of E-DNA in a tumor. Following exposure to reagents used in treatments, a large portion of cells within a tumor die and release their intracellular contents, including DNA, into the extracellular space. Hence, E-DNA concentration in tumors increases following chemotherapy. In summary the E-DNA of untreated and higher concentrations of E-DNA following treatment of cancers provide ideal targets for the binding of therapeutic molecules. [006] Increases in E-DNA as a molecular target for cancer treatments stands in stark contrast to the current state of the art. To date, cancer treatments have focused on the properties responsible for enhanced growth and cell division or the disabled pathways for cellular senescence. In either case, following administration of effective

chemotherapeutics, the target population of living cells decreases. In contrast, a therapeutic agent that binds E-DNA benefits from an increasing target population following the administration of cytotoxic agents. A cancer treatment that causes and exploits increases in the molecular target stands to greatly enhance the efficacy of treatment and diminish toxicity to healthy tissues.

[007J The targeting of E-DNA also provides novel inroads into the imaging and diagnosis of multiple diseased states associated with necrosis. For example, following myocardial infarction or stroke, ischemic tissues die and expose their DNA contents to the extracellular space. As shown here, a membrane-impermeable DNA-binding moiety coupled to a fluorescent imaging agent allows for the specific and sensitive imaging of necrotic tissues generated by myocardial infarction. SUMMARY OF THE INVENTION

[008] Described in this disclosure are compositions comprise a targeting moiety linked to one or more therapeutic moieties, one or more diagnostic moieties, or multiple therapeutic and diagnostic moieties. The compositions have numerous applications in diagnosis and therapeutic management of disease. [009] Among other things, this disclosure provides for compositions for the improved delivery and efficacy of therapeutic agents comprising:

(B)_n-L-(T)_n,

wherein B is a targeting moiety is chemically linked via a linker, L, which is chemically linked to T, wherein T is a therapeutic moiety, and wherein n and m may be independently varied from 1-3.

[0010] In another aspect, the disclosure provides for compositions for the improved diagnosis of disease comprising:

(B)_n-L-(I)_n,

wherein B is a targeting moiety is chemically linked via a linker, L, which is chemically linked to 1, wherein [ is a diagnostic moiety, and wherein n and m may be independently varied from 1-3.

[0011] In a further aspect, the disclosure provides for compositions for both the improved delivery and efficacy of therapeutic agents, and improved diagnosis of disease comprising:

(B)_n-L'-(I)_m-L"-(T)_x

wherein B is a targeting moiety is chemically linked via a linker, L', which is chemically linked to I, wherein I is a diagnostic moiety, which is chemically linked via a linker, L", to T, wherein T is a therapeutic moiety, and wherein n,m, and x may be independently varied from 1-3.

[0012] In a further aspect, the disclosure provides for compositions for both the improved delivery and efficacy of therapeutic agents, and improved diagnosis of disease comprising:

(BVL'-CIVLMDm

wherein B is a targeting moiety chemically linked via a linker, L', to T, wherein T is a therapeutic moiety which is chemically linked via a linker, L", to I, wherein l is a diagnostic moiety, and wherein /ι, m, and x may be independently varied from 1-3.

[0013] Accordingly, one aspect of the disclosure provides for compositions wherein the targeting moiety, B, is a DNA binding agent. In a further aspect, the DNA binding agent has DNA minor groove binding activity. In another aspect, the DNA binding agent is a DNA intercalating agent. In an additional aspect, the DNA binding agent is a derivative or fragment of Hoechst 33258 and related DNA binding agents. In a further aspect, the targeting moiety, B, may be an optionally substituted acyclic, polycyclic, polyaromatic, heterocyclic, or other hydrophobic moiety.

[0014| In one aspect, the compound may include a linker, such as L, L' or L" in the preceding formulas, which can be cleaved under physiological conditions. In another aspect, the linker is an optionally substituted polyether or other biocompatible chemical linker which may further comprise branching moieties. In a further aspect, the linker may comprise amide, ester or other sites which may undergo hydrolysis under physiological conditions. In yet a further aspect, the linker may comprise a disulfide bond which may undergo sulfhydryl exchange under physiological conditions. Moieties may be added to the linker to modify the rate of cleavage, including hydrolysis or sulfhydryl exchange, under physiological conditions.

[OOISJ In certain aspects, the diagnostic moiety, I, can be an imaging agent or a ligand for coordinating the imaging agent. The term "imaging agent" is defined herein as any agent or compound that increases or enhances the ability of cells or tissue to be imaged or viewed using imaging techniques known in the art when compared to visualizing the cells or tissue without the imaging agent. Imaging agents known in the art can be used herein. In one aspect, the imaging agent comprises a chromophore, fluorophore, a denoptical dye, a MRl contrast agent, a PET probe, a SPECT probe, a CT contrast agent, a radiodiagnostic agent, or an ultrasound contrast agent. In one aspect, imaging agents useful in magnetic resonance imaging include Gd^{^3}, Eu⁺³, Tm⁺³, Dy⁺³, Yb"³, Mn⁺², or Fe"³ ions or complexes. In another aspect, imaging agents useful in PET and SPECT imaging include ⁵⁵Co, ⁶⁴Cu, ⁶⁷Cu, ⁴⁷Sc, ⁶⁶Ga, ⁶⁸Ga, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ¹¹¹In, ¹⁰⁹Pd, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, or ¹⁸⁸Re. In other aspects, the imaging agent is a radioisotope of a halide including, but not limited to, ¹⁸F, ¹²V³¹^ ⁷⁷Br₁ ⁸V Or ²¹¹At.

|0016J Also disclosed are dosage forms comprising a therapeutically effective amount of at least one product of a disclosed synthetic method and a pharmaceutically acceptable carrier.

[0017] Also disclosed are dosage forms for administration to a subject comprising a therapeutically effective amount of one or more disclosed compounds and a

pharmaceutically acceptable carrier. [0018] Also disclosed is a method of treating a disorder of uncontrolled cellular proliferation in a subject comprising the step of administering to the subject a disclosed dosage form, thereby treating the disorder of uncontrolled cellular proliferation.

[0019) Additional advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice. Other advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed

BRIEF DESCRIPTION OF THE FIGURES

[0020] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description serve to explain the principles of certain embodiments of this disclosure.

[0021] FIG. 1 provides a schematic illustration of one embodiment of the present disclosure showing a targeting moiety connected via a cleavable chemical linker to a therapeutic moiety.

[0022] FlG. 2 provides a schematic illustration of another embodiment of the present disclosure showing a targeting moiety connected via a non-cleavable chemical linker to a diagnostic moiety. [0023] FlG. 3 illustrates one aspect of the present disclosure wherein the targeting moiety linked to a therapeutic moiety (the "autocatalytic delivery vehicle" or "ADV") is in equilibrium between an unbound state and bound to either serum albumin or a target in the circulatory system, and the bound ADV molecules are in equilibrium with binding to the target at the site of therapeutic action. [0024] FIG. 4 illustrates another aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is in equilibrium with either serum albumin or DNA in the circulatory system, and these bound ADV molecules in the circulatory system are in equilibrium with binding to DNA at the therapeutic site. [0025] FIG. 5 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is in equilibrium with either serum albumin or DNA in the circulatory system, and these bound ADV molecules in the circulatory system are in equilibrium with localization to the disease site. [0026] FIG. 6 illustrates a further aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is in equilibrium with serum albumin in the circulatory system, and these bound ADV molecules in the circulatory system are in equilibrium with localization to the disease site.

[0027] FlG. 7 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is bound to a target at the disease site, undergoes cleavage outside of the disease cell resulting in a bound targeting moiety and a released therapeutic moiety, which is internalized into the disease cell.

|0028| FIG. 8 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is bound to a target at the disease site and is then internalized intact into the disease cell, where it undergoes cleavage a targeting moiety and a therapeutic moiety which are no longer linked.

[0029] FIG. 9 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is bound to a target at the disease site, undergoes cleavage outside of the disease cell resulting in a bound targeting moiety and a released therapeutic moiety, which are both internalized into the disease cell.

[00301 FIG. 10 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is bound to a target at the disease site and the ADV with bound target is internalized intact into the disease cell, where it undergoes cleavage a targeting moiety bound to target and a therapeutic moiety which are no longer linked.

[0031] FIG. 11 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is localized at the disease site, undergoes cleavage outside of the disease cell resulting in a targeting moiety and a released therapeutic moiety, which are both internalized into the disease cell.

[0032] FIG. 12 illustrates an aspect of the present disclosure wherein the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, is localized at the disease site and is then internalized intact into the disease cell, where it undergoes cleavage a targeting moiety and a therapeutic moiety which are no longer linked.

[0033] FIG. 13 illustrates an aspect of the present disclosure wherein repeated therapeutic treatment of tumor cells with the ADV, comprising a targeting moiety linked via a cleavable linker to a therapeutic moiety, results in increased extracellular DNA at the tumor site, thus increasing the amount of potential binding sites for the ADV.

[0034] FIG. 14 shows an exemplary use of the present disclosure wherein the compound of Example 8 is used to image cultured cells. The figure shows in panels A-C: RAW 264.7 macrophage cells were incubated for 30 min with either the compound of Example 8 (20 uM) or Hoechst 33258 (20 μM) and imaged by fluorescence microscopy, using an excitation of 340 nm. (Panel A) Live cells treated with the compound of Example 8, (Panel B) Fixed cells treated with the compound of Example 8, and (Panel C) live cells treated with Hoechst 33258. (0035) FiG. 15 shows an exemplary use of the present disclosure wherein the compound of Example 8 is used to image tissue necrosis after myocardial infarction. (Panel A) Micrograph of a heart isolated from a mouse subjected to ischemia reperfusion injury and treated with the compound of Example 8 showing accumulation in the necrotic zone of the infarct. (Panel B) Micrographs of mouse hearts subjected to ischemia reperfusion injury ("infarct") + the compound of Example 8, sham surgery treatment + the compound of Example 8, or sham surgery treatment + saline injection ("No Hoechst-IR"). (Panel C) Heart tissue with infarct injury show an approximately two-fold increase in accumulation of the compound of Example 8 compared to sham treated heart tissue.

[0036] FIG. 16 shows an exemplary use of the present disclosure wherein the compound of Example 8 is used to image tissue necrosis caused by LPS-induced tissue necrosis. (Panel A) (i) Representative fluorescent image of mouse treated with an intraperitoneal injection of LPS (2.5 μg/kg), followed by an i.v. injection of the compound of Example 8 (5 mg/kg), (ii) representative fluorescent image of mouse treated with an intraperitoneal injection of saline, followed by an i.v. injection of the compound of Example 8 (5 mg/kg). (Panel B) Quantification of fluorescence intensities of mice treated with LPS and saline. Mice treated with LPS have a 3-4 fold increase in fluorescence over mice treated with saline (*P < 0.05, n = 5).

[0037J FIG. 17 shows an exemplary DNA binding plot of the compound of Example 8 against oligonucleotide. [0038] FIG. 18 shows an exemplary ¹H NMR of the compound of Example 8.

[0039| FlG. 19 shows exemplary data and uses of the compound of Example 14. (Panel A) Live or methanol fixed monolayer cells (HT29) were treated with either the compound of Example 14 ("H-gemcitabine") or Hoechst 33258. (Panels B and C) HT29 monolayer cell cultures were treated with either H-gemcitabine, free gemcitabine or the compound of Example 12 ("gemcitabine-SH") for 4 or 24 hours (as indicated) and cell viability was determined by the MTT assay (mean ± STD, n=9). (Panel D) Extracellular DNA of spheroids with different diameters (-100 μm, -300 μm, and -400 μm) were measured using the fluorescence intensity of Hoechst 33258, after treatment with H-gemcitabine, (mean ± STD, n=5). (Panel E) Confocal fluorescence Z-sliced image of the middle section of a tumor spheroid treated with H-gemcitabine (10 μM for 2hr). Dashed line represents area of spheroid, images of spheroid taken at two different radial locations (square images).

[0040] FIG. 20 shows exemplary data and uses of the compound of Example 14. (Panel A) Spheroids were treated with either 10 μM of the compound of Example 14 ("H- gemcitabine") or gemcitabine and E-DNA (extracellular DNA or DNA in dead cells) was measured using propidium iodide (PI) (mean ± STD, n=9, *p<0.05 vs. gemcitabine). (Panel B) Concentration of H-gemcitabine was measured by Hoechst 33258 fluorescence. (Panel C) Tumor spheroids were treated with one or four consecutive doses of H- gemcitabine; each dose was given at a 10 μM concentration for 2 hours. Spheroids were imaged by confocal microscopy to measure H-gemcitabine accumulation. (Panel D) Cell killing efficacy of H-gemcitabine in tumor spheroids as a function of dose number (10 μM) (mean^'± STD, n=9, *p<0.05 vs. gemcitabine).

[0041] FIG. 21 shows exemplary data and uses of the compound of Example 14. (Panel A) Comparison of the amount of E-DNA (extracellular DNA or DNA in dead cells) determined from propidium iodide fluorescence as a result of treatment with either the compound of Example 14 ("H-gemcitabine") or gemcitabine (mean ± STD, n=7, *p<0.05 vs. gemcitabine). (Panel B) Tumor bearing nude mice (implanted HT29 tumor cells) were injected with 10 mg/kg of gemcitabine equivalents once every three days for 12 days (mean ± STD, n=9, *p<0.05 vs gemcitabine). (Panel C) Tumor growth inhibition results plotted as relative tumor growth wherein the tumor size of each group was compared with its starting tumor size. (Panel D) Survival rates for the treatments as shown (mean ± STD, n=15,*p<0.05 vs gemcitabine on 30 days).

|0042] FIG. 22 shows exemplary data for the binding of the compound of Example 14 to oligomeric DNA. [0043] Additional advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. Other advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0045] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described. [0046] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entireties, for the purpose of describing and disclosing the constructs and methodologies presented in those publications, which might be used in connection with the methods of this disclosure. Any publications and patents discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure, for example, by virtue of prior invention. To the extent that any definition or usage provided by any document incorporated herein by reference conflicts with the definition or usage provided herein, the definition or usage provided herein controls.

[0047] In any application before the United States Patent and Trademark Office, the Abstract of this application is provided for the purpose of satisfying the requirements of 37 CF R. § 1.72 and the purpose stated in 37 C.F.R. § 1.72(b) "to enable the United States Patent and Trademark Office and the public generally to determine quickly from a cursory inspection the nature and gist of the technical disclosure." Therefore, the Abstract of this application is not intended to be used to construe the scope of the claims or to limit the scope of the subject matter that is disclosed herein. Moreover, any headings that are employed herein are also not intended to be used to construe the scope of the claims or to limit the scope of the subject matter that is disclosed herein. Any use of the past tense to describe an example otherwise indicated as constructive or prophetic is not intended to reflect that the constructive or prophetic example has actually been carried out.

Definitions

[0048] To define more clearly the terms used herein, the following definitions are provided. Unless otherwise indicated, the following definitions are applicable to this disclosure. If a term is used in this disclosure but is not specifically defined herein, the definition from the IUPAC Compendium of Chemical Terminology, 2^nd Ed (1997) can be applied, as long as that definition does not conflict with any other disclosure or definition applied herein, or render indefinite or non-enabled any claim to which that definition is applied. To the extent that any definition or usage provided by any document incorporated herein by reference conflicts with the definition or usage provided herein, the definition or usage provided herein controls. [0049] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component," "a compound," or "a dosage form" includes mixtures of two or more such components, compounds, or dosage forms, and the like. |0050] Unless indicated otherwise, when a range of any type is disclosed or claimed, for example a range of the number of carbon atoms in a particular structure, a range of temperatures, and the like, it is intended to disclose or claim individually each possible number that such a range could reasonably encompass, including any sub-ranges encompassed therein. For example, when describing a range of the number of carbon atoms, each possible individual integral number and ranges between integral numbers of atoms that the range includes are encompassed therein. Thus, by disclosing a Ci to Cio alky I group or an alkyl group having from 1 to 10 carbon atoms or "up to" 10 carbon atoms, Applicants' intent is to recite that the alkyl group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, and these methods of describing such a group are interchangeable.

When describing a range of measurements such as temperatures, every possible number that such a range could reasonably encompass can, for example, refer to values within the range with one significant digit more than is present in the end points of a range. In this example, the disclosure that a temperature can be between 5O⁰C and 6O⁰C is intended to individually recite temperatures of 50⁰C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59⁰C, and 6O⁰C. Applicants' intent is that these two methods of describing the range are interchangeable. Moreover, when a range of values is disclosed or claimed, which Applicants intent to reflect individually each possible number that such a range could reasonably encompass, Applicants also intend for the disclosure of a range to reflect, and be interchangeable with, disclosing any and all sub-ranges and combinations of sub- ranges encompassed therein. In this aspect, Applicants' disclosure of a Ci to Cio alkyl group is intended to literally encompass a Ci to Cβ alkyl, a C₄ to Q alkyl, a combination of a Ci to C3 and a C5 top C7 alkyl, and so forth. Accordingly, Applicants reserve the right to proviso out or exclude any individual members of any such group, including any subranges or combinations of sub-ranges within the group, if for any reason Applicants choose to claim less than the full measure of the disclosure, for example, to account for a reference that Applicants are unaware of at the time of the filing of the application. [0051] For any particular compound disclosed herein, the general structure presented is also intended to encompasses all conformational isomers and stereoisomers that can arise from a particular set of substituents, unless indicated otherwise. Thus, the general structure encompasses all enantiomers, diastereomers, and other optical isomers whether in enantiomeric or racemic forms, as well as mixtures of stereoisomers, as the context permits or requires. For any particular formula that is presented, any general formula presented also encompasses all conformational isomers, regioisomers, and stereoisomers that can arise from a particular set of substituents.

[0052] As used herein, the terms "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the term "optionally substituted," as used herein, means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent the substituents can be the same or different.

[0053) As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described hereinbelow. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms "substitution" or "substituted with" include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0054] A "residue" of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Examples of chemical species include therapeutic agents, imaging agents or diagnostic agents.

IOO55| Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or seal em ic mixtures.

[0056] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis/trans (EIZ) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

[0057] The term "substantially" as used herein can be applied to modify any quantitative representation which could permissibly vary without resulting in a change in the basic function to which it is related. For example, the term "substantially pure" is intended to refer to a mixture wherein the desired compound is present in from about 70% to about 100% parts by weight, e.g., 75%, 80%, 90%, 95%, 99%. The term "substantially enantiopure" is intended to refer to a mixture of chiral isomers (e.g., enantiomers, diastereomers, meso compounds, and the like) wherein one compound in the mixture is present in about 70%, or about 80%, or about 85%, or about 90%, or about 95% parts by weight.

[0058] The term "enantiomeric excess" is intended to refer to the absolute difference between the mole fraction of each enantiomer in an enantiomeric mixture. Thus, enantiomeric excess exists when one enantiomer in a mixture of enantiomers is present in a greater amount than the other(s). As an example, a sample with 70% of an R isomer and 30% of an S will have an enantiomeric excess of 40%. This can also be thought of as a mixture of 40% pure R with 60% of a racemic mixture (which contributes 30% R and 30% S to the overall composition).

[0059] The term "hydrolysable residue" is meant to refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, "Protective Groups in Organic Synthesis", T. W. Greene, P. G. M. Wuts, Wiley-lnterscience, 1999). [0060] The term "leaving group" is meant to refer to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, brosylate, and halides.

[0061] Certain instances of the above defined terms can occur more than once in the structural formulae, and upon such occurrence each term shall be defined independently of the other. For example, if more than one compound is represented by a formula comprising an Rl substituent, each Rl of each formula shall be treated independently. Thus, if Rl is defined as alkyl in one instance, Rl is not necessarily alkyl in another instance.

|0062| As used herein, and without limitation, the term "derivative" is used to refer to any compound which has a structure derived from the structure of the compounds disclosed herein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected, by one skilled in the art, to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. For example, a derivative can be a prodrug, a metabolite, or a pharmaceutically acceptable derivative.

[0063] The term "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., without causing any undesirable biological effects or interacting in a deleterious manner.

[0064] The term "pharmaceutically acceptable derivative" refers to any homolog, analog, or fragment corresponding to the disclosed compounds which can have positive therapeutic effect at the disease site in a manner similar to the parent or related compound. In one aspect, a "pharmaceutically acceptable derivative" corresponding to the disclosed which can target the compounds of the present disclosure to a desired disease site, have the desired therapeutic effect at a disease site, or provide appropriate diagnostic effects at the desired disease site. In a further aspect, a "pharmaceutically acceptable derivative" may bind DNA. In another aspect, a "pharmaceutically acceptable derivative" may bind serum albumin. In yet another aspect, a "pharmaceutically acceptable derivative" may have cytotoxic effects. A "pharmaceutically acceptable derivative," for example, includes any pharmaceutically acceptable salζ ester, amide, salt of an ester or amide, or other derivative of a disclosed compound.

[0065J The term "modulate" or "modulating" refers to the ability of an agent to regulate a desired response, e.g., inhibiting cellular proliferation including cell killing. Modulate, as used herein, can refer to a process by which an agent elevates or reduces, or increases or decreases, a desired response. Modulate refers to the ability of an agent to regulate a response either directly or indirectly. Modulate can refer to a process by which an agent substantially inhibits, stabilizes, or prevents a response when a response would otherwise increase. Modulate can also refer to a process by which an agent substantially stabilizes, enhances, or maintains a response when a response would otherwise decrease. Thus, compounds disclosed herein as tumor modulators, can function as inhibitory agents, for example. Included within "inhibitory agents" is a preventative agent, i.e. a compound capable of eliminating uncontrolled cellular proliferation.

[0066] A "cleavable linker" is intended to refer to a chemical moiety within the linker which may be cleaved a number of different chemical reactions under physiological conditions comprising chemical reduction, hydrolysis under physiological conditions, disulfide exchange, nucleophilic substitution or by the action of various proteins, enzymes, or other biological materials in either the intracellular or extracellular environment.

[0067| A "cytotoxic" substance is intended to refer to a substance that imparts a toxic effect on a cell. In one aspect, a toxic effect can be cell killing. In a further aspect, a toxic effect can induce cellular necrosis. In yet a further aspect, a toxic effect can induce cellular apoptosis. In a further aspect, a cytotoxic substance is toxic against a certain cell (e.g., a tumor cell) and non-toxic, or not as toxic, against other cells (e.g., non-tumor cells).

[0068] As used herein, "ICjo," is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, an IC50 can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein.

[0069] The term "contacting" as used herein refers to bringing a disclosed compound and a cell or other biological entity together in such a manner that the compound can affect the progression of the disease state (e.g., a cell, a tissue, etc.), either directly; i.e., by interacting with a living cell which is causing in part in whole the pathophysiological state itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent. In one aspect, a biological entity that is contacted is DNA. In a further aspect, DNA is accessible either in the extracellular space, in the circulatory system, or in non-intact cells. In yet a further aspect, a biological entity which is contacted is serum albumin.

[0070] As used herein, the teπn "cancer" means any condition characterized by cells displaying uncontrolled growth, invasion of normal tissue, and/or metastasis.

[0071] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0072] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side affects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a

"prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.

[0073] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of

microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0074 j As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0075J As used herein, the term "prevent" or "preventing" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0076] As used herein, "diagnosed" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, "diagnosed with a disorder associated with uncontrolled cellular proliferation" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can favorably affect uncontrolled cellular proliferation (e.g., a spliceosome modulator). [0077J Disclosed are the components to be used to prepare the compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds can not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C- E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods.

[0078] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

[0079] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order.

Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

Mode of Action

[0080] The compounds of the present disclosure may act in one or modes of action when delivered to the body. In one aspect, the compounds of the present disclosure comprise a targeting moiety is linked to one or more of a therapeutic moiety and/or diagnostic moiety. The entire compound may be referred to as either an "autocatalytic drug delivery vehicle" or an "autocatalytic delivery vehicle" (collectively or individually referred to as "ADV"). In one aspect, the ADV may comprise a targeting moiety linked via a cleavable linker to one or more of a therapeutic moiety and/or diagnostic moiety. In another aspect, the ADV may comprise a targeting moiety linked via a non-cleavable or very slowly cleavable linker to one or more of a therapeutic moiety and/or diagnostic moiety. In a further aspect, the diagnostic moiety may comprise an imaging agent. As discussed in the present disclosure, these compounds may be administered to a human or an animal via oral, injection, inhalation and multiple other routes of delivery which result in distribution of some portion of the delivered compound into the circulatory system of the human or animal.

[0081] In one aspect, the ADV is in equilibrium between an unbound state and bound to either serum albumin or a target in the circulatory system, and the bound ADV molecules are in equilibrium with binding to the target at the disease site. An embodiment comprising this mode of action is shown in Figure 3. In another aspect of the present disclosure the ADV is in equilibrium with either serum albumin or DNA in the circulatory system, and these bound ADV molecules in the circulatory system are in equilibrium with binding to DNA at the disease site. An embodiment comprising this mode of action is shown in Figure 4. In a further aspect of the present disclosure the ADV is in equilibrium with either serum albumin or DNA in the circulatory system, and these bound ADV molecules in the circulatory system are in equilibrium with localization to the disease site, wherein the ADV molecules may preferentially localize or distribute to the disease site. An embodiment comprising this mode of action is shown in Figure 5. In a further aspect of the present disclosure, the ADV is in equilibrium with serum albumin in the circulatory system, and these bound ADV molecules in the circulatory system are in equilibrium with localization to the disease site. An embodiment comprising this mode of action is shown in Figure 6.

[0082] Tn one aspect of the present disclosure, the ADV is bound to a target at the disease site, undergoes cleavage outside of the disease cell resulting in a bound targeting moiety and a released therapeutic moiety, which is internalized into the disease cell. An embodiment comprising this mode of action is shown in Figure 7. In a further aspect of the present disclosure the ADV is bound to a target at the disease site and is then internalized intact into the disease cell, where it the cleavable linker undergoes cleavage to release a targeting moiety and a therapeutic moiety which are no longer linked. An embodiment comprising this mode of action is shown in Figure 8. In yet another aspect of the present disclosure, the ADV is bound to a target at the disease site, undergoes cleavage outside of the disease cell resulting in a bound targeting moiety and a released therapeutic moiety, which are both internalized into the disease cell. An embodiment comprising this mode of action is shown in Figure 9. In another aspect of the present disclosure the ADV is bound to a target at the disease site and the ADV with bound target is internalized intact into the disease cell, where it undergoes cleavage a targeting moiety bound to target and a therapeutic moiety which are no longer linked. An embodiment comprising this mode of action is shown in Figure 10. In a further aspect of the present disclosure, the ADV is localized at the disease site, undergoes cleavage outside of the disease cell resulting in a targeting moiety and a released therapeutic moiety, which are both internalized into the disease cell. An embodiment comprising this mode of action is shown in Figure 1 1. In yet a further aspect of the present disclosure, the ADV is localized at the disease site and is then internalized intact into the disease cell, where it undergoes cleavage a targeting moiety and a therapeutic moiety which are no longer linked. An embodiment comprising this mode of action is shown in Figure 12.

[0083] In another aspect of the present disclosure, it may be important that the linker is not cleavable or only slowly cleavable under physiological. In a further aspect of the present disclosure, the linker provides linkage between a targeting moiety and a diagnostic moiety. In yet a further aspect, the diagnostic moiety is an imaging agent. [0084] In one aspect of the present invention, the ADV is bound to at least one of serum albumin or a target. In another aspect, the target comprises DNA. In a further aspect, the binding of ADV to serum albumin and/or target provides a reservoir which maintains an elevated and constant concentration of ADV. In this aspect, the therapeutic and/or diagnostic moiety present in the ADV is maintained a more beneficial, efficacious, or useful level in the circulatory system compared to free therapeutic and/or diagnostic agent. In a further aspect, the therapeutic and/or diagnostic moiety present in the ADV is maintained for a longer period of time in the circulatory system compared to free therapeutic and/or diagnostic agent. In yet a further aspect, the bound ADV is less toxic or has fewer adverse effects to the patient or animal receiving the ADV compared to administration of the free therapeutic and/or diagnostic agent. [0085] In one aspect, the action of a therapeutic agent at the disease, either present in the ADV or as a therapeutic agent co-administered with the ADV, enhances the effect of the ADV with each treatment of the therapeutic agent. In another aspect, the therapeutic agent, either present in the ADV or as a therapeutic agent co-administered with the ADV, causes increased necrosis of cells at the disease site. In a further aspect, the increased necrosis results in increased levels of the target to which the targeting moiety of the ADV is directed. In another aspect, the therapeutic agent, either present in the ADV or as a therapeutic agent co-administered with the ADV, causes increased apoptosis of cells at the disease site. In yet another aspect, the increased apoptosis results in increased levels of the target to which the targeting moiety of the ADV is directed. In a further aspect, the target which is increased by necrosis and/or apoptosis is DNA. An embodiment comprising the enhanced effect of the ADV with each treatment of the therapeutic agent is shown in Figure 13. In a further aspect, the necrotic cells are within a tumor or cancer site. In yet a further aspect, the cells most refractory to treatment of the tumor or cancer are in proximity to the necrotic cells. In still a further aspect, the ADV provides enhanced treatment of cells in a tumor or cancer that are normally refractory to treatment. The figure illustrates embodiment of the present disclosure wherein repeated therapeutic treatment of tumor cells with the ADV results in increased extracellular DNA at the tumor site, thus increasing the amount of potential binding sites for the ADV.

Methods of making the compounds

[0086] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or synthesized using techniques generally known to those of skill in the art or by methods disclosed herein. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N. J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fiese^s Reagents for Organic Synthesis, Volumes 1- 17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH

Publishers Inc., 1989).

[0087] In the reaction schemes of the present disclosure, the conditions may be altered as needed to optimize yields as would be determined by one skilled in the art. Unless otherwise noted, the reactions take place overnight (for about 12-18 hours) at room temperature (about 15-30°C) with stirring. The molar ratio of TBTU and/or DlEA to reactant is about 1:1, but can vary from 0.25-2 to 1. When reaction products are isolated, flash chromatography on silica gel or preparative HPLC may be used. Other isolation methods may be used as determined by one skilled in the art.

[0088J In one aspect, the synthesis of compounds in the present disclosure can be accomplished in one aspect starting with the following:

The commercially available 4-cyanophenol 26 and bromo-PEG-phthalimide 27 are mixed under argon atmosphere, and then anhydrous K2CO3 is added followed by 18-crown-6. The reaction mixture is heated at 8O⁰C for 5 h, and was then allowed to cool to room temperature. The solvent was concentrated in vacuo and the resulting residue was partitioned between water and ethyl acetate. The methods used are generally described by

W. S. Yeo, D. H. Min, R. W. Hsieh, G. L. Greene, and M. Mrlcsich, (Angew Chew Int Ed Engl 2005, 44, 5480). The imidate ester 29 was synthesized by bubbling anhydrous HCl gas into a solution of 28 in anhydrous EtOH for 30 min. The general conditions for the formation of the imidate ester have been described by D. P. Arya and B. Willis {J Am Chem Soc 2003, 125, 12398). The subsequent steps and general reaction conditions are given in the scheme above, and also described in the exemplary embodiments.

[0089J In another aspect, the compounds of the present invention may generally be synthesized as described in the following reaction scheme:

[009Oj In a further aspect, compounds of the present disclosure may be synthesized as described by the following, wherein n may vary from about 1 to 60:

[0091] In a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following, wherein an amine functional group on R may be modified as shown:

wherein R is a residue comprising a therapeutic agent or a diagnostic agent. In a further aspect, the therapeutic agent comprises an agent for the treatment of a tumor or a cancer. In yet another aspect, the diagnostic agent comprises a fluorescent, PET, MRI, or infrared imaging agent.

(0092) In a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following, wherein an amine functional group on R may be modified as shown:

wherein R is a residue comprising a therapeutic agent or a diagnostic agent, and n may vary from about O to 60. In a further aspect, the therapeutic agent comprises an agent for the treatment of a tumor or a cancer. In yet another aspect, the diagnostic agent comprises a fluorescent, PET, MRl, or infrared imaging agent.

[0093] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[0094J In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[0095] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[0096] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[0097] In a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[0098] In yet a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[0099] In one aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[OOIOOJ In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00101] In still another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00102] In yet a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00103] In an additional aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00104] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following.

[001051 1° Y^et another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00106] In yet further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00107] In an additional aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00108] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00109] In yet an additional aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00110] In a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00111] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00112] In yet another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00113] In yet an additional aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00114] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00115) 1° ^a further aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00116] In another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

[00117] In yet another aspect, the synthesis of compounds of the present disclosure may utilize the synthetic steps described in the following:

Compounds

1. Structure

[00118] As summarized above, the present disclosure compositions for the improved delivery and efficacy of therapeutic agents comprising in one aspect compounds comprising the formula below:

(B)n-L-(T)m

wherein B is a targeting moiety is chemically linked via a linker, L₁ which is chemically linked to T, wherein T is a therapeutic moiety, and wherein n and m may be independently varied from about 1-3. A schematic representation of an embodiment of a compound of the formula above is shown in Figure 1. [00119] In another aspect, the disclosure provides for compositions for the improved diagnosis of disease comprising in another aspect compounds comprising the formula below:

(B)n-L-(I)m

[00120] wherein B is a targeting moiety is chemically linked via a linker, L, which is chemically linked to I, wherein I is a diagnostic moiety, and wherein n and m may be independently varied from about 1-3. A schematic representation of an embodiment of a compound of the formula above is shown in Figure 1.

[00121] In a further aspect, the disclosure provides for compositions for both the improved delivery and efficacy of therapeutic agents, and improved diagnosis of disease comprising:

(B)n-L'-(I)m-L"-(T)x

[00122] wherein B is a targeting moiety is chemically linked via a linker, L', which is chemically linked to I, wherein I is a diagnostic moiety, which is chemically linked via a linker, L", to T, wherein T is a therapeutic moiety, and wherein n,m, and x may be independently varied from about 1-3.

[00123] In one aspect, the compound may include a linker, such as L, L' or L" in the preceding formulas, which can be cleaved under physiological conditions. In another aspect, the linker is an optionally substituted polyether or other biocompatible chemical linker which may further comprise branching moieties. In a further aspect, the linker may comprise amide, ester or other sites which may undergo hydrolysis under physiological conditions. In yet a further aspect, the linker may comprise a disulfide bond which may undergo sulfhydryl exchange under physiological conditions. Moieties may be added to the linker to modify the rate of cleavage, including hydrolysis or sulfhydryl exchange, under physiological conditions.

[00124] In another aspect of the present disclosure comprises a compound of the structure represented by the formula:

wherein R is a residue comprising a therapeutic agent or a diagnostic agent and n may be varied from 0 to about 60. In a further aspect, the therapeutic agent comprises an agent for the treatment of a tumor or a cancer. In yet another aspect, the diagnostic agent comprises a fluorescent, PET, MRI, or infrared imaging agent.

[00125] In a further aspect of the present disclosure comprises a compound of the structure represented by the formula:

[00126] In yet a further aspect of the present disclosure comprises a compound of the structure represented by the formula:

[00127] In another aspect of the present disclosure comprises a compound of the structure represented by the formula:

wherein R is a residue comprising a therapeutic agent or a diagnostic agent and n may be varied from 0 to about 60. In a further aspect, the therapeutic agent comprises an agent for the treatment of a tumor or a cancer. In yet another aspect, the diagnostic agent comprises a fluorescent, PET, MRl, or infrared imaging agent.

[00128] In one aspect, the compound can be present as:

In a further aspect, the compound can be present as:

In one aspect, the compound can be present as:

131] In another aspect, the compound may be present as:

r

2. Anti-Cancer Activity

[00132| The disclosed compounds can have anticancer activity, and thus can be effective at treating one or more cancers and/or related disorders. In one aspect, the compound is capable of inhibiting the proliferation of at least one cell (e.g., a cancer cell). A disclosed compound can also be cytotoxic against at least one cancer cell line (e.g., a lymphoma cell line). For example, the compound can be effective at treating

overproliferation solid tumor such as HT-29 cells..

[00133] The disclosed compounds can be efficacious against a variety of cancer and related cell lines. In one aspect, the disclosed compounds can be used to treat solid tumors. If a cell line is selected from HT-29 cells, for example, a compound disclosed herein can exhibit an IC_J0 value of about 0.010, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.100, 0.500, 1, 5, 10, 20, 30, 50, 75, 100, or 250 μM against the cell line. In another aspect, the cells are cultured in vitro. In a further aspect, the cultured cells are treated with a disclosed compound for 4 hours. In an additional aspect, the cultured cells are treated with a disclosed compound for 24 hours. In yet another aspect, the cultured cells are treated for about 24, 48, 72 or 96 hours. In one aspect, the compound exhibits an ICso value of about 0.1 μM against a cancer or tumor cell line after treatment for 24 hours. In one aspect, the compound exhibits an IC50 value of about 0.5 μM against a cancer or tumor cell line after treatment for 24 hr. In a further aspect, the compound exhibits an IC50 value of about 1.0 μM against a cancer or tumor cell line after treatment for 24 hr. In yet a further aspect, the compound exhibits an IC50 value of about 5 μM against a cancer or tumor cell line after treatment for 24 hr. In an additional aspect, the compound exhibits an IC 50 value of about 10 μM against a cancer or tumor cell line after treatment for 24 hr. In one aspect, the compound exhibits an IC50 value of about 20 μM against a cancer or tumor cell line after treatment for 24 hr. In another aspect, the compound exhibits an IC₅₀ value of about 50 μM against a cancer or tumor cell line after treatment for 24 hr. In a further aspect, the compound exhibits an IC₅0 value of about 100 μM against a cancer or tumor cell line after treatment for 24 hr.

[00134] In another aspect, the activity of the disclosed compounds are tested in cell lines cultured in vitro, and the IC₅₀ dose obtained from such experiments are used to determine appropriate doses for human use. In another aspect, the data from such experiments are used to assess the relative activity of the disclosed compounds. In a further aspect, the activity of the disclosed compounds are tested in xenograft tumor models utilized nude mice. The methods for such studies are known to one skilled in the art. In one aspect, the xenograft tumors are established by subcutaneous injection. In another aspect, the xenograft tumors are established orthotopically. In a further aspect, the xenograft tumors may be established with HT-29 cells.

[00135] In another aspect, the activity of a compound can exhibit an IC50 value of about 0.010, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.100, 0.500, 1, 5, 10, 20, 30, 50, 75, 100, or 250 mg/kg in a xenograft tumor model after about 1, 2, 3, 4 or 5 treatments. In one aspect, the treatment is daily. In a further aspect, the xenograft tumor model is established in nude mice. In yet another aspect, the xenograft tumor model is established using HT-29 cells. In one aspect, the compound exhibits an IC50 value of about 0.01 mg/kg against in a xenograft tumor model. In a further aspect, the compound exhibits an IC₅₀ value of about 0.05 mg/kg against in a xenograft tumor model. In another aspect, the compound exhibits an IC₅₀ value of about 0.1 mg/kg against in a xenograft tumor model. In an additional aspect, the compound exhibits an IC50 value of about 5 mg/kg against in a xenograft tumor model. In one aspect, the compound exhibits an IC jo value of about 10 mg/kg against in a xenograft tumor model. In one aspect, the compound exhibits an IC₅₀ value of about 20 mg/kg against in a xenograft tumor model. In yet another aspect, the compound exhibits an IC50 value of about 50 mg/kg against in a xenograft tumor model. In a further aspect, the compound exhibits an IC$o value of about 100 mg/kg against in a xenograft tumor model.

3. Diagnostic Activity

[00136] The disclosed compounds can be used for diagnosis of disease. In one aspect, the disease results in cell necrosis. Necrotic tissue is a central characteristic of numerous diseases including sepsis, cancer, cardiac dysfunction, atherosclerosis and several others. In one aspect, cell necrosis results in the disruption of the cell membrane in pathophysiologic conditions, which causes the release of the cellular contents, including genomic DNA, into the extracellular space. In another aspect, the disrupted cell membrane resulting from necrosis allows increased access to intracellular components, including genomic DNA. In a further aspect, the accessible DNA in the extracellular space or in cells with disrupted membranes increases with necrosis and can be target for the disclosed compounds. In another aspect, the disclosed compounds show a preferential localization and binding to accessible DNA in the extracellular space or in cells with disrupted membranes in necrotic tissue. In an additional aspect, the disclosed compounds are used to diagnosis the presence of necrotic tissue. In a further aspect, the disclosed compounds are used for diagnosis in the human to provide clinically useful information on the disease state. In an additional aspect, the disease state is sepsis, cancer, cardiac dysfunction, or atherosclerosis. In another aspect, the disease state is associated with cellular necrosis.

[00137] In another aspect, the disease results in increased cell apoptosis at the disease site. In one aspect, cell apoptosis results in the disruption of the cell membrane in certain pathophysiologic conditions, which causes the release of the cellular contents, including genomic DNA, into the extracellular space. In another aspect, the disrupted cell membrane resulting from pathophysiological, increased or abnormal apoptosis allows increased access to intracellular components, including genomic DNA, at the disease site. In a further aspect, the accessible DNA in the extracellular space or in cells with disrupted membranes increases with pathophysiological, increased or abnormal apoptosis and can be target for the disclosed compounds. In another aspect, the disclosed compounds show a preferential localization and binding to accessible DNA in the extracellular space or in cells with disrupted membranes in tissue with pathophysiological, increased or abnormal apoptosis. In an additional aspect, the disclosed compounds are used to diagnosis the presence of pathophysiological, increased or abnormal apoptosis. In a further aspect, the disclosed compounds are used for diagnosis in the human to provide clinically useful information on the disease state. In an additional aspect, the disease state is sepsis, cancer, cardiac dysfunction, or atherosclerosis. In another aspect, the disease state is associated with pathophysiological, increased or abnormal apoptosis. [00138] In a further aspect, the diagnostic moiety may comprise an imaging agent further comprising an isotope provided in Table I below.

Table 1

Pharmaceutical Compositions

[00139] Also disclosed are pharmaceutical compositions, including dosage forms, comprising one or more disclosed compounds. The disclosed compounds can be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

[00140] In one aspect, a dosage form can comprise a therapeutically effective amount of at least one product of the synthetic methods disclosed herein. In a further aspect, a dosage form for administration to a subject can comprise a therapeutically effective amount of any compound disclosed herein and a pharmaceutically acceptable carrier. In a specific aspect, the dosage form can comprise a therapeutically effective amount that can be effective for a mammal, e.g., a human or a mouse. [00141] In one aspect, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions can contain a therapeutically effective amount of one or more spliceosome inhibitors preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should typically suit the mode of administration.

[00142] In one aspect, a composition can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[00143] The pharmaceutical compositions can be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route, the oral route being preferred. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.

[00144] Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings or they can be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art.

[00145] Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.

[00146] Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable formulations are also contemplated as being within the scope of the present invention.

[00147] Other suitable administration forms include suppositories, sprays, ointments, creams, gels, inhalants, dermal patches, implants etc.

[00148] For parenteral administration, solutions of the compounds in sterile aqueous solution, aqueous propylene glycol or sesame or peanut oil can be employed. Such aqueous solutions should be suitable buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

[00149] Lt is understood that the pharmaceutical compositions can be used in connection with the methods and compounds disclosed herein.

Methods of Using the Compounds and Compositions

1. Treatment of a disorder

[00150] Diseases and disorders involving uncontrolled cellular proliferation or cell overproliferation that can be treated or prevented include but are not limited to cancers, premalignant conditions (e.g., hyperplasia, metaplasia, dysplasia), benign tumors, hyperproliferative disorders, and benign dysproliferative disorders. Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne. Malignancies and related disorders that can be treated, prevented, managed, amerliorated, particularly metastatic cancer, by administration of a compound of the invention that inhibits ceramidase function as discussed below (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

[00151] In one aspect, the disclosed compounds and/or compositions can be useful for the treatment of a cancer, including, but not limited to, Leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, Lymphoma, Hodgkin's disease, non-Hodgkin's disease, Multiple myeloma, Waldenstrom's macro globulinemia, Heavy chain disease, Solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,

angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, curvical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

[00152] In a further aspect, cancers and related disorders that can be treated or prevented by methods and compositions disclosed herein include but are not limited to the following: Leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic,

myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary

plasmacytoma; Waldenstrom's inacroglobulinemia, monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget 's disease, and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor, pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget' s disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, muco epidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphom, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to pappillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocyte, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer); Wilms 'tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions : The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U. S. A., Inc., United States of America).

2. Determining a therapeutically effective amount

[00153J Toxicity and therapeutic efficacy of the disclosed compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e. g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5O/ED5O. Compounds that exhibit large therapeutic indices can be desirable. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00154] Data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in disclosed herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture experiments. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[00155] Suitable daily doses for the treatment or prevention of a disorder described herein can be readily determined by those skilled in the art. A recommended dose of a compound of a compound disclosed herein can be from about 0.1 mg to about 1000 mg per day, e.g., from about 0.1 to about 500 mg/kg/day, 0.1 to about 250 mg/kg/day, or 0.1 to about 100 mg/kg/day, per kg of body weight, given as a single dose, a single once-a-day dose, or as divided doses throughout a selected time period.

[00156] The anti-cancer activity of the disclosed therapies can be determined by using various experimental animal models of such as the SCID mouse model or nude mice with human tumor grafts known in the art and described in Yamanaka, 2001, Microbiol Immunol 2001; 45 (7): 507-14.

[00157] The disclosed protocols and compositions can be tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific therapeutic protocol is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a protocol, and the effect of such protocol upon the tissue sample is observed.

[00158] A lower level of proliferation or survival of the contacted cells can indicate that the therapy can be effective to treat a selected disorder in a subject. Alternatively, instead of culturing cells from a patient, protocols can be screened using cells of a tumor or malignant cell line. Many assays known in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring 3H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes or cell cycle markers; cell viability can be assessed by trypan blue staining, while differentiation can be assessed visually based on changes in morphology, etc.

[00159] Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, etc. The principle animal models for cancer known in the art and widely used include mice, such as described in Hann et al, 2001, Curr Opin Cell Biol 2001, 13 (6): 778-84, which is incorporated herein by reference in its entirety.

[00160] Further, any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of the combinatorial therapies disclosed herein for treatment, prophylaxis, management or amelioration of one or more symptoms associated with the disease or disorder as described hereinabove. 3. Co-therapeutic use

[00161] [00164] In one aspect, other cancer treatments can be used in combination with the administration of one or more compounds disclosed herein. Such treatments include the use of one or more molecules, or compounds for the treatment of cancer (i. e., cancer therapeutics). Some examples include, but are not limited to, chemoagents,

immunotherapeutics, cancer vaccines, anti-angiogenic agents, cytokines, hormone therapies, gene therapies, biological therapies, and radiotherapies. While maintaining or enhancing efficacy of treatment, preferably the methods of the present invention increase patient compliance, improve therapy and/or reduce unwanted or adverse effects.

[00162] In one aspect, the methods of the invention includes the administration of one or more angiogenesis inhibitors such as but not limited to: Angiostatin (plasminogen fragment); antiangio genie antithrombin III; Angiozyme; ABT-627; Bay 12- 9566;

Benefin; Bevacizumab; BMS-275291 ; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP- 7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIII fragment); Fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin (hCG); 1M-862; Interferon alpha/beta/gamma; Interferon inducible protein (IP-IO); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinase inhibitors (TIMPs); 2- Methoxyestradiol; MMI 270 (CGS 27023 A); MoAb IMC-ICl 1; Neovastat; NM-3;

Panzem; PI-88; Placenta ribonuclease inhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-related protein (PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SUl 1248; Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1 (TSP- 1); TNP- 470; Transforming growth factor-beta (TGF-β); Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474; farnesyl transferase inhibitors (FTI); and bisphosphonates.

(00163] Additional examples of anti-cancer agents that can be used in the various aspects disclosed herein, including pharmaceutical compositions and dosage forms disclosed herein, include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busuifan; cactinomycin; calusterone; caracemide; carbetimer, carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interieukin 11, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;

mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;

perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin;

plomestane; porfϊmer sodium; porfϊromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin;

spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride;

temoporftn; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa;

tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;

zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to:

20-epi-l, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin 111 derivatives; balanol; batimastat; BCR/ ABL antagonists; benzochlorins; benzoyl staurosporine; beta lactam derivatives; beta-alethine; betaclamycin

B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;

bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox 1L-2;

capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN

700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS);

castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide;

cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;

cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone;

dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;

diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;

fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-I receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane;

iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin

B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum

compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;

losoxantrone; lovastatin; loxoribine; lurtotecan; lutecium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin;

methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogues;

mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;

molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor I- based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;

paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol;

panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol;

phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine

hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RlI retinamide; rogletimide; rohitukine;

romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofϊran; sobuzoxane; sodium borocaptate; sodium phenyl acetate; solverol; somatomedin binding protein; sonermin; sparfosic acid;

spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squal amine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene;

tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm;

temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors;

tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus- derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfrn;

vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Preferred additional anti-cancer drugs are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and a topoisomerase inhibitor.

[00164] Fn a further aspect, the treatment methods disclosed herein includes the administration of one or more immunotherapeutic agents, such as antibodies and immunomodulators, which include, but are not limited to, HERCEPTINS, RITUXANS, OVAREX™, PANOREX@, BEC2, IMC-C225, VITAMIN, CAMPATH@ I/H, Smart MI95, LYMPHOCIDE™, Smart I DlO, and ONCOL YM™, rituximab, gemtuzumab, or trastuzumab.

[00165] In a still further aspect, the treatment methods disclosed herein includes administering one or more anti-angiogenic agents, which include, but are not limited to, angiostatin, thalidomide, kringle 5, endostatin, other Serpins, anti-thrombin, 29 kDa N- terminal and 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a 13- amino acid peptide corresponding to a fragment of platelet factor-4 (Maione et al., 1990, Cancer Res. 51 : 2077), a 14-amino acid peptide corresponding to a fragment of collagen 1 <BR>

<BR> (Tolma et al., 1993, J. Cell Biol. 122; 497), a 19 amino acid peptide corresponding to a<BR> fragment of Thrombospondin I (Tolsma et al., 1993, J Cell Biol. 122: 497), a 20-amino acid peptide corresponding to a fragment of SPARC (Sage et al., 1995, J : Cell. Biochem.57: 1329-), or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable derivatives thereof.

[00166] In one aspect, the treatment methods disclosed herein can comprise the use of radiation.

[00167] In a further aspect, the treatment methods further comprises the administration of one or more cytokines, which includes, but is not limited to, lymphokines, tumor necrosis factors, tumor necrosis factor-like cytokines, lymphotoxin-a, lymphotoxin-b, interferon-a, interferon-b, macrophage inflammatory proteins, granulocyte monocyte colony stimulating factor, interleukins (including, but not limited to, inter! eukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CDl 37 ligands, Fas-Fas ligand, 4- IBBL, endothelial monocyte activating protein or any fragments, family members, or derivatives thereof, including

pharmaceutically acceptable salts thereof.

[00168] In a further aspect, the treatment method comprises hormonal treatment.

Hormonal therapeutic treatments comprise hormonal agonists, hormonal antagonists (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRONTM), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, steroids (e. g., dexamethasone, retinoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e. g., mifepristone, onapristone), antiandrogens (e. g., cyproterone acetate), and the like.

|00169] In a further aspect, the disclosure also relates to kits comprising at least one disclosed compound and one or more other therapeutically active compounds, which are usually applied in the treatment of the above mentioned conditions. For example, the disclosed kits can comprise therapeutically effective amounts of one or more disclosed compound and one or anti-cancer agents. The kits can be co-packaged, co-formulated, and/or co-delivered with the anti-cancer agents. For example, a drug manufacturer, a drug reseller, a physician, or a pharmacist can provide a disclosed kit for delivery to a patient. 4. Prophylatic treatment

[00170] In a further aspect, the disclosed compounds, compositions, and methods can be used prophylactically, i.e., to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed above. Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W B.

Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell.

Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of nonneoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder. S. Subjects

[00171] The compounds, compositions, and methods disclosed herein can be useful for the treatment or prevention of one or more disorders associated with elevated cell necrosis and/or apoptosis in a subject, as discussed hereinabove. In general, a subject can be any age, including a fetus. A subject to which a compound or compositions disclosed herein can be administered can be an animal, including but not limited to a mammal, such as a non-primate mammal (e.g., cows, pigs, sheep, goats, horses, chickens, dogs, rats, etc.) and a primate (e.g., a monkey such as a acynomolgous monkey and a human). A subject can also be a laboratory animal (e.g, a mouse, rabbit, guinea pig, fruit fly, etc.).

[00172] In one aspect, a subject can be diagnosed with one or more disorders as discussed herein elsewhere. In a specific aspect, a subject can be diagnosed with one or more disorders as discussed herein elsewhere before the step of administering to the subject a therapeutically effective amount of one more compounds disclosed herein.

[00173] In a further aspect, a subject can be a subject in need of treatment for disorder of uncontrolled cellular proliferation, e.g., cancer. In a still further aspect, a subject can have cancer or a related disorder, as discussed hereinbefore. In one aspect, a subject can be treated prophylactically with a compound or composition disclosed herein, as discussed herein elsewhere.

[00174] One or more compounds or compositions disclosed herein can be utilized for the prevention of a variety of cancers, e. g, in individuals who are predisposed as a result of familial history or in individuals with an enhanced risk to cancer due to environmental factors.

[00175] The methods and compositions of the invention can be used in patients who are treatment naive, in patients who have previously received or are currently receiving treatment with other pharmaceutical agents or combinations, including but not limited to anti-cancer agents. Other subjects can include patients that have metastasis or no metastasis.

[00176] The methods and compositions of the invention are useful not only in untreated patients but are also useful in the treatment of patients partially or completely un- responsive to other treatments. In various aspects, the disclosure provides methods and compositions useful for the treatment of diseases or disorders in patients that have been shown to be or can be refractory or non-responsive to therapies comprising the administration of other agents.

[00177] Fn one aspect, subjects that can be treated with the compositions disclosed herein include those subjects displaying the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a subject, can indicate the desirability of

prophylactic/therapeutic administration of a compound or composition disclosed herein. As mentioned hereinabove, such characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein, etc.

[00178] In a further aspect, a subject that exhibits one or more of the following predisposing factors for malignancy can be treated by administration of an effective amount of a compound disclosed herein: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and

pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular

melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-1 13) etc.).

EXAMPLES

[00179J The present disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort can be had to various other aspects, embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present invention or the scope of the appended claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. If an example below is a prophetic example, it is identified as such.

1. General Methods

[00180| General chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were used as received, unless otherwise specified. S-acetyl-dPEG™8-alcohol was obtained from Quanta BioDesign Ltd. (Powell, OH, USA). 2-Deoxy-2',2'-difluorocytidine was purchased from Carbosynth Ltd. (Berkshire, UK). Dimethyl formamide, ethanol, dichloromethane and methanol and other solvents were purified using standard methods. Flash chromatography was carried out using silica gel (230-400 mesh). NMR spectra were recorded on a (Varian (400 MHz) or Bruker (500 MHz) for ¹H and 100 MHz for ¹³C) spectrometer with chloroform (7.26) or methanol (3.30) as internal reference. Chemical shifts are reported in ppm and multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), and comp (composite).

Coupling constants, J, are reported in hertz (Hz). High-resolution mass spectra (HRMS) were obtained on a Karatos MS9 and are reported as m/z (relative intensity). Accurate masses are reported for the molecular ion (M+) or a suitable fragment ion. Fluorescence spectroscopic studies were performed using Shimadzu (RF-53O1-PC) spectrofluorometer. Unless indicated otherwise, parts are parts by weight, temperature is in ⁰C or is at ambient temperature, and pressure is at or near atmospheric. Treatment groups were analyzed for statistical significance (*p < 0.05) by a one-way analysis of variance (ANOVA), using a post-test analysis where necessary. 2. Synthesis of 4-(2-(2-(2-(l ,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy) benzonitrile:

[00181] To a solution of 4-cyanophenol (1.18 g, 9.9 mmol) and bromo-PEG- phthalimide (3.40 g, 9.9 mmol) in anhydrous DMF (25 mL) under argon atmosphere, was added anhydrous K₂CO₃ (2.74 g, 19.8 mmol) followed by 18-crown-6 (0.26 g, 0.9 mmol). The reaction mixture was heated at 80 ⁰C for 5 h, and was then allowed to cool to room temperature. The solvent was concentrated in vacuo and the resulting residue was partitioned between water and ethylacetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried (NaJSO₄), filtered and concentrated in vacuo. Flash chromatography on silica gel using ethyl acetate-hexanes as an eluant provided the benzonitrile derivative (3.5 g, 9.21 mmol, 92.6% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.75 - 7.71 (m, 2H), 7.64 - 7.61 (m, 2H), 7.47 (d, J= 5.7 Hz, 2H), 6.85 (d, J= 8.5 Hz, 2H), 3.99 (t, J = 4.6 Hz, 2H), 3.80 (t, J = 5.7 Hz, 2H), 3.73 - 3.71 (m, 2H), 3.64 (t, J= 5.8 Hz, 2H), 3.57 (s, 4H); ¹³C NMR (IOO MHZ, CDCl₃): 6 168.0, 161.9, 133.8, 133.7, 131.9, 123.0, 119.0, 115.1, 103.8, 70.6, 70.0, 69.2, 67.8, 67.5, 37.1 ; HRMS (FAB): m/z calcd for C₂₁H₂₁N₂O5 [M+Hf : 381.14505, found: 381.14573.

3. Ethyl 4-(2-(2-(2-(l ,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)benzimidate hydrochloride:

[00182] The imidate esterwas synthesized by bubbling anhydrous HCl gas into a solution of benzonitrile derivate (from Example 2) in anhydrous EtOH for 30 min as previously described by D. P. Arya and B. Willis (J Am Chem Soc 2003, 125, 12398). The reaction mixture was stored at 4⁰C overnight and excess acid was removed by bubbling nitrogen gas through the reaction mixture into a saturated NaHCCh solution. The ethanol was removed under reduced pressure to obtain a gummy residue, which was triturated with diethylether (2 x 10 mL), the resulting solid was collected by filtration, dried in vacuo to obtain the imidate ester as a white solid, which was used for the next reaction without further purification.

4. 2-(2-(2-(2-(4-(5-(4-methylpiperazin-l-yl)-l//,rH-2,5'-bibenzo[</Jimidazol-2'- yl)phenoxy)ethoxy) ethoxy)ethyl)isoindoline-l,3-dione:

[00183] To a solution of imidate ester (from Example 3, 0.12 g, 0.3 mmol) in glacial acetic acid (5 mL) was added 2-(3,4-diaminophenyl)-6-(l-methyl-4- piperazinyl)benzimidazole (0.17 g, 0.3 mmol) and the reaction mixture was refluxed for 15 h under N₂ atmosphere. The benzimidazole compound was prepared as previously described by D. P. Arya and B. Willis (J Am Chem Soc 2003, 125, 12398). The reaction mixture was allowed to cool to room temperature and the acetic acid was removed in vacuo. The resulting reddish brown solid was purified by flash chromatography on silica gel using 12 % MeOH in CH₂Cl₂ containing 0.1 % Et₃N as the eluant to obtain 9 (0.1 g, 0.1 mmol, 38.9% yield) as a pale yellow powder. ¹H NMR (400 MHz, CD₃OD): δ 8.26 (s, IH), 8.04 (d, J = 8.7 Hz, 2H), 7.96 (d, J- 8.7 Hz, IH), 7.81 (dd, J= 5.6, 3.0 Hz, 2H), 7.75 - 7.69 (m, 3H), 7.51 (d, J= 8.8 Hz, IH), 7.15 (s, IH), 7.08 - 7.04 (m, 3H), 4.09 (t, J= 4.9 Hz, 2H), 3.86 (t, J = 5.7 Hz, 2H), 3.79 (t, J= 4.9 Hz, 2H), 3.74 (t, J= 5.7 Hz, 2H), 3.65 (s, 4H), 3.27 - 3.23 (m, 4H), 2.74 (t, J = 4.5 Hz, 4H), 2.42 (s, 3H); ¹³C NMR (100 MHz, CD₃OD): 5 178.3, 168.4, 160.9, 153.9, 152.8, 148.1, 134.0, 132.0, 128.2, 124.3, 122.8, 121.7, 121.2, 1 15.1, 114.8, 70.4, 70.0, 69.4, 67.6, 67.4, 54.7, 50.2, 44.5, 37.2; HRMS (FAB): m/z calcd for C₃₉H₄₀N₇O₅ [M+H]⁺: 686.30909, found: 686.31 119. 5. 2-(2.(2-(4-(5-(4-methylpiperazin-l-yl)-l//,rH-2,5'-bibenzoIr/]iraida2ol-2'- yl)phenoxy)ethoxy) ethoxy)ethanamine hydrochloride:

[00184] To a solution of isoindoline-l,3-dione derivative (from Example 4, 0.10 g, 0.1 mmol) in ethanol (3 mL) was added hydrazine monohydrate (82.6 uL, 1.4 mmol) and the mixture was refluxed for 2 h. After being cooled to it, the solvent was removed in vaaio, generating a brown solid. The excess hydrazine monohydrate was removed by co- evaporating with ethanol (2 x 10 mL). The resulting solid was dissolved in IM HCl (2 mL) and refluxed for 1 h. The reaction mixture was allowed to cool to rt and filtered to remove solid precipitate. The filtrate was concentrated in vacuo to obtain the ethanamine derivative (0.06 g, 0.09 mmol, 97% yield) as a brown solid. ¹H NMR (400 MHz, CD₃OD): δ 8.62 - 8.59 (m, IH), 8.29 (dd, J= 9.0, 1.7 Hz, IH), 8.23 (d, J= 9.0 Hz, 2H), 8.11 (d, J= 8.7 Hz, IH), 7.79 (d, J= 9.1 Hz, I H), 7.48 (dd, J = 9.2, 2.2 Hz, IH), 7.39 - 7.36 (m, 3H), 4.36 - 4.31 (m, 2H), 4.05 - 3.92 (m, 4H), 3.80 - 3.66 (m, 6H), 3.41 - 3.20 (m, 6H), 3.16 - 3.10 (m, 2H), 3.01 (s, 3H); ¹³C NMR (IOO MHz, CD₃OD). δ 165.4, 153.2, 150.7, 148.5, 135.6, 134.0, 133.2, 131.7, 127.1, 126.7, 121.4, 119.6, 117.2, 116.5, 1 15.6, 1 14.9, 1 14.4, 100.3, 71.7, 71.3, 70.4, 69.3, 67.8, 54.3, 48.0, 43.5, 40.6; HRMS (FAB): m/z [M+H]⁺: calcd for C₃iH₃₈N₇O₃556.3031, found: 556.3025.

6. 2-((£>-2-((£)-2-(23-hydroxy-3,6,9,12α5,18,21-heptaoxatricosylthio)-3-((£)-2- (1,3-3-trimethyl indolin-2-ylidene)ethylidene)cyclohex-l-enyl)vinyl)-l_T3,3-trimethyl- 3H-indolium iodide:

[00185] To a solution of IR-786 iodide (0.10 g, 0.16 mmol) in anhydrous CH₂Cl₂ (5 mL) was added thio-PEGg-alcohol (69.4 mg, 0.18 mmol) followed by triethylamine (25 μL, 0.18 mmol). IR-786 iodide is 2-[2-[2-Chloro-3-[(l,3-dihydro-l,3,3-trimethyl-2H- indol-2-ylidene)ethylidene]-l-cyclohexen-l -yl]ethenyl]-l, 3, 3-trimethylindolium iodide . The reaction mixture was stirred at room temperature overnight, and the reaction mixture was then diluted with CHzCb(IO mL) and washed with water (2 x 10 mL). The combined organic layers were dried (Na₂SO₄), filtered and concentrated in vacuo to obtain a solid. The solid was purified by flash chromatography on silica gel using 2-5% of MeOH in CH₂CI₂ as an eluant to obtain 12 (92 mg, 0.095 mmol, 60% yield) as a green solid. ¹H IMMR (400 MHz, CDCl₃): δ 8.77 (d, J= 14.2 Hz, 2H), 7.36 - 7.32 (m, 4H), 7.18 (t, J= 7.5 Hz, 2H), 7.13 (d, J= 8.4 Hz, 2H), 6.12 (d, J = 14.1 Hz, 2H), 3.69 - 3.48 (m, 30H), 2.93 (t, J= 6.6 Hz, 2H), 2.87 (s, OH), 2.60 (t, J= 5.8 Hz, 4H), 1.93 - 1.80 (m, 2H), 1.69 (s, 12H); ¹³C NMR (100 MHz, CDCl₃): δ 172.8, 157.5, 146.0, 143.0, 140.9, 134.1, 128.9, 125.3,

122.3, 1 10.8, 101.4, 72.6, 71.8, 61.7, 49.2, 36.8, 31.7, 28.1, 26.4, 21.0; HRMS (FAB): m/z calcd for C₄₈H₆₉N₂O₈S [M]⁺: 833.47746, found: 833.48094. The thio-PEGg-alcohol was obtained by the acetyl deprotection of commercially available S-acetyl-dPEG™8-alcohol as described by E. Klein, P. Kerth, and L. Lebeau (Biomaterials 2008, 29, 204).

7. l,3,3-trimethyI-2-((£)-2-((£)-2-(l-(4-nitrophenoxy>-l-oxo-2,5,8,l 1,14,17,20,23- octaoxapentacosan-25-yltliio)-3-((£)-2-(l,3^-triinethylindoIin-2- ylidene)ethylidene)cyclohex-l-enyl)vinyl)-3H-indolium iodide:

[00186] To a solution of the trimethyl-3H-indolium iodide derivative (from Example 6, 55 mg, 0.057 mmol) in anhydrous CH₂Cl₂ (5 mL) at 0⁰C was added Et₃N (36.6 μL, 0.26 mmol) followed by 4-nitrophenyl chloroformate (40 mg, 0.19 mmol). After being stirred for 30 mi n at 0⁰C, the reaction mixture was allowed to warm to rt and stirred for an additional 5 h. The reaction mixture was diluted with CH₂Cl₂ (5 mL) and washed with IN HCl (2 x 5 mL) and then 5% NaHCO₃ (3 x 5 mL). The combined organic layers were dried (Na₂SO₄), filtered and concentrated in vacuo to obtain a solid residue. Flash chromatography on silica gel using 0-4 % MeOH in CH₂Cl₂ as eluant provided 3 (50 mg, 0.044 mmol, 78% yield) as a green solid. ¹H NMR (400 MHz, CDCl₃): δ 8.78 (d, J = 14.0 Hz, 2H), 8.27 (d, J= 9.3 Hz, 2H), 7.44 - 7.32 (m, 6H), 7.28 - 7.14 (m, 4H), 6.28 (d, J = 14.2 Hz, 2H), 4.46 - 4.39 (m, 2H), 3.83 - 3.77 (m, 2H), 3.76 (s, 6H), 3.70 - 3.47 (m, 26H), 2.95 (t,J= 7.0 Hz, 2H), 2.69 (t,J = 5.8 Hz, 4H), 1.91 (m, 2H), 1.73 (s, 12H); ¹³C NMR(IOO MHz, CDCl₃): 5172.7, 157.5, 155.6, 152.5, 146.0, 145.3, 142.9, 140.8, 134.1, 128.8, 126.0, 125.2, 122.2, 121.9, 116.1, 110.6, 101.3,71.3,71.0,70.2,70.0,68.6,68.4, 49.1, 42.9, 36.7, 31.5, 29.7, 28.0, 26.3, 20.9; HRMS (FAB): m/z calcd for Cs₅H₇₂N₃Oi₂S [M]⁺: 998.4831, found: 998.4865.

8. Synthesis of Hoechst-IR786:

(00187J To a mixture of ethanamine hydrochloride derivative (from Example 5, 15 mg, 0.021 mmol) and JH-indolium iodide derivative (from Example 7, 35.4 mg, 0.031 mmol) was added Et₃N (58.4 μL, 0.42 mmol) followed by anhydrous DMF (0.5 mL) under an argon atmosphere. The reaction mixture was stirred overnight at it, and the solvent was then evaporated in vacuo to obtain a residue. The residue was purified by silica gel preparative TLC chromatography using 12 % MeOH in CH_∑Ch containing 0.1 % Et₃N as an eluant to obtain 1 (10 mg, 0.006 mmol, 31%) as a blue solid. ¹HNMR(SOOMHZ, CD3OD): δ 8.84 (d,J= 14.0 Hz, 2H), 8.28 (s, IH), 8.06 (d, J= 8.4 Hz, 2H), 7.96 (d,J = 8.4 Hz, IH), 7.77 (d, J= 8.0 Hz, IH), 7.62 (d, J= 8.4 Hz, IH), 7.46 (d, J= 7.6 Hz, 2H), 7.38 (t,J= 7.6 Hz, 2H), 7.27-7.24 (m, 7H), 7.10 (d,J= 8.4 Hz, 2H), 6.19 (d,J = 13.6 Hz, 2H), 4.19-4.17 (m, 2H), 4.11 -4.09(m, 2H), 3.86-3.84 (m, 4H), 3.71 - 3.68 (m, 4H), 3.64-3.50 (m, 44H), 3.00 (s, 3H), 2.93 (t, J = 6.4 Hz, 2H), 2.61 (m, 4H), 1.89 - 1.84 (m, 2H), 1.71 (s, 12H); ¹³C NMR (125 MHz, CDCl₃): δ 174.4, 162.8, 158.8, 155.9, 152.1, 149.6, 147.1, 144.3, 142.3, 134.5, 129.9, 129.7, 126.1, 123.3, 123.0, 122.2, 121.2, 118.2, 116.3, 115.9, 111.7, 102.1, 102.0,71.59,71.51,71.4,71.3,71.2,71.0,70.7,70.5,68.9, 65.0, 54.8, 53.6, 50.3, 49.8, 49.6, 47.9, 43.6, 41.7, 38.1, 31.6, 28.2, 27.1, 22.2; HRMS (MALDI): m/z calculated for C₈₀HI₀₄N₉O_I2S [M]^" : 1414.7525, found: 1414.7598. 9. 7V-(2-(2-(4-(5-(4-raethyIpiperazine-l-yl)-lH-l'H-2^'-bibenzo[(/limidazoI-2'-yl) phenoxy)ethoxy)ethyl)-3-(tritylthio)propanamide:

[00188] 3-(Tritylthio) propionic acid (50.0 mg, 0.1 mmol) was dissolved in anhydrous DMF (1 mL) and brought to O⁰C, and O-Benzotriazol-l-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) (54.9 mg, 0.1 mmol) was added to this solution followed by diisopropylethylamine (0.6 mL, 3.5 mmol). The reaction mixture was stirred for 0.5 h, before ethanamine hydrochloride derivative (from Example 5, 0.1 g, 0.1 mmol) was added and stirred for an additional 0.5 h at O⁰C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was evaporated under reduced pressure and crude product was purified by flash column chromatography, using a gradient of 5-10% of methanol in dichloromethane containing 0.1 % Et3N. The compound 6 was obtained as a light yellow solid (0.1 g, 56%). ¹H NMR (400 MHz, CDCl₃): δ 8.24 (s, IH), 8.01 (d, J= 8.8 Hz, 2H), 7.93 (dd, J= 8.4, 1.6 Hz, IH), 7.76 (d, J= 7.6 Hz, IH), 7.69 - 7.66 (m, 2H), 7.51 (d, J = 8.8 Hz, IH), 7.40 - 7.20 (m, 12H), 7.17 - 7.15 (m, 3H), 7.05 (d, J= 8.8 Hz, IH), 4.14 (t, J= 4.4 Hz, 2H), 3.81 (t, J= 4.4 Hz, 2H), 3.67 - 3.58 (m, 4H), 3.51 (t, J= 5.2 Hz, 2H), 3.46 - 3.35 (m, 8H), 3.32 (m, 2H), 2.91 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 2.1 (t, J = 6.8 Hz, 2H); ¹³C NMR (100 MHz, CD₃OD): δ 172.4, 160.9, 154.0, 152.5, 146.9, 144.7, 144.3, 142.2, 129.2, 128.2, 127.8, 127.4, 126.3, 125.2, 124.7, 121.1, 1 17.2, 1 15.4, 1 14.8, 110.5, 70.4, 69.9, 69.3, 69.1, 67.3, 66.3, 48.4, 46.4, 42.4, 39.0, 34.3, 27.5; HRMS (ESI) m/z: [M+Hf calculated for C₅₃H₅₆N₇O₄S, 886.4109; found, 886.405.

10. 3-mercapto-iV-(2-(2-(4-(5-(4-methylpiperazin-l-yl)-lH,l'H-2,5'- bibenzo(d]imidazol-2'-yl) phenoxy)ethoxy)ethyl) propanamide:

[00189] The 3«(tritylthio)propanamide derivative (from Example 9, 149 mg, 0.1 mmol) was added to 0.5 mL of TFA, containing triethylsilane (134 μL, 0.8 mmol), and stirred for 0.5 h. The TFA was removed under reduced pressure to obtain an oil, which was washed with diethyl ether (2 x 5 mL) and dried under reduced pressure to obtain 3 as a solid (96 mg, 88%).

11. N-(l-((2R,4R,5R)-33-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran- 2-yl)-2-oxo-l^-dihydropyrimidin-4-yl)-3-(trityltnio)propananiide:

[00190] 3-(Tritylthio) propionic acid (0.24 g, 0.6 mmol) was dissolved in anhydrous DMF (2 mL) and brought to 0⁰C was added 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethyiaminium tetrafluoroborate (TBTU) (0.26 g, 0.8 mmol) and

diisopropylethylamine (0.2 mL, 1.3 mmol). After 0.5 h, 2-Deoxy-2',2',-difluoro-D- cytidine 7 (0.2 g, 0.7 mmol) was added and stirred for another 0.5 h at O⁰C. The reaction mixture was then allowed to warm to room temperature and stirred for 18 h. The solvents were evaporated under reduced pressure and the crude solid was purified by silica gel column chromatography, using 0 - 4% methanol as a gradient in dichloromethane, to obtain pure 8 as a white solid (0.2 g, 50%). ¹H NMR (400 MHz, CDCl₃): 8.27 (d, J= 7.2 Hz, IH), 7.42 - 7.23 (m, 15H), 7.18 (d, J = 7.2 Hz, IH), 6.14 (t, J= 5.6 Hz, IH), 4.55 - 4.47 (m, IH), 4.13 - 3.92 (m, 3H), 2.47 (t, J = 6.4 Hz, 2H), 2.37 (t, J= 6.4 Hz, 2H); ¹³C NMR (IOO MHz, CDCl₃) S 172.0, 162.9, 156.2, 144.9, 144.7, 129.7, 128.1, 126.9, 121.4, 97.7, 81.6, 67.1, 59.3, 55.1, 43.2, 36.5, 29.8. HRMS (FAB) m/z: [M+H]^f calculated for C_3IH₁₀F₂N₃O₅S, 594.1869; found, 594.1935. 12. yV-(l-((2Λ,4Λ,5/?)-3>κliπuoro-4-hydroxy-5-(hydroxymethyI)tetrahydrofuran- 2-yl)-2-oxo-l,2-dihydropyriniidin-4-yl)-3-mercaptopropanamide:

[00191] The 3-(tritylthio)propanamide derivative (from Example 11 , 0.3 g, 0.5 mmol) was added to 2.0 mL of TFA:CH₂C1₂ (1 to 1 ratio), containing triethylsilane (0.2 mL, 1.2 mmol) as a carbocation scavenger, and stirred for 1 h. The TFA÷CttCb was removed under reduced pressure to obtain an oil, which was washed with diethyl ether (2 x 5 mL) and dried under reduced pressure to obtain 9 as a solid (0.14 g, 97%), which was used without further purification.

13. yV-(l-((2Λ,4Λ,5/?)-3,3-dinuoro-4-hydroxy-5-(hydroxyιnethyl)tetrahydrofuran-

2-yI)-2-oxo-l£-dihydropyrimidin-4-yl)-3-(pyridine-2-yldisulfanyl)propanamide:

[00192] The 3-mercaptopropanamide derivative (from Example 12, 0.12 g, 0.3 mmol) and 2, 2'-dithiodipyridine 10 (0.1 g, 0.7 mmol) were dissolved in anhydrous methanol (4 mL) containing glacial acetic acid (10.3 μL, 0.1 mmol). The reaction mixture was stirred at room temperature for 18 h, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (using a 0 - 7% methanol gradient in dichloromethane), generating 4 as a white solid (0.09 g, 41%). ¹H NMR (400 MHz, CDCl₃): 8.53 (d, J = 4.0 Hz, IH), 8.14 (d, J = 7.6 Hz, IH), 7.64 - 7.60 (m, 2H), 7.43 (d, J= 7.6 Hz, IH), 7.13 - 7.10 (m, IH), 6.21 (t, J= 5.6 Hz, IH), 4.40 - 4.31 (m, IH),

4.00- 3.97 (m, 2H), 3.85 - 3.83 (m, IH), 3.07 (t, J= 6.4 Hz, 2H), 2.85 (t, J= 6.4 Hz, 2H) ; ¹³C NMR (100 MHz, CDCl₃) δ 171.7, 162.8, 158.9, 155.9, 149.2, 144.7, 137.4, 121.1, 120.2, 97.6, 81.3, 58.9, 54.7, 42.8, 36.4, 33.7. HRMS (FAB) m/z: [M+Hf calculated for Ci₇H₁₉N₄O₅ F₂S₂, 461.0759; found, 461.0742.

14. Synthesis of Hoechst-gemcitabine conjugate

[00193] An oven dried 25 mL single neck round bottom flask containing a magnetic stir bar was fitted with a rubber septum and kept under an argon atmosphere. To this flask mercapto-propanamide derivative (from Example 10, 0.08 g, 0.1 mmol) and 3-(pyridine-2- yldisulfanyOpropanamide derivative (from Example 13, 0.12 g, 0.2 mmol) were added followed by anhydrous DMF (1.4 mL). The reaction mixture was stirred at room temperature overnight. The solvents were evaporated under reduced pressure, and the crude prodcut was purified by flash chromatography using silica gel, with a gradient of 10- 15 % methanol in dichloromethane containing 0.1 % Et3N. The product 1 was obtained as a light yellow solid (yield 0.04 g, 30%). ¹H NMR (400 MHz, CD₃OD+DMSO): δ 8.33 (d, J= 7.2 Hz, 2H), 8.14 (d, J= 8.8 Hz, 2H), 8.03 - 7.09 (m, IH), 7.77 - 7.71 (m, IH), 7.56 - 7.53 (m, IH), 7.43 (d, J= 7.6 Hz, IH), 7.19 - 7.17 (m, 3H), 7.08 (dd, J = 9.2, 2.0 Hz, IH), 6.25 (t, J= 7.2 Hz, IH), 4.42 (s, IH), 4.31 - 4.25 (m, 2H), 3.81 - 3.73 (m, 3H), 3.68 - 3.65 (m, 2H), 3.56 (t, J = 5.6 Hz, 2H), 3.38 (t, J= 5.6 Hz, 2H), 3.20 (s, 3H), 3.12 (t, J= 5.2 Hz, 4H), 2.82 (t, J= 6.8 Hz, 4H), 2.76 - 2.74 (m, 2H), 2.55 (t, J= 5.2 Hz, 4H), 2.46 (t, J = 6.8 Hz, 2H), 2.25 (s, 3H); ¹³C NMR (100 MHz, DMSO): δ 172.6, 170.5, 163.2, 160.5, 154.6, 153.2, 148.1, 145.3, 128.7, 125.9, 124.8, 123.4, 122.9, 118.6, 115.3, 114.0, 111.6, 96.4, 84.5, 84.2, 81.4, 70.3, 70.0, 69.5, 69.3, 69.0, 68.7, 68.5, 67.8, 59.2, 55.3, 50.3, 49.0, 46.2, 46.0, 39.0, 36.7, 35.3, 34.3, 33.1, 33.0, 21.6; HRMS (ESI) m/z: [M+H]⁺calcd for C₄₆H₅₅N₁₀O₉ F₂S₂, 993.3557; found, 993.3596.

IS. In Vitro Imaging

[00194] RAW 264.7 macrophage cells were incubated with either the compound of Example 8 or Hoechst 33258 (20 μM) in medium containing 10% serum for 30 minutes at 37°C. The cell media was then replaced by warm Krebs-Ringer buffer and the cellular fluorescence was recorded using a fluorescence microscope (Nikon E600, Nikon, Melville, NY), using an excitation wavelength of 340 nm and an emission filter between 400-500 nm. In case of fixed cells, RAW 264.7 macrophage cells were treated with pre- cooled methanol at -2O⁰C for 10 minutes. The cells were washed with PBS followed by incubation with Hoechst-IR for 30 min at 37°C. The cell media was then replaced by Krebs-Ringer buffer and the cellular fluorescence was recorded using a fluorescence microscope, using an excitation wavelength of 340 nm and an emission filter between 400-500 nm.

16. In Vivo Imaging of Myocardial Infarction

[00195] The left anterior descending coronary artery of the mouse was ligated with a suture for a period of 30 minutes. The suture was then released and the heart was reperfused, generating ischemia/reperfusion injury. The chest was closed and the animals were allowed to recover for 2 h. After 2 h, 10 nmol of the compound of Example 8 in 0.1 mL saline, supplemented with 5% ethanol and 5% cremophor EL (PEG-functionalized castor oil), was injected intravenously via retro-orbital injection. The compound was allowed to circulate for 1 h before the animals were sacrificed and perfused for 5 min with saline and 5 min with 4% paraformaldehyde in PBS buffer. Organs were then harvested and imaged in an in vivo imager (Olympus OVlOO).

17. In Vivo Imaging of Sepsis

[00196J Ten BALB/6 mice were divided into two groups, one group was given an intraperitoneal (i.p.) injection of 100 μL of LPS (2.5 μg/kg) and D-galactosamine (GaIN) (700 mg/kg) and second group (control) was given an i.p. injection of a 100 μL of saline. Animals were anesthetized by isofluorane inhalation and the abdominal fur was removed using hair removal cream. After 24 h, the LPS and saline treated mice were injected intravenously with 100 μL of the compound of Example 8 (5 mg/kg). 24 h later, the mice were imaged as doublets one from each group, using an IVIS imaging system (Carestream Healthcare, New Haven CT), using an excitation wavelength of 745 nm, an emission filter of 820 nm and bright-field settings. The mean fluorescence intensity of each mouse was measured with Image-Pro software and a one-way ANOVA with repeated measures. 18. Determination of dissociation constant for the compound of Example 8 with oligomeric DNA

[00197] The duplex 22 bp oligonucleotide (sense strand: 5'- AGTTGAGGGGACTTTCCC AGGC-3') was purchased from Integrated IDT

Technologies (Coralville, IA). The double-stranded oligonucleotide was dissolved in buffer (10 mM phosphate buffer, pH = 7.4) to generate a 20 nM concentration. Various quantities of the compound of Example 8 were added to this DNA solution, to generate the compound of Example 8 concentrations ranging from (0.4 nM - 20 nM). The solution was allowed to stand for 5 minutes and the fluorescence spectra of the solution was measured with a Shimadzu spectrophotometer, using an excitation wavelength of 340 nm and an emission wavelength of 440 nm. AH measurements were corrected from the fluorescence intensity of the compound of Example 8 by itself. The corrected

fluorescence intensity data points were fitted using a non-linear regression analysis (Graph Pad software), to obtain the saturation binding curve and the dissociation constant Kd.

19. Cell permeability

[00198] The cell permeability of the compound of Example 14 ("H-gemcitabine") was measured in vitro using HT29 human colon cancer cells (ATCC, Manassas, VA, USA). HT29 cells were cultured in a humidified atmosphere at 37°C, 5 % CO2 in RPMI-medium supplemented with 10 % fetal bovine serum (FBS), 2 mM glutamine and 0.2 % penicillin/streptomycin (all purchased from Gibco, California, USA). Cells were incubated with either 10 μM of H-gemcitabine or hoechst 33258 in medium containing 10% serum for 30 min at 37°C. The cell media was then replaced by PBS and the cellular fluorescence was visualized with a fluorescence microscope (Nikon E600, Nikon, Melville, NY, USA), using an excitation wavelength of 340 nm and an emission filter between 400-500 nm. In addition, HT29 cells were also fixed with methanol (10 % v/v) to permeabilize their membranes, and then incubated with either H-gemcitabine or hoechst 33258 (10 μM) for 30 min at 37°C. The cell media was then replaced by PBS and the cellular fluorescence was visualized as described above.

20. Cell toxicity

[00199] The time dependent cell killing efficacy of the compound from Example 14

("H-gemcitabine"), gemcitabine, and the compound from Example 12 ("gemcitabine-SH") were tested in vitro. T75 flasks containing HT29 human colon cells were treated with a 0.2 % (w/v) trypsin-0.1 % (w/v) EDTA solution (Gibco, California, USA) and were seeded onto 96 well plates at a density of 5x 10⁵ cells/mL. The cells were allowed to grow for one day and then incubated with, free gemcitabine, gemcitabine-SH, or H-gemcitabine at concentrations ranging from 10 μM-1 mM for either 4 or 24 hours. The cell viability was assayed using the MTT assay.

21. Accumulation of compound in tumor spheroids

[00200] HT29 cells were cultured in T75 flasks and detached using 0.25 %

trypsin/EDTA, 1 mL aliquots of cellular suspensions containing 5xlO³ cells/mL, 5xlO⁴ cells/mL, and 5x 10³ cells/mL were transferred to twelve-well plates coated with 0.5 mL of 0.5 % agarose, and allowed to grow into spheroids. After 3 days, spheroids of different sizes (100, 300, and 400 μm) were transferred into 12 wells plates and incubated with 1 mL of the compound from Example 14 ("H-gemcitabine", 10 μM) for 2 hours. The quantity of H-gemcitabine targeting the tumor spheroids was determined by measuring the hoechst fluorescence in the spheroids. Spheroids treated with H-gemcitabine were washed with PBS three times, and then incubated with a 0.2 % (w/v) trypsin-0.1 % (w/v) EDTA solution (1 mL) for 10 min to disintegrate the spheroids into individual cells. The resulting cell suspensions were harvested and an additional 2 mL of PBS was added to the 12 well plates to recover any residual cells. The cell suspensions (3 mL) were treated with 1 M NaOH (1 mL) to hydrolyze cellular DNA, and the fluorescence intensity of the solutions were measured with a spectrofluorometer (Shimadzu RF-53O1PC, Durham, NC, USA), using an excitation wavelengdi of 340 nm and an emission wavelength of 461 nm. The concentration of H-gemcitabine in the spheroids was calculated by fitting the fluorescence intensity of the spheroids to a H-gemcitabine calibration curve. The volume of the spheroid was estimated from the equation (r = radius of spheroid).

22. Amplification of compound and E-DNA in tumor spheroids

[00201] Tumor spheroids composed of HT29 cells (5χlO⁶ cells/mL) were prepared as described above and transferred into 12 wells plates. Tumor spheroids were treated with one, two, three or four consecutive doses of either the compound from Example 14 ("H- gemcitabine") or free gemcitabine, as described above. The E-DNA (i.e. extracellular DNA or DNA in dead cells) in the spheroids was measured with propidium iodide ("PI"). After the final H-gemcitabine or free gemcitabine dose, spheroids were washed three times and then treated with 1 mL of propidium iodide (1 mM) for 10 min. The spheroids were then washed with PBS three times to remove unbound PI and trypisinized into individual cells, harvested and processed, as described above. The PI fluorescence intensity in the spheroid solutions was measured with a Shimadzu RF-5301PC spectrofluorometer, using an excitation wavelength of 488 nm and an emission wavelength of 588 nm. E-DNA fold increase was determined by dividing the fluorescence of treated spheroids with the fluorescence of untreated spheroids. In a separate set of experiments, the accumulation of H-gemcitabine in tumor spheroids was measured. Tumor spheroids composed of HT29 cells (5x10⁶ cells/mL) were prepared as described above and transferred into 12 wells plates. The tumor spheroids were treated with one, two, three or four consecutive doses of H-gemcitabine, each dose was given at a 10 μM concentration for 2 hours. After each dose, the media was removed and the spheroids were washed with PBS three times, and allowed to grow for 24 hours. After the final dose, the spheroids were harvested and analyzed for hoechst fluorescence as described above.

23. Cytotoxicity to tumor spheroids

[00202] Tumor spheroids composed of HT29 cells (5xlO⁶ cells/mL) were incubated with one, two, three or four consecutive doses of the compound from Example 14 ("H- gemcitabine", 10 μM), or free gemcitabine (10 μM) for 2 hours, each dose was given 24 hours apart. After the final dose, the spheroids were then washed with PBS three times, trypsinized as described above, and resuspended in cell culture medium and seeded into 96 wells. After 24 hours, the cell viability was assayed using the MTT assay.

24. Tumor development in mice

[00203] HT29 tumors were grown in Balb/c nude mice (Simonsen Lab Inc., Gilroy, CA). The mice were accommodated in autoclaved micro-isolator cages that were housed in a positive pressure containment rack and maintained under the guidelines of an approved animal protocol from the Georgia Institute of Technology Institutional Animal Care and Use Committee (animal protocol #: A08051). Xenografts of human colon HT29 carcinoma cells were developed by subcutaneously implanting 2 * 10⁶ cell/mL in the right rare flanks of nude mice. When the tumor volume reached between 50- 150 mm³, mice were randomly assigned to experimental and control groups.

25. Amplifⁱcation of E-DNA in tumors in vivo

[00204] HT29 tumors were grown in mice as described above. Mice with tumors between 50-150 mm³ were treated with one, two, three or four consecutive doses of the compound from Example 14 ("H-gemcitabine"), or free gemcitabine, each given 3 days apart. All formulations were injected intravenously via the jugular vein, at a dose of 10 mg/kg equivalents of gemcitabine, through 25G5/8 needles. Twenty four hours after each dose, the E-DNA (i.e. extracellular DNA or DNA in dead cells) of the tumors was stained by injecting 50 μL of propidium iodide ("PI", 10 mM) directly on the tumor (mice were anesthetized for the tumor injection). Twenty four hours after the PI injection, the mice were euthanized with CO₂ and the tumor tissue was harvested. The tumor tissues (100 mg) were transferred into 12 well plates, incubated with 1 mL of 0.2 % (w/v) trypsin-0.1 % (w/v) EDTA solution for 4 hours, chopped, and pipetted. Each well then was incubated with I mL of NaOH (1 mM) and 2 mL of PBS for 30 min and centrifuged to remove residual tumor tissue. The supernatant was isolated and the fluorescence intensity was measured with a Shimadzu spectrofluorometer, using an excitation wavelength of 488 nm and emission wavelength of 588 nm.

26. Inhibition of tumor growth

[00205] The ability of the compound from Example 14 ("H-gemcitabine") to reduce tumor growth and increase the survival rate of nude mice bearing HT29 tumors was measured. Mice were given HT29 tumors as described above, and were given 5 consecutive doses of H-gemcitabine or gemcitabine, each dose was given 3 days apart (day 0, 3, 6, 9, and 12). All formulations were injected intravenously via the jugular vein at a dose of 10 mg/kg equivalents of gemcitabine through 25G5/8 needles. The tumor volume of the mice was calculated by the following equation: V = (w)² x (/)/2, where (H') and (/) are the width and length of the tumor, as measured by a caliper. The animals were sacrificed when they met any of the following conditions: (1) 15 % loss in initial body weight (2) the size of the tumor > 1.5 cm in any dimension, (3) the mouse became lethargic, sick or unable to feed, (4) the mouse developed an ulcerated tumor. 27. Determination of the dissociation constant (Kd) with DNA

[00206] The 21bp oligonucleotide (5'-GCGACTGCAATTTCGACGTCCO') was dissolved in deionized water to generate a 20 nM concentration. Various quantities of the compound from Example 14 ("H-gemcitabine") were added to this DNA solution, to generate H-gemcitabine concentrations ranging from (2 nM - 100 nM). The solution was allowed to stand for 5 minutes and the fluorescence spectra of the solution was measured with a Shimadzu spectrophotometer, using an excitation wavelength of 340 nm and an emission wavelength of 440 nm. All measurements were corrected for the fluorescence intensity of H-gemcitabine by itself. The corrected fluorescence intensity data points were fitted using a non-linear regression analysis (Graph Pad software), to obtain the saturation binding curve and the dissociation constant (Kj).

Claims

CLAIMS What is claimed is:

1. A compound having the formula

(B)_n-L-(T)_n,, wherein

each B is independently a targeting moiety;

L is a cleavable linker;

each T is independently a therapeutic moiety; and

at least one B comprises a DNA binding agent; at least one B is chemically bonded to L, L is chemically bonded to at least one T, and // and m are independently from 1 to 3.

2. A compound having the formula

(B)_n-L-(I)_n,, wherein

each B is independently a targeting moiety;

L is a cleavable linker;

each I is independently a diagnostic moiety; and

at least one B comprises a DNA binding agent; at least one B is bonded to L, L is bonded to at least one 1, and /; and m are independently from 1 to 3.

3. A compound havi ng the formul a

(B)_n-L'-(l)_m-L"-(T)_Xl wherein

each B is independently a targeting moiety;

L' and L" are independently cleavable linkers;

each 1 is independently a diagnostic moiety;

each T is independently a therapeutic moiety; and

at least one B comprises a DNA binding agent; at least one B is bonded to L\ U is bonded to at least one I, at least one I is bonded to L", L" is bonded to at least one T, and n, m, and x are independently from 1 to 3.

4. A compound having the formula

(B)_n-L'-(T)_x-L"-(I)_ra, wherein

each B is independently a targeting moiety;

L' and L" are independently cleavable linkers;

each T is independently a therapeutic moiety; each I is independently a diagnostic moiety; and

at least one B comprises a DNA binding agent; at least one B is bonded to L', L/ is bonded to at least one T, at least one T is bonded to L", L" is bonded to at least one I, and n, m, and x are independently from 1 to 3.

5. The compound according to any one of claims 1-4, wherein the DNA binding agent has DNA minor groove binding activity or is a DNA intercalating agent.

6. The compound according to any one of claims 1-4, wherein the DNA binding agent is a derivative or fragment of Hoechst 33258.

7. The compound according to any one of claims 1-4, wherein the DNA binding agent is an optionally substituted acylic, polycyclic, polyaromatic, or heterocyclic hydrophobic moiety.

8. The compound according to any one of claims 1-4, wherein L, L', and L" can be cleaved under physiological conditions.

9. The compound according to any one of claims 1-4, wherein L, L', and L" independently comprise an optionally substituted polyether, an amide, an ester, a disulfide bond, or a combination thereof.

10. The compound according to any one of claims 2-4, wherein I is an imaging agent or a ligand capable of coordinating to an imaging agent.

11. The compound according to any one of claims 2-4, wherein I is an imaging agent comprising a chromophore, a fluorophore, a denoptical dye, a magnetic resonance imaging

(MRJ) contrast agent, a PET probe, a SPECT probe, a CT contrast agent, a radiodiagnostic agent, or an ultrasound contrast agent.

12. The compound according to any one of claims 2-4, wherein each I is selected independently from:

a) a magnetic resonance imaging agent comprising Gd '\ Eu⁺³, Tm^"3, Dy⁺³, Yb⁺³,

Mn⁺², or Fe⁺³;

b) a PET or SPECT imaging agent comprising ⁵⁵Co, ⁶⁴Cu, ⁶⁷Cu, ⁴⁷Sc, ⁶⁶Ga, ⁶⁸Ga, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ¹¹¹In, ¹⁰⁹Pd, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, or ¹⁸⁸Re; and c) a radioisotope selected from 1¹8⁸τF-, 1 "24⁴τI, I "31¹ _TI, 7 "7Bo_r, 8 "2Bτ r, and "¹At.

13. A compound having the formula:

wherein R is a residue comprising a therapeutic agent or a diagnostic agent, n is from 0 to 60, the therapeutic agent when present comprises an agent for the treatment of a tumor or a cancer, and the diagnostic agent when present comprises a fluorescent, PET, MRI, or infrared imaging agent.

14. A compound having the formula:

15. A compound having the formula:

16. A compound having the formula:

wherein R is a residue comprising a therapeutic agent or a diagnostic agent, n is from O to 60, the therapeutic agent when present comprises an agent for the treatment of a tumor or a cancer, and the diagnostic agent when present comprises a fluorescent, PET, MRI, or infrared imaging agent.

17. A compound having the formula:

18. A compound having the formula:

19. A compound having the formula:

20. A compound having the formula:

21. A compound having the formula

22. A compound having the formula:

23. A compound having the formula:

24. A compound having the formula:

25. A compound having the formula:

26. A compound having the formula:

27. A compound having the formula:

28. A compound having the formula:

29. A compound having the formula:

30. A compound having the formula:

31. A compound having the formula:

32. A compound having the formula:

33. A compound having the formula:

34. A compound having the formula:

35. A compound having the formula:

36. A compound having the formula:

37. A compound having the formula:

38. A compound having the formula:

39. A compound having the formula:

40. A compound having the formula:

41. A compound having the formula:

42. A compound having the formula:

43. A compound having the formula:

44. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1-43 and a pharmaceutically acceptable carrier.

45. A method of treating a disorder of uncontrolled cellular proliferation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the compound according to any of claims 1-43 and a pharmaceutically acceptable carrier.

46. The method according to claim 45, the method further comprising co- administering a therapeutically effective amount of a cancer therapeutic compound or composition.

47. The method according to claim 46, the method further comprising coadministering a therapeutically effective amount of a composition comprising at least one chemoagent, immunotherapeutic, cancer vaccine, anti-angiogenic agent, cytokine, hormone therapy, gene therapy, biological therapy, or radiotherapy.

48. The method according to claim 46, the method further comprising coadministering a therapeutically effective amount of 5-fluorouracil or leucovorin.

49. The method according to claim 46, the method further comprising coadministering a therapeutically effective amount of a composition comprising at least one immunotherapeutic agent.

50. The method according to claim 46, the method further comprising coadministering a therapeutically effective amount of a composition comprising at least one immunotherapeutic agent, antibody or immunomodulator.

51. The method according to claim 46, the method further comprising coadministering a therapeutically effective dose of radiation.