Compounds and constructs useful for targeting fibroblast activation protein

Abstract

Described herein are methods for identifying small molecules capable of binding to a protein of interest using in silico rigid and flexible docking analysis, which may be followed by in vitro binding assays. Also described herein are constructs comprising a compound that may be coupled to a chelating agent, which may in turn be bound to a radionuclide. Further described herein are methods of treatment for cancer and / or methods of detecting a tumor, comprising administering a construct as described herein to a subject in need thereof.

Description

[0001] Compounds and Constructs Useful for Targeting Fibroblast Activation Protein

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63 / 490,805 filed March 17, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Fibroblast activation protein (FAP) is a type 2 transmembrane serine proline peptidase that is only transiently expressed during normal development and rarely expressed in healthy adult tissues. It is highly upregulated in the stromal cells at sites of active tissue remodeling such as wound healing, fibrosis, arthritis, atherosclerosis, and in over 90% of human epithelial cancers, such as breast, colorectal, prostate, pancreatic, and skin tumors (Garin-Chesa et al., PNAS, 1990, 87.18:7235; Park et al., J Biol Chem, 1999, 274: 36505; Pure et al., Oncogene, 2018, 37: 4343; Rettig et al. Cancer Res., 1993, 53.14:3327; Kratochwil J Nuc Med 2019, 60: 801). These tumors can actively recruit fibroblasts, mesenchymal stem cells, smooth muscle cells, epithelial and endothelial cells. Once recruited, the fibroblasts are often transformed into cancer associated fibroblasts (CAFs), which then express FAP and provide support to the tumors by secreting cytokines, chemokines, and growth factors (Madar et al., Trends Mol Med, 2013, 19: 447; Kalluri, NatRev Cancer, 2016, 16: 582; Pure et al., Oncogene, 2018, 37: 4343; Quail et al., NatMed, 2013, 19: 1423). Across a wide range of human tumor indications, FAP expression is described to correlate with higher tumor grade and worse overall survival (Pure et al., Oncogene, 2018, 37: 4343).

[0006] With the expression patterns described above, FAP is considered to be a suitable marker for radiopharmaceutical diagnostics and a suitable target for radiopharmaceutical therapy for various epithelial cancers (Siveke, J Nucl Med, 2018, 59: 1412; Christiansen et al., Neoplasia, 2013, 15: 348; Zi et al., Mol Med Rep, 2015, 11: 3203). However, current agents targeting FAP do not have sufficient specificity when tested on homologous proteins, such as other dipeptidyl peptidases, and suffer from poor pharmacokinetic characteristics and cellular retention. Specificity of FAP targeting is sufficient for diagnostic use, however improved pharmacokinetic properties of FAP targeting agents for radiopharmaceutical therapy is desired. The potential clinical uses for FAP could also extend into other disease indications, such as rheumatoid arthritis, idiopathic pulmonary fibrosis, Crohn’s disease, liver fibrosis and atherosclerosis. (Bauer et al., Arthritis Res Ther, 2006, 8: R171; Milner et al., Arthritis Res Ther, 2006, 8:R23; Truffi et al., Inflamm Bowel Dis, 2018, 24: 332; Monslow et al., Circulation, 2013, 128: A17597; Fan et ai., J Biol Chem, 2016, 291 :8070; Santacroce et al., Cells, 2022 11 :429; Fitzgerald and Weiner, Cancer Metastasis Rev, 2020, 39:783).

[0007] SUMMARY

[0008] Provided herein according to some aspects of the present invention is a method for identifying a small molecule capable of binding to a protein of interest. In some embodiments, the method includes some or all of the steps of: a) providing a small molecule library; b) performing an in silico rigid docking analysis for each member of the small molecule library to i) the surface of the protein of interest and to ii) the surface of a second protein that is similar in structure and / or function to the protein of interest (e.g., a homolog), to select a first subset of the small molecule library; c) performing an in silico flexible docking analysis for each member of the first subset to i) the surface of the protein of interest and to ii) the surface of the second protein, to select a second subset of the small molecule library; and d) performing an in vitro binding assay (e.g., a surface plasmon resonance (SPR) assay) for each member of the second subset to i) the surface of the protein of interest and to ii) the surface of the second protein, to generate a binding signal.

[0009] In some embodiments, the in vitro binding assay is a surface plasmon resonance (SPR) assay. In some embodiments, the method further includes: e) correcting for non-specific binding in the in vitro binding assay; and optionally may further include f) generating a concentrationresponse curve for each member of the second subset from the concentration-dependent binding signal; and optionally g) calculating the binding dissociation constant (Kd) from the concentrationresponse curve.

[0010] In some aspects, selecting the first subset of the small molecule library in step b) comprises selecting small molecules that have a binding affinity for the protein of interest that is at least 100- fold more favorable than for the second protein.

[0011] In some aspects, selecting the second subset of the small molecule library in step c) further comprises selecting small molecules that have a high predicted binding affinity for the protein of interest and a low predicted binding affinity for the second protein. In some aspects, performing the in vitro binding assay in step d) further comprises repeatedly performing the assay with increasing concentrations of each member in the second subset to generate a concentration-dependent binding signal.

[0012] In some aspects, a member of the second subset is identified as capable of binding to the protein of interest based on the Kd value.

[0013] Also provided herein according to some aspects of the present invention is a compound of Formula VIII:

[0014] Formula VIII or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0015] R1is selected from the group consisting of H, alky, and cycloalkyl; n is 0, 1, or 2;

[0016] R2is selected from the group consisting of H, alky, and cycloalkyl; and

[0017] R3is selected from the group consisting of NR', OR', alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or wherein R2and R3join to form a heterocycle (e.g., azetidinone, pyrrolidinone, imidazolidinone, oxazolidinone, piperidinone, piperazinone, morpholinone, azepanone, diazepanone, or oxazepanone).

[0018] In some embodiments, the compound of Formula VIII is in a compound of Formula VIIIA:

[0019] Formula VIIIA or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0020] R1is selected from the group consisting of H, alky, and cycloalkyl;

[0021] W, X and Y are each independently selected from the group consisting of CR4, O, N, and

[0022] NR5; R4and R6are each independently selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; and

[0023] R5is selected from the group consisting of H, acyl, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; or two of R4, R5and R6join to form a carbocycle, heterocycle, aryl, or heteroaryl.

[0024] In some embodiments, the compound of Formula VIIIA is a compound of Formula VIIIA(a):

[0025] Formula VIIIA(a) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0026] R1is selected from the group consisting of H, alky, and cycloalkyl;

[0027] A is selected from the group consisting of N and C;

[0028] B is selected from the group consisting of O, S, and N; and

[0029] R5is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl.

[0030] In some embodiments, R7is alkyl, A is C, and B is S.

[0031] In some embodiments, the compound of Formula VIII or Formula VIIIA is a compound of Formula VIIIA(b):

[0032] Formula VIIIA(b) 2 or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0033] R1is selected from the group consisting of H, alky, and cycloalkyl;

[0034] Y is selected from the group consisting of CR4b, N, and NR5; and R4a, R4band R6are each independently selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or two of R4a, R4b, R5and R6join to form a cycloalkyl, heterocycle, aryl, or heteroaryl.

[0035] In some embodiments, the compound of Formula VIIIA(b) is a compound of Formula VIIIA(b)(i): or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0036] R1is selected from the group consisting of H, alky, and cycloalkyl; and

[0037] R4a, R4b, and R7are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

[0038] In some embodiments, R7is halo.

[0039] In some embodiments, the compound of Formula VIIIA(b) is a compound of Formula wherein R1is selected from the group consisting of H, alky, and cycloalkyl;

[0040] R6and R7are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and

[0041] Z1and Z2are each independently selected from the group consisting of N and CR', wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0042] In some embodiments, the compound of Formula VIIIA(b)(ii) is a compound of Formula VIIIA(b)(ii)(a): wherein R1is selected from the group consisting of H, alky, and cycloalkyl;

[0043] R6and R8are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and

[0044] Z1and Z2are each independently selected from the group consisting of N and CR', wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0045] In some embodiments, R8is halo.

[0046] In some embodiments, R8is alkyl.

[0047] In some embodiments, the compound of Formula VIII is a compound of Formula VIIIB: or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0048] R1is selected from the group consisting of H, alky, and cycloalkyl;

[0049] R2is selected from the group consisting of H, alky, and cycloalkyl; and

[0050] R3is selected from the group consisting of NR', OR', alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or wherein R2and R3join to form a heterocycle (e.g., azetidinone, pyrrolidinone, imidazolidinone, oxazolidinone, piperidinone, piperazinone, morpholinone, azepanone, diazepanone, or oxazepanone).

[0051] In some embodiments, the compound of Formula VIIIB is a compound of Formula VIIIB(a): or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0052] R1is selected from the group consisting of H, alky, and cycloalkyl;

[0053] R2is selected from the group consisting of H, alky, and cycloalkyl; and

[0054] R4is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl.

[0055] In some embodiments, the compound of Formula VIIIB is a compound of Formula VIIIB(b): or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0056] R1is selected from the group consisting of H, alkyl, and cycloalkyl;

[0057] R6is selected from the group consisting of H, alkyl, and cycloalkyl; and

[0058] R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl.

[0059] Also provided is a compound of Formula IX:

[0060] Formula IX or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0061] R1and R2are each independently selected from the group consisting of H, NR', OR', alkyl, cycloalkyl, aminoalkyl, hydroxyalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl;

[0062] R3is selected from the group consisting of H, alkyl, and cycloalkyl; or wherein two of R1, R2and R3join to form a cycloalkyl, heterocycle, aryl, or heteroaryl;

[0063] R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl; R6is selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; and

[0064] R7is selected from the group consisting of H, alkyl, and cycloalkyl; or wherein two of R4, R5, R6and R7join to form a cycloalkyl, heterocycle, aryl, or heteroaryl.

[0065] In some embodiments, the compound of Formula IX is a compound of Formula IXA:

[0066] Formula IXA or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0067] R1is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl;

[0068] R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and n is 0, 1, 2, or 3.

[0069] In some embodiments, the compound of Formula IXA is a compound of Formula IXA(a):

[0070] Formula IXA(a) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0071] X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and

[0072] R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

[0073] In some embodiments, the compound of Formula IXA is a compound of Formula IXA(b):

[0074] Formula IXA(b) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0075] X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and

[0076] R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

[0077] In some embodiments, X is CR8and R8is halo.

[0078] In some embodiments, the compound of Formula IX is a compound of Formula IXB:

[0079] Formula IXB or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:

[0080] R1and R2are each independently selected from the group consisting of H, NR', OR', alkyl, cycloalkyl, aminoalkyl, hydroxyalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or wherein R1and R2join to form a cycloalkyl, heterocycle, aryl, or heteroaryl;

[0081] R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl; or wherein R4and R5join to form a cycloalkyl, heterocycle, aryl, or heteroaryl; and

[0082] R7is independently selected from the group consisting of H, alkyl, and cycloalkyl.

[0083] In some embodiments, the compound of Formula IXB is a compound of Formula IXB(a): or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein: X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl;

[0084] R2is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and

[0085] R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl.

[0086] In some embodiments, the compound of Formula IXB is a compound of Formula IXB(b):

[0087] Formula IXB(b) wherein R8is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and

[0088] R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl.

[0089] In some embodiments, R8is haloaryl or haloarylalkyl (e.g., halobenzyl).

[0090] Also provided is a construct comprising a compound as described herein coupled to a chelating agent. In some embodiments, the chelating agent is bound to a radionuclide, which may be suitable for optical imaging, PET imaging, SPECT imaging, or radiotherapy. In some embodiments, the chelating agent is coupled to the compound by a linker.

[0091] Further provided is a method of treatment for cancer comprising administering a construct as described herein to a subject in need thereof, or the use of the construct in a method for treating cancer, or for preparing a medicament for use in treating cancer.

[0092] Also provided is a compound as described herein coupled to an imaging agent. In some embodiments, the imaging agent is coupled to the compound by a linker.

[0093] Further provided is a method of detecting a tumor in a subject in need thereof comprising administering a construct described herein to the subject and detecting the imaging agent, or the use of the construct in a method of detecting a tumor in a subject, or for preparing a medicament useful for detecting a tumor.

[0094] Also provided is a composition comprising a compound or construct as taught herein and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 is a flowchart showing an example method for identifying a small molecule capable of binding to a protein of interest.

[0096] DETAILED DESCRIPTION

[0097] The disclosures of all patent references cited herein are hereby incorporated by reference to the extent they are consistent with the disclosure set forth herein. As used herein in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0098] A. Definitions

[0099] “H” refers to a hydrogen atom. “C” refers to a carbon atom. “N” refers to a nitrogen atom. “O” refers to an oxygen atom. “S” refers to a sulfur atom. “Halo” refers to F, Cl, Br or I. The term “hydroxy,” as used herein, refers to an -OH moiety. “Br” refers to a bromine atom. “Cl” refers to a chlorine atom. “I” refers to an iodine atom. “F” refers to a fluorine atom. The term “cyano” or “nitrile” refers to a -CN moiety (i.e., -ON).

[0100] An “acyl” is intended to mean a group -C(O)-R, where R is a suitable substituent, such as alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. Examples of acyl include, but are not limited to, an acetyl group, a propionyl group, a butyroyl group, a benzoyl group, etc.

[0101] “Alkyl,” as used herein, refers to a straight or branched chain hydrocarbon containing from 1 or 2 to 10 or 20 or more carbon atoms (e.g., C2, C3, C4, C5, C6, C7, C8, C9, CIO, Cl l, C12, C13, C14, C15, etc.). In some embodiments the alkyl can be a lower alkyl. "Lower alkyl" refers to a straight or branched chain alkyl having from 1 to 3, or from 1 to 5, or from 1 to 8 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3- methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, alkyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. Representative examples of halo substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl and trifluoromethyl.

[0102] As generally understood by those of ordinary skill in the art, “saturation” refers to the state in which all available valence bonds of an atom (e.g., carbon) are attached to other atoms. Similarly, “unsaturation” refers to the state in which not all the available valence bonds are attached to other atoms; in such compounds the extra bonds usually take the form of double or triple bonds (usually with carbon). For example, a carbon chain is “saturated” when there are no double or triple bonds present along the chain or directly connected to the chain (e.g., a carbonyl), and is “unsaturated” when at least one double or triple bond is present along the chain or directly connected to the chain (e.g., a carbonyl). Further, the presence or absence of a substituent depending upon chain saturation will be understood by those of ordinary skill in the art to depend upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon).

[0103] The term “optionally substituted” indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents. A “substituent” that is “substituted” is an atom or group which takes the place of a hydrogen atom on the parent chain or cycle of an organic molecule, for example, H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0104] “Alkenyl,” as used herein, refers to a straight or branched chain hydrocarbon containing from 1 or 2 to 10 or 20 or more carbons, and containing at least one carbon-carbon double bond, formed structurally, for example, by the replacement of two hydrogens. Representative examples of “alkenyl” include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l -heptenyl, 3-decenyl and the like. In some embodiments, alkenyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0105] “Alkynyl,” as used herein, refers to a straight or branched chain hydrocarbon group containing from 1 or 2 to 10 or 20 or more carbon atoms, and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1- propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-butynyl and the like. In some embodiments, alkynyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0106] The term “cycloalkyl,” as used herein, refers to a saturated cyclic hydrocarbon group containing from 3 to 8 carbons or more. Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. “Heterocycle,” as used herein, refers to a monocyclic, bicyclic or tricyclic ring system. Monocyclic heterocycle ring systems are exemplified by any 4-, 5-, 6- or 7-member ring containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of: O, N, and S. The 4-member ring has 0 to 1 double bond, the 5-member ring has from 0 to 2 double bonds, and the 6 and 7 member rings have from 0 to 3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, diazepine, 1,3- dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadi azole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3 -benzodi oxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. In some embodiments, heterocyclo groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0107] “Aryl” as used herein refers to a ring system having one or more aromatic rings. Representative examples of aryl include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The aryl groups of this invention can be substituted with 1, 2, 3, 4, or 5 substituents independently selected from alkenyl, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfinyl, alkylsulfonyl, alkylthio, alkynyl, aryl, aryloxy, azido, arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl, halogen, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl, sulfo, sulfonate, -NR’R” (wherein, R’ and R” are independently selected from hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl and formyl), and -C(O)NR’R” (wherein R’ and R” are independently selected from hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl, and formyl). In some embodiments, aryl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0108] “Heteroaryl” means a cyclic, aromatic hydrocarbon in which one or more carbon atoms have been replaced with heteroatoms. If the heteroaryl group contains more than one heteroatom, the heteroatoms may be the same or different. Examples of heteroaryl groups include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, benzofuranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, and benzo[b]thienyl. Preferred heteroaryl groups are five and six membered rings and contain from one to three heteroatoms independently selected from the group consisting of: O, N, and S. The heteroaryl group, including each heteroatom, can be unsubstituted or substituted with from 1 to 4 suitable substituents, as chemically feasible. For example, the heteroatom S may be substituted with one or two oxo groups, which may be shown as =0. In some embodiments, heteroaryl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0109] “Alkoxy,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like. In some embodiments, alkoxy groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy.

[0110] An “amine” or “amino” is intended to mean the group -NH2. “Optionally substituted” amines refers to -NH2 groups wherein none, one or two of the hydrogens is replaced by a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, carbonyl, carboxy, etc. In some embodiments, one or two of the hydrogens are optionally substituted with independently selected, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. Disubstituted amines may have substituents that are bridging, i.e., form a heterocyclic ring structure that includes the amine nitrogen.

[0111] An “amide” as used herein refers to an organic functional group having a carbonyl group (C=0) linked to a nitrogen atom (N), or a compound that contains this group, generally depicted as: wherein, R and R’ can independently be any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0112] A “thiol” or “mercapto” refers to an -SH group.

[0113] A “sulfide” or “thioether” as used herein refers to a group -S-R, where R is a suitable substituent, such as alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0114] A “sulfone” as used herein refers to a sulfonyl functional group, generally depicted as: wherein, R can be any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0115] A “sulfoxide” as used herein refers to a sulfinyl functional group, generally depicted as: wherein, R can be any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0116] “Carbonyl” is a functional group having a carbon atom double-bonded to an oxygen atom (C=O). “Carboxy” as used herein refers to a -COOH functional group, also written as -CO2H or -(C=O)-OH.

[0117] A “pharmaceutically acceptable salt” is intended to mean a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bi sulfates, sulfites, bi sulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, mal onates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4- dioates, hexyne- 1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxybutyrates, glycollates, tartrates, methane-sulfonates, propanesulfonates, naphthalene- 1 -sulfonates, naphthal ene-2-sulfonates, and mandelates.

[0118] A “pharmaceutically acceptable hydrate” or “pharmaceutically acceptable hydrated salt” is intended to mean a pharmaceutically acceptable salt of the definition herein that has one or more molecule of water included in its crystalline lattice.

[0119] “Form a ring” as used herein with respect to two substituents, e.g., R7and R8together forming a ring, refers to the two groups being linked together via one or more atoms (e.g., carbon) to form ring atoms making up a cycloalkyl, heterocyclo, aryl or heteroaryl as described herein. Rings may be part of a monocyclic, bicyclic or tricyclic moiety, each of such ring being a saturated or unsaturated member of the monocyclic, bicyclic or tricyclic moiety.

[0120] “Radioisotope,” as used herein, refers to synthetic and / or naturally occurring atoms that have excess nuclear energy and where this excess energy is emitted as radiation. Examples of this type of radiation energy are alpha rays, beta rays, and gamma rays. Examples of radioisotopes includenC,13N,150,68Ga,18F, "mTc,212Pb,133Xe,2O1T1,n iIn,123I,131I,90Y,64Cu,67Cu,186Re,177LU,149Tb ,152Tb ,155Tb, and161Tb. “Xe” refers to a xenon atom. “Tl” refers to a thallium atom. “In” refers to an indium atom. “Y” refers to a yttrium atom. “Cu” refers to a copper atom. “Re” refers to a rhenium atom. “Lu” refers to a lutetium atom. “Tb” refers to a terbium atom. “Ga” refers to a gadolinium atom. “Tc” refers to a technetium atom. “Pb” refers to a lead atom.

[0121] The terms “chelate,” “chelating agent” or “chelator” are intended to mean a molecule with two or more functional groups that are able to donate at least two electron pairs and so bind a metal ion. It is common for the chelating agent to be an organic molecule. One chelating agent will often use its electron pairs to form a coordinate bond with one metal ion, though it is possible for a chelating agent to bond more than one metal ion. Examples of chelating compounds include, but are not limited to, dimercaptopropanol, ethylenediaminotetraacetic acid (EDTA), di ethylenetriaminepentaacetic acid (DTP A), octadentate macrocyclic bifunctional 1,4,7,10- tetraazacyclododacane- 1,4,7, 10 -tetraacetic acid (DOTA), hexadentate macrocyclic bifunctional l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), hydroxy ethylidene diphosphonic acid (HEDP), ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) (EDTMP) and 1,4,7, 10-tetraazacy clododecane- 1,4,7, 10-tetraaminomethylenephosphonic acid (DOTMP), mercaptoacetyltriglycine (MAGs), salicylic acid, triethanolamine, ferrioxamines, and ionophores.

[0122] The term "linker" is intended to mean a chemical group or molecule that is capable of linking together two or more of the same or different other chemical groups or moieties. In some embodiments, the linker is positioned between, or flanked by, two groups, molecules, or moieties and connected to each one via a covalent bond, thus connecting the two. Linkers include, but are not limited to polyethylene glycol (PEG, [e.g., PEG4, PEGe, or PEGs]), a sequence of PEG molecules, an amino acid, a triazole, a sequence of amino acids, an organic molecule, an organic molecule group, a saturated or unsaturated branched or unbranched alkyl chain, or other chemical moiety.

[0123] The terms “cancer and “cancerous” are intended to mean the physiological condition typically characterized by unregulated cell growth in a portion of a multicellular organism. Often it is intended to mean a disease where the unregulated cell growth has the potential to spread and invade into multiple regions or tissues of the host organism (e.g., metastasis). A “tumor comprises one or more cancerous cells. Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.

[0124] The terms “radioactive label” or “radiotracer” are used herein to refer to a radioisotope that is used to generate an image that is detectable often using an appropriate instrument. Examples of techniques that use radiotracers include positron emission tomography (PET) and single photon emission computed tomography (SPECT). Examples of radiation emitted by radiotracers include gamma rays and X-rays.

[0125] The terms “radiation therapy” or “radiotherapy” are used herein to refer to a method of treating cancer whereby one or more tumors are destroyed using the radiation emitted from a radioisotope. The amount and delivery method of the radioisotope are often controlled in such a way as to produce as minimal harm as possible to the host organism. Examples of radiation used in radiotherapy include alpha rays, beta rays, and Auger electrons.

[0126] The term “imaging agent” as used herein refers to any moiety useful for the detection, tracing, or visualization of a compound of the invention when coupled thereto. Imaging agents include, but are not limited to, an enzyme, a fluorescent dye (e.g., carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane, Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag- S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS), a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a metallic particle (e.g., a gold particle), a nanoparticle, and a radioisotope. An imaging agent can be coupled to a compound of the invention by, for example, a covalent bond, ionic bond, van der Waals interaction or a hydrophobic bond. An imaging agent of the invention can be a radiolabel coupled to a compound of the invention, or a radioisotope incorporated into the chemical structure of a compound of the invention. Methods of detecting such imaging agents include, but are not limited to, positron emission tomography (PET), X-ray computed tomography (CT), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT).

[0127] B. Active Compounds

[0128] Active compounds useful for FAP targeting / binding are set forth below. In some embodiments, active compounds include those of Formula VIII, or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof.

[0129] In some embodiments of Formula VIII, R1is selected from the group consisting of H, alkyl, and cycloalkyl; n is 0, 1, or 2; R2is selected from the group consisting of H, alkyl, and cycloalkyl; and R3is selected from the group consisting of NR', OR', alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. In some embodiments, R2and R3join to form a ring such as a heterocycle (e.g., azetidinone, pyrrolidinone, imidazolidinone, oxazolidinone, piperidinone, piperazinone, morpholinone, azepanone, diazepanone, or oxazepanone).

[0130] One example compound of Formula VIII, in which n is 0, and R2and R3join to form a heterocycle, is a compound of Formula VIIIA.

[0131] Formula VIIIA

[0132] In some embodiments of Formula VIIIA, R1is selected from the group consisting of H, alkyl, and cycloalkyl; W, X and Y are each independently selected from the group consisting of CR4, O, N, and NR5; R4and R6are each independently selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; and R5is selected from the group consisting of H, acyl, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl. In some embodiments, two of R4, R5and R6join to form a carbocycle, heterocycle, aryl, or heteroaryl. One example compound of Formula VIIIA, in which X is NR5, W is CR4a, Y is CR4bwherein R4bis carbonyl, and R4aand R6join to form a heteroaryl, is a compound of Formula VIIIA(a).

[0133] Formula VIIIA(a)

[0134] In some embodiments of Formula VIIIA(a), R1is selected from the group consisting of H, alkyl, and cycloalkyl; A is selected from the group consisting of N and C; B is selected from the group consisting of O, S, and N; and R5is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl. Example compounds of Formula VIIIA(a), in which R1is H, A is C, B is S, and R5is alkyl, are shown below.

[0135] Formula VIIIA(a)(i) Formula VIIIA(a)(ii) Formula VIIIA(a)(i) One example compound of Formula VIIIA, in which W is N and X is CR4a, is a compound of Formula VIII A(b).

[0136] Formula VIIIA(b)

[0137] In some embodiments of Formula VIIIA(b), R1is selected from the group consisting of H, alkyl, and cycloalkyl; Y is selected from the group consisting of CR4b, N, and NR5; and R4a, R4and R6are each independently selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. In some embodiments, two of R4a, R4b, R5and R6join to form a ring such as a cycloalkyl, heterocycle, aryl, or heteroaryl.

[0138] One example compound of VIIIA(b), in which Y is CR4band R6is aryl, is a compound of Formula VIIIA(b)(i).

[0139] In some embodiments of Formula VIIIA(b)(i), R1is selected from the group consisting of

[0140] H, alkyl, and cycloalkyl; and R4a, R4b, and R7are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl. Example compounds of Formula VIIIA(b)(i), in which R4aand R4bare each H, and R7is halo, are shown below.

[0141]

[0142] One example compound of Formula VIIIA(b), in which Y is N5, and R4aand R5join to form a heteroaryl, is a compound of Formula VIIIA(b)(ii).

[0143] In some embodiments of Formula VIIIA(b)(ii), R1is selected from the group consisting of H, alkyl, and cycloalkyl; R6and R7are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and Z1and Z2are each independently selected from the group consisting of N and CR', wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

[0144] One example compound of Formula VIIIA(b)(ii), in which R7is aryl, is a compound of Formula VIIIA(b)(ii)(a).

[0145] In some embodiments of Formula VIIIA(b)(ii)(a), R1is selected from the group consisting of H, alkyl, and cycloalkyl; R6and R8are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and Z1and Z2are each independently selected from the group consisting of N and CR', wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. Example compounds of Formula VIIIA(b)(ii)(a), in which R1and R6are each H, and R8is halo or alkyl, are shown below.

[0146] Formula VIIIA(b)(ii)(5) Formula VIIIA(b) (H)(6)

[0147] One example compound of Formula VIII is a compound of Formula VIIIB.

[0148] Formula VIIIB

[0149] In some embodiments of Formula VIIIB, R1is selected from the group consisting of H, alkyl, and cycloalkyl; R2is selected from the group consisting of H, alkyl, and cycloalkyl; and R3is selected from the group consisting of NR', OR', alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. In some embodiments, R2and R3join to form a ring such as a heterocycle (e.g., azetidinone, pyrrolidinone, imidazolidinone, oxazolidinone, piperidinone, piperazinone, morpholinone, azepanone, diazepanone, or oxazepanone).

[0150] One example compound of Formula VIIIB is a compound of Formula VIIIB(a).

[0151] In some embodiments of Formula VIIIB(a), R1is selected from the group consisting of H, alkyl, and cycloalkyl; R2is selected from the group consisting of H, alkyl, and cycloalkyl; and R4is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl. Example compounds of Formula VIIIB(a), in which R1and R2are each H, and R3is aryl, are shown below.

[0152] One example compound of Formula VIIIB is a compound of Formula VIIIB(b).

[0153] Formula VIIIB(b) In some embodiments of Formula VIIIB(b), R1is selected from the group consisting of H, alkyl, and cycloalkyl; R6is selected from the group consisting of H, alkyl, and cycloalkyl; and R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl. Example compounds of Formula VIIIB(b), in which R1, R4and R5are each alkyl, and R6is H, are shown below. Active compounds of the current invention include those of Formula IX, or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof.

[0154] Formula IX

[0155] In some embodiments of Formula IX, R1and R2are each independently selected from the group consisting of H, NR', OR', alkyl, cycloalkyl, aminoalkyl, hydroxyalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; R3is selected from the group consisting of H, alkyl, and cycloalkyl; R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl; R6is selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; and R7is selected from the group consisting of H, alkyl, and cycloalkyl. In some embodiments, two of R1, R2and R3join to form a cycloalkyl, heterocycle, aryl, or heteroaryl. In some embodiments, two of R4, R5, R6and R7join to form a cycloalkyl, heterocycle, aryl, or heteroaryl.

[0156] One example compound of Formula IX, in which R2 and R3 join to form a heterocycle, and R7 is H, is a compound of Formula IXA.

[0157] Formula IXA

[0158] In some embodiments of Formula IXA, R1is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and n is 0, 1, 2, or 3.

[0159] One example compound of Formula IXA, in which R1is aryl or heteroaryl and n is 1, is a compound of Formula IXA(a).

[0160] Formula IXA(a)

[0161] In some embodiments of Formula IXA(a), X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

[0162] One example compound of Formula IXA, in which R1is aryl or heteroaryl and n is 2, is a compound of Formula IXA(b).

[0163] Formula IXA(b)

[0164] In some embodiments of Formula IXA(b), X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl. Example compounds of Formula IXA(a), in which X is CR8awherein R8ais halo, Y is CR8bwherein R8bis H, R4and R6are each alkyl, and R5is H, are shown below.

[0165] One example compound of Formula IX is a compound of Formula IXB.

[0166] Formula IXB

[0167] In some embodiments of Formula IXB, R1and R2are each independently selected from the group consisting of H, NR', OR', alkyl, cycloalkyl, aminoalkyl, hydroxyalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, or alkynyl; and R7is selected from the group consisting of H, alkyl, and cycloalkyl. In some embodiments, R1and R2join to form a ring such as a cycloalkyl, heterocycle, aryl, or heteroaryl. In some embodiments, R4and R5join to form a ring such as a cycloalkyl, heterocycle, aryl, or heteroaryl.

[0168] One example compound of Formula IXB is a compound of Formula IXB(a).

[0169] In some embodiments of Formula IXB(a), X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; R2is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl. Example compounds of Formula IXB(a), in which X is N, Y is CR8wherein R8is H, R2and R5are each alkyl and R4is H, are shown below.

[0170] One example compound of Formula IXB is a compound of Formula IXB(b).

[0171] Formula IXB(b)

[0172] In some embodiments of Formula IXB(b), R8is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; and R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl. Example compounds of Formula IXB(b), in which R8is substituted alkyl (haloarylalkyl such as halobenzyl), R4is H and R5is alkyl, are shown below.

[0173] Formula IXB(b)(i) Formula IXB(b)(ii) Formula IXB(b)(iii)

[0174] C. Constructs

[0175] In one aspect, the current invention is directed to a construct comprising a compound of the current invention that is coupled to a therapeutic, detectable group and / or a chelating agent.

[0176] In some embodiments, the chelating agent coupled to the compound of the current invention is bound to a radionuclide that is suitable for use as an imaging agent (e.g., optical imaging, PET imaging, SPECT imaging). In some embodiments, the chelating agent coupled to the compound of the current invention is bound to a radionuclide that is suitable for use as a radiotherapeutic.

[0177] In some embodiments, the chelating agent is coupled to the compound of the current invention by a linker, such as:

[0178] Particular non-limiting examples of such constructs include, but are not limited to: (with FAP Binder of Formula VIII), such as: , such as: In another aspect, the current invention is directed to a construct comprising a compound of the current invention that is coupled to an imaging agent.

[0179] D. Methods of Use

[0180] In one aspect, the current invention is directed to a method for treating cancer in a subject. In some embodiments, the method comprises administering a therapeutically effective amount of a construct of the current invention to a subject in need thereof, wherein the construct comprises a therapeutic, thereby treating a cancer.

[0181] In another aspect, the current invention is directed to a method of detecting a tumor in a subject. In some embodiments, the method comprises administering a construct of the current invention to a subject in need thereof and detecting an imaging agent.

[0182] The present invention is explained in greater detail in the following non-limiting examples.

[0183] EXAMPLES

[0184] Example 1: Identification of FAP specific binding molecules

[0185] Computational screening of small molecules was carried out in order to identify compound structures that would selectively bind to fibrinogen activation protein (FAP). Protein structures for FAP as well as dipeptidyl peptidase IV (DPP4), a FAP homolog, were downloaded from the Protein Data Bank (PDB). Binding affinity and FAP selectivity were predicted using AutoDock Vina (vina.scripps.edu / ).

[0186] A starting compound list was selected from the ZINC15 database (zincl5.docking.org / ), which resulted in a virtual library of 2,444,661 molecules that were commercially available to purchase (zincl5.docking.org / catalogs / ). Rigid docking analysis to FAP narrowed this list down to 145,000 compounds with an AutoDock Vina rigid binding affinity score <10'9. Selectivity was determined by comparing predicted binding affinities to FAP and DPP4. Compounds with predicted binding affinities to FAP that were at least 100-fold more favorable than the predicted binding affinities to DPP4 were selected for further evaluation. These 594 compounds were then analyzed using flexible docking techniques, and 31 compounds were identified with high predicted FAP binding affinity and low predicted binding affinity to DPP4. Further chemical structure comparisons between these molecules were assessed using Tanimoto Similarity scores. Example 2: Surface plasmon resonance binding assays

[0187] Surface plasmon resonance (SPR) assays identified several chemically diverse structures that had suitable starting affinity profiles. SPR is a phenomenon commonly employed in drug development programs to measure binding (affinity and kinetics) of a soluble analyte to a biomolecule of interest that is immobilized on a metal surface, or SPR biosensor. SPR occurs upon photoexcitation (using a single wavelength light source) of electrons in a thin metal surface that directs them to a parallel metal surface with a specific angle of incidence. This angle of incidence is dependent upon the refractive index of the metal surface, or SPR biosensor. Accordingly, changes in the refractive index of the surface upon binding of a soluble analyte to an immobilized biomolecule of interest is quantitatively detected by changes in SPR. This technique can rapidly and accurately measure binding affinity and kinetics of drug candidates to drug targets.

[0188] SPR binding assays are better suited to detect binders to the protein of interest than the majority of functional assays, which are often designed to measure only stimulation or inhibition of protein function. Accordingly, SPR-based binding assays were used for the evaluation of top virtual screening hits and ultimate identification of novel FAP binders with high FAP affinity and selectivity over DPP-IV.

[0189] To implement these assays, purified FAP and DPP-IV proteins were immobilized on carboxylic acid-functionalized SPR biosensors. Commercially available compounds from the list of top virtual screening hits were then tested (sourced from MolPort or Sigma) by Malvern Panalytical (Malvern, United Kingdom) for their abilities to bind to FAP and DPP-IV separately in a concentration-dependent manner. Compounds showing increased binding to the protein- functionalized biosensor over increasing compound concentrations were considered binders. After correcting for non-specific binding, concentration-response curves were generated by plotting the SPR signal versus the log of the compound concentration. Binding dissociation constants (Kd values) were then calculated using non-linear regression.

[0190] A flow chart of the screening process as described in Examples 1 and 2 above is presented in FIG. 1. To summarize, as a proof-of-principal, we performed in silicio docking analysis using a target system (human FAP alpha). The in silico docking studies versus a second protein (DPP4) identified a series of novel target binding structures which were subsequently assessed using the in vitro binding assay SPR, as reported below. This demonstrates that our approach is both feasible and productive. (Comparative docking was also performed with another protein, PREP. However, ligand library binding to DPP4 was found to be more similar to FAPa that PREP was to FAPa.) Our approach is beneficial because designing against a single target may not account for the possible binding of ligand candidates to other gene products, potentially reducing effective dosing and increasing off-target side-effects. Selecting against ligands with high affinity to biophysically similar yet non-target surface during the screening process may address these concerns. Traditional in silico docking is a time-consuming computational task, so to speed up the process, it is state-of-the-art to use rigid docking to reduce the degrees of freedom. We used this to help filter the ligands down to a more manageable number, but then performed the added step of flexible docking to assess potentially more realistic energetics within the docking simulation. The following structures selected from the identification pipeline were found to bind to

[0191] FAP with SPR assays, with indicated dissociation constants and standard deviations from fitted steady state curves. The fold Rmax is the experimental Rmax divided by the theoretical Rmax calculated from the protein immobilization levels and molecular weights of protein and test compound. Fold Rmax values that are either 0 or very high may be indicative of invalid results, as Fold Rmax 0 indicates very little extent of binding whereas high Fold Rmax values (e.g., >20) indicate significant nonspecific binding.

[0192]

[0193] The following structures did not show FAP binding or had FAP Kd >1,000 with SPR assays.

[0194] 10

[0195]

[0196] Example 3: Activity assays (prophetic)

[0197] To test the efficacy of radioisotope therapy for cancer with constructs described in the current invention, cell viability assays are carried out.

[0198] Target cell lines may include both epithelial cancer cells (e.g., breast tumors, colorectal tumors, pancreatic tumors, melanoma, etc.) and non-cancerous cells, both epithelial (e.g., primary cell lines) and non-epithelial (e.g., 293T) cells. These cell lines are cultured in 24-, 96-, or 384- well plates with the appropriate medium and then incubated for up to 48 hours with varying concentrations of test constructs as described herein, with a compound as described herein coupled to a chelating agent that is bound to a therapeutic radionuclide. Live cells are counted using either colorimetric staining (e.g., with crystal violet), ATP quantitation (e.g., CellTiter-Glo® Luminescent Cell Viability Assay), and / or tetrazolium reduction assays (e.g., 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assays). Percent of cell viability is calculated by comparing the total cell count to the live cell count.

[0199] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That which is claimed is:

1. A compound of Formula VIII:Formula VIII or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl; n is 0, 1, or 2;R2is selected from the group consisting of H, alky, and cycloalkyl; andR3is selected from the group consisting of NR', OR', alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or wherein R2and R3join to form a heterocycle (e.g., azetidinone, pyrrolidinone, imidazolidinone, oxazolidinone, piperidinone, piperazinone, morpholinone, azepanone, diazepanone, or oxazepanone).

2. The compound of claim 1, wherein the compound of Formula VIII is in a compound of Formula VIIIA:Formula VIIIA or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl;W, X and Y are each independently selected from the group consisting of CR4, O, N, andNR5;R4and R6are each independently selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; andR5is selected from the group consisting of H, acyl, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; or two of R4, R5and R6join to form a carbocycle, heterocycle, aryl, or heteroaryl.

3. The compound of claim 2, wherein the compound of Formula VIIIA is a compound of Formula VIII A(a):Formula VIIIA(a) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl;A is selected from the group consisting of N and C;B is selected from the group consisting of O, S, and N; andR5is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl.

4. The compound of claim 3, wherein R7is alkyl, A is C, and B is S.

5. The compound of claim 4, wherein the compound is selected from the group consisting of:Formula VIIIA(a)(i)6. The compound of claim 2, wherein the compound of Formula VIII or Formula VIIIA is a compound of Formula VIIIA(b):Formula VIIIA(b) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl;Y is selected from the group consisting of CR4b, N, and NR5; andR4a, R4band R6are each independently selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or two of R4a, R4b, R5and R6join to form a cycloalkyl, heterocycle, aryl, or heteroaryl.

7. The compound of claim 6, wherein the compound of Formula VIIIA(b) is a compound of Formula VIIIA(b)(i):or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl; andR4a, R4b, and R7are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

8. The compound of claim 7, wherein R7is halo.

9. The compound of claim 8, wherein the compound is selected from the group consisting of:

10. The compound of claim 6, wherein the compound of Formula VIIIA(b) is a compound of Formula VIIIA(b)(ii):wherein R1is selected from the group consisting of H, alky, and cycloalkyl;R6and R7are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl; andZ1and Z2are each independently selected from the group consisting of N and CR', wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

11. The compound of claim 10, wherein the compound of Formula VIIIA(b)(ii) is a compound of Formula VIIIA(b)(ii)(a):wherein R1is selected from the group consisting of H, alky, and cycloalkyl;R6and R8are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl; andZ1and Z2are each independently selected from the group consisting of N and CR', wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl.

12. The compound of claim 11, wherein R8is halo.

13. The compound of claim 11, wherein the compound is selected from the group consisting of:Formula VIIIA(b)(ii)(3)14. The compound of claim 11, wherein R8is alkyl.

15. The compound of claim 14, wherein the compound is selected from the group consisting of:Formula VIIIA(b)(ii)(6)16. The compound of claim 1, wherein the compound of Formula VIII is a compound of F ormula VIIIB :or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl;R2is selected from the group consisting of H, alky, and cycloalkyl; andR3is selected from the group consisting of NR', OR', alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or wherein R2and R3join to form a heterocycle (e.g., azetidinone, pyrrolidinone, imidazolidinone, oxazolidinone, piperidinone, piperazinone, morpholinone, azepanone, diazepanone, or oxazepanone).

17. The compound of claim 16, wherein the compound of Formula VIIIB is a compound of Formula VIIIB(a):Formula VIIIB(a) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alky, and cycloalkyl;R2is selected from the group consisting of H, alky, and cycloalkyl; andR4is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl.

18. The compound of claim 17, wherein the compound is selected from the group consisting of:

19. The compound of claim 16, wherein the compound of Formula VIIIB is a compound of Formula VIIIB(b):Formula VIIIB(b) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alkyl, and cycloalkyl;R6is selected from the group consisting of H, alkyl, and cycloalkyl; andR4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl.

20. The compound of claim 19, wherein the compound is selected from the group consisting of:Formula VIIIB(b)(i) Formula VIIIB(b)(ii) Formula VIIIB(a)(iii) and21. A compound of Formula IX:Formula IX or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1and R2are each independently selected from the group consisting of H, NR', OR', alkyl, cycloalkyl, aminoalkyl, hydroxyalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl;R3is selected from the group consisting of H, alkyl, and cycloalkyl; or wherein two of R1, R2and R3join to form a cycloalkyl, heterocycle, aryl, or heteroaryl;R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl;R6is selected from the group consisting of H, NR', OR', carbonyl, carboxy, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; andR7is selected from the group consisting of H, alkyl, and cycloalkyl; or wherein two of R4, R5, R6and R7join to form a cycloalkyl, heterocycle, aryl, or heteroaryl.

22. The compound of claim 21, wherein the compound of Formula IX is a compound of Formula IXA:Formula IXA or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl;R4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; and n is 0, 1, 2, or 3.

23. The compound of claim 22, wherein the compound of Formula IXA is a compound of Formula IXA(a):Formula IXA(a) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; andR4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

24. The compound of claim 22, wherein the compound of Formula IXA is a compound of Formula IXA(b):Formula IXA(b) or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl; andR4, R5, and R6are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl.

25. The compound of claim 24, wherein X is CR8and R8is halo.

26. The compound of claim 24, wherein the compound is selected from the group consisting of:and27. The compound of claim 21, wherein the compound of Formula IX is a compound of Formula IXB:Formula IXBor a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:R1and R2are each independently selected from the group consisting of H, NR', OR', alkyl, cycloalkyl, aminoalkyl, hydroxyalkyl, heterocycle, aryl, and heteroaryl, wherein R' is alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl; or wherein R1and R2join to form a cycloalkyl, heterocycle, aryl, or heteroaryl;R4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl; or wherein R4and R5join to form a cycloalkyl, heterocycle, aryl, or heteroaryl; andR7is independently selected from the group consisting of H, alkyl, and cycloalkyl.

28. The compound of claim 27, wherein the compound of Formula IXB is a compound of Formula IXB(a):or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein:X and Y are each independently selected from the group consisting of N and CR8, wherein R8is selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, alkyl, alkenyl, and alkynyl;R2is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; andR4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl.

29. The compound of claim 28, wherein X is N.

30. The compound of claim 29, wherein the compound is selected from the group consisting of:Formula IXB(a)(iii)31. The compound of claim 27, wherein the compound of Formula IXB is a compound of Formula IXB(b):Formula IXB(b) wherein R8is selected from the group consisting of H, alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl; andR4and R5are each independently selected from the group consisting of H, hydroxy, halo, alkoxy, cyano, acyl, alkyl, alkenyl, and alkynyl.

32. The compound of claim 31, wherein R8is haloaryl or haloarylalkyl (e.g., halobenzyl).

33. The compound of claim 32, wherein the compound is selected from the group consisting of:Formula IXB(b)(iii)34. A construct comprising the compound of any one of claims 1-33 coupled to a chelating agent.

35. The construct of claim 34, wherein the chelating agent is selected from the group consisting of: dimercaptopropanol, ethylenediaminotetraacetic acid (EDTA), di ethylenetriaminepentaacetic acid (DTP A), octadentate macrocyclic bifunctional 1,4,7,10- tetraazacyclododacane-1,4,7,10 -tetraacetic acid (DOTA), hexadentate macrocyclic bifunctional l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), hydroxy ethylidene diphosphonic acid (HEDP), ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) (EDTMP) and 1,4,7, 10-tetraazacy clododecane- 1,4,7, 10-tetraaminomethylenephosphonic acid (DOTMP), mercaptoacetyltriglycine (MAGs), salicylic acid, triethanolamine, ferrioxamines, and ionophores.

36. The construct of claim 34 or 35, wherein the chelating agent is bound to a radionuclide.

37. The construct of claim 36, wherein the radionuclide is suitable for optical imaging, PET imaging, SPECT imaging, or radiotherapy38. The construct of claim 36 or 37, wherein the radionuclide isnC,13N,15O,68Ga,18F, "mTc,123I,2O1T1,mIn,177LU,90Y,213Bi,212Pb,149Tb,152Tb,155Tb, and / or161Tb.

39. The construct of any one of claims 34-38, wherein the chelating agent is coupled to the compound by a linker.

40. The construct of claim 39, wherein the linker comprises a polyethylene glycol (PEG) or a polypeptide.

41. The construct of claim 39 or 40, wherein the linker comprises PEG4, PEGe, or PEGs.

42. A method of treatment for cancer comprising administering the construct of any one of claims 34-41 to a subject in need thereof wherein said composition is provided in a therapeutically effective amount.

43. A construct comprising the compound of any one of claims 1-33 coupled to an imaging agent.

44. The construct of claim 43, wherein the imaging agent is a fluorescent dye selected from the group consisting of: carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane, Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

45. A method of detecting a tumor in a subject in need thereof comprising administering a construct of any one of claims 34-41 and 43-44 to the subject and detecting the imaging agent.

46. A composition comprising a compound of any one of claims 1-33 or a construct of any one of claims 34-41 and 43-44, and a pharmaceutically acceptable carrier.

47. A method for identifying a small molecule capable of binding to a protein of interest, comprising: a) providing a small molecule library; b) performing an in silico rigid docking analysis for each member of the small molecule library to i) the surface of the protein of interest and to ii) the surface of a second protein that is similar in structure and / or function to the protein of interest, to select a first subset of the small molecule library; c) performing an in silico flexible docking analysis for each member of the first subset to i) the surface of the protein of interest and to ii) the surface of the second protein, to select a second subset of the small molecule library; and d) performing an in vitro binding assay for each member of the second subset to i) the surface of the protein of interest and to ii) the surface of the second protein, to generate a binding signal.

48. The method of claim 47, wherein the second protein is a homolog of the protein of interest.

49. The method of claim 47 or 48, wherein selecting the first subset of the small molecule library in step b) comprises selecting small molecules that have a binding affinity for the protein of interest that is at least 100-fold more favorable than for the second protein.

50. The method of any one of claims 47-49, wherein selecting the second subset of the small molecule library in step c) further comprises selecting small molecules that have a high predicted binding affinity for the protein of interest and a low predicted binding affinity for the second protein.

51. The method of any one of claims 47-50, wherein the method further comprises: e) correcting for non-specific binding in the in vitro binding assay.

52. The method of any one of claims 47-51, wherein performing the in vitro binding assay in step d) further comprises repeatedly performing the assay with increasing concentrations of each member in the second subset to generate a concentration-dependent binding signal.

53. The method of claim 52, wherein the method further comprises: f) generating a concentration-response curve for each member of the second subset from the concentration-dependent binding signal.

54. The method of claim 53, wherein the method further comprises: g) calculating the binding dissociation constant (Kd) from the concentration-response curve.

55. The method of claim 54, wherein a member of the second subset is identified as capable of binding to the protein of interest based on the Kd value.

56. The method of any one of claims 47-55, wherein the in vitro binding assay in step d) is a surface plasmon resonance (SPR) assay.

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