Pyrazole compound and method of making and using the same

Abstract

Disclosed herein is a compound. Also disclosed are compositions comprising the compound and a pharmaceutically acceptable excipient, methods for making the compound, and methods of using the compound as a therapy for subjects suffering from, or susceptible to, a disease involving a vascular pathology.

Description

[0001] 8123-112871-03 11 / 21 / 25 06880

[0002] PYRAZOLE COMPOUND AND METHOD OF MAKING AND USING THE SAME CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to the earlier filing date of U. S. Provisional Application No. 63 / 726,007, filed November 27, 2024; and U. S. Provisional Application No. 63 / 845,962, filed July 17, 2025, which are herein incorporated by reference in their entirety.

[0004] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under Contract No. EY032632 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] The present application is directed to a compound for use as an anti-angiogenic agent, including methods of making and using the compound.

[0008] BACKGROUND

[0009] Angiogenesis, the process of forming new blood vessels from pre-existing vasculature, plays a critical role in both non-pathological physiological processes, such as wound healing and embryonic development, and in pathological conditions, including cancer, age-related macular degeneration, and diabetic retinopathy. Aberrant angiogenesis is a hallmark of many diseases including cancer. Endothelial cell migration and / or proliferation are essential processes of angiogenesis that are highly dependent on the action of actin cytoskeleton. Actin monomer-binding protein Profilinl (Pfnl) plays an important role in facilitating actin polymerization in cells, and depletion of Pfnl results in reduced cellular level of polymerized actin (F-actin), and defects in proliferation and migration in a variety of cell types including endothelial cells (ECs).

[0010] There exists a need in the art for new compounds that can act as anti-angiogenesis agents.

[0011] SUMMARY

[0012] Disclosed herein is a compound, having a structure: 8123-112871-03 11 / 21 / 25 06880

[0013]

[0014] In certain aspects, Ri is not 4-bromophenyl. In certain aspects, R2 is hydrogen or a methyl group. In certain aspects, R3 is hydrogen, a phenyl group, a substituted phenyl group, a heterocycle or a substituted heterocycle.

[0015] Also disclosed herein is a compound, having a structure:

[0016] HN-N

[0017]

[0018] F

[0019] which is named 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol).

[0020] Also disclosed is a pharmaceutically acceptable composition, comprising: 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol); and a therapeutically acceptable excipient.

[0021] Also disclosed is a method, comprising administering 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) to a subject exhibiting, or susceptible to exhibiting, a vascular pathology, or a pharmaceutically acceptable composition thereof.

[0022] Also disclosed is a method, comprising administering 4.4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) to a subject, wherein the subject is suffering from, or is susceptible to, a disease involving a vascular pathology.

[0023] In yet additional aspects, a method of making 4,4'-((4-(trifhioromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) is disclosed, which comprises reacting 3-methyl-5-pyrazolone with 4-(trifhroromethyl)benzaldehyde in the presence of a surfactant.

[0024] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. 8123-112871-03 11 / 21 / 25 06880

[0025] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A shows images of whole mount fluorescein isothiocyanate (FITC)-lectin stained P6 retinae from Profilinl (Pfnl) + / + and vascular endothelial cell (VEC)-specific Pfnl - / - mice (generated by breeding Pfnl flox / flox mice with tamoxifen-inducible vascular endothelial (VE)-cadherin-specific Cre driver mouse), including close-up images (bottom) showing hypovascularization in Pfnl - / - retinae (n = 12 retinae per group).

[0026] FIG. IB is a plot quantifying retinal VEC coverage area in the images of FIG. 1 (n = 12 retinae per genotype).

[0027] FIG. 1C is a plot quantifying endothelial sprouting in the micrographs of FIG. 1 (n = 12 retinae per genotype).

[0028] FIG. ID shows images with CD31 (a marker of VEC) staining showing hypovascularization in developing kidney in Pfnl -I- mice (n = 5 mice / genotype).

[0029] FIG. IE is a plot quantifying CD31+ expression in the images of FIG. ID.

[0030] FIG. 2A is a plot of relative transcript values for Pfnl in CD31+ VEC from proliferative diabetic retinopathy (PDR) patients (n = 7) vs control (n = 4) human retinal samples (transcriptome GEO dataset: GSE94019).

[0031] FIG. 2B shows images from an oxygen-induced retinopathy assay in mouse retinae. FIG. 2C shows additional images from the oxygen-induced retinopathy assay of FIG.

[0032] 2B.

[0033] FIG. 2D shows a plot quantifying vaso-obliteration in the central retinae of FIG. 2B. FIG. 2E shows a plot quantifying neovascularization after the oxygen-induced retinopathy assay of FIG. 2B.

[0034] FIG. 2F shows images of choroidal explant angiogenesis culture under vehicle (dimethyl sulfoxide (DMSO)) and C74-treated conditions (inset shows explants; red outline shows the edge of the vascular outgrowth).

[0035] FIG. 2G shows a plot quantifying vascular outgrowth in the images of FIG. 2F. FIG. 2H shows images of CD31 staining of subcutaneous tumors established by renal carcinoma (RENCA) cancer cells in Balb / C mice.

[0036] FIG. 21 shows a plot quantifying choroidal neovascularization (CNV) area in the images of FIG. 2H (“**” represents a p-value < 0.05)

[0037] FIG. 2J shows an image (4x magnification) of CD31 staining of subcutaneous tumors established by RENCA renal cancer cells in Balb / C mice subjected to DMSO control treatment. 8123-112871-03 11 / 21 / 25 06880

[0038] FIG. 2K shows an image (4x magnification) of CD31 staining of subcutaneous tumors established by RENCA renal cancer cells in Balb / C mice subjected to daily intra-tumoral injection of C74 for 2 weeks (n = 5 mice / group).

[0039] FIG. 2L shows a plot quantifying CD31+ in the subcutaneous RENCA tumor grafts of FIGS. 2J-2K (“**” represents a p-value < 0.01; n = 5 mice / group).

[0040] FIG. 3A shows a plot of pyrene-actin polymerization assays (fluorescence measures actin polymerization), in which C74 reverses Pfnl’s inhibition of actin polymerization (A = actin, P = Pfnl, the numbers in the parentheses indicate relative stoichiometry).

[0041] FIG. 3B shows representative images of matrigel cord-morphogenesis assays illustrating relative anti-angiogenic efficacies of C2 and C74 in dermal micro- VECs.

[0042] FIG. 3C shows representative images of matrigel cord assays demonstrating the anti-angiogenic effect of C74 (25 pM) on human retinal micro-VECs.

[0043] FIG. 3D shows a plot quantifying the matrigel cord assays of FIG. 3C (three experiments, “**” indicates a p-value < 0.01).

[0044] FIG. 4A shows representative proximity -ligation assay (PLA) images.

[0045] FIG. 4B is a plot quantifying a number of Pfnl -actin PLA spots in the images of FIG.

[0046] 4A (“**” indicates a p-value < 0.01).

[0047] FIG. 4C is a plot quantifying migration of VECs in a culture (“**” indicates a p-value < 0.01).

[0048] FIG. 4D is a plot of migration of Pfnl knockdown (KD) VEC when rescued with fluorescent (GFP)-Pfnl but not actin-binding deficient mutant GFP-H119E-Pfnl “**” indicates a p-value of < 0.01, “NS” = not significant).

[0049] FIG. 4E is a plot quantifying proliferation of VECs.

[0050] FIG. 5 shows cord morphogenesis assay images of dermal VECs following treatment with indicated concentrations of illustrated compounds.

[0051] FIG. 6A illustrates a representative scheme for synthesizing a compound according to aspects of the present disclosure.

[0052] FIG. 6B is another illustration of a representative scheme for synthesizing a compound according to aspects of the present disclosure.

[0053] FIG. 6C illustrates another representative scheme for synthesizing a compound, along with representative yields of selected compounds, according to aspects of the present disclosure. 8123-112871-03 11 / 21 / 25 06880

[0054] FIG. 7A illustrates an example of a compound according to aspects of the present disclosure.

[0055] FIG. 7B is a plot of binding behavior between Pfnlwr and actin according to aspects of the present disclosure.

[0056] FIG. 7C is a plot of binding behavior between Pfnluii9E and actin according to aspects of the present disclosure.

[0057] FIG. 7D is a plot of binding behavior between Pfnlwr and C74 according to aspects of the present disclosure.

[0058] FIG. 7E is a plot of binding behavior between PfnluiwE and C74 according to aspects of the present disclosure.

[0059] FIG. 8 is a mass spectrogram of microbubble (MB) -encapsulated C74 according to aspects of the present disclosure.

[0060] FIG. 9 A shows a series of images of a control (no treatment (NT)), C74 treatment, MB treatment with ultrasound (US), MB-encapsulated C74 treatment (C74 MB), treatment with MB-encapsulated C74 and US (C74 MB US), and C74 treatment with US, illustrating that ultrasound-targeted microbubble destruction (UTMD) of C74-encapsulated MBs inhibits cord morphogenesis of endothelial cells (ECs).

[0061] FIG. 9B is a plot of relative cord formation with each of the treatments of FIG. 9A. FIG. 10 is a series of images depicting UTMD within tumors according to aspects of the present disclosure.

[0062] FIG. 11 A is an image of tumors treated with control MBs and C74-containing MBs according to aspects of the present disclosure.

[0063] FIG. 1 IB is a plot of tumor mass after treatments with control MBs and C74-containing MBs according to aspects of the present disclosure.

[0064] FIG. 12A shows micrographs of blood vessels in tissue after treatments with control MBs and C74-containing MBs according to aspects of the present disclosure.

[0065] FIG. 12B is a plot of blood vessel count in tissue after treatments with control MBs and C74-containing MBs according to aspects of the present disclosure.

[0066] FIG. 13 A shows a single-dose in vitro angiogenesis assay of UP-6 according to aspects of the present disclosure.

[0067] FIG. 13B is a plot of cord length for the assay of FIG. 13 A.

[0068] FIG. 14A shows images of another cord formation assay according to aspects of the present disclosure. 8123-112871-03 11 / 21 / 25 06880

[0069] FIG. 14B is a plot of cord length for the assay of FIG. 14A.

[0070] FIG. 14C shows a protein thermal shift (PTS) assay of the Pfnl protein with compound UP-6, C74, and a DMSO control according to aspects of the present disclosure.

[0071] FIG. 15 A shows CD31 -immunostaining after injection with growth-factor-reduced matrigel co-injected with C74, UP-6, or DMSO control according to aspects of the present disclosure.

[0072] FIG. 15B is a plot of relative CD31+ area in the assay of FIG. 15 A.

[0073] DETAILED DESCRIPTION

[0074] Overview of Terms

[0075] The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

[0076] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

[0077] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment and may be applied to any embodiment disclosed.

[0078] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification 8123-112871-03 11 / 21 / 25 06880

[0079] or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and / or limits of detection under standard test conditions / methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about" is recited. Furthermore, not all alternatives recited herein are equivalents.

[0080] When chemical structures are depicted or described, unless explicitly stated otherwise, all carbons are assumed to include implicit hydrogens such that each carbon conforms to a valence of four. For example, in the structure on the left-hand side of the schematic below there are nine hydrogen atoms implied. The nine hydrogen atoms are depicted in the right-hand structure.

[0081] H H H

[0082]

[0083] Sometimes a particular atom in a structure is described in textual formula as having a hydrogen or hydrogen atoms, for example -CH2CH2-. It will be understood by a person of ordinary skill in the art that the aforementioned descriptive techniques are common in the chemical arts to provide brevity and simplicity to description of organic structures.

[0084] Certain functional group terms include a symbol which is used to show how the defined functional group attaches to, or within, the compound to which it is bound. A person of ordinary skill in the art would recognize that the definitions provided below and the compounds and formulas included herein are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. In formulas and compounds disclosed herein, a hydrogen atom is present and completes any formal valency requirements (but may not necessarily be illustrated) wherever a functional group or other atom is not illustrated. For example, a phenyl ring that is drawn as (

[0085] comprises a hydrogen atom attached to each carbon atom of the phenyl ring other than the “a” carbon, even though such hydrogen atoms are not illustrated. Any functional group disclosed herein and / or defined above can be substituted or unsubstituted, unless otherwise indicated herein.

[0086] If a group R is depicted as “floating” on a ring system, as for example in the group: 8123-112871-03 11 / 21 / 25 06880

[0087]

[0088] then, unless otherwise defined, a substituent R can reside on any atom of the fused bicyclic ring system, so long as a stable structure is formed that conforms to standard valence conditions as understood by a person of ordinary skill in the art. In the example depicted, the R group can reside on an atom in either the 5-membered or the 6-membered ring of the indolyl ring system, including the heteroatom by replacing the explicitly recited hydrogen, but excluding the atom carrying the bond with the

[0089]

[0090] ” symbol and the bridging carbon atoms.

[0091] When there are more than one such depicted “floating” groups, as for example in the formulae:

[0092] H H

[0093] N

[0094]

[0095] where there are two groups, namely, the R and the bond indicating attachment to a parent structure; then, unless otherwise defined, each “floating” group can reside on any atoms of the ring system, again assuming each replaces a depicted, implied, or expressly defined hydrogen on the ring system and a chemically stable compound would be formed by such an arrangement.

[0096] When a group R is depicted as existing on a ring system containing saturated carbons, for example as in the formula:

[0097]

[0098] where, in this example, y can be more than one, and assuming each R replaces a currently depicted, implied, or expressly defined hydrogen on the ring; then, unless otherwise defined, two R’s can reside on the same carbon. A simple example is when R is a methyl group. The depicted structure can exist as a geminal dimethyl on a carbon of the depicted ring (an “annular” carbon). In another example, two R’s on the same carbon, including that same carbon, can form a ring, thus creating a spirocyclic ring (a “spirocyclyl” group) structure. For example, shown below two Rs can form a piperidine ring in a spirocyclic arrangement with the cyclohexane, as

[0099]

[0100] 8123-112871-03 11 / 21 / 25 06880

[0101] As used herein, the term “substituted” refers to all subsequent modifiers in a term, for example in the term “substituted arylCusalkyl,” substitution may occur on the “Ci-salkyl” portion, the “aryl” portion or both portions of the arylCi-salkyl group.

[0102] “Substituted,” when used to modify a specified group or moiety, means that at least one, and perhaps two or more, hydrogen atoms of the specified group or moiety is independently replaced with the same or different substituent groups as defined below. In a particular embodiment, a group, moiety or substituent may be substituted or unsubstituted, unless expressly defined as either “unsubstituted” or “substituted.” Accordingly, any of the groups specified herein may be unsubstituted or substituted. In particular embodiments, the substituent may or may not be expressly defined as substituted, but is still contemplated to be optionally substituted. For example, an “alkyl” or a “pyrazolyl” moiety may be unsubstituted or substituted, but an “unsubstituted alkyl” or an “unsubstituted pyrazolyl” is not substituted.

[0103] “Substituents” or “substituent groups” for substituting for one or more hydrogen atoms on saturated carbon atoms in the specified group or moiety are, unless otherwise specified, -R60, halo, =0, -OR70, -SR70, -N(R80)2, haloalkyl, perhaloalkyl. -CN, -NO2, =N2, -N3, -SO2R70, -SO3 M+, -SO3R70, -OSO2R70, -OSO3 M+, -OSO3R70, -P(O)(O )2(M+)2, -P(O)(O )2M2+, -P(O)(OR70)O M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2 M+. -CO2R70, -C(S)OR70, -C(O)N(R80)2, -C(NR7O)(R8O)2, -OC(O)R70, -OC(S)R70, -0C02M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR7()CO2

[0104]

[0105] M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(0)N(R80)2, -NR7OC(NR7O)R70or -NR70C(NR70)N(R80)2, where R60is Ci-ioaliphatic, heteroaliphatic, or cycloaliphatic, typically, Ci-ealiphatic, more typically Ci-ealkyl, where R60optionally may be substituted; each R70is independently for each occurrence hydrogen or R60; each R80is independently for each occurrence R70or alternatively, two R80groups, taken together with the nitrogen atom to which they are attached, form a 3- to 7-membered heterocycloaliphatic, which optionally includes from 1 to 4 of the same or different additional heteroatoms selected from O, N and S, of which N optionally has R70substitution, such as H or Ci-Chalkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+is independently for each occurrence, for example, an alkali metal ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R70)4; a protonated amino acid ion, such as a lysine ion, or an arginine ion; or an alkaline metal earth ion, such as [Ca2+]o.s, [Mg2+lo.5, or [Ba2+lo.5 (a subscript “0.5” means, for example, 8123-112871-03 11 / 21 / 25 06880

[0106] that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other is a typical counter ion such as chloride, or two ionized compounds can serve as counter ions for such divalent alkali earth ions, or alternatively, a doubly ionized compound can serve as the counter ion for such divalent alkali earth ions). As specific examples, -N(R80)2 includes -NH2, -NH-alkyl, -NH-pyrrolidin-3-yl, A-pyrrolidinyl, A'-piperazinyl, 4A-methyl-piperazin- 1 -yl, / V-morpholinyl and the like. Any two hydrogen atoms on a single carbon also can be replaced with, for example, =0, =NR70, =N-OR70, =N2or =S.

[0107] Substituent groups for replacing hydrogen atoms on unsaturated carbon atoms in groups containing unsaturated carbons are, unless otherwise specified, -R60, halo, -O’ M+, -OR70. -SR70, -S M+.

[0108] -N(R80)2, perhaloalkyl, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3

[0109] M+, -SO3R70, -OSO2R70,

[0110] -OSO, M+. -OSO3R70, -PO3’2(M+)2, -PO3’2M2+, -P(O)(OR70)O M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2 M+, -CO2R70, -C(S)OR70, -C(O)NR80R80. -C(NR70)N(R80)2, -OC(O)R70, -OC(S)R70, -OCO2 M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(0)N(R80)2, -NR70C(NR70)R70or -NR70C(NR70)N(R80)2, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O’ M+, -OR70, -SR70, or -S M+.

[0111] Substituent groups for replacing hydrogen atoms on nitrogen atoms in groups containing such nitrogen atoms are, unless otherwise

[0112] specified, -R60, -O’M+, -OR70, -SR70, -S’M+, -N(R80)2, perhaloalkyl, -CN, -NO, -NO2, -S(O)2R70, -SO3‘M+, -SO3R70, -OS(O)2R70, -OSO3’M+, -OSO3R70, -PO32‘(M+)2, -PO32’ M2+, -P(O)(OR70)O’M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C( S)R70, -NR70CO2R70, -NR70C(S)OR70, -NR70C(0)N(R80)2, -NR70C(NR70)R70o

[0113]

[0114] r -NR70C(NR70)N(R80)2, where R60, R70, R80and M+are as previously defined. 8123-112871-03 11 / 21 / 25 06880

[0115] In one embodiment, a group that is substituted has at least one substituent up to the number of substituents possible for a particular moiety, such as 1 substituent, 2 substituents, 3 substituents, or 4 substituents.

[0116] Additionally, in embodiments where a group or moiety is substituted with a substituted substituent, the nesting of such substituted substituents is limited to three, thereby preventing the formation of polymers. Thus, in a group or moiety comprising a first group that is a substituent on a second group that is itself a substituent on a third group, which is attached to the parent structure, the first (outermost) group can only be substituted with unsubstituted substituents. For example, in a group comprising -(aryl-1 )-(aryl-2)-(aryl-3), ary 1-3 can only be substituted with substituents that are not themselves substituted.

[0117] Any group or moiety defined herein can be connected to any other portion of a disclosed structure, such as a parent or core structure, as would be understood by a person of ordinary skill in the art, such as by considering valence rules, comparison to exemplary species, and / or considering functionality, unless the connectivity of the group or moiety to the other portion of the structure is expressly stated, or is implied by context.

[0118] Particular examples of the presently disclosed compounds include one or more asymmetric centers; thus these compounds can exist in different stereoisomeric forms.

[0119] Accordingly, compounds and compositions may be provided as individual pure enantiomers or as stereoisomeric mixtures, including racemic mixtures. In certain aspects, the compounds disclosed herein are synthesized in or are purified to be in substantially enantiopure form, such as in a 90% enantiomeric excess, a 95% enantiomeric excess, a 97% enantiomeric excess or even in greater than a 99% enantiomeric excess, such as in enantiopure form.

[0120] Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.), Hawley’s Condensed Chemical Dictionary, published by lohn Wiley & Sons, Inc., 2016 (ISBN 978-1-118-13515-0). The presently disclosed compounds also include all isotopes of atoms present in the compounds, which can include, but are not limited to, deuterium, tritium,18F,14C, etc. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, lohn & Sons, Inc., 1995 (ISBN 0471186341); and other similar references. 8123-112871-03 11 / 21 / 25 06880

[0121] In order to facilitate review of the various aspects of the disclosure, the following explanations of specific terms are provided.

[0122] Active agent: A drug, medicament, pharmaceutical, therapeutic agent, nutraceutical, or other compound that may be administered to a subject to effect a change, such as treatment, amelioration, or prevention of a disease or disorder or at least one symptom associated therewith. The active agent may be a “small molecule,” generally having a molecular weight of about 2000 daltons or less. The active agent may also be a “biological active agent.” Biological active agents include proteins, antibodies, antibody fragments, peptides, oligonucleotides, vaccines, and various derivatives of such materials.

[0123] “Acyl” refers to the group -C(O)R, where R is H, aliphatic, heteroaliphatic, heterocyclic or aromatic. Exemplary acyl moieties include, but are not limited to, -C(O)H, -C(O)alkyl, -C(O)Ci-C6alkyl, -C(O)Ci-C6haloalkyl, -C(O)cycloalkyl, -C(O)alkenyl, -C(O)cycloalkenyl, -C(O)aryl, -C(O)heteroaryl, or -C(O)heterocyclyl. Specific examples include -C(O)H, -C(O)Me, -C(O)Et, or -C(O)cyclopropyl.

[0124] Administration / administering: “Administration of’ and “administering a” compound should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition can be administered by another person to the subject (e.g., intravenously) or it can be selfadministered by the subject (e.g., tablets). Co-administration or co-administering means administering two or more therapeutic agents or modalities. Co-administration may occur simultaneously or sequentially in any order, and may occur by the same or different routes of administration. When administering simultaneously, the two or more therapeutic agents may be present in a single pharmaceutical composition or in separate pharmaceutical compositions.

[0125] Alcohol: An organic compound including at least one hydroxyl group. Alcohols may be monohydric (including one -OH group), dihydric (including two -OH groups; diols, such as glycols), trihydric (including three -OH; triols, such as glycerol) groups, or polyhydric (including three or more -OH groups; polyols). The organic portion of the alcohol may be aliphatic, cycloaliphatic (alicyclic), heteroaliphatic, cycloheteroaliphatic (heterocyclic), polycyclic, aryl, or heteroaryl, and may be substituted or unsubstituted.

[0126] Aldehyde: A carbonyl-bearing functional group having a formula 8123-112871-03 11 / 21 / 25 06880

[0127]

[0128] where the line drawn through the bond indicates that the functional group can be attached to any other moiety, but that such moiety simply is not indicated.

[0129] An “adjuvant” is an excipient that modifies the effect of other agents, typically the active ingredient. Adjuvants are often pharmacological and / or immunological agents. An adjuvant may modify the effect of an active ingredient by increasing an immune response. An adjuvant may also act as a stabilizing agent for a formulation. Exemplary adjuvants include, but are not limited to, aluminum hydroxide, alum, aluminum phosphate, killed bacteria, squalene, detergents, cytokines, paraffin oil, and combination adjuvants, such as Freund’s complete adjuvant or Freund’s incomplete adjuvant.

[0130] Aliphatic: A substantially hydrocarbon-based compound, or a radical thereof (e.g., C6H13, for a hexane radical), including alkanes, alkenes, alkynes, including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Unless expressly stated otherwise, an aliphatic group contains from one to twenty-five carbon atoms; for example, from one to fifteen, from one to ten, from one to six, or from one to four carbon atoms. The term "lower aliphatic" refers to an aliphatic group containing from one to ten carbon atoms. An aliphatic chain may be substituted or unsubstituted. Unless expressly referred to as an “unsubstituted aliphatic,” an aliphatic group can either be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in an aliphatic chain, or up to one substituent for each carbon of a -C=C- double bond in an aliphatic chain, or up to one substituent for a carbon of a terminal methine group). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or other functionality.

[0131] Alkoxy: A radical (or substituent) having the structure -OR, where R is a substituted or unsubstituted alkyl. Methoxy (-OCH3) is an exemplary alkoxy group. In a substituted alkoxy, R is alkyl substituted with a non-interfering substituent. “Thioalkoxy” refers to -S-R, where R is substituted or unsubstituted alkyl. “Haloalkyloxy” means a radical -OR where R is a haloalkyl. 8123-112871-03 11 / 21 / 25 06880

[0132] Alkyl: A hydrocarbon group having a saturated carbon chain. The chain may be cyclic, branched or unbranched. Examples, without limitation, of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. The term lower alkyl means the chain includes 1-10 carbon atoms. The terms alkenyl and alkynyl refer to hydrocarbon groups having carbon chains containing one or more double or triple bonds, respectively.

[0133] Alkylamino: A chemical functional group -N(H)R, where R is an alkyl group. Alkylammonium: A cation having a formula [N(H)(R')3]+where each R' independently is H or alkyl.

[0134] Allyl: A hydrocarbon group with the structural formula H2C=CH-CH2-. Allylation is a reaction that adds an allyl group to a compound and / or forms an allyl group in a compound.

[0135] Allyloxycarbonyl (Aloe, Alloc, Aoc): A functional group with the general formula:

[0136]

[0137] CH2

[0138] Amide: An organic compound characterized by a carbonyl group (C=O) linked to a nitrogen atom and having the following general formula, where R, R' and R" are the same or different, and typically are selected from hydrogen, aliphatic, and aryl.

[0139] Amido: A chemical functional group -C(O)N(R)(R’) where R and R’ are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, or other functionality.

[0140] Amino: A chemical functional group -N(R)R' where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, or other functionality. A “primary amino” group is -NH2. “Mono-substituted amino” means a radical -N(H)R substituted as above and includes, e.g., methylamino, (l-methylethyl)amino, phenylamino, and the like. “Di-substituted amino” means a radical -N(R)R' substituted as above and includes, e.g., dimethylamino, methylethylamino, di(l-methylethyl)amino, and the like. 8123-112871-03 11 / 21 / 25 06880

[0141] Amino Acid: An organic acid containing both a basic amino group (-NH2) and an acidic carboxyl group (-COOH). The 25 amino acids that are protein constituents are a-amino acids, i.e., the -NH2 group is attached to the carbon atom next to the -COOH group.

[0142] Amino acid transporter: A membrane transport protein capable of transporting amino acids across the membrane.

[0143] Aminoalkyl: A chemical functional group -RNH2 where R is an alkyl group.

[0144] Analog or Mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound.

[0145] Antibody: “Antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules [including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any chordate such as a vertebrate, for example, in mammals such as humans, goats, rabbits and mice] and fragments thereof that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. An “antibody” typically comprises a polypeptide ligand having at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of an antigen. Immunoglobulins are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the immunoglobulin. Exemplary immunoglobulin fragments include, without limitation, proteolytic immunoglobulin fragments [such as F(ab’)2 fragments, Fab’ fragments, Fab’-SH fragments and Fab fragments as are known in the art], recombinant immunoglobulin fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”). Other examples of antibodies include diabodies, and triabodies (as are known in the art), and camelid antibodies. " Antibody" also 8123-112871-03 11 / 21 / 25 06880

[0146] includes genetically engineered molecules, such as chimeric antibodies (for example, humanized murine antibodies), and heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rdEd., W. H. Freeman & Co., New York, 1997.

[0147] Aralkyl / arylalkyl: An aryl group (such as a phenyl group) appended to an alkyl radical including, but not limited to, benzyl, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene, and the like. Conversely the term "phenylalkyl" refers to a phenyl group appended to an alkyl radical. Aralkyl groups, such as benzyl groups, may be unsubstituted or substituted with one, two or three substituents, with substituent(s) independently selected from alkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, haloalkyl (such as -CF3), halo, nitro, cyano, -OR (where R is hydrogen or alkyl), -N(R)R’ (where R and R’ are independently of each other hydrogen or alkyl), -COOR (where R is hydrogen or alkyl) or -C(O)N(R’)R” (where R’ and R” are independently selected from hydrogen or alkyl).

[0148] Non-limiting examples, include o-, m-, and / or p-chlorobenzyl, o-, m-, and / or

[0149] p-methoxybenzyl, and o-, m-, and / or p-(trifluoromethyl)benzyl.

[0150] Aromatic: Unsaturated, cyclic hydrocarbons having alternate single and double bonds. Benzene, a 6-carbon ring containing three double bonds, is a typical aromatic compound.

[0151] Aryl: A monovalent aromatic carbocyclic group of, unless specified otherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., indole, benzodioxole, and the like), provided that the point of attachment is through an atom of an aromatic portion of the aryl group and the aromatic portion at the point of attachment contains only carbons in the aromatic ring. If any aromatic ring portion contains a heteroatom, the group is a heteroaryl and not an aryl. Aryl groups are monocyclic, bicyclic, tricyclic or tetracyclic.

[0152] Arylalkyl: An acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylalkenyl and / or arylalkynyl may be used.

[0153] Azole: A 5-membered heterocyclic compound containing a nitrogen atom and at least one other heteroatom (nitrogen, sulfur, or oxygen) as part of the ring. Exemplary azoles 8123-112871-03 11 / 21 / 25 06880

[0154] include imidazole, pyrazole, 1,2, 3 -tri azole, 1,2,4-triazole, tetrazole, pentazole, oxazole, isoxazole, oxadiazole, furazan, 1,3,4-oxadiazole, thiazole, isothiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole.

[0155] Bioisostere: A molecule resulting from the exchange of an atom or group of atoms with another atom or group of atoms to produce a molecule with similar biological properties to the parent compound. For example, a hydrogen atom may be replaced by a deuterium or fluorine atom, a carbon atom may be replaced by a Si atom, or a methyl group may be replaced by an amino group, a hydroxy group, a fluorine atom, or a chlorine atom.

[0156] Biomolecule: Any molecule that may be included in a biological system, including but not limited to, a synthetic or naturally occurring protein, glycoprotein, lipoprotein, amino acid, nucleoside, nucleotide, nucleic acid, oligonucleotide, DNA, RNA, carbohydrate, sugar, lipid, fatty acid, hapten, and the like.

[0157] Bis: A prefix meaning "twice" or "again." It is used in chemical nomenclature to indicate that a chemical group or radical occurs twice in a molecule. For example, a bis-ester has two ester groups.

[0158] Carbohydrate: An organic compound that consists only of carbon, hydrogen, and oxygen, usually with a hydrogen: oxygen atom ratio of 2:1. Carbohydrates include monosaccharides, disaccharides, and polysaccharides. Monosaccharides are aldehydes or ketones with two or more hydroxyl groups, and a general chemical formula of (C H2O)n. Disaccharides are formed from two monosaccharides linked together by a glycosidic bond. A polysaccharide is a polymer of monosaccharides linked together by glycosidic bonds.

[0159] Carbonyl: A radical of the formula -C(O)-. Carbonyl -containing groups include any substituent containing a carbon-oxygen double bond (C=O), including acyl groups, amides, carboxy groups, esters, ureas, carbamates, carbonates and ketones and aldehydes, such as substituents based on -COR or -RCHO where R is an aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine.

[0160] Carboxyalkyl: A functional group with the formula -COOR where R is alkyl.

[0161] Carboxyl: A -COOH radical. Substituted carboxyl refers to -COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or a carboxylic acid or ester.

[0162] Carboxylic Acid: A carbonyl-bearing functional group having a formula RCOOH where R is aliphatic, heteroaliphatic, alkyl, or heteroalkyl. 8123-112871-03 11 / 21 / 25 06880

[0163] “Carboxyl Ester” or “carboxy ester” refers to the group -C(O)OR, where R is aliphatic, heteroaliphatic, cyclicaliphatic, heterocyclic, and aromatic, including both aryl and heteroaryl.

[0164] Combination: A combination comprises two or more components that are administered such that the effective time period of the first component overlaps with the effective time period of the second and subsequent components. A combination may be a composition comprising the components, or it may be two or more individual components administered substantially simultaneously, or sequentially in any order Condensation: A type of chemical reaction in which two or more molecules combine with the separation of water, alcohol, or other simple substance.

[0165] Conjugate: Two or more moieties directly or indirectly coupled together. For example, a first moiety may be covalently or noncovalently (e.g., electrostatically) coupled to a second moiety. Indirect attachment is possible, such as by using a "linker" (a molecule or group of atoms positioned between two moieties).

[0166] Control: A sample or procedure performed to assess test validity. In one example, a control is a quality control, such as a positive control. For example, a positive control is a procedure or sample, such as a tissue or cell, that is similar to the actual test sample, but which is known from previous experience to give a positive result. A positive control confirms that the basic conditions of the test produce a positive result, even if none of the actual test samples produce such result. In a particular example, a positive control is a sample known by previous testing to contain the suspected antigen.

[0167] In other examples, a control is a negative control. A negative control is a procedure or test sample known from previous experience to give a negative result. The negative control demonstrates the base-line result obtained when a test does not produce a measurable positive result; often the value of the negative control is treated as a "background" value to be subtracted from the test sample results. In a particular example, a negative control is a reagent that does not include the specific primary antibody. Other examples include calibrator controls, which are samples that contain a known amount of a control antigen. Such calibrator controls have an expected signal intensity, and therefore can be used to correct for inter- or intra-run staining variability.

[0168] Conjugating, joining, bonding or linking: Coupling a first unit to a second unit. This includes, but is not limited to, covalently bonding one molecule to another molecule, noncovalently bonding one molecule to another (e.g. electrostatically bonding) (see, for 8123-112871-03 11 / 21 / 25 06880

[0169] example, U. S. Patent No. 6,921,496, which discloses methods for electrostatic conjugation), non-covalently bonding one molecule to another molecule by hydrogen bonding, non-covalently bonding one molecule to another molecule by van der Waals forces, and any and all combinations of such couplings.

[0170] Counterstaining: A method of post -treating samples after they have already been stained with agents to detect one or more targets, such that their structures can be more readily visualized under a microscope. For example, a counterstain is optionally used prior to coverslipping to render a immunohistochemical stain more distinct. Counterstains differ in color from a primary stain. Numerous counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, 4',6-diamidino-2-phenylindole (DAPI), and Nuclear Fast Red. In some examples, more than one stain can be mixed together to produce the counterstain. This provides flexibility and the ability to choose stains. For example, a first stain, can be selected for the mixture that has a particular attribute, but yet does not have a different desired attribute. A second stain can be added to the mixture that displays the missing desired attribute. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.

[0171] Coupled: The term "coupled" means joined together, either directly or indirectly. A first atom or molecule can be directly coupled or indirectly coupled to a second atom or molecule. A secondary antibody provides an example of indirect coupling. One specific example of indirect coupling is a rabbit anti-hapten primary antibody that is bound by a mouse anti -rabbit IgG antibody, which is in turn bound by a goat anti -mouse IgG antibody that is covalently linked to a detectable label.

[0172] “Cyano” refers to the group -CN.

[0173] Cyclic: Designates a substantially hydrocarbon, closed-ring compound, or a radical thereof. Cyclic compounds or substituents also can include one or more sites of unsaturation, but does not include aromatic compounds. One example of such a cyclic compound is cy clopentadienone.

[0174] “Cycloaliphatic” refers to a cyclic aliphatic group having a single ring (e.g., cyclohexyl), or multiple rings, such as in a fused, bridged or spirocyclic system, at least one of which is aliphatic. Typically, the point of attachment to the parent structure is through an aliphatic portion of the multiple ring system. Cycloaliphatic includes saturated and unsaturated systems, including cycloalkyl, cycloalkenyl and cycloalkynyl. A cycloaliphatic group may contain from three to twenty-five carbon atoms: for example, from three to fifteen, 8123-112871-03 11 / 21 / 25 06880

[0175] from three to ten, or from three to six carbon atoms. Unless otherwise stated, a cycloaliphatic group may be substituted or unsubstituted. Exemplary cycloaliphatic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, or cyclohexenyl.

[0176] Cycloalkyl: A saturated monovalent cyclic hydrocarbon radical of three to seven ring carbons, e.g.. cyclopentyl, cyclohexyl, cycloheptyl and the like.

[0177] Degree of branching (DOB): The term “degree of branching” refers to the number of branches per molecule and is usually provided as a percentage. Percent DOB is defined as (T+Z) / (T+Z+L) x 100, where T is the average number of terminally bonded monomer units, Z is the average number of monomer units forming branches, and L is the average number of linearly bonded monomer units.

[0178] Derivative: A compound that is derived from a similar compound or a compound that can be imagined to arise from another compound, for example, if one atom is replaced with another atom or group of atoms. The latter definition is common in organic chemistry. In biochemistry, the word is used for compounds that at least theoretically can be formed from the precursor compound. In some examples, a derivative is a biologically active molecule derived from the base structure.

[0179] Detect: To determine if an agent (such as a signal or particular antigen, protein or nucleic acid) is present or absent, for example, in a sample. In some examples, this can further include quantification, and / or localization, for example localization within a cell or particular cellular compartment. “Detecting” refers to any method of determining if something exists, or does not exist, such as determining if a target molecule is present in a biological sample. For example, “detecting” can include using a visual or a mechanical device to determine if a sample displays a specific characteristic. In certain examples, detection refers to visually observing a probe bound to a target, or observing that a probe does not bind to a target. For example, light microscopy and other microscopic means are commonly used to detect chromogenic precipitates for methods described here.

[0180] Detectable Label: A detectable compound or composition that is attached directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Nanoparticles provide one, non-limiting example of a class of detectable labels.

[0181] Diastereomers: Optically active isomers containing two or more asymmetric carbons with differing configurations at one or more of the stereocenters and are not mirror images of each other: 8123-112871-03 11 / 21 / 25 06880

[0182] H2O

[0183] HO — C H H - C - OH

[0184] H - C - OH

[0185]

[0186] CH2OH CH2OH

[0187] Diastereomers that differ at only one stereocenter are also known as epimers.

[0188] “Effective amount” with respect to a compound or composition refer to an amount of the compound or composition sufficient to achieve a particular desired result, such as to inhibit a protein or enzyme, particularly an interleukin-1 receptor-associated kinase; to elicit a desired biological or medical response in a tissue, system, subject or patient; to treat a specified disorder or disease; to ameliorate or eradicate one or more of its symptoms; and / or to prevent the occurrence of the disease or disorder. The amount of a compound which constitutes an “effective amount” may vary depending on the compound, the desired result, the disease state and its severity, the age of the patient to be treated, and the like.

[0189] Enzyme: A protein molecule that is capable of catalyzing a chemical reaction. For example, cellulase is an enzyme capable of converting cellulose to glucose.

[0190] Epoxide: A cyclic ether with a 3 -membered ring having a general formula

[0191] R1,0 R3

[0192]

[0193] ,2

[0194] where R'-R4independently are H or aliphatic.

[0195] Ester: A chemical compound derived from an organic acid (general formula:

[0196] RCO2H) where the hydrogen of the -OH (hydroxyl) group is replaced by an aliphatic, alkyl or aryl group. A general formula for an ester derived from an organic acid is shown below:

[0197]

[0198] K OR'

[0199] where R and R' denote virtually any group, including aliphatic, substituted aliphatic, aryl, arylalkyl, heteroaryl, etc.

[0200] Ether: A class of organic compounds containing an ether group, that is an oxygen atom connected to two aliphatic and / or aryl groups, and having a general formula R-O-R', where R and R' may be the same or different.

[0201] Excipient: A physiologically inert substance that is used as an additive in a pharmaceutical composition. As used herein, an excipient may be incorporated within 8123-112871-03 11 / 21 / 25 06880

[0202] particles of a pharmaceutical composition, or it may be physically mixed with particles of a pharmaceutical composition. An excipient can be used, for example, to dilute an active agent and / or to modify properties of a pharmaceutical composition. Examples of excipients include but are not limited to polyvinylpyrrolidone (PVP), tocopheryl polyethylene glycol 1000 succinate (also known as vitamin E TPGS, or TPGS), dipalmitoyl phosphatidyl choline (DPPC), trehalose, sodium bicarbonate, glycine, sodium citrate, and lactose.

[0203] Fatty acid: A carboxylic acid having a long, unbranched, aliphatic chain or tail. Fatty acids contain from 4 to 22 carbon atoms (usually an even number). Fatty acids can be represented by the general formula RCOOH, where R is a saturated or unsaturated aliphatic chain. Saturated fatty acids can be described by the general formula CH3(CH2)XCOOH. Many natural fatty acids have an aliphatic chain that has at least eight carbon atoms and an even number of carbon atoms (including the carbon atom in the carboxyl group). The fatty acid may be a liquid, semisolid, or solid.

[0204] Fluorescence: The emission of visible radiation by an atom or molecule passing from a higher to a lower electronic state, wherein the time interval between absorption and emission of energy is 10sto 10’3second. Fluorescence occurs when the atom or molecule absorbs energy from an excitation source (e.g., an ultraviolet lamp) and then emits the energy as visible radiation.

[0205] Free radical: An atom, molecule, or ion with an unpaired electron. Free radicals are formed by splitting a chemical bond within a molecule, and are usually short-lived and highly reactive. Free radicals are capable of initiating chemical chain reactions, e.g., polymerization. They also act as initiators or intermediates in oxidation, combustion, and photolysis.

[0206] Functional group: A specific group of atoms within a molecule that is responsible for the characteristic chemical reactions of the molecule. Exemplary functional groups include, without limitation, alkyl, alkenyl, alkynyl, aryl, halo (fluoro, chloro, bromo, iodo), epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate ester, carboxylate, carboxyl, ether, ester, peroxy, hydroperoxy, carboxamide, amino (primary, secondary, tertiary), ammonium, imide, azide, cyanate, isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkyl, nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl), disulfide.

[0207] Glycidyl ether: A functional group having the general structure:

[0208] o

[0209]

[0210] - / -o

[0211] “Halo,” “halide” or “halogen” refers to fluoro, chloro, bromo or iodo. 8123-112871-03 11 / 21 / 25 06880

[0212] Haloalkyl: An alkyl group substituted with one or more same or different halo atoms, e.g., -CH2CI, -CF3, -CH2CF3, -CF2CF3, -CH2CCI3, and the like.

[0213] Hapten: A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule. Examples of haptens include, but are not limited to fluorescein, biotin, nitroaryls, including, but, not limited to, dinitrophenol (DNP), digoxigenin, oxazole, pyrazole, thiazole, benzofuran, triperpene, urea, thiourea, rotenoid, coumarin and cyclolignan.

[0214] Heteroalkyl: An alkyl or cycloalkyl radical having at least one carbon atom in the chain and containing at least one heteroatom, such as N, O, S, or S(O)n(where n is 1 or 2).

[0215] Heteroaliphatic: An aliphatic compound or group having at least one carbon atom in the chain and at least one heteroatom, i.e., one or more carbon atoms has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. Heteroaliphatic compounds or groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" groups.

[0216] Heteroaryl: An aromatic compound or group having at least one heteroatom, i.e., one or more carbon atoms in the ring has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. In specific examples, the term heteroaryl includes, but is not limited to pyridyl, pyrrolyl, thiophene, pyrazolyl, thiazolyl, imidazolyl, pyrimidinyl, thiadiazolyl, indolyl, carbazolyl, azaindolyl, benzofuranyl, benzimidazolyl, benzthiazolyl, quinoxalinyl, benzotriazolyl, benzisoxazolyl, purinyl, quinolinyl, isoquinolinyl, benzopyranyl, and derivatives thereof.

[0217] Heterobifunctional: Cross-linking agents contain at least two different reactive groups at each end, which are reactive towards numerous groups, including but not limited to sulfhydryls and amines, and create chemical covalent bonds between two or more molecules, for example between specific binding agent or moiety (such as an antibody) and an enzyme (such as horseradish peroxidase).

[0218] “Heterocyclyl,” “heterocyclo” and “heterocycle” refer to both aromatic and nonaromatic ring systems, and more specifically refer to a stable three- to fifteen-membered ring moiety comprising at least one carbon atom, and typically plural carbon atoms, and at least one, such as from one to five, heteroatoms. The heteroatom(s) may be nitrogen, phosphorus, oxygen, silicon or sulfur atom(s). The heterocyclyl moiety may be a monocyclic moiety, or 8123-112871-03 11 / 21 / 25 06880

[0219] may comprise multiple rings, such as in a bicyclic or tricyclic ring system, provided that at least one of the rings contains a heteroatom. Such a multiple ring moiety can include fused or bridged ring systems as well as spirocyclic systems; and any nitrogen, phosphorus, carbon, silicon or sulfur atoms in the heterocyclyl moiety can be optionally oxidized to various oxidation states. For convenience, nitrogens, particularly, but not exclusively, those defined as annular aromatic nitrogens, are meant to include their corresponding N-oxide form, although not explicitly defined as such in a particular example. Thus, for a compound having, for example, a pyridinyl ring, the corresponding pyridinyl-N-oxide is included as another compound of the invention, unless expressly excluded or excluded by context. In addition, annular nitrogen atoms can be optionally quaternized. Heterocycle includes heteroaryl moieties, and heteroal icyclyl or heterocycloaliphatic moieties, which are heterocyclyl rings that are partially or fully saturated. Examples of heterocyclyl groups include, but are not limited to, azetidinyl, oxetanyl, acridinyl, benzodioxolyl, benzodioxanyl, benzofuranyl, carbazoyl, cinnolinyl, dioxolanyl, indolizinyl, naphthyridinyl, perhydroazepinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrazoyl, tetrahydroisoquinolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, dihydropyridinyl, tetrahydropyridinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolinyl, oxazolidinyl, triazolyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, isoindolyl, indolinyl, isoindolinyl, octahydroindolyl, octahydroisoindolyl, quinolyl, isoquinolyl, decahydroisoquinolyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzo thiazolyl, benzoxazolyl, furyl, di azabicycloheptane, diazapane, diazepine, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothieliyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, dioxaphospholanyl, and oxadiazolyl.

[0220] Heteropolyacid: An acid that includes hydrogen, oxygen, metal(s) and non-metal(s). Typical non-metals include, e.g., silicon, phosphorus, and arsenic. Typical metals include, e.g., tungsten, molybdenum, and vanadium. Exemplary heteropolyacids include H4Wi2SiO40, ^MonSiCUo, H3PM012O40, H3PW12O40, H3PM06V6O40, and H3PM010V2O40.

[0221] Hydrocarbon: An organic compound consisting of the elements carbon and hydrogen. Hydrocarbons typically are derived from petroleum, coal tar, and plant sources. 8123-112871-03 11 / 21 / 25 06880

[0222] Hydrocarbons include aliphatic compounds (alkanes, alkenes, alkynes, and cyclic versions thereof, including straight- and branched-chain arrangements), aromatic compounds (unsaturated, cyclic hydrocarbons having alternate single and double bonds), and combinations thereof (e.g., arylalkyl compounds).

[0223] “Hydroxyl” refers to the group -OH.

[0224] Hydroxyalkyl: An alkyl group as defined above substituted with at least one hydroxyl group, provided that if two or more hydroxyl groups are present no two hydroxyl groups are on the same carbon atom.

[0225] Imine: An organic compound containing a -C=NR group, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, or other functionality.

[0226] Immunohistochemistry (IHC): A method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with a specific binding agent or moiety, such as an antibody. A sample including an antigen (such as a target antigen) is incubated with an antibody under conditions permitting antibody-antigen binding. Antibody-antigen binding can be detected by means of a detectable label conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody, which is raised against the primary antibody (e.g., indirect detection). Detectable labels include, but are not limited to, radioactive isotopes, fluorochromes (such as fluorescein, fluorescein isothiocyanate, and rhodamine), enzymes and chromogenic molecules.

[0227] Indole: An aromatic heterocyclic organic compound with a bicyclic structure consisting of a six-membered benzene ring fused to a five -membered, nitrogen-containing pyrrole ring.

[0228]

[0229] Isomer: One of two or more molecules having the same number and kind of atoms, but differing in the arrangement or configuration of the atoms. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are

[0230] non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, if a carbon atom is bonded to four 8123-112871-03 11 / 21 / 25 06880

[0231] different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and

[0232] S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-) isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.” E / Z isomers are isomers that differ in the stereochemistry of a double bond. An E isomer (from entgegen, the German word for "opposite") has a trans-configuration at the double bond, in which the two groups of highest priority are on opposite sides of the double bond. A Z isomer (from zusammen, the German word for "together") has a cA-configuration at the double bond, in which the two groups of highest priority are on the same side of the double bond. The E and Z isomers of 2-butene are shown below:

[0233] H\ / CH3 H3C CH3

[0234] yC=C\ \=C

[0235] H

[0236]

[0237] 3C H / \

[0238] (Ej-but-2-ene (Z)-but-2-ene

[0239] Ketone: A carbonyl-bearing substituent having a formula

[0240] where R is virtually any group, including aliphatic, substituted aliphatic, aryl, arylalkyl, heteroaryl, etc.

[0241] Ligand: A molecule that binds to a receptor, having a biological effect.

[0242] Linker: A molecule or group of atoms positioned between two moieties. For example, a quantum dot-DNA binding molecule conjugate may include a linker between the quantum dot and the DNA binding molecule. Typically, linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two moieties. The two functional groups may be the same, i.e., a homobifunctional linker, or different, i.e., a heterobifunctional linker.

[0243] Lipid: An inclusive term for fats and fat-derived materials. It includes all substances that are (1) relatively insoluble in water but soluble in organic solvents; (2) related either actually or potentially to fatty acid esters, fatty alcohols, sterols, waxes, etc.; and (3) utilizable by a living organism. 8123-112871-03 11 / 21 / 25 06880

[0244] Moiety: A moiety is a fragment of a molecule, or a portion of a conjugate.

[0245] Molecular weight: The sum of the atomic weights of the atoms in a molecule. As used herein with respect to polymers, the terms molecular weight, average molecular weight, and mean molecular weight refer to the number-average molecular weight, which corresponds to the arithmetic mean of the molecular weights of individual macromolecules. The number-average molecular weight may be determined by any method generally known by persons of ordinary skill in the art, such as chromatographic methods.

[0246] Molecule of interest or target molecule: A molecule for which the presence, location and / or concentration is to be determined. Examples of molecules of interest include proteins and nucleic acid sequences present in tissue samples.

[0247] Monomer: A molecule or compound, usually containing carbon, that can react and combine to form polymers. For example, polystyrene is formed by polymerization of the monomer styrene. Polyvinyl acetate is formed by polymerization of the monomer vinyl acetate. Common monomers include, for example and without limitation, alkenes, amides, amino acids, arenes, monosaccharides (e.g., glucose and fructose, among others), and unsaturated carboxylic acids (e.g., acrylic acid, adipic acid, and muconic acid, among others). Molecules formed by the combination of monomers can be characterized by the number of monomers. For example, a dimer is a molecule formed from two monomers, a trimer is a molecule formed from three monomers, etc. A molecule with more than 10 monomers is typically referred to using the number of monomeric units, e.g., a 20-mer is a molecule having 20 monomeric units.

[0248] “Nitro” refers to the group -NO2.

[0249] Nucleophile: An ion or molecule that donates an electron pair to an atomic nucleus to form a covalent bond.

[0250] Nucleoside: A compound containing a purine or pyrimidine base linked to either d-ribose, forming a riboside, or -deoxyribose, forming a deoxyriboside. Nucleosides are nucleotides minus the phosphorus group.

[0251] Number average molecular weight: The statistical average molecular weight of all the polymer chains in a sample, defined as:

[0252]

[0253] where M, is the molecular weight of a chain, and Ni is the number of chains of that molecular weight. Mncan be predicted by polymerization mechanisms and is measured by methods that 8123-112871-03 11 / 21 / 25 06880

[0254] determine the number of molecules in a sample of a given weight, e.g., gel permeation chromatography, viscometry, colligative methods such as vapor pressure osmometry, end group assays, or proton NMR.

[0255] Olefin: An unsaturated aliphatic hydrocarbon having one or more double bonds. Olefins with one double bond are alkenes; olefins with two double bonds are alkadienes or diolefins. Olefins typically are obtained by cracking petroleum fractions at high temperatures.

[0256] Oligo-: Prefix meaning "a few."

[0257] Oligomer: A general term for a polymeric molecule consisting of relatively few monomers.

[0258] Oligonucleotide: A plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid molecules.

[0259] Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15, or 20 bases.

[0260] Paraffin: A saturated hydrocarbon having the general formula CnH2n+2.

[0261] “Patient” or “Subject” refers to mammals and other animals, particularly humans. Thus disclosed methods are applicable to both human therapy and veterinary applications.

[0262] Pharmaceutically acceptable: A substance that can be taken into a subject without significant adverse toxicological effects on the subject. The term "pharmaceutically acceptable form" means any pharmaceutically acceptable derivative or variation, such as stereoisomers, stereoisomer mixtures, enantiomers, solvates, hydrates, isomorphs, polymorphs, pseudomorphs, neutral forms, salt forms, and prodrug agents.

[0263] Pharmaceutically acceptable carrier: An excipient that is a carrier or vehicle, such as a suspension aid, solubilizing aid, or aerosolization aid. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, 8123-112871-03 11 / 21 / 25 06880

[0264] Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005). describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some examples, the pharmaceutically acceptable carrier is a non-naturally occurring or synthetic carrier. The carrier also can be formulated in a unit-dosage form that carries a preselected therapeutic dosage of the active agent, for example in a pill, vial, bottle, or syringe.

[0265] “Pharmaceutically acceptable excipient” refers to a substance, other than the active ingredient, that is included in a formulation of the active ingredient. As used herein, an excipient may be incoiporated within particles of a pharmaceutical composition, or it may be physically mixed with particles of a pharmaceutical composition. An excipient can be used, for example, to dilute an active agent and / or to modify properties of a pharmaceutical composition. Excipients can include, but are not limited to, antiadherents, binders, coatings, enteric coatings, disintegrants, flavorings, sweeteners, colorants, lubricants, glidants, sorbents, preservatives, adjuvants, carriers or vehicles. Excipients may be starches and modified starches, cellulose and cellulose derivatives, saccharides and their derivatives such as disaccharides, polysaccharides and sugar alcohols, protein, synthetic polymers, crosslinked polymers, antioxidants, amino acids or preservatives. Exemplary excipients include, but are not limited to, magnesium stearate, stearic acid, vegetable stearin, sucrose, lactose, starches, hydroxypropyl cellulose, hydroxypropyl methylcellulose, xylitol, sorbitol, maltitol, gelatin, polyvinylpyrrolidone (PVP), polyethyleneglycol (PEG), tocopheryl polyethylene glycol 1000 succinate (also known as vitamin E TPGS, or TPGS), carboxy methyl cellulose, dipalmitoyl phosphatidyl choline (DPPC), vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben, sugar, 8123-112871-03 11 / 21 / 25 06880

[0266] silica, talc, magnesium carbonate, sodium starch glycolate, tartrazine, aspartame, benzalkonium chloride, sesame oil, propyl gallate, sodium metabisulphite or lanolin.

[0267] Pharmaceutically acceptable salt: A biologically compatible salt of a compound that can be used as a drug, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically acceptable acid addition salts are those salts that retain the biological effectiveness of the free bases while formed by acid partners that are not biologically or otherwise undesirable, e.g., inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, benzene sulfonic acid (besylate), cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (See, for example, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19, which is incorporated herein by reference.)

[0268] “Phosphate” refers to the group -O-P(O)(OR’)2, where each -OR’ independently is -OH; -O-aliphatic, such as -O-alkyl or -O-cycloalkyl; -O-aromatic, including both -O-aryl and -O-heteroaryl; -O-aralkyl; or 8123-112871-03 11 / 21 / 25 06880

[0269] -OR’ is -O'M+, where M+is a counter ion with a single positive charge. Each M+may be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R”)4 where each R” independently is H, aliphatic, heterocyclyl or aryl; or an alkaline earth ion, such as [Ca2+]o.5, [Mg2+]o.s, or [Ba2+]o,5- Phosphonooxyalkyl refers to the group -alkyl-phosphate, such as, for example, -CH2OP(O)(OH)2, or a salt thereof, such as -CH2OP(O)(O Na+)2, and (((dialkoxyphosphoryl)oxy)alkyl) refers to the dialkyl ester of a phosphonooxyalkyl group, such as, for example, -CH2OP(O)(O-tert-butyl)2.

[0270] “Phosphonate” refers to the group -P(O)(OR’)2, where each -OR’ independently is -OH; -O-aliphatic such as -O-alkyl or -O-cycloalkyl; -O-aromatic, including both -O-aryl and -O-heteroaryl; or -O-aralkyl; or

[0271] -OR’ is -O'M+, and M+is a counter ion with a single positive charge. Each M+is a positively charged counterion and may be, by way of example, an alkali metal ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R”)4 where each R” independently is H, aliphatic, heterocyclyl or aryl; or an alkaline earth metal ion, such as [Ca2+]o.s, [Mg2+]o.s, or [Ba2+]o.s.

[0272] Phosphonoalkyl refers to the group -alkyl-phosphonate, such as, for

[0273] example, -CH2P(O)(OH)2, or -CH2P(O)(O Na+)2, and ((dialkoxyphosphoryl)alkyl) refers to the dialkyl ester of a phosphonoalkyl group, such as, for example, -CH2P(O)(O-tert-butyl)2.

[0274] Polymer: A molecule of repeating structural units (e.g., monomers) formed via a chemical reaction, i. e., polymerization.

[0275] Polymerization: A chemical reaction, usually carried out with a catalyst, heat or light, in which a large number of relatively simple molecules (monomers) combine to form a chainlike macromolecule (a polymer). The chains further can be combined, or crosslinked, by the addition of appropriate chemicals. The monomers typically are unsaturated or otherwise reactive substances. Polymerization commonly occurs by addition or condensation. Addition polymerization occurs when an initiator, usually a free radical, reacts with a double bond in the monomer. The free radical adds to one side of the double bond, producing a free electron on the other side. This free electron then reacts with another monomer, and the chain becomes self-propagating. Condensation polymerization involves the reaction of two monomers, resulting in the splitting out of a water molecule.

[0276] Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified 8123-112871-03 11 / 21 / 25 06880

[0277] sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0278] Probe: A substance used to detect or identify another substance in a sample. Some examples of probes include an isolated compound or molecule attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992).

[0279] “Prodrug” refers to compounds that are transformed in vivo to yield a biologically active compound, particularly the parent compound, for example, by hydrolysis in the gut or enzymatic conversion. Common examples of prodrug moieties include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety. Examples of pharmaceutically acceptable esters of the compounds of this invention include, but are not limited to, esters of phosphate groups and carboxylic acids, such as aliphatic esters, particularly alkyl esters (for example Ci -ealkyl esters ). Other prodrug moieties include phosphate esters, such as -CH2-O-P(O)(OR')2 or a salt thereof, wherein R’ is H or Ci ealkyl. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl. Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides, and secondary and tertiary alkyl amides (for example with between about one and about six carbons).

[0280] Amides and esters of disclosed exemplary embodiments of compounds according to the present invention can be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.

[0281] Proton: Hydrogen ions (H+) and water-solvated hydrogen ions, e.g., HAY, [H5O2]+, [H9O4]+.

[0282] Providing a compound or composition comprising the compound: Refers to a 8123-112871-03 11 / 21 / 25 06880

[0283] person, entity or other manufacturer who makes the compound or composition comprising the compound and provides instructions for its use, such as by establishing the manner and / or timing of using the compound or composition; a supplier who supplies the compound or composition and provides instructions for its use, establishing the manner and / or timing of using the compound or composition; a facility that uses the compound or composition; and / or a subject who uses the compound or composition themselves. The manufacturer, supplier, facility and / or subject may act jointly or as a joint enterprise by agreement, by a common purpose, a community of pecuniary interest, and / or substantially equal say in direction of using the compound or composition. Alternatively, or additionally, the manufacturer, supplier, facility and / or subject may condition participation in an activity or receipt of a benefit upon performance of a step or steps of the method of using the compound or composition disclosed herein, and establish the manner and / or timing of that performance.

[0284] Purine: A heterocyclic aromatic organic compound including a pyrimidine ring fused to an imidazole ring. Purines found in nucleosides include adenine and guanine.

[0285] NH?0

[0286] L!i / k k > k O

[0287]

[0288] n n n purine adenine guanine

[0289] Pyrimidine: A heterocyclic aromatic organic compound having the general formula C4H4N2 with nitrogen atoms at positions 1 and 3 in the ring. Pyrimidines found in nucleosides include cytosine, thymine, and uracil.

[0290]

[0291] cytosine thymidine uracil

[0292] Pyrrole: A 5-membered heterocyclic compound with the formula C4H4NH.

[0293] Racemization: The partial conversion by heat or chemical reaction of one isomer into a mixture of isomers. Racemization refers particularly to the conversion of enantiomers, or optically active isomers. Enantiomers are pairs of stereoisomers that are nonsuperimposable mirror images of one another.

[0294] Sample: The term “sample” refers to any liquid, semi-solid or solid substance (or material) in or on which a target can be present. In particular, a sample can be a biological 8123-112871-03 11 / 21 / 25 06880

[0295] sample or a sample obtained from a biological material. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a non-pathological joint or a joint affected by disease). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. In some examples, a biological sample is a nuclear extract. In some examples, a biological sample is bacterial cytoplasm. In other examples, a sample is a test sample. For example, a test sample is a cell, a tissue or cell pellet section prepared from a biological sample obtained from a subject. In an example, the subject is one that is at risk or has acquired a particular condition or disease.

[0296] Silyl: A functional group comprising a silicon atom bonded to different functional groups, and typically having a formula

[0297] where R1-R3 independently are selected from various g groups including, by way of example, hydrogen, aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0298] Silyl ester: A functional group with the formula:

[0299] O / Rl

[0300] II /

[0301] — c-o— Si — R2

[0302]

[0303] R3

[0304] where R1-R3 independently are selected from various groups, including by way of example aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl. 8123-112871-03 11 / 21 / 25 06880

[0305] Silyl ether: A functional group with the formula:

[0306] — O— S / i -1R2

[0307] R3

[0308] where R1-R3 independently are selected from various groups, including by way of example aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0309] Soluble: Capable of becoming molecularly or ionically dispersed in a solvent to form a homogeneous solution. U. S. Pharmacopeia definitions: very soluble: more than 1000 mg / ml, freely soluble: 100-1000 mg / ml, soluble: 30-100 mg / ml, sparingly soluble: 10-30 mg / ml, slightly soluble: 1-10 mg / ml, very slightly soluble: 0.1-1 mg / ml, practically insoluble or insoluble: < 0.1 mg / ml.

[0310] Solution: A homogeneous mixture composed of two or more substances. A solute (minor component) is dissolved in a solvent (major component). A plurality of solutes and / or a plurality of solvents may be present in the solution.

[0311] “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of a solute. The solvent can be an organic solvent, an inorganic solvent, or a mixture of both. Exemplary solvents include, but are not limited to, alcohols, such as methanol, ethanol, propanol: amides such as N, N-dialiphatic amides, such as N, N-dimethylformamide; tetrahydrofuran; alkylsulfoxides, such as dimethylsulfoxide; water; and combinations thereof. The compounds described herein can exist in un-solvated as well as solvated forms when combined with solvents, pharmaceutically acceptable or not, such as water, ethanol, and the like. Solvated forms of the presently disclosed compounds are within the scope of the embodiments disclosed herein.

[0312] Specifically binds: A term that refers to the binding of agent that preferentially binds to a defined target (such as an antibody to a specific antigen or a nucleic acid probe to a specific nucleic acid sequence). With respect to an antigen, “specifically binds” refers to the preferential association of an antibody or other ligand, in whole or part, with a specific polypeptide. With respect to a nucleic acid sequence, “specifically binds” refers to the preferential association of a nucleic acid probe, in whole or part, with a specific nucleic acid sequence

[0313] A specific binding agent or moiety binds substantially only to a defined target. It is recognized that a minor degree of non-specific interaction may occur between a molecule, 8123-112871-03 11 / 21 / 25 06880

[0314] such as a specific binding agent or moiety, and a non-target polypeptide or non-target nucleic acid sequence. Although a selectively reactive antibody binds an antigen, it can do so with low affinity. Antibody to antigen specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a target polypeptide, as compared to a non-target polypeptide. A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0315] Nucleic acid probe to nucleic acid sequence specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound nucleic acid probe to a target nucleic acid sequence, as compared to a non-target nucleic acid. A variety of in situ hybridization (ISH) conditions are appropriate for selecting nucleic acid probes that bind specifically with a particular nucleic acid sequence.

[0316] Stereochemistry: The three-dimensional spatial configuration of a molecule.

[0317] Stereoisomers: Isomers that have the same molecular formula and sequence of bonded atoms, but which differ only in the three-dimensional orientation of the atoms in space.

[0318] Substituent: An atom or group of atoms that replaces another atom in a molecule as the result of a reaction. The term "substituent" typically refers to an atom or group of atoms that replaces a hydrogen atom, or two hydrogen atoms if the substituent is attached via a double bond, on a parent hydrocarbon chain or ring. The term “substituent” may also cover groups of atoms having multiple points of attachment to the molecule, e.g., the substituent replaces two or more hydrogen atoms on a parent hydrocarbon chain or ring. In such instances, the substituent, unless otherwise specified, may be attached in any spatial orientation to the parent hydrocarbon chain or ring. Exemplary substituents include, for instance, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thio, and thioalkoxy groups. 8123-112871-03 11 / 21 / 25 06880

[0319] Substituted: A fundamental compound, such as an aryl or aliphatic compound, or a radical thereof, having coupled thereto one or more substituents, each substituent typically replacing a hydrogen atom on the fundamental compound. A person of ordinary skill in the art will recognize that compounds disclosed herein may be described with reference to particular structures and substituents coupled to such structures, and that such structures and / or substituents also can be further substituted, unless expressly stated otherwise or context dictates otherwise. Solely by way of example and without limitation, a substituted aryl compound may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene. Again solely by way of example and without limitation, a long-chain hydrocarbon may have a hydroxyl group bonded thereto. Groups which are substituted (e.g. substituted alkyl), may in some embodiments be substituted with a group which is substituted (e.g. substituted aryl). In some embodiments, the number of substituted groups linked together is limited to two (e.g. substituted alkyl is substituted with substituted aryl, wherein the substituent present on the aryl is not further substituted). In some embodiments, a substituted group is not substituted with another substituted group (e.g. substituted alkyl is substituted with unsubstituted aryl).

[0320] Substrate: A molecule acted upon by a catalyst, such as an enzyme.

[0321] “Sulfonamide” refers to the group or moiety -SChamino, or -N(R)sulfonyl, where R is H, aliphatic, heteroaliphatic, cyclic, heterocyclic, including aromatic, both aryl and heteroaryl.

[0322] “Sulfanyl” refers to the group or -SH, -S-aliphatic, -S-heteroaliphatic, -S-cyclic, -S-heterocyclyl, including -S-aromatic, both-S-aryl and -S-heteroaryl.

[0323] “Sulfinyl” refers to the group or moiety -S(O)H, -S(O)aliphatic, -S(O)heteroaliphatic, -S(O)cyclic, -S(O)heterocyclyl, including aromatic, both-S(O)aryl and -S(O)heteroaryl.

[0324] Sulfonyl: A functional group with the general formula:

[0325] R

[0326]

[0327] v R'

[0328] where R and R' independently are selected from various groups, including by way of example aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0329] Therapeutically effective amount or dose: An amount sufficient to provide a 8123-112871-03 11 / 21 / 25 06880

[0330] beneficial, or therapeutic, effect to a subject or a given percentage of subjects.

[0331] Therapeutic time window: The length of time during which an effective, or therapeutic dose, of a compound remains therapeutically effective in vivo.

[0332] Thioalkoxyalkyl: A thioalkoxy group appended to an alkyl radical.

[0333] Thiol: A functional group with the formula -SH.

[0334] Thioester: A functional group with the general formula:

[0335] O II

[0336]

[0337] R SR'

[0338] where R and R' independently are selected from various groups, including by way of example aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0339] Thioether: A functional group with the general formula: R-S-R' where R and R' independently are selected from various groups, including by way of example aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl. A thioether is similar to an ether, except that it contains a sulfur atom in place of the oxygen.

[0340] Tissue: A collection of interconnected cells that perform a similar function within an organism. Any collection of cells that can be mounted on a standard glass microscope slide including, without limitation, sections of organs, tumor sections, bodily fluids, smears, frozen sections, cytology preps, and cell lines.

[0341] Titrate: As used herein, the term “titrate” refers to adjusting the dose of an administered therapeutic agent (e.g., an estrogenic compound) to achieve a desired result.

[0342] “Treating” or “treatment” as used herein concerns treatment of a disease or condition of interest in a patient or subject, particularly a human having the disease or condition of interest, and includes by way of example, and without limitation:

[0343] (i) preventing the disease or condition from occurring in a patient or subject, in particular, when such patient or subject is predisposed to the condition but has not yet been diagnosed as having it;

[0344] (ii) inhibiting the disease or condition, for example, arresting or slowing its development;

[0345] (iii) relieving the disease or condition, for example, causing regression of the disease or condition or a symptom thereof; or

[0346] (iv) stabilizing the disease or condition. 8123-112871-03 11 / 21 / 25 06880

[0347] As used herein, the terms “disease" and “condition" can be used interchangeably or can be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been determined) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, where a more or less specific set of symptoms have been identified by clinicians.

[0348] Tumor: An abnormal growth of cells, which can be benign or malignant. Cancer is a malignant tumor, which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. “Metastatic disease” refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system. Thus, a metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.”

[0349] The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and / or can metastasize is referred to as “malignant.” Examples of hematological tumors include leukemias, including acute leukemias (such as llq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0350] Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast 8123-112871-03 11 / 21 / 25 06880

[0351] carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, salivary gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma).

[0352] An “established” or “existing” tumor is an existing tumor that can be discerned by diagnostic tests. In some embodiments, an established tumor can be palpated. In some embodiments, an “established tumor” is at least 500 mm3, such as at least 600 mm3, at least 700 mm3, or at least 800 mm3in size. In other embodiments, the tumor is at least 1 cm long. With regard to a solid tumor, an established tumor generally has a robust blood supply, and has induced Tregs and myeloid derived suppressor cells (MDSCs).

[0353] Tyramine: A compound having the formula CsHnNO, also known as 4-(2-aminoethyl)phenol.

[0354] Tyramide: A tyramine derivative, wherein the amine functional group of a tyramine molecule has formed an amide bond with a carbonyl-containing functional group.

[0355] Tyramine Derivative: A compound having the following formula

[0356]

[0357] where Ri is selected from ether, hydroxyl, -(nitrogen-Rs)-, where Rs is selected from aliphatic, heteroaliphatic, aryl, heteroaryl, and hydrogen; R2 is selected from aliphatic, heteroaliphatic, aryl, and heteroaryl; n is from 1 to 20; and Y is selected from oxygen, sulfur, and -(nitrogen-Rs )-, where R3 is selected from aliphatic, heteroaliphatic, aryl, heteroaryl, and hydrogen.

[0358] Ultraviolet (UV): Electromagnetic radiation having a wavelength ranging from 10 nm to 400 nm.

[0359] Unit Dose: A drug or pharmaceutical composition in a single or metered dose form, such as a table, capsule, powder or solution to be administered as a single dose, or multiple preselected doses. In some examples, the unit dose is a liquid contained in a sterile vial, or a powder in a sterile vial capable of being reconstituted for administration by introduction of a 8123-112871-03 11 / 21 / 25 06880

[0360] liquid into the vial. In other examples, the unit dosage form is provided in a syringe suitable for administration, for example injection into a subject.

[0361] Weight average molecular weight:

[0362] ShlM2

[0363]

[0364] where M, is the molecular weight of a chain, and Ni is the number of chains of that molecular weight. Weight average molecular weight is determined by methods that are sensitive to molecular size, such as light-scattering techniques, small angle neutron scattering, X-ray scattering, and sedimentation velocity.

[0365] Introduction

[0366] As introduced above, aberrant angiogenesis can be a hallmark of many diseases including cancer. For example, renal cell carcinoma (RCC) is characterized by a highly vascularized tumor microenvironment.

[0367] An exploratory in vivo study indicated that intratumoral injection of C74 reduces subcutaneous tumor growth of renal carcinoma (RENCA, a widely used murine RCC cell line) cells when implanted in a syngeneic mouse host. However, since in vitro studies also showed that C74 treatment reduces RENCA cell proliferation, the relative contributions of tumor-intrinsic vs. tumor-extrinsic effects of C74 underlying its anti-tumor action were not able to be discerned. Therefore, an end-point assessment of tumor angiogenesis was first performed by performing immunohistochemistry of tumor sections for CD31 (a marker of vascular endothelial cells) and quantifying the relative CD31 -positive areas in vehicle control (dimethyl sulfoxide (DMSO)) versus C74-treated RENCA tumors. C74 treatment resulted in reduction of CD31 -positive area by -30% in a statistically significant manner, providing the initial evidence for an anti-angiogenic action of C74 in an in vivo tumor setting.

[0368] In some instances, clinical efforts to develop anti-angiogenic therapies focus on inhibiting the action of vascular endothelial growth factor (VEGF). However, spontaneous or acquired resistance to anti-VEGF therapies can occur in certain patient populations, which may be due to involvement of other pro-angiogenic mediators. Therefore, there is a technical need for alternative anti-angiogenic targets and new compounds that can act as antiangiogenesis agents that could pave the way for novel therapeutic avenues.

[0369] Vascular endothelial cell (VEC) migration and proliferation can lead to angiogenesis. In some aspects, VEC activation by pro-angiogenic cues allows a selective population of 8123-112871-03 11 / 21 / 25 06880

[0370] VECs (e.g., tip cells) to extend F-actin-rich filopodial protrusions, migrate toward guidance cues, and initiate vessel sprouting, while VECs trailing behind the tip cells (e.g., stalk cells) proliferate to elongate the sprouts. Dynamic remodeling of the endothelial actin cytoskeleton is one component of angiogenesis. Pfnl, a G-actin binding protein that also interacts with many actin assembly factors in cells (e.g. formins, Ena / vasodilator-stimulated phosphoprotein (VASP) proteins) can mediate actin polymerization in cells. Pfnl -actin interaction in VECs may be stimulated by VEGF through a site-specific (Y129) phosphorylation of Pfnl that leads to increased angiocrine factor (VEGF, bFGF, Hb-EGF) production in a feed-forward manner, blocking of which reduces angiogenesis during tissue repair and tumor progression, suggesting Pfnl's regulation has a large role in angiogenesis in physiological and pathological settings. In some aspects, endothelial Pfnl depletion leads to reduced angiogenic activity of VECs in vitro and ex vivo, and hypo-vascularization during development of various organs including retinae in vivo (FIGS. 1A-1E), underscoring the role of Pfnl for angiogenesis. Thus, Pfnl is a clinically relevant and viable target for therapeutic intervention against pathological angiogenesis, such as in ocular settings.

[0371] In some aspects, Pfnl is associated with vascular related pathology in diabetes. In certain aspects, endothelial Pfnl expression is elevated in diabetic patients and in response to factors that promote diabetes-associated pathology e.g. atherosclerosis, advanced glycation end-products) in vitro and in vivo (including human patients). Pfnl elevation can exacerbate VEC dysfunction (e.g., compromised barrier function) in the diabetic milieu, and, conversely, Pfnl depletion may offer protection against oxidative damage, inflammation, and VEC dysfunction in diabetic animals in vivo. Elevated endothelial Pfnl expression can occur in experimentally induced abnormal angiogenesis in mouse retinae and angiogenesis-associated human diseases including proliferative diabetic retinopathy (PDR) (FIG. 2A) and clear cell renal cell carcinoma. Pfnl may be a viable target for therapeutic intervention for several reasons. First, with the exception of global absence of Pfnl at the embryonic level, complete loss of Pfnl can be tolerated in many tissues in mammals. In certain aspects, when Pfnl disruption in VECs is induced after birth, animals are viable and do not exhibit any discernible complications even up to adult life. Second, in certain aspects, molecular disruption of key ligand interactions of Pfnl does not cause lethality in cultured cells including VEC, suggesting that differentiated cells tolerate loss of Pfnl function reasonably well and therefore Pfnl inhibition will likely not result in systemic toxicities. Further, in certain aspects, mice do not exhibit any weight loss or changes in serum analytes (liver and 8123-112871-03 11 / 21 / 25 06880

[0372] kidney enzymes, electrolytes, waste products) when subjected to daily intraperitoneal administration of Pfnl -actin inhibitor C74 (at 16 mg / kg) for 2 weeks, which is suggestive of in vivo safety.

[0373] Two structurally distinct non-cytotoxic small molecule compounds (C2 (8-(3-hydroxyphenyl)-10-phenyl-2,4,5,6,7,ll,12-heptaazatricyclo[7.4.0.03,7] trideca-l(13),3,5,9,ll-pentaen-13-ol) and C74 (4-[(4-bromophenyl)(5-hydroxy-3-methyl-lH-pyrazol-4-yl)methylJ-3-methyl-lH-pyrazol-5-ol)) were identified that reversed Pfnl’s effect on actin polymerization in pyrene-actin polymerization assays, reduced cellular Pfnl-actin interaction in proximity-ligation assays, and inhibited cell migration and proliferation. It was demonstrated that both compounds C2 and C74 exhibit anti-angiogenic action in vitro, ex vivo and in vivo. (FIGS. 2B-2L). Comparative assessment of these two components in cell migration and proliferation assays indicated that C74 might be biologically more potent than C2 in some aspects. Accordingly, inhibition of the Pfnl-actin interaction by small molecules may be able to address aberrant angiogenesis in the eye, and associated diseases.

[0374] Pfnl presents a unique point of interference because it is utilized by multiple actin assembly pathways and therefore occupies a critical node of convergent pathways. The nodal positioning of Pfnl in actin assembly may reduce the susceptibility of Pfnl -oriented therapies to resistance from the compensatory response of alternative pathways, as is seen with VEGF inhibitors. High-quality chemical probes can help to understand the role of Pfnl in aberrant angiogenesis and anti-VEGF resistance.

[0375] Furthermore, VEGF-induced vascular leakage leads to visual complications in diabetic retinopathy (DR). Silencing Pfnl expression confers protection against VEGF-induced junctional disruption in VEC in vitro and accordingly reduces retinal vascular leakage in vivo, suggesting that Pfnl may be a central regulator for endothelial barrier function. Accordingly, the compounds disclosed herein can serve as an endothelial barrier stabilizing agent and / or provide chemical tools for modulating endothelial barrier function.

[0376] Therefore, a new C74-derivative with improved anti-angiogenic capability in vivo could be a promising therapeutic tool compound either as a stand-alone agent or in combination with other therapies for future preclinical testing, such as in RCC. The therapeutic potential of targeting Pfnl-actin interaction extends beyond cancer to other vascular-related pathology (e.g., atherosclerosis). Excessive or abnormal angiogenesis is also a hallmark of several other diseases, including age-related macular degeneration (AMD), DR, and psoriasis. In these conditions, unchecked angiogenesis leads to the formation of 8123-112871-03 11 / 21 / 25 06880

[0377] dysfunctional blood vessels that contribute to disease progression and tissue damage.

[0378] Pharmacokinetics, toxicology, and preclinical efficacy studies involving UP6 in the future will determine its clinical utility as a potential anti-angiogenic therapeutic strategy.

[0379] Compounds and Methods

[0380] Disclosed herein is a compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, having a structure:

[0381]

[0382] In some aspects, the compound is based on a C74 scaffold, which is a small molecule compound with the ability to interrupt the Pfnl -actin interaction in cells as demonstrated by the PLA data described in more detail below with reference to FIGS. 4A-4E. There are several potential sites for introducing chemical modifications in C74, which include the bromoaryl ring and the two hydroxypyrazole rings. A preliminary structural-activity relationship (SAR) of C74 was established around the bromine (Br) substituent by comparing the biological activity four commercial C74 analogs where the R(Br) group in C74 was substituted by either halogens (e.g., Cl, F) or H or CH3. While all five compounds reduced morphogenetic ability of HmVECs in an in vitro cord formation assay when cells were treated with these compounds at a 25 mM concentration, the anti-angiogenic efficacy was highest when Br was replaced by either F or CH3 substitution. When the compound concentration was reduced to 10 mM, only those two substitutions were effective in conferring an anti-angiogenic ability to C74. These initial findings suggested that chemical modifications of the structural scaffold of C74 could potentially open avenues for deriving compounds with increased biological potency. In some aspects, smaller ligands (larger than H but smaller than Br) improve the anti-angiogenic potency of C74 derivatives. In some aspects, derivatives maintain similar electronegative properties as the Br ligand but are also smaller in size. In some aspects, the compounds disclosed herein have sub-pM potency in both biochemical and cellular assays and demonstrated target engagement and selectivity in cells.

[0383] In some aspects, Ri has any one of the following structures or a derivative thereof: 8123-112871-03 11 / 21 / 25 06880

[0384]

[0385] In some aspects, Ri is not phenyl.

[0386] In some aspects, Ri is a substituted phenyl group, a heterocycle, or a substituted heterocycle.

[0387] In certain aspects, Ri has the following structure or a derivative thereof:

[0388]

[0389] R5

[0390] In some aspects, Rs is not bromine at the para- position of the phenyl group of Ri (i.e. Ri is not 4-bromophenyl).

[0391] In some aspects, Rs is not a methyl group at the para- position of the phenyl group of Ri (i.e., Ri is not 4-methylphenyl).

[0392] In some aspects, R.s is not a halogen at the para- position of the phenyl group of Ri (e.g., Ri is not 4-chlorophenyl nor 4-fluorophenyl).

[0393] In some aspects, Rs is a trifluoromethyl group.

[0394] In certain aspects, Ri has any one of the following structures or a derivative thereof:

[0395] CF:

[0396]

[0397] In some aspects, Ri has any one of the following structures or a derivative thereof:

[0398]

[0399] In some aspects, Ri has any one of the following structures or a derivative thereof:

[0400] N N

[0401]

[0402] In some aspects, Ri has the following structure or a derivative thereof: 8123-112871-03 11 / 21 / 25 06880

[0403]

[0404] In some aspects, Ri is located at the edge of a pocket in which R3 is located when the compound interacts with Pfnl and / or actin. In some aspects, Ri is approximately five A away from residues Hl 19, R74 and 173 of Pfnl and is in a similar position as the phenyl moiety of compound C2. In some aspects, and without wishing to be bound by theory, substituents at the Ri position can interact with these residues to modulate the interaction between the compound and Pfnl.

[0405] In some aspects, Ri comprises a photoreactive moiety. In certain aspects, the term “photoreactive moiety” generally refers to a moiety in which a structural change occurs in response to being exposed to light. Examples of photoreactive moieties include phenylazides, phenyldiazirines, benzophenones, trifluoromethylphenyldiazirine, and tetrafluorophenylazide. Incorporation of a photoreactive moiety can enable the compound to serve as a photoaffinity probe.

[0406] Referring again to the structure:

[0407] ■2

[0408]

[0409] In some aspects, the hydroxypyrazole (to which R2 is attached in the compound of the structure provided above) makes four hydrogen bonds with Pfnl (e.g., with R88, N99, Hl 19 and G120).

[0410] In some aspects, R2 is hydrogen or a methyl group.

[0411] In some aspects, R3 is hydrogen, a phenyl group, a substituted phenyl group, a heterocycle or a substituted heterocycle.

[0412] In some aspects, R3 is a pyrazole, or a substituted pyrazole (such as a hydroxypyrazole).

[0413] In certain aspects, R3 has the following structure or a derivative thereof:

[0414] HN-N

[0415] HO

[0416]

[0417] In certain aspects, R4 is hydrogen or a methyl group. 8123-112871-03 11 / 21 / 25

[0418] In some aspects, R3 forms hydrogen bonds with D86 and the carbonyl of G 120 in Pfnl. In certain aspects, R3 is also located in a pocket that is formed by 173, R74, D86, L87, N99 and T101, but does not fully occupy it. Derivatives that could maintain the interactions with D86 and G120 but also could extend further into the pocket (e.g., as seen with compound C2) could further increase potency of the compound. Examples of such derivatives may include substituted hydroxypyrazoles or larger hydroxypyrazole isosteres, such as indoles or oxindoles.

[0419] In some aspects, R3 has any one of the following structures or a derivative thereof:

[0420]

[0421] In some aspects, R3 has any one of the following structures or a derivative thereof:

[0422]

[0423] In some aspects, the compound is chemically stable, has water solubility > 100 pM, adheres to Lipinski / Veber parameters, is acceptable for ophthalmic administration, or a combination thereof.

[0424] Additional aspects of binding and / or other interactions between the compound and Pfnl are described in more detail below with reference to FIGS. 5 and 6A-6C.

[0425] Also disclosed herein is a compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, having a structure:

[0426] HN-N

[0427] N

[0428] F- F

[0429]

[0430] Also disclosed herein is a compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, having a structure: 8123-112871-03 11 / 21 / 25 06880

[0431] A HC OH

[0432] N; A!> -'LA N

[0433]

[0434] t: N / ~'HH

[0435] Also disclosed herein is a compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, having a structure:

[0436] Cl..o

[0437] HO '■J" OH

[0438] <

[0439]

[0440] T T >

[0441] HN'A / -NH

[0442] Pharmaceutical Formulations

[0443] The compounds described herein can be used to prepare therapeutic pharmaceutical compositions. The compounds may be added to the compositions in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and b-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

[0444] Pharmaceutically acceptable salts may be obtained using procedures known to persons of ordinary skill in the art, for example by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

[0445] The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human or veterinary patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes. 8123-112871-03 11 / 21 / 25 06880

[0446] The compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1 % of active compound. The percentage of the compositions and preparations can vary and may conveniently be from about 2% to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level can be obtained.

[0447] The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0448] The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms. 8123-112871-03 11 / 21 / 25 06880

[0449] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and / or gelatin.

[0450] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0451] For topical administration, compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0452] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, DMSO, alcohols, glycols, or water-alcohol / glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pumptype or aerosol sprayer. 8123-112871-03 11 / 21 / 25 06880

[0453] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0454] Examples of dermatological compositions for delivering active agents to the skin are known to the art; for example, see U. S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition.

[0455] Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

[0456] The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg / m2, conveniently 10 to 750 mg / m2, most conveniently, 50 to 500 mg / m2of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0457] Pharmaceutical Dosage Forms

[0458] The following formulations illustrate exemplary pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a compound of a formula described herein, a compound specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Compound X'):

[0459] (i) Tablet 1 mg / tablet

[0460] 'Compound X' 100.0

[0461] Eactose 77.5

[0462] Povidone 15.0

[0463] Croscarmellose sodium 12.0 8123-112871-03 11 / 21 / 25 06880

[0464] Microcrystalline cellulose 92.5

[0465] Magnesium stearate 3.0

[0466] 300.0

[0467] (ii) Tablet 2 mg / tablet

[0468] 'Compound X' 20.0

[0469] Microcrystalline cellulose 410.0

[0470] Starch 50.0

[0471] Sodium starch glycolate 15.0

[0472] Magnesium stearate 5.0

[0473] 500.0

[0474] (iii) Capsule mg / capsule

[0475] 'Compound X' 10.0

[0476] Colloidal silicon dioxide 1.5

[0477] Lactose 465.5

[0478] Pregelatinized starch 120.0

[0479] Magnesium stearate 3.0

[0480] 600.0

[0481] (iv) Injection 1 (1 mg / mL) mg / mL

[0482] 'Compound X' (free acid form) 1.0

[0483] Dibasic sodium phosphate 12.0

[0484] Monobasic sodium phosphate 0.7

[0485] Sodium chloride 4.5

[0486] 1.0 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injection q.s. to 1 mL

[0487] (v) Injection 2 (10 mg / mL) mg / mL

[0488] 'Compound X' (free acid form) 10.0

[0489] Monobasic sodium phosphate 0.3

[0490] Dibasic sodium phosphate 1.1

[0491] Polyethylene glycol 400 200.0

[0492] 0.1 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injection q.s. to 1 mL

[0493] (vi) Aerosol mg / can

[0494] 'Compound X' 20

[0495] Oleic acid 10

[0496] Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000 Dichlorotetrafluoroethane 5,000

[0497] (vii) Topical Gel 1 wt. %

[0498] 'Compound X' 5%

[0499] Carbomer 934 1.25%

[0500] Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben 0.2%

[0501] Purified water q.s. to 100g 8123-112871-03 11 / 21 / 25 06880

[0502] (viii) Topical Gel 2 Wt. %

[0503] 'Compound X' 5%

[0504] Methylcellulose 2%

[0505] Methyl paraben 0.2%

[0506] Propyl paraben 0.02%

[0507] Purified water q.s. to 100g

[0508] (ix) Topical Ointment wt. %

[0509] 'Compound X' 5%

[0510] Propylene glycol 1%

[0511] Anhydrous ointment base 40%

[0512] Polysorbate 80 2%

[0513] Methyl paraben 0.2%

[0514] Purified water q.s. to 100g

[0515] (x) Topical Cream 1 wt. %

[0516] 'Compound X' 5%

[0517] White bees wax 10%

[0518] Liquid paraffin 30%

[0519] Benzyl alcohol 5%

[0520] Purified water q.s. to 100g

[0521] (xi) Topical Cream 2 wt. %

[0522] 'Compound X' 5%

[0523] Stearic acid 10%

[0524] Glycerylmonostearate 3%

[0525] Polyoxyethylenestearyl ether 3%

[0526] Sorbitol 5%

[0527] Isopropyl palmitate 2 %

[0528] Methyl Paraben 0.2%

[0529] Purified water q.s. to 100g

[0530] These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

[0531] Overview of Several Aspects

[0532] Disclosed herein is a compound, having a structure: 8123-112871-03 11 / 21 / 25 06880

[0533] AR2

[0534] Ri

[0535]

[0536] HOAN'H

[0537] wherein Ri is not phenyl, Ri is not 4-bromophenyl, Ri is not 4-chlorophenyl, Ri is not 4-fluorophenyl, and Ri is not a methyl group;

[0538] wherein R2 is hydrogen or a methyl group: and

[0539] wherein R3 is hydrogen, a phenyl group, a substituted phenyl group, a heterocycle or a substituted heterocycle.

[0540] In some aspects, Ri is a pyrazole or a substituted pyrazole.

[0541] In any or all of the above aspects, R3 has a structure:

[0542] HN-N

[0543] HO^ JVVAV R4

[0544]

[0545] wherein R4 is hydrogen or a methyl group.

[0546] In any or all of the above aspects, R3 has a structure:

[0547]

[0548] wherein R5 is not bromine, chlorine, or fluorine at the para- position of R3.

[0549] In any or all of the above aspects, Rs is a trifluoromethyl group.

[0550] In any or all of the above aspects, the compound is

[0551]

[0552] In any or all of the above aspects, the compound is used as a vascular therapy.

[0553] Also disclosed is a pharmaceutically acceptable composition, comprising a compound according to aspects of the present disclosure, and a therapeutically acceptable excipient.

[0554] Also disclosed is a method, comprising administering a compound, or a pharmaceutically acceptable composition thereof, according to aspects of the present disclosure, to a subject exhibiting, or susceptible to exhibiting: a vascular pathology or a disease involving a vascular pathology. 8123-112871-03 11 / 21 / 25 06880

[0555] In some aspects, the disease is a cancer, age-related macular degeneration (AMD), diabetic retinopathy, psoriasis, atherosclerosis, or a combination thereof.

[0556] Also disclosed is a compound, having a structure:

[0557] HN-N

[0558]

[0559] F

[0560] In some aspects, the compound is used as a vascular therapy.

[0561] Also disclosed is a pharmaceutically acceptable composition, comprising: 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-niethyl-lH-pyrazol-5-ol); and a therapeutically acceptable excipient.

[0562] In some aspects, the composition further comprises an additional therapeutic.

[0563] In any or all of the above aspects, the additional therapeutic is a cancer therapy, a vascular therapy, or a combination thereof.

[0564] Also disclosed is a method, comprising administering 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) to a subject exhibiting, or susceptible to exhibiting, a vascular pathology, or a pharmaceutically acceptable composition thereof.

[0565] In some aspects, the subject exhibits a loss of Von-Hippel Lindau (VHL) gene. In any or all of the above aspects, the subject exhibits an upregulation of Pfnl.

[0566] Also disclosed is a method, comprising administering 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) to a subject, wherein the subject is suffering from, or is susceptible to, a disease involving a vascular pathology.

[0567] In some aspects, the disease is a cancer, age-related macular degeneration (AMD), diabetic retinopathy, psoriasis, or a combination thereof.

[0568] In any or all of the above aspects, the cancer is clear cell renal cell carcinoma.

[0569] Also disclosed is a method of making the compound according to any or all of the above aspects, comprising reacting 3-methyl-5-pyrazolone with 4-(trifluoromethyl)benzaldehyde in the presence of a surfactant.

[0570] In some aspects, reacting 3-methyl-5-pyrazolone with the benzaldehyde comprises reacting the 3-methyl-5-pyrazolone with 4-(trifluoromethyl)benzaldehyde.

[0571] In any or all of the above aspects, the surfactant is an anionic surfactant. 8123-112871-03 11 / 21 / 25 06880

[0572] In any or all of the above aspects, the surfactant is sodium dodecyl sulfate.

[0573] In any or all of the above aspects, the 3-methyl-5-pyrazolone and the benzaldehyde or the nicotinaldehyde are heated in the presence of the surfactant.

[0574] Examples

[0575] Cell Culture and Reagents - RENCA renal cell carcinoma (RCC) cells (ATCC, CRL-2947) were cultured in Roswell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Human microvascular endothelial cells (HmVECs, ATCC, CRL-3243) were cultured in MCDB-131 media with growth supplements. Initial screening experiments used HmVEC-1 cells and confirmatory results with the top hits were obtained with human renal epithelial cells (HREC).

[0576] Statistics - All experiments were repeated at least three times with multiple technical replicates. ANOVA was performed using the Bonferroni post-hoc test for comparison of means with > 2 groups. Student’s t-test was used when two groups were involved. The Mann- Whitney test was used for small sample sizes when normality of data was violated.

[0577] Liquid Microbubble Encapsulation of C74 - Lipid microbubbles were prepared with modifications to a previously described method (PMID: 27551081). DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine; 850365C, Avanti Polar Lipids, Alabaster, AL, USA), DSPE-mPEG 2000 (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly -ethylene glycol)-2000] ammonium salt; 880120C, Avanti Polar Lipids), and poly-oxyethylene (40) stearate (P3440, Millipore Sigma) dissolved in chloroform were mixed with C74 (dissolved in ethanol) in a 2:1:1:0.5 (weight) ratio and dried under argon at room temperature for 25 to 30 minutes, followed by overnight vacuum drying. The dried lipid film was hydrated with saline and the resulting dispersion was sonicated with a Misonix XL2020 Sonicator Ultrasonic Processor XL (Bioventus, Farmingdale, NY, USA) at power level 5.25 in the presence of perfluorobutane (PFB) gas (FluoroMed, Round Rock, TX, USA) for 1 minute 15 seconds to form microbubbles. These were washed twice in a saline bag, incubating at room temperature for 60 minutes after each wash, then resuspended in saline. The final formulation was aliquoted into vials with PFB-filled headspace and stored at 4 °C until use. This method produced microbubbles with an average concentration of ~0.5 x 109 / mL and a mean diameter of 3 pm, as measured using a Coulter counter (Multisizer 4e, Beckman Coulter, Indianapolis, IN, USA) with a 30 pm aperture tube. 8123-112871-03 11 / 21 / 25 06880

[0578] In Vitro Anti-Proliferation and Anti-Angiogenesis Screening - For initial screening of small molecule compounds in cell proliferation assay, human VECs (HmVECs) were seeded in the wells of a 24-well plate and treated with various compounds at a 20 mM concentration or equivalent concentration of DMSO (solvent control), and cell count was measured well-by-well by imaging and extrapolating the overall number of cells / well using a Digital Cell Imager (Millipore). For initial screening of anti-angiogenic capability of small molecule compounds, HmVECs were plated on growth factor-reduced matrigel (R& D Systems, 3433-005-01) and treated with various compounds at a 20 mM concentration or equivalent concentration of DMSO. Endothelial cord formation was imaged and quantified after 18 hours, with the length of branch segments used as a measure of angiogenic activity.

[0579] For in vitro testing of microbubble-encapsulated C74, HmVECs were seeded for cord formation as described herein and allowed to grow for 24 h. Microbubbles loaded as indicated were added to the culture media to achieve a final ratio of 10 microbubbles per cell. The wells were covered with cell media, sealed with polycarbonate membrane and inverted. To disrupt microbubbles and release C74, Ultrasound was delivered with a single-element immersion transducer (A302S, 25.4 mm in diameter, Olympus NDT, Center Valley, PA), driven by an arbitrary function generator (AFG3252, Tektronix, Beavertona, OR) connected to a gated radio frequency power amplifier (250A250AM8, Amplifier Research, Souderton, PA). The ultrasound field was calibrated with a 200- pm capsule hydrophone (HGL-0200, Onda Corp, Sunnyvale, CA). Ultrasound pulses were delivered at 1 MHz, 0.50 MPa peak negative pressure (spatial peak temporal peak), 10 ps pulse duration, and pulse interval 1 ms (duty cycle 1%), for a total of 10 s. The media was removed after UTMD, and cells were replenished with 10% 4',6-diamidino-2-phenylindole (DMEM) and incubated for 48 hr for cell viability assay.

[0580] In vivo studies - All animal experiments were conducted in compliance with an approved IACUC protocol, according to University of Pittsburgh Division of Laboratory Animal Resources guidelines.

[0581] RCC tumor model studies - 1 million RENCA cells were injected in a 1:1 phosphate-buffered saline (PBS) / matrigel mix subcutaneously into the flanks of 6-7-week-old male immunocompetent Balb / C mice to establish RCC tumors. These mice were subjected to daily injections of C74 (16 mg / kg) or equivalent amount of DMSO dissolved in saline at the tumor cell inoculation site over a course of 19 days starting on day one before sacrificing the animals for end-point tumor angiogenesis assessment by CD31 staining of tumor 8123-112871-03 11 / 21 / 25 06880

[0582] histosections. For studies involving microbubble-based delivery of C74, subcutaneous RENCA tumors were established as described above. 100 pL suspension of microbubbles in PBS was retro-orbitally injected into animals. Ultrasound pulses were delivered for 25 minutes with the ultrasound system used for in vitro studies (S3 probe, Sonos 7500). The transducer probe was placed on the tumor and the ultrasound system was operated in ultraharmonic mode (center frequency of 1.3 MHz) with an on-screen mechanical index of 1.6. The system was time-triggered, with 4 frames per burst and a burst interval of 2 s to allow reperfusion of the tumor with microbubbles in between bursts for a period of 5 minutes. The treatment was monitored by imaging the tumor using a 15L8 transducer probe on a Sequoia 512 ultrasound imaging system (Siemens Ultrasound, Issaquah, WA, USA) operated in Contrast Pulse Sequencing (CPS) mode (mechanical index = 0.20 and frame rate of 5 Hz) to confirm MB destruction by therapy pulses and subsequent reperfusion of MBs in the tumor. For tumor growth inhibition studies (N=6-8 mice per group), treatment was initiated after 5 days post-inoculation and animals received a total of three UTMD treatments at two to three -day intervals. Mice were euthanized 2 days after the last treatment and tumors were harvested.

[0583] Matrigel plug angiogenesis assay - A single dose of 0.5 mL of growth-factor-reduced matrigel plugs supplemented with bFGF (500 ng, a stimulator of angiogenesis) and either DMSO or a small molecule compound disclosed herein (200 mM) was subcutaneously implanted bilaterally into the flanks of C57 / B6 mice (5-6-week-old male mice). After 10 days, the plugs were excised and infiltrating endothelial cells (ECs) into the plugs were identified by CD31 immunostaining and quantified to assess the extent of vascularization.

[0584] CD31 immunostaining of plugs harvested 10 days after injection revealed that treatment of both C74 and UP6 significantly reduced EC infiltration into the plugs, but the reduction in CD31 -positivity of plugs treated with UP6 was more (-50% of control) prominent than that achieved by the treatment of C74 compound (-25% of control), and this difference was statistically significant. These data suggest that UP6 has a more potent anti-angiogenic activity than C74, providing the first in vivo proof-of-concept of enhancement of anti-angiogenic efficacy of an existing Pfnl -actin interaction inhibitor by chemical optimization.

[0585] Toxicity - Toxicity of compounds as assessed by a live-dead assay (Life

[0586] Technologies). 8123-112871-03 11 / 21 / 25 06880

[0587] Imaging - Live-cell imaging of random cell motility or collagen invasion was used to quantify the effect of Pfnl -actin inhibitors on the migration speed of VECs. Cell migration was used as an indicator of the selectivity of compounds by using actin-binding-deficient mutation Hl 19E-Pfnl.

[0588] Immunohistochemistry - Tissue sections were deparaffinized and rehydrated before blocking and incubating overnight with an anti-CD31 antibody (77699, Cell Signaling Technology, 1: 100). For immunodetection of the primary antibody, a secondary biotin-labeled anti -rabbit antibody (ab97049, Abeam) and streptavidin-peroxidase conjugates (11089153001, Roche) were used. The staining was detected with the Pierce DAB substrate kit (34002, Thermo Fisher). For some experiments, the slides were counterstained with hematoxylin before dehydration and mounting.

[0589] Protein Purification and Actin Polymerization Assay - Recombinant wild-type (WT) Pfnl was expressed in BL21(DE3) Codon + RPIL E. Coli as an N-terminal fusion with a purification tag containing both av 8X-His and the fluorescent protein, mRuby2. After lysis, the fusion protein was purified using immobilized metal affinity chromatography (IMAC) and the His8-mRuby2 tag liberated from the fusion protein by overnight digestion with tobacco etch virus (TEV) protease. The purification tag and TEV protease were then separated from Pfnl using a second round of IMAC. The resulting Pfnl was then subjected to ion exchange and size exclusion chromatography. The resulting purity is >99% as judged by SDSPAGE. For PTS assays, Pfnl was used at a concentration of 0.3 mg / ml in a buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 1 mM P-mercaptoethanol, and 5X SYPRO Orange. Fluorescence of SYPRO Orange was measured and plotted as a function of temperature to quantify protein unfolding. The Tm was defined as the temperature with the maximum rate of change in fluorescence. Poly-proline binding activity was confirmed for Pfnl (Kd ~ lOpM) and for actin to polymerize. A 96-well format pyrene-based actin polymerization assay (cytoskeleton, BK003) was used for initial hit identification. A proximity -ligation assay (PLA - Duolink kit; Sigma, DUO9210) was used to quantitatively evaluate the extent of reduction of direct Pfnl -actin interaction in cells by treatment with the compounds disclosed herein.

[0590] Surface Plasmon Resonance (SPR) - SPR analysis was conducted using a Reichert 4SPR instrument (Reichert Technologies) with 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20, and 3 mM ethylenedi aminetetraacetic acid (EDTA) as the running buffer. An anti-glutathione-S-transferase (GST) antibody (Sigma Aldrich, G7781) was immobilized 8123-112871-03 11 / 21 / 25 06880

[0591] first on carboxymethyl dextran hydrogel biosensor chips (Reichert) using standard amine coupling chemistry with l-ethyl-3 -(-3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS). Purified recombinant GST-Pfnl proteins or GST alone as a negative control were then captured by the immobilized antibodies on separate channels on the chip. Analytes (purified actin and C74) were then injected over a range of concentrations at 50 pL / min with a 60 s association phase and 120 s dissociation phase. The chip surface was regenerated with 1 mM NaOH after each analyte injection. Each analyte concentration was measured in duplicate (Actin) or triplicate (C74), and the resulting sensorgrams were corrected for buffer effects. Kinetic and binding constants were calculated by fitting the sensorgrams with a 1: 1 Langmuir binding model using the TraceDrawer software (Reichert).

[0592] Isothermal Titration Calorimetry (ITC) - ITC was used to measure direct interactions between Pfnl and compounds. Pfnl-C74 interactions were measured by titrating 0 to 2.5 molar ratio C74 into 100 pM Pfnl. Titration of C74 was used to measure background, and a titration against actin was a control for indirect interactions within the Pfnl -actin system. C74 binding was housed within the actin-binding site, as demonstrated by titrating C74 into a Hl 19E-Pfnl mutant. These measurements validate the binding of compounds to Pfnl in solution, inform on the location of the binding site, and also serve as the baseline of comparison between C74 and other compounds disclosed herein. Titrations were performed in triplicate to determine binding constants. Differential scanning fluorimetry was also used to monitor Pfnl / compound interactions. Microscale thermophoresis was also used to measure Pfnl -polyproline interactions.

[0593] Anti-Angiogenic Action of Pfnl-Actin Interaction Inhibitors - FIG. 2A shows relative transcript values for Pfnl in CD31+ VEC from PDR patients (N = 7) vs control (N = 4) human retinal samples (transcriptome GEO dataset: GSE94019). FIGS. 2B-2C shows images of oxygen-induced retinopathy (OIR) - an in vivo model for pathological retinal NV. In this assay, mouse pups were housed in a 75% oxygen (hyperoxic) environment from P7-P12 which causes vaso-obliteration in the central retina. Subsequent housing of pups in a normoxic environment from P12-P17 creates a relative ischemia (hypoxia) causing VEGF upregulation and NV (known as tufts) driven by proliferation of superficial vascular plexi of retinae. FIGS. 2F-2L show the effects of two rounds (on P12 and P14) of intravitreal (IVT) injection of C2 (50 pM) vs DMSO (contralateral eye) on P17 retinal vasculature detected by lectin staining in the OIR assay. Although both groups have a similar percentage of vaso- 8123-112871-03 11 / 21 / 25 06880

[0594] obliteration, C2-treated pups show reduced NV (expressed as NV area relative to total area). Panel C confirms that C2 does not affect vascular density in the non-NV regions, suggesting lack of vascular toxicity. FIG. 2F shows images of choroidal explant angiogenesis culture on D6 under vehicle (DMSO) and C74-treated conditions. The inset shows explants on D3, while the red outline shows the edge of the vascular outgrowth. FIG. 2H shows laser-induced choroidal NV (CNV - an in vivo model that induces sub-retinal NV in response to laser injury in mice and is widely used to study NV aspects of wet AMD) in the settings of 1VT injection of either DMSO (VEH) or C74 (50 |1M), and associated quantification is shown in FIG. 21. FIGS. 2J-2K show CD31 staining of subcutaneous tumors established by RENCA renal cancer cells in Bal / C mice, which show reduced tumor angiogenesis after daily intra-tumoral injection of C74 for 2 weeks relative to DMSO treatment control (n = 5 mice / group).

[0595] Validation - Compound C2 was discovered as part of a structure -based virtual screen. C2 was active in an actin polymerization assay, where it reversed Pfnl’s inhibitory effect on actin polymerization but did not alter actin polymerization without the presence of Pfnl. Although Pfnl promotes actin assembly in cells, it acts as an inhibitor of actin polymerization in biochemical assays because of its actin-monomer-sequestering action. To explore the SAR, all commercially available analogs of C2 were acquired. This revealed that the phenol of C2 had a central role in binding, which is consistent with a predicted binding mode where the hydroxyl group on this phenol forms a hydrogen bond with Hl 19, which is an amino acid residue involved in actin-Pfnl interaction. Additionally, this phenyl group was sensitive to modification: additional substituents that extend towards solvent could maintain or remove activity (e.g., a methyl in the para- position maintained activity, while a methoxy in the meta position reduced it). This may be due to interactions with R74, which exhibits sufficient flexibility in NMR studies (PDB: 1PFL) to interact with this phenyl substituent. Because of the limited number of commercially available analogs of C2 and the inherent limitations of its scaffold (low solubility, high aromatic ring count, and chemical complexity), pharmacophore-driven scaffold hopping was performed. This resulted in identification of compound C74, which is predicted to maintain the hydrogen bond network of C2 while also interacting with R88.

[0596] Pyrene-actin polymerization assays (fluorescence measures actin polymerization) show that C74 reverses Pfnl’s inhibition of actin polymerization (FIG. 4A). In FIG. 3A, “A” represents actin, “P” represents Pfnl, and the numbers in parentheses indicate relative 8123-112871-03 11 / 21 / 25 06880

[0597] stoichiometry. C74 does not affect actin polymerization kinetics without the presence of Pfnl.

[0598] FIG. 3B compares relative anti-angiogenic efficacies of C2 and C74 in dermal micro-VEC matrigel cord-morphogenesis assays. As shown in FIG. 4B, both C2 and C74 demonstrated reproducible dose responses in angiogenesis assays. C74 inhibits angiogenesis starting at 10 pM while C2 exhibits a discernable anti-angiogenic effect at ~50 pM, suggesting that C74 is biologically more potent than C2. Both compounds inhibit angiogenesis in a dose-dependent fashion. FIG. 2C shows example images of matrigel cord assays and FIGS. 2D-2E shows associated quantification, demonstrating the anti-angiogenic effect of C74 (25 pM) on human retinal micro- VEC. The data shown in FIGS. 2D-2E are summarized from three experiments. “**” indicates a p-value < 0.01, which was considered statistically significant.

[0599] Target engagement of C2 and C74 was verified in cells by proximity ligation assay (PLA) where treatment with the compounds substantially diminished the Pfnl -actin interaction in VECs (FIGS. 4A-4B - data shown for C2 only). Live-dead assays confirmed that neither C2 nor C74 exhibited cellular toxicity at the concentrations tested (up to 100 pM). Consistent with the effect of Pfnl knockdown, both compounds inhibited proliferation and migration of various cells including VECs (FIGS. 4C-4E - data shown for C2 only). Using engineered microvascular VEC sublines stably expressing either the wild-type (WT) form or the actin-binding deficient Hl 19E mutant of Pfnl as GFP-fusion proteins with endogenous Pfnl suppressed by siRNA treatment further showed that C2 inhibits migration of WT- but not Hl 19E-Pfnl expressers, further suggesting anti-migration phenotype specific to inhibition of the Pfnl -actin interaction (FIG. 4D). FIG. 4D shows that C2 inhibits migration of Pfnl KD VEC when rescued with GFP-Pfnl but not actin-binding deficient mutant GFP-H119E-Pfnl. All experiments were repeated 2-3 times with multiple technical replicates. “**” denotes a p-value < 0.01, which was considered statistically significant.

[0600] SAR -Up to 40 analogs of C74 were purchased. Compounds were template docked and scored against the Pfn l structure using both Autodock Vina and a convolutional neural network scoring scheme using similar consensus scoring protocols to those that were used in the identification of C74. Receptor flexibility was accounted for by docking against an ensemble of Pfnl structures generated through molecular dynamics simulation. Those predicted to have additional binding interactions, or those that will help validate a key binding hypothesis were selected. This early SAR exercise helped to develop an 8123-112871-03 11 / 21 / 25 06880

[0601] understanding of structural requirements for potency and generated data points to increase the predictive ability of research models. Compounds were evaluated in the actin polymerization assay as described above, then in ITC to determine an IC50.

[0602] In some aspects, compounds that are predicted to occupy the putative binding site compared to C74 are not commercially available. Specific initial analogs included those in which the where the methyl of the hydroxypyrazole is substituted or the hydroxypyrazole is replaced by isosteres. In these cases, information from exploration of one or both hydroxypyrazole sites was used to incorporate alternative substituents. In some aspects, one or more compounds disclosed herein have a potency in ITC <1 pM, and cord morphogenesis assay activity < 1 pM.

[0603] FIG. 5 shows preliminary SAR results for C74. Cord-morphogenesis of dermal VEC following treatment with the indicated concentrations of various C74 analogs (chemical structures shown above images) showed increased potency of C74-C and C74-D analogs relative to the original C74 compound. Compounds C74-C and -D have CH3 and fluorinated substitutions in place of the Br present in C74. These two analogs also inhibit VEC migration more potently that C74-P.

[0604] Molecular docking was performed using GNINA (PMID: 34108002) which performs Monte Carlo search over possible ligand conformations and poses and ranks the resulting poses using a convolutional neural network. Default settings were used and the protein receptor structure was kept rigid. The protein structure used for docking includes a cryptic pocket at the Pfnl: actin interface that was identified through molecular dynamics simulations.

[0605] Small Molecule Compounds - Compounds disclosed herein were purchased from commercial sources or chemically synthesized. Reagents and solvents used in chemical synthesis were purchased from commercial sources at the highest commercial quality. Thin layer chromatography (TLC) was performed using Millipore Sigma™ silica gel 60 F254 coated aluminum-backed TLC sheets. TLC spots were detected using 254 nm UV light.1H-NMR was used to confirm the purity of all the final products. Methanol-D and DMSO-D were used as the solvents.1H-NMR and13C-NMR spectra were recorded on a Broker Avance 500MHz spectrometer and a Broker Avance NEO 400 MHz spectrometer.1H and13C chemical shifts were reported relative to the residual solvent’s peak. Nominal mass accuracy LCMS data were obtained by use of a Waters Acquity UPLC system equipped with a Waters TUV detector (254 nm) and a Waters SQD single quadrupole mass analyzer with 8123-112871-03 11 / 21 / 25 06880

[0606] electrospray ionization. LC gradient 500 pL / min: 30 second hold, 95:5 (water:acetonitrile 0.1% v / v formic acid), 2 minute gradient to 5:95, and 30 second hold. Acquity UPLC HSS C18, 1.7um, 2. lx 50 mm column. All small molecule compounds were dissolved in DMSO. The details of chemical synthesis of various C74 analogs and structural confirmation are listed herein.

[0607] Synthetic Routes - C74 derivatives of general structure 4 (Scheme 1; routes 1 and 2) can be synthesized via several different methods.

[0608] Scheme 1 - synthetic methods for derivative preparation.

[0609] -

[0610]

[0611] The reaction of a pyrazolone with an aldehyde in a Knoevenagel, Michael tandem reaction sequence affords symmetric bis-hydroxypyrazole derivatives, such as C74, in which the same hydroxypyrazole flanks the Ri substituent (Scheme 1, route 1), if two equivalents (eq.) of pyrazolone are used. Such symmetric derivatives can also be synthesized in a multicomponent reaction format (Scheme 1, route 2) in which an aldehyde, 2 eq. of hydrazine or hydrazine hydrochloride and 2 eq. of ethyl acetoacetate react in one pot. The commercial availability of many diverse aldehydes assures access to key starting materials. Asymmetric derivatives in which there are two differently substituted hydroxypyrazoles can also be accessed via route 1. Knoevenagel condensation products 3 are easily prepared by a desired aldehyde and 1 eq. of methylpyrazolone and can be subjected to Michael reaction conditions with a second differently substituted pyrazolone intermediate under the conditions used for the preparation of symmetric derivatives. Analogs in which one of the hydroxypyrazoles has been replaced by another moiety / isostere (general structure 7, scheme 1, routes 3 & 4) can be prepared through a combination of protocols. For example, ketones, like aldehydes, react in a Knoevenagel fashion with pyrazolones to afford the corresponding condensation products with exocyclic double bond (general structure 6, scheme 1, route 3) and these products are known to be selectively reduced with NaBFU or Hantzsch ester hydride. Alternatively, these derivatives can be prepared by the reaction of ethylacetoacetate with substrates of the general structure 8 to provide ketoesters 9 which in turn can be 8123-112871-03 11 / 21 / 25 06880

[0612] converted to desired derivatives by treatment with hydrazine (scheme 1, route 4). In the cases of asymmetric analogs, racemic products were prepared and analyzed in the primary assay (actin polymerization). Active compounds (which may include one or more active agent(s)) were then separated by chiral HPLC and re-assayed to evaluate if a chiral preference exists. Evaluation for configurational stability involved HPLC analysis of the chiral product under assay conditions, and only those that are configurationally stable advanced.

[0613] FIGS. 6A-6C illustrate aspects of various synthesis schemes.

[0614] General procedure for the synthesis of water-soluble products (3UP1, 3UP4, 3UP5, 3UP7, 3UP9, 3UP10, 3UP11, 3UP12, and 3UP13): To a round-bottom flask was added aldehyde 1 (1 equiv), 3 -methy 1-5 -pyrazolone (2, 1.7 equiv), sodium dodecyl sulfate (0.3 equiv) and H2O (30 mL). The reaction mixture was stirred at room temperature for 30 min and then refluxed until thin-layer chromatography shows starting material 2 is gone. The reaction mixture was concentrated in vacuo, and the residue was washed with acetone (15 mL x 3). The resulting crude product was purified using silica gel chromatography (Combi flash Rf, 60% MeOH-DCM) to give solid product.

[0615] General procedure for the synthesis of insoluble compounds (3UP2, 3UP3, 3UP6, and 3UP8): to a round-bottom flask was added aldehyde 1 (1 eq.), 3-methyl-5-pyrazolone (2, 2.4 eq.), sodium dodecyl sulfate (0.3 eq.) and H2O (30 mL). The reaction mixture was stirred at room temperature for 30 min and then refluxed until thin-layer chromatography shows starting material 1 is gone. Solid was formed in the reaction mixture. The reaction mixture was filtered, and the solution was disposed. The remaining residue washed with H2O (15 mL x 3) to give solid product.

[0616] Aspects of certain compounds are described below:

[0617]

[0618] 4,4'-(pyridin-4-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP1):

[0619] Yield 65%: red powder;

[0620] LC / MS (m / z): 286 (M + H); 8123-112871-03 11 / 21 / 25 06880

[0621] ’H NMR (500 MHz, MeOD-d); 58.46 (d, J = 6 Hz, 2H), 7.38 (d, J = 6 Hz, 2H), 5.09 (s, 1H), 2.30 (s, 6H);

[0622] 13C NMR (500 MHz, DMSO-d6): 5 161.2, 153.2, 149.1, 140.7, 123.6, 103.5, 32.7, 10.7.

[0623]

[0624] 4,4'-((3-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP2) Yield 93%; light orange sticky solid;

[0625] LC / MS (m / z): 353 (M + H);

[0626] ’H NMR (400 MHz, DMSO-d6); 57.51-7.42 (m, 4H), 4.94 (s, 1H), 2.10 (s, 6H);

[0627] 13C NMR (400 MHz, DMSO-d6): 5 145.3, 132.3, 129.2, 128.8. 126.3, 124.2, 123.6, 122.8, 104.1, 33.1, 10.8.

[0628]

[0629] 4,4'-((2-(trifluoromethyl)phenyl)methylene)bis(3 -methyl- 1 H-pyrazol-5 -ol) (3UP3) Yield 75%; light yellow sticky solid;

[0630] ’H NMR (500 MHz. MeOD-d): 57.88 (d. J = 8 Hz. 1H), 7.66 (d. J = 7.5 Hz. 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 7 Hz, 1H), 5.45 (s, 1H), 2.10 (s, 6H);

[0631] 13C NMR and LC / MS are consistent with literature.

[0632]

[0633] 8123-112871-03 11 / 21 / 25 06880

[0634] 4,4'-(pyridin-3-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP4)

[0635] Yield 40%; light yellow sticky solid;

[0636] ’H NMR (500 MHz, MeOD-d): 58.34 (s, 2H), 7.69 (d, J = 8 Hz, 1H), 7.35 (dd, J = 4.5, 7.5 Hz, 1H), 5.06 (s, 1H), 2.28 (s, 6H);

[0637] 13C NMR and LC / MS are consistent with literature.

[0638]

[0639] 4,4'-(pyridin-2-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP5)

[0640] Yield 75%; light yellow sticky solid;

[0641] ’H NMR (500 MHz, MeOD-d): 58.44 (d, J = 5 Hz, 1H), 7.79 (t, J = 8 Hz, 1H), 7.51 (d, J = 8 Hz, 1H), 7.28 (t, J = 6 Hz, 1H), 5.15 (s, 1H), 2.20 (s, 6H);

[0642] 13C NMR and LC / MS are consistent with literature.

[0643]

[0644] 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP6) Yield 80%; light orange sticky solid;

[0645] LC / MS (m / z): 353 (M + H);

[0646] ’H NMR (400 MHz, DMSO-d6): 57.60 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 4.91 (s, 1H), 2.10 (s, 6H);

[0647] 13C NMR (400 MHz, DMSO-d6): 5148.8, 128.7, 126.5, 126.4, 125.1, 33.2, 10.8. 8123-112871-03 11 / 21 / 25 06880

[0648]

[0649] 1 -(4-(bis(5-hydroxy-3 -methyl- lH-pyrazol-4-yl)methyl)phenyl)ethan-l -one (3UP7) Yield 61%; orange powder;

[0650] LC / MS (m / z); 327 (M + H);

[0651] 1H NMR (400 MHz, DMSO-d6): 87.82 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 4.84 (s, 1H), 2.08 (s, 6H);

[0652] 13C NMR (400 MHz, DMSO-d6): 6 198.0, 161.4, 150.0, 135.0. 128.3, 104.2, 89.3. 33.3, 27.1, 11.6, 10.8.

[0653]

[0654] 4,4'-((4-(benzyloxy)phenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP8)

[0655] Yield 46% (180 mg); light yellow powder;

[0656] LC / MS (m / z); 392 (M + 2H);

[0657] 1H NMR (400 MHz, DMSO-d6); 87.44 (d, J = 6.8 Hz, 2H), 7.38 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8 Hz, 2H), 4.75 (s, 1H), 2.07 (s, 6H);13C NMR (400 MHz, DMSO-d6): 8 156.8, 137.8, 136.0, 128.9, 128.2, 114.5, 69.6, 32.4, 10.8 8123-112871-03 11 / 21 / 25 06880

[0658]

[0659] 4,4'-((4-aminophenyl)methylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP9)

[0660] Yield 78%; light yellow sticky solid;

[0661] LC / MS (m / z); 300 (M + H);

[0662] XH NMR (500 MHz, MeOD-d): 5

[0663] 13C NMR (400 MHz, DMSO-d6): 5 146.6, 130.9, 128.3, 114.0, 32.4, 10.9.

[0664]

[0665] 4,4'-(isoquinolin-4-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP10)

[0666] Yield 84%; light yellow sticky solid;

[0667] LC / MS (m / z); 336 (M + H);

[0668] ’H NMR spectrum of 3n (400 MHz, DMSO-d6): 59.16 (s, 1H), 8.27 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.72 (t, J = 7.2 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 5.54 (s.

[0669] 1H), 1.81 (s, 6H);

[0670] 13C NMR spectrum of 3n (400 MHz, DMSO-d6): 5151.5, 142.9, 134.1, 131.8, 130.6, 128.6, 127.1, 123.7, 102.7, 29.2, 10.9.

[0671]

[0672] 4,4’-(isoquinolin-8-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP11) 8123-112871-03 11 / 21 / 25 06880

[0673] Yield 81%; red solid;

[0674] LC / MS (m / z): 336 (M + H);

[0675] ‘H NMR spectrum of 3k (400 MHz, DMSO-d6): 59.30 (s, 1H), 8.45 (d, J = 5.6 Hz, 1H), 7.80 (m, 2H), 7.65 (t, J = 8 Hz, 1H), 7.42 (s, 1H), 5.76 (s, 1H), 1.76 (s, 6H);

[0676] 13C NMR spectrum of 3k (400 MHz, DMSO-d6): 5 160.3, 149.2, 142.5, 140.4, 138.4, 136.4, 130.3, 127.4, 126.4, 125.6, 121.4, 103.3, 30.5, 10.8.

[0677]

[0678] 4,4'-(isoquinolin-5-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP12)

[0679] Yield 88%; red solid;

[0680] LC / MS (m / z): 336 (M + H);

[0681] ’H NMR spectrum of 31 (400 MHz, DMSO-d6): 89.27 (s, 1H), 8.43 (d, J = 6 Hz, 1H), 7.96 (m, 1H), 7.69 (d, J = 6.4 Hz, 1H), 7.58 (m, 2H), 5.55 (s, 1H), 1.77 (s, 6H);

[0682] 13C NMR spectrum of 31 (400 MHz, DMSO-d6): 5 160.4, 153.4, 143.1, 138.7, 138.1, 134.1, 130.1, 129.1, 127.1, 126.4, 117.3, 102.9, 30.7, 10.9

[0683]

[0684] 4,4'-(quinolin-5-ylmethylene)bis(3-methyl-lH-pyrazol-5-ol) (3UP13)

[0685] Yield 92%; red solid;

[0686] LC / MS (m / z): 336 (M + H);

[0687] ’H NMR spectrum of 3m (400 MHz, DMSO-d6): 58.84 (d, J = 3.2 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.47 (dd, J = 6.4, 10.4 Hz, 1H), 7.38 (d, J = 6.8 Hz, 1H), 5.61 (s. 1H), 1.73 (s, 6H);

[0688] 13C NMR spectrum of 3m (400 MHz, DMSO-d6): 5 150.1, 148.6, 140.1. 132.6, 129.1, 127.8, 126.3, 121.3, 30.6, 10.8. 8123-112871-03 11 / 21 / 25 06880

[0689] C74 Binding to Pfnl - While Pfnl’s action enhances actin polymerization in cells, in biochemical assays without the presence of any other actin-assembly factors, Pfnl inhibits actin polymerization through preventing actin incorporation onto the pointed ends of growing actin filaments as well as by inhibiting spontaneous actin nucleation. The discovery of C74 (FIG. 7 A) as an inhibitor of the Pfnl -actin interaction was based on the results of a pyreneactin polymerization screening assay where C74 reversed recombinant GST-Pfnl’s inhibitory effect on actin polymerization but had no discernible effect on actin polymerization on its own. Further, C74 treatment reduces proximity -ligand interactions of endogenous Pfnl and actin in cells and induces other phenotypic changes (i.e. reduced cell spreading, migration, and proliferation) that are expected upon inhibition of cellular Pfnl -actin interaction.

[0690] However, whether C74 directly binds to Pfnl remained unexplored. GST epitope-tagging enabled us to immobilize all purified proteins with an anti-GST antibody on the SPR chip thereby reducing the masking of any binding site of Pfnl for potential C74 interaction. As an initial test for the utility of the SPR assay to detect known ligand interactions of Pfnl, GST-tagged wild-type (WT) Pfnl interacted with G-actin while GST-tagged Hl 19E-Pfnl (a pointmutant of Pfnl that has ~25-fold reduction in actin-binding compared to WT-Pfnl) did not exhibit any detectable interaction with G-actin as expected (FIGS. 7B-7C, Table 1). The estimated dissociation constant (Kd) of GST-Pfnl: actin interaction in the SPR assay was approximately 5.3 pM (FIG. 7E). This value is an order of magnitude higher than previously reported (0.15 pM) for Pfnl:actin interaction measured in an isothermal calorimetric assay. This discrepancy could be due to either differences between tagged- and untagged-Pfn 1 in terms of their binding affinities for actin and / or assay-specific differences in the Kd measurements. C74’s interaction with Pfnl was detected with an estimated Kd value of approximately 60 pM, consistent with its potency in previous anti-angiogenesis assays, and Hl 19E substitution did not impair the interaction of C74 with Pfnl (FIGS. 7D-7E, Table 1). These results suggest that C74 is a low-affinity binder of Pfnl.

[0691] Table 1: Pfnl (WT and FfnlniwE) binding properties with actin and C74.

[0692] Ligand Analyte Ka(M-'s-' ) Kd(s-1) Kp (M) Chi2(RU2) WT-Pfnl Actin 5.58 x lO32.95 x 10'25.29 x IO’60.67 PfnlH119E Actin No interaction.

[0693] WT-Pfnl C74 6.29 x 1033.72 x 10'15.91 x W50.29 Pfnlnii9E C74 4.93 x 1032.36 x 1044.79 x 10’50.14

[0694]

[0695] 8123-112871-03 11 / 21 / 25 06880

[0696] Intratumoral Release of Pfnl Inhibitor Diminishes Neovascularization of RCC Tumors - In a RENCA-based RCC mouse model, intratumoral administration of C74 leads to reduced tumor growth in a subcutaneous implant setting as described in Allen, A. et al., Actin-binding protein profilinl promotes aggressiveness of clear-cell renal cell carcinoma cells, J Biol Chem 295, 15636 (2020). However, since in vitro studies also showed that C74 treatment reduces RENCA cell proliferation, the relative contributions of tumor-intrinsic vs -extrinsic effects of C74 underlying its anti -tumor action were not discernable. Therefore, an end-point assessment of tumor angiogenesis was performed using immunohistochemistry (IHC) of tumor sections for CD31 (a marker of vascular ECs) and quantifying the relative CD31-positive areas in DMSO (vehicle control)- vs. C74-treated RENCA tumors collected from in vivo studies. As per these analyses, C74 treatment reduced the CD31 -positive area by -30% in a statistically significant manner (FIGS. 2J-2K).

[0697] Based on these results, microbubble-based delivery of C74 was pursued to overcome potential issues associated with systemic and direct injection approaches. Unlike subcutaneous tumors, access to internal organ tumors (such as RCC) is challenging for direct injection, which can make localized administration challenging. Moreover, since C74 is a low-affinity binder of Pfnl, systemic delivery of C74 may require high doses to achieve therapeutic concentrations at the tumor site, potentially leading to off-target effects and toxicity. Lipid microbubbles serve as efficient carriers of small molecules and nucleic acids and can release their payloads for localized therapeutic applications when subjected to ultrasound-targeted microbubble destruction (UTMD). Therefore, C74 was encapsulated within lipid microbubbles (-3 pm in diameter), as confirmed by mass-spectrometry evaluation of microbubbles (FIG. 8).

[0698] A cord morphogenesis (an indicator of angiogenic potential) assay served as a proof-of-concept in vitro study with HmVECs, which demonstrated that similar to the action of unencapsulated C74 (a positive control), UTMD of C74-encapsulated microbubbles prominently inhibits cord morphogenesis of ECs (FIGS. 9A-9B). The

[0699] extent of angiogenesis inhibition was more pronounced when C74 was released from microbubbles compared to the setting where C74 was directly added to the culture. This may be due to possible differences of C74 concentrations between these two settings. To establish specificity, several negative control groups were included in the same experimental setting where either C74-encapsulated microbubbles were not subjected to UTMD or C74 was 8123-112871-03 11 / 21 / 25 06880

[0700] transiently released by UTMD followed by immediate C74 washout and replacement with regular culture media or UTMD of microbubbles was performed without C74 as a payload (to rule out any possible detrimental effect of ultrasound cavitation on cells). No adverse effect on cord formation by ECs was observed in any of these negative control settings when compared to the control DMSO treatment group (FIGS. 9A-9B).

[0701] Given these results demonstrating the feasibility of UTMD to release C74 and achieve anti-angiogenic action in cell culture settings, exploratory in vivo tumor model studies were performed involving subcutaneous implantation of RENCA cells in Balb / c mice. After allowing these tumors to reach palpable size, tumor-bearing mice received retro-orbital injections of C74-loaded microbubbles three times over the course of a week, followed by ultrasound application to target the tumor site. This precision delivery approach led to a significant reduction in tumor growth and a concomitant decrease in tumor angiogenesis (FIGS. 10, 11A-1 IB, and 12A-12B), consistent with the above-described in vitro findings. These results support the utility of lipid microbubble-based delivery of C74 as a promising targeted therapy to diminish neovascularization and tumor growth.

[0702] Optimization of C74 Identifies Analogs with Anti-Angiogenic Activity - The bromine (Br) substituent in C74 can be effectively replaced with either F or CH3, and these analogs exhibited anti-angiogenic activity at 10 pM, which is more potent than C74. Based on these results, additional analogs with greater potency than C74, efficacy and drug-like properties were identified. Based on docking studies of C74 the aryl group was selected for modification as its binding pocket was not fully occupied, modification at this position was not only tolerated but led to improvements in potency, possibly through interactions with the R74 and Hl 19 residues of Pfnl.

[0703] Initially, the pyrazole groups were predicted to engage in multiple H-bonding interactions with Pfnl. Therefore, these groups were not modified, replaced, or removed in C74 analogs UP1-UP13, as changes to the pyrazole groups may have led to less active compounds by reducing the number of H bonds between the C74 analog and Pfnl. It will also be appreciated that one or both pyrazole groups can be modified, replaced, or removed to develop other analogs of C74.

[0704] Based on available SAR (e.g., F and CH3 substitution led to greater efficacy than Cl or H) potential new analogs were based on: 1) docking studies that suggested the aryl bromine pocket was not fully occupied, 2) exploring whether lipophilicity, polarity or electronegativity contributed to activity, and 3) optimizing the physical chemical properties 8123-112871-03 11 / 21 / 25 06880

[0705] such as solubility and permeability. A series of potential analogs were docked using the GNINA molecular docking software. Docking scores and other factors (e.g., predicted interactions and physical chemical properties) were used to prioritize compounds for synthesis. Examples are shown in Table 2.

[0706] Table 2

[0707] Inhibits EC Inhibits EC Inhibits EC Inhibits EC

[0708] Compound Cord Compound Cord Proliferation Proliferation

[0709] Formation Formation UP-1 No N / A UP-8 Yes ++

[0710] UP-2 Yes ++ UP-9 No N / A

[0711] UP-3 Yes + UP- 10 No N / A

[0712] UP-4 No N / A UP- 11 No N / A

[0713] UP-5 No N / A UP- 12 No N / A

[0714] UP-6 Yes +++ UP- 13 No N / A

[0715] UP-7 No N / A

[0716]

[0717] The aryl halide was replaced with various pyridine isomers (UP-1, -4, and -5) to evaluate if electronegativity contributed to binding and to potentially enhance solubility; CF3 (UP-2, -3, and -6) was introduced at various positions to probe size constraints and electronegativity, as well as enhance any metabolic instability. Several nitrogen-containing bicyclic systems (UP-10 through UP-13) were introduced to probe the size of the binding site. The ketone (UP -7) and benzyl ether (UP-8) were designed to probe the size of the binding site and a potential hydrogen bond with R74, and the aniline (UP-9), which was predicted to be more soluble, tested whether polarity or electronegativity contributed to binding.

[0718] The compounds of Table 2 were tested in EC proliferation assays at a single dose of 20 pM, and then those compounds (UP-2, -3, -6 and -8) that inhibited EC proliferation relative to DMSO control were selected for qualitative evaluation of anti -angiogenic activity in EC cord formation assays. Based on this initial qualitative screening, compound UP-6 4,4'-((4-(trifluoromethyl)phenyl)methylene)bis(3,5-dimethyl-l H-pyrazole) was the most effective analog in terms of its ability to inhibit angiogenic activity of ECs in vitro (Table 2). Single-dose in vitro angiogenesis assay results involving compound UP-6 are shown in FIGS.

[0719] 13A-13B. The near-complete blockade of cord formation by 20 pM UP-6 was similar to that observed in ECs when subjected to a much higher concentration (50 pM) of C74. C74, when 8123-112871-03 11 / 21 / 25 06880

[0720] applied at 10 pM, is completely ineffective in reducing cord formation of EC.

[0721] Encouragingly, at a 10 pM concentration, UP6 is still effective in inhibiting EC cord formation (by -50%) while C74 is not (FIGS. 14A-14B).

[0722] To further characterize UP-6, a protein thermal shift (PTS) assay compared the themral stability of the Pfnl protein with compound UP-6, C74, and a DMSO control. Both C74 and UP -6 produced negative themral shifts of Pfnl relative to the DMSO control, but the extent of the negative thermal shift was more pronounced for UP6 than C74 (FIG. 14C). Accordingly, wishing to be bound by theory, these results suggest that small molecule interactions can either destabilize Pfnl to some extent or induce a conformational change that facilitates SYPRO fluorescence at lower temperatures with UP-6 having a more pronounced impact than C74. Given that UP6 outperforms C74 in terms of its ability to reduce cord formation by ECs at a lower concentration than C74 in vitro, matrigel plug angiogenesis assay was used to further determine whether this is also true in an in vivo setting: bFGF (a stimulator of angiogenesis)-supplemented growth-factor-reduced matrigel was co-injected with either C74 or UP-6 (at 200 pM) or equivalent DMSO control bilaterally into the flanks of C57 / B16 mice. CD31 immunostaining of plugs harvested 10 days after injection revealed that treatment of

[0723] either C74 or UP-6 significantly reduced EC infiltration into the plugs, but the reduction in CD31 -positivity of plugs treated with UP-6 was more prominent (>50% decrease relative to control) than that achieved by the treatment of C74 compound (-25% decrease vs control), and this difference was statistically significant (FIGS. 15A-15B). These data suggest that UP-6 has a more potent antiangiogenic activity than C74, providing the first in vivo proof-of-concept that anti-angiogenic efficacy based on inhibition of the Pfnl -actin interaction can be optimized through medicinal chemistry strategies.

[0724] In view of the many possible embodiments to which the principles of the present disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be taken as limiting the scope of the present disclosure. Rather, the scope is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. 8123-112871-03 11 / 21 / 25 068802.We claim:

1. A compound, having a structure:

5. 7.wherein Ri is not phenyl, Ri is not 4-bromophenyl, Ri is not 4-chlorophenyl, Ri is not 4-fluorophenyl, and Ri is not a methyl group;8.wherein R2 is hydrogen or a methyl group; and9.wherein R3 is hydrogen, a phenyl group, a substituted phenyl group, a heterocycle or a substituted heterocycle.

2. The compound of claim 1, wherein R3 is a pyrazole or a substituted pyrazole.

3. The compound of claim 1 or claim 2, wherein R3 has a structure:12.HN-N13.HO^y^R414.•~i~16. 18.wherein R4 is hydrogen or a methyl group.

4. The compound of any one of claims 1-3, wherein Ri has a structure:

21. 23.wherein R5 is not bromine, chlorine, or fluorine, at the para- position of Ri.

5. The compound of claim 4, wherein R5 is a trifluoromethyl group.

6. The compound of any one of claims 1-5, wherein the compound is8123-112871-03 11 / 21 / 25 0688026.HN-N27.L 'N29.

7. The compound of any one of claims 1-6 for use as a vascular therapy.32.A pharmaceutically acceptable composition, comprising:33.the compound of any one of claims 1-6; and34.a therapeutically acceptable excipient.

9. A method, comprising administering the compound of any one of claims 1-6, or a pharmaceutically acceptable composition thereof, to a subject exhibiting, or susceptible to exhibiting: a vascular pathology or a disease involving a vascular pathology.

10. The method of claim 9, wherein the disease is a cancer, age-related macular degeneration (AMD), diabetic retinopathy, psoriasis, atherosclerosis, or a combination thereof.

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