Bisphosphonate-linked compounds

Abstract

To provide novel compounds useful in the treatment of cancer.SOLUTION: The present invention provides compounds represented by formula (I), and pharmaceutically acceptable salts, cocrystals, polymorphs, solvates, hydrates, and as thereof, where Z represents a (poly)ethylene oxide group or the like, X represents (-CH2-)n2, R1 represents H or C1-C3 alkyl, R2 represents H or OH, n2 represents an integer of 0-3, n3 represents 0 or 1, and Y represents bisphosphonate.SELECTED DRAWING: Figure 5F

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel compounds useful in the treatment of cancer, particularly compounds linking a biphosphonate moiety to KBU2046 [Steve, a novel use of the compounds is to treat a side effect of cancer, namely bone destruction, should we mention that here too?], as well as pharmaceutically acceptable salts, co-crystals, polymorphs, solvates, hydrates, and enantiomers thereof. [Background technology]

[0002] 2. Background of the Invention 3-(4-fluorophenyl)chroman-4-one (KBU2046), a first-in-class HSP90 activity modulator, may be synthesized as described in US Patent Application Publication No. 2016 / 0128973 (Bergan et al. - Northwestern University) entitled Inhibition of Cancer Cell Motility. KBU2046 inhibits metastasis throughout mouse models of human prostate and breast cancer, reduces bone destruction, and extends survival after oral administration at nanomolar concentrations.

[0003] There remains a need for improved delivery and efficacy of oncological agents to treat bone cancers, such as metastatic cancers located in bone, such as metastatic cancers of the breast, prostate, lung, kidney, thyroid, bladder, esophagus, stomach, liver, pancreas, testis, skin, head and neck cancers from soft tissue or bone, lymphoma, multiple myeloma, and sarcoma. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2016 / 0128973 Summary of the Invention

[0005] Provided is a compound of the formula: TIFF2025028945000002.tif42161, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof; During the ceremony, Z is Selected from the group TIFF2025028945000003.tif17128; n1 in each instance is an integer selected from the group of 1, 2, 3, 4, 5, and 6; X is (-CH2-) n2 and; R1 is selected from the group of H and C1-C3 alkyl; R2 is selected from the group of H and OH; n2 is an integer selected from the group of 0, 1, 2, and 3; n3 is an integer selected from the group of 0 and 1; and Y is a bisphosphonate compound.

[0006] In some embodiments, R1 is selected from the group of methyl and ethyl.

[0007] In some embodiments, R1 is methyl.

[0008] In some embodiments, R1 is ethyl.

[0009] In other embodiments, R1 is H.

[0010] In some embodiments, R2 is H.

[0011] In other embodiments, R2 is OH.

[0012] In some embodiments, n1 is an integer selected from the group of 1, 2, 3, 4, and 5.

[0013] In some embodiments, n1 is an integer selected from the group of 1, 2, 3, and 4.

[0014] In some embodiments, Y is selected from the following groups, as represented by their corresponding bisphosphonate entities: It is selected from the structure of TIFF2025028945000004.tif166166.

[0015] In the structures herein, wavy or oscillating lines crossing solid lines TIFF2025028945000005.tif9128 shows a bond through which adjacent atoms are connected. [The present invention 1001] formula: The compound of TIFF2025028945000006.tif42161, During the ceremony, Z is Selected from the group TIFF2025028945000007.tif17128; n1 in each instance is an integer selected from the group of 1, 2, 3, 4, 5, and 6; X is (-CH2-)n2; R1 is selected from the group of H and C1-C3 alkyl; R2 is selected from the group of H and OH; n2 is an integer selected from the group of 0, 1, 2, and 3; n3 is an integer selected from the group of 0 and 1; and Y is a bisphosphonate compound; The compound, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1002] A bisphosphonate compound represented by Y, The compound of the present invention 1001 selected from the group consisting of TIFF2025028945000008.tif203144, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1003] The compound of formula (I) may be represented by formula (II): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, n1 is an integer selected from the group of 1, 2, 3, and 4; and n3 is an integer selected from the group of 0 and 1; 1001 compounds of the present invention. [The present invention 1004] formula: The compound of the present invention 1003 having the TIFF2025028945000010.tif52128, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1005] formula: The compound of the present invention 1003 having the TIFF2025028945000011.tif50128, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1006] formula: The compound of the present invention 1003 having the formula TIFF2025028945000012.tif52133, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1007] formula: The compound of the present invention 1003 having the formula TIFF2025028945000013.tif55163, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1008] The compound of formula (I) has the formula (Ib): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, Z is Selected from the group TIFF2025028945000015.tif17128; n1 is an integer selected from the group of 1, 2, 3, and 4; X is (-CH2-)n2; R1 is selected from the group of H and C1-C3 alkyl; R2 is selected from the group of H and OH; n2 is an integer selected from 0, 1, 2, and 3; n3 is an integer selected from the group of 0 and 1; and Y is a bisphosphonate compound; The compound of the present invention 1001, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1009] The compound of formula (Ib) may be represented by formula (Ic): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, R1 is selected from H and methyl; n1 is an integer selected from the group of 1, 2, and 3; n3 is an integer selected from the group of 0 and 1; and Z is TIFF2025028945000017.tif17128, The compound of the present invention 1008, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1010] formula: The compound of the present invention 1009 having the TIFF2025028945000018.tif35128, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1011] formula: The compound of the present invention 1009 having TIFF2025028945000019.tif36128, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [The present invention 1012] A pharmaceutical composition comprising a pharma- ceutical effective amount of a compound of the present invention or a pharma- ceutical acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, and a pharma- ceutical acceptable carrier or excipient. [The present invention 1013] 1. A method for treating osteoclast-mediated bone degradation in a subject, comprising: The method comprises the step of administering to a subject in need thereof a pharma- ceutical effective amount of a compound of the present invention 1001 or a pharma- ceutical acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof. [Brief description of the drawings]

[0016] [Figure 1A] 1 graphically depicts the effect of chemotherapy on the growth of androgen-independent PC3-M PCa cells in vitro. [Figure 1B] 1 graphically depicts the effect of chemotherapy on androgen-independent PCa cell growth in vivo. [Figure 2A] A graph shows lung metastases measured by qPCR for human Alu sequences and expressed as a percentage of control. [Figure 2B] Weekly IVIS imaging of the tumor is shown. [Figure 2C] The corresponding tumor weights in the mice tested are shown. [Figure 3A] Shows the effect on AR-responsive gene selection in LNCaP cells treated with R1881(R), KBU2046(46), and / or enzalutamide. [Figure 3B] Shows the effect on AR-responsive gene selection in VCaP cells treated with R1881(R), KBU2046(46), and / or enzalutamide. [Figure 3C] 1 shows the effect of enzalutamide on LNCaP cell proliferation. [Figure 3D] 1 shows the effect of enzalutamide on VCaP cell proliferation. [Figure 3E]Effects on AR activation are shown by Western blots of nuclear (N) and cytoplasmic (C) preparations of cell extracts probed for AR, laminin B1, or α-tubulin. [Figure 3F] 13 graphically depicts the effect on tumor growth after androgen deprivation therapy in LNCaP / AR-luc cells implanted subcutaneously in castrated mice, followed by KBU2046 treatment. [Figure 3G] 13A-C graphically depict the effect on tumor growth after androgen deprivation therapy in orthotopically implanted LNCaP / AR-luc cells in castrated mice, followed by KBU2046 treatment. [Figure 4A] 1 shows a Western blot of protein production following KBU2016 treatment in RAW 267.4 cells. [Figure 4B] 1 shows the effect of treating RAW 267.4 cells with RANKL and KBU2046 on cell morphology. [Figure 4C] 4 shows tartrate-resistant acid phosphatase expression in treated cells. [Figure 4D] Bone degradation quantified by bone surface area is shown graphically. [Figure 5A] 13A-C graphically depict bone degradation, as quantified by bone surface area, upon treatment of RAW 267.4 cells with KBU2046, zoledronic acid, or the combination. [Figure 5B] 1 shows the effect of treating RAW 267.4 cells with KBU2046, zoledronic acid, or the combination on bone degradation. [Figure 5C] A graph showing cell viability, as measured by the Triplex Assay, of RAW 267.4 cells treated with KBU2046, zoledronic acid, or the combination. [Figure 5D] RAW 267.4 cells were treated with KBU2046, zoledronic acid, or the combination, and the cytotoxicity was measured by Triplex Assay. [Figure 5E]Apoptosis, as measured by the Triplex Assay, is shown graphically in RAW 267.4 cells treated with KBU2046, zoledronic acid, or the combination. [Figure 5F] Figure 1 shows the effect on bone degradation, as measured by computed tomography, of treating mice with KBU2046, zoledronic acid, or the combination after intracardiac injection of human PC3 prostate cancer cells. [Figure 5G] 13A-C graphically depict the effect on mandibular bone degradation, as quantified by computed tomography, of treating mice with KBU2046, zoledronic acid, or the combination following intracardiac injection of human PC3 prostate cancer cells. [Figure 5H] 13A-C graphically depict the effect on femoral bone degradation, as quantified by computed tomography, of treating mice with KBU2046, zoledronic acid, or the combination following intracardiac injection of human PC3 prostate cancer cells. [Figure 6-1] 6A and 6B show the effect of compound E (n1=1) herein in inhibiting osteoclast-mediated bone degradation. [Figure 6-2] 6C and 6D show the effect of compound E (n1=2) herein in inhibiting osteoclast-mediated bone degradation. [Figure 6-3] 6E and 6F show the effect of compound E (n1=3) herein in inhibiting osteoclast-mediated bone degradation. [Figure 6-4] Figures 6G and 6H show the effect of compound G herein in inhibiting osteoclast-mediated bone degradation. [Figure 6-5] 6I and 6J show the effect of compound H herein in inhibiting osteoclast-mediated bone degradation. [Figure 6-6] Figures 6K and 6L show the effect of compound I herein in inhibiting osteoclast-mediated bone degradation. [Figure 7] 1 shows the effect of experimental compounds in inhibiting PCa cell motility. [Figure 8A] Figure 1 shows transwell migration of PC3 cells treated with 10 micromolar concentration of compound E (n=1). [Figure 8B] 1 shows transwell invasion of PC3 cells treated with 10 micromolar compound E (n=1). [Figure 9] FIG. 1 shows PC3 cell viability after 1 day of treatment with 10 micromolar concentration of compound E (n=1) under exponential growth. [Figure 10] Shown are raw 267.4 cells treated with RANKL under exponential growth and treated with micromolar concentrations of compound E (n=1). [Figure 11A] 4 shows the extent of mouse jaw bone destruction quantified after treatment with compound E (n=1). [Figure 11B] 4 shows the extent of mouse jaw bone destruction quantified after treatment with compound E (n=1). [Figure 12] FIG. 1 shows mouse survival after treatment with compound E (n=1) one week prior to cardiac injection of PC3-luc cells. [Figure 13] 1 shows that KBU2046 does not inhibit the activity of adriamycin in MDA-MB-231 cells. [Figure 14] 1 shows that KBU2046 does not inhibit the activity of adriamycin in PC3-M cells. [Figure 15] 1 shows that KBU2046 does not inhibit the activity of vinblastine in MDA-MB-231 cells. [Figure 16] 1 shows that KBU2046 does not inhibit the activity of vinblastine in PC3-M cells. [Figure 17] 1 shows that KBU2046 does not inhibit the activity of taxol in MDA-MB-231 cells. [Figure 18] 1 shows that KBU2046 does not inhibit the activity of taxol in PC3-M cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The following description sets forth example methods, parameters, etc. However, it should be understood that such description is not intended as a limitation on the scope of the present disclosure, but is provided as a description of example embodiments.

[0018] Also provided is a compound of formula (Ia): TIFF2025028945000020.tif39154, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein R1, R2, n1, X, Y, and Z are as defined above for formula (I).

[0019] Also provided is a compound of formula (Ib): TIFF2025028945000021.tif40147, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein R1, R2, n1, X, Y, and Z are as defined above for formula (I).

[0020] Also provided is a compound of formula (Ic): TIFF2025028945000022.tif38149, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein R1, R2, n1, n3, X, Y, and Z are as defined above for formula (I).

[0021] Another embodiment provides a compound of formula (Ic), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein R1 is selected from the group of H and methyl; and n1 is an integer selected from the group of 1, 2, and 3; n3 is an integer selected from the group of 1 and 2; and Z is TIFF2025028945000023.tif17128.

[0022] Within each of the above embodiments relating to a compound of formula (Ic), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, there exists a further embodiment wherein n3 is 0 and R1 is hydrogen.

[0023] Within each of the above embodiments relating to a compound of formula (Ic), or a pharma- ceutically acceptable salt, cocrystal, polymorph, solvate, hydrate, or enantiomer thereof, there exists a further embodiment wherein n3 is 0 and R1 is methyl.

[0024] Within each of the foregoing aspects relating to a compound of formula (Ic), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, Z is There is a further embodiment, which is TIFF2025028945000024.tif16128.

[0025] Within each of the foregoing aspects relating to a compound of formula (Ic), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, Z is There is a further embodiment, which is TIFF2025028945000025.tif16128.

[0026] Within each of the above embodiments directed to a compound of Formula (Ic), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, there exists a further embodiment, wherein n3 is 0.

[0027] Within each of the above embodiments directed to a compound of Formula (Ic), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, there exists a further embodiment, wherein n3 is 1.

[0028] Provided is a compound of formula (Id): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, n1 is an integer selected from the group of 1, 2, 3, 4, 5, and 6; X is (-CH2-)n2; n2 is an integer selected from the group of 0, 1, 2, and 3; and Y is a bisphosphonate compound.

[0029] A further embodiment provides a compound of formula (Id) as above, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, 4, and 5; and n2, X, and Y are as defined for formula (Id) above.

[0030] A further embodiment provides a compound of formula (Id) as above, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, and 4; and n2, X, and Y are as defined for formula (Id) above.

[0031] Also provided is a compound of formula (II): TIFF2025028945000027.tif75150, or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof; During the ceremony, Z is Selected from the group TIFF2025028945000028.tif17128; n1 is an integer selected from the group of 1, 2, 3, 4, 5, and 6; and n3 is an integer selected from the group of 0 and 1.

[0032] A further embodiment provides a compound of formula (II) as above, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, 4, and 5; and n2 is an integer selected from the group of 0 and 1.

[0033] Yet a further aspect provides a compound of formula (II) as above, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, and 4; and n2 is an integer selected from the group of 0 and 1.

[0034] Also provided is a compound of formula (IIa): TIFF2025028945000029.tif69142, or a pharma- ceutically acceptable salt thereof, wherein n1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, and 6.

[0035] A further embodiment provides a compound of formula (IIa), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, 4, and 5.

[0036] A further embodiment provides a compound of formula (IIa), or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, and 4.

[0037] Specific examples of compounds of formula (II) include: TIFF2025028945000030.tif63148 Compound G: (2-(3-(3-(3-(2-(2-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)acetamido)ethoxy)propanamido)-2-(l1-oxidanyl)propyl)-1H-3l4-imidazol-1-yl)-1-hydroxyethane-1,1-diyl)bis(hydrogen phosphonate) TIFF2025028945000031.tif66156 Compound H: (2-(3-(1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-15-(l1-oxidanyl)-2,12-dioxo-6,9-dioxa-3,13-diazahexadecan-16-yl)-1H-3l4-imidazol-1-yl)-1-hydroxyethane-1,1-diyl)bis(hydrogen phosphonate) TIFF2025028945000032.tif63155 Compound I: (2-(3-(1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-18-(l1-oxidanyl)-2,15-dioxo-6,9,12-trioxa-3,16-diazanonadecan-19-yl)-1H-3l4-imidazol-1-yl)-1-hydroxyethane-1,1-diyl)bis(hydrogen phosphonate) TIFF2025028945000033.tif57156 (2-(3-(1-((3-(4-Fluorophenyl)-4-oxochroman-7-yl)oxy)-21-(l1-oxidanyl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazadocosan-22-yl)-1H-3l4-imidazol-1-yl)-1-hydroxyethane-1,1-diyl)bis(hydrogen phosphonate). Includes:

[0038] In the structures relating to compounds of formula (II), it is understood that a positive charge may also be shown on the 3-position nitrogen in the imidazolyl ring as follows: TIFF2025028945000034.tif40128

[0039] Further provided in another embodiment are compounds of formula (III), formula (IV), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), and formula (XI): TIFF2025028945000035.tif36164TIFF2025028945000036.tif211168TIFF2025028945000037.tif126170, or a pharma- ceutically acceptable salt thereof; In each of formulas (III) to (XI), n1 is an integer selected from the group of 1, 2, 3, 4, 5, and 6; X is (-CH2-)n2; and n2 is an integer selected from the group of 0, 1, 2, and 3.

[0040] A further embodiment provides a compound of formula (Id) as above, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, 4, and 5; and n2 and X are as defined in formulas (III)-(XI) above.

[0041] A further embodiment provides a compound of formula (Id) as above, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, wherein n1 is an integer selected from the group of 1, 2, 3, and 4; and n2 and X are as defined in formulas (III)-(XI) above.

[0042] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment in which the integer represented by n1 is 1, 2, or 3. Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another distinct embodiment in which the integer represented by n1 is 1 or 2. Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exist further embodiments wherein the integer represented by n1 is 3 or 4.

[0043] Within each of the compound embodiments described herein using (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment in which the integer represented by n1 is 1.

[0044] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n1 is 2.

[0045] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n1 is 3.

[0046] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n1 is 4.

[0047] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n1 is 5.

[0048] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n1 is 6.

[0049] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n3 is 0.

[0050] Within each of the compound embodiments described herein with formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI), there exists another embodiment wherein the integer represented by n3 is 1.

[0051] References herein to formulas (I)-(XI) are understood to include all formulas, including formulas (I), (Ia), (Ib), (Ic), (Id), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI).

[0052] Within each of the above descriptions of compounds for formulae (I)-(XI), there are alternative embodiments having the same descriptions except that X is 0. Within each of the above descriptions of compounds for formulae (I)-(XI), there are alternative embodiments having the same descriptions except that X is 1 or 2. Within each of the above descriptions of compounds for formulae (I)-(XI), there are alternative embodiments having the same descriptions except that X is 2 or 3. Within each of the above descriptions of compounds for formulae (I)-(XI), there are alternative embodiments having the same descriptions except that X is 3 or 4.

[0053] Further provided herein is a pharmaceutical composition comprising a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, and a pharma- ceutically acceptable carrier or excipient.

[0054] Also provided is a method of treating osteoclast-mediated bone degradation in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

[0055] Also provided is a method of treating bone cancer in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

[0056] Also provided is a method of treating cancer metastasis to bone in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt, cocrystal, polymorph, solvate, hydrate, or enantiomer thereof. Additional methods include, among others, methods for bone cancer, which is a cancer arising as and metastatic from breast cancer, prostate cancer, lung cancer, kidney cancer, thyroid cancer, bladder cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, testicular cancer, skin cancer, head and neck cancer, lymphoma, multiple myeloma, and soft tissue- or bone-derived sarcoma.

[0057] Also provided is a method of treating bone cancer, or cancer metastasis to bone, in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, and a second therapeutic agent, such as, but not limited to, a hormonal therapy or a chemotherapeutic agent. In specific non-limiting embodiments, the hormone therapy agent can be an anti-estrogen (e.g., tamoxifen, toremifene, fulvestrant, raloxifene, droloxifene, idoxifene, etc.), a progestogen (e.g., megestrol acetate, etc.), an aromatase inhibitor (e.g., anastrozole, letrozole, exemestane, vorozole, exemestane, fedrozole, aminoglutethimide, exemestane, 1-methyl-1,4-androstadiene-3,17-dione, etc.), an anti-androgen (e.g., bicalutamide, nilutamide, flutamide, cyproterone acetate, etc.), a luteinizing hormone releasing hormone agonist (LHRH agonist) (e.g., goserelin, leuprolide, buserelin, etc.); a 5-α-reductase inhibitor such as finasteride.In specific non-limiting embodiments, the cytotoxic chemotherapeutic agent is (i) an alkylating agent, such as procarbazine, ifosfamide, cyclophosphamide, melphalan, chlorambucil, decarbazine, busulfan, thiotepa; (ii) a platinum chemotherapeutic agent, such as cisplatin, carboplatin, oxaliplatin, eloxatin; (iii) an antimetabolite, such as methotrexate, 5-fluorouracil (e.g., capecitabine), gemcitabine (2'-deoxy-2',2'-difluorocytidine monohydrochloride (β-isomer), Eli Lilly), 6-mercaptopurine, 6-thioguanine, fludarabine, cladribine, cytarabine, tegafur, raltitrexed, cytosine arabinoside, (iv) anthracyclines such as daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, adriamycin, bleomycin, mitomycin-C, dactinomycin, mithramycin, (v) taxanes, It can be, for example, paclitaxel, docetaxel, taxotere, taxol, taxazm, 7-epipaclitaxel, t-acetylpaclitaxel, 10-desacetyl-paclitaxel, 10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, 10-desacetyl-7-epipaclitaxel, 7-N-N-dimethylglycylpaclitaxel, 7-L-alanylpaclitaxel.

[0058] Also provided is a method for treating osteoporosis in a subject, comprising administering to a subject in need thereof a pharmacologic effective amount of the compound of formula (I) or its pharmacologic acceptable salt, cocrystal, polymorph, solvate, hydrate or enantiomer.In one embodiment, the subject in need of osteoporosis treatment is a postmenopausal woman.In another embodiment, the subject in need of osteoporosis treatment is a man with osteoporosis.

[0059] Also provided is a method of preventing or inhibiting osteoporosis in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

[0060] Also provided is a method of treating glucocorticoid-induced osteoporosis in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

[0061] Also provided is a method of treating Paget's disease of bone in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

[0062] Also provided is a method of treating osteogenesis imperfecta of bone in a subject, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

[0063] Also provided is a compound of the formula: It is an intermediate compound of TIFF2025028945000038.tif37144, wherein n1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, and 6.

[0064] Further provided is a compound of the formula: TIFF2025028945000039.tif38134, wherein n1 is an integer selected from the group of 1, 2, 3, 4, 5, and 6.

[0065] synthesis The compounds described herein may be prepared by methods known in the art.Specific examples of synthetic routes are provided.The following method for preparing 3-(4-fluorophenyl)-7-hydroxychroman-4-one (compound A) can also be found in US 2016 / 0128973 A1. TIFF2025028945000040.tif57164

[0066] Compound A may be converted to 2-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)acetic acid, compound B, by the method described in Chemical & Pharmaceutical Bulletin, 39(10), 2681-90; 1991, as follows. TIFF2025028945000041.tif58169

[0067] Compound A may be converted to the corresponding carbonate compound, phenylcarbonate 3-(4-fluorophenyl)-4-oxochroman-7-yl, compound C, as follows at 0° C. to RT (room temperature) for about 1 hour. TIFF2025028945000042.tif28132

[0068] Zolendronic acid can be converted to 3-(3-amino-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium, compound D, as follows: TIFF2025028945000043.tif53150

[0069] To reduce inorganic residues, 1.1 equivalents of Na2CO3 was used instead of saturated Na2CO3 solution for the quaternization [step 2] reaction, from which 3-(3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium, compound 9, was synthesized and deprotected. This freshly prepared compound D was used in the final amidation reaction without further purification.

[0070] The synthesis of (1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-21-hydroxy-2,16-dioxo-7,10,13-trioxa-3,17-diazahenicosane-21,21-diyl)bis(phosphonic acid) was then achieved by the following steps. The synthesis of the three E compounds, (n=1), (n=2), (n=3), and (n=4), are achieved with the corresponding starting materials. TIFF2025028945000044.tif181128TIFF2025028945000045.tif116170

[0071] Compound E (n1=1) TIFF2025028945000046.tif36141(4-(3-(2-(2-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)acetamido)ethoxy)propanamido)-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) - obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization) on HPLC: 97.14%. NMR: (1H); Mass Spec: [M+H]: 663. TIFF2025028945000047.tif25162

[0072] Compound E (n1=2) TIFF2025028945000048.tif32158(1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-17-hydroxy-2,12-dioxo-6,9-dioxa-3,13-diazaheptadecane-17,17-diyl)bis(phosphonic acid) - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization) on HPLC: 92.35%. NMR: (1H); Mass Spec: [M+H]: 707. TIFF2025028945000049.tif18164

[0073] Compound E (n1=3) TIFF2025028945000050.tif26162(1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-21-hydroxy-2,16-dioxo-7,10,13-trioxa-3,17-diazahenicosane-21,21-diyl)bis(phosphonic acid) - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization) on HPLC: 95.59%. NMR: (1H); Mass Spec: [M+H]: 751. TIFF2025028945000051.tif25160

[0074] The synthesis of compounds G (n1=1), H (n1=3), and I (n1=4) is accomplished by the following reaction scheme. TIFF2025028945000052.tif174164

[0075] Compound G(n 1 =1) TIFF2025028945000053.tif431643-(3-(3-(2-(2-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)acetamido)ethoxy)propanamido)-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization): 98.37%. NMR: (1H); Mass Spectrometry: [M+H]: 759. TIFF2025028945000054.tif26165

[0076] Compound H(n 1 =3) TIFF2025028945000055.tif381523-(1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-18-hydroxy-2,15-dioxo-6,9,12-trioxa-3,16-diazanonadecan-19-yl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization) on HPLC: 89.88%. NMR: (1H); Mass Spec: [M+]: 803. TIFF2025028945000056.tif26165

[0077] Compound I(n 1 =4) TIFF2025028945000057.tif381613-(1-((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)-21-hydroxy-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazadocosane-22-yl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization) on HPLC: 94.61%. NMR: (1H); Mass Spectrometry: [M+]: 847. TIFF2025028945000058.tif26163

[0078] Synthesis of compound L TIFF2025028945000059.tif206170 Compound L: (4-(3-(2-((((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)carbonyl)(methyl)amino)ethoxy)propanamido)-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) Nosyl group protection was carried out on a 0.5 g scale with PEG-1 followed by N-alkylation, and 0.4 g of compound 3 was isolated. Nosyl deprotection of compound 3 was carried out on a 0.4 g [PhSH (1 eq) / K2CO3 (2.5 eq) / DMF / RT] scale. Workup and subsequent purification gave 0.14 g of the desired compound 4. 0.7 g of compound 4 was isolated after two steps with PEG-1 on a 2 g scale. Coupling of compound 4 with compound F was carried out on a 0.25 g [F (1.1 eq) / triphosgene (0.5 eq) / DIPEA (2 eq) / DCM / 0°C / 1 h] scale. Workup and subsequent purification gave 0.11 g of the desired compound 5. Tert-butyl deprotection followed by preparation of Osu derivative (Int-7) on a 0.11 g [TFA / DCM / RT / 5 h followed by N-hydroxysuccinimide (1.5 eq) / EDC.HCl (3.0 eq) / DCM / 0°C to RT / 16 h] scale. Work-up afforded 0.2 g of the desired Osu derivative 7. Final coupling of intermediate 7 with alendronic acid (compound H) was carried out on a 0.05 g [alendronic acid (1.7 eq) / KHCO3 (2.0 eq) / DMF / water] scale, which was subjected to preparative HPLC purification. Analysis is currently being carried out. Final coupling of intermediate 7 with alendronic acid (compound H) was carried out on a 0.350 g [alendronic acid (1.7 eq) / KHCO3 (2.0 eq) / DMF / water] scale. - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization) on HPLC: 97.39%. NMR: (1H); Mass Spec: [M+H]: 663. TIFF2025028945000060.tif26164

[0079] Synthesis of Compound M TIFF2025028945000061.tif197169 Compound M: (4-(2-(3-((((3-(4-fluorophenyl)-4-oxochroman-7-yl)oxy)carbonyl)(methyl)amino)propoxy)acetamido)-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) CBZ protection (step 1) with 3-(methylamino)propanol (SM 1) followed by O-alkylation (step 2) was carried out on a 5 g scale, and 4.5 g of compound 5 was isolated. CBZ deprotection (step 2) of compound 5 was carried out on a 4.5 g [Pd-C / H2 / MeOH / RT] scale to give 2.6 g of compound 6. Coupling reaction between amine intermediate 6 and triphosgene was attempted on a 500 mg [triphosgene (0.5 equiv.) / NaHCO3 / CHCl2 / DMF / H2O / 0°C to RT] scale. SM remains unreacted even after 12 h. Coupling reaction between compound F and triphosgene was attempted on a 700 mg [triphosgene (0.5 equiv.) / DIPEA / CHCl2 / DMF / 0°C to RT] scale to isolate 400 mg of compound 7. After deprotection of compound 7 on a 0.4 g [TFA / DCM] scale, 0.380 g of compound 8 was obtained as a crude product. Preparation of the OSu derivative (compound 9) was attempted on a 0.38 g [N-hydroxysuccinimide (1.5 eq.) / EDC.HCl (3.0 eq.) / DCM / 0°C to room temperature / 2 h] scale. Workup afforded 0.4 g of the desired compound 9 as a crude product. Finally, the coupling reaction of compound 9 with alendronic acid was carried out on a 0.4 g [alendronic acid (1.7 eq.) / KHCO3 (2.0 eq.) / DMF / H2O / room temperature] scale. LCMS of the crude product suggested the formation of the desired compound M, which was purified by preparative HPLC to isolate 0.66 g of the final compound M. - Obtained as a white solid. Wavelength (λ 210nm Qualitative chromatographic purity by HPLC (% by area normalization): 99.00%. NMR: (1H); Mass Spec: [M+H]: 663. TIFF2025028945000062.tif18163

[0080] Additional information is available in the article A Multifunctional Therapy Approach for Cancer: Targeting Raf1-Mediated Inhibition of Cell Motility, Growth, and Interaction with Microenvironment, Zhang et al., Molecular Cancer Therapeutics, 19(1), January 2020, pp. 39-51, which is incorporated herein by reference in its entirety.

[0081] test KBU2046 does not interfere with chemotherapy activity Chemotherapy is widely used in the treatment of androgen-independent PCa, with docetaxel and cabazitaxel both shown to have a survival benefit (Tannock et al., N Engl J Med 351, 1502-1512 (2004); Sweeney et al., N Engl J Med 373, 737-746 (2015); and Bono et al., Lancet 376, 1147-1154 (2010)), both taxanes that target microtubule function, thereby inhibiting cell proliferation (Lyseng-Williamson et al., Drugs 65, 2513-2531, 2005). Combining chemotherapy with KBU2046 did not affect the ability of either docetaxel or cabazitaxel to inhibit the proliferation of androgen-independent PC3-M cells in vitro (Figure 1A).

[0082] No interference with cytotoxicity was observed when KBU2046 was co-administered with agents spanning different pharmacological classes, including adriamycin, taxol, and vinblastine, and the effect was demonstrated in both PC3-M and MDA-MB-231 human breast cancer cells. To evaluate whether KBU2046 inhibits chemotherapy efficacy in vivo, mice were implanted subcutaneously with PC3-M cells and treated with docetaxel ± KBU2046 (Figure 1B). Compared to control mice, docetaxel significantly inhibited tumor growth (P < 0.01). However, KBU2046 did not significantly affect growth when added to control or docetaxel-treated mice. These findings indicate that KBU2046 does not inhibit the efficacy of cytotoxic agents.

[0083] FIG. 1 shows that KBU2046 does not inhibit the efficacy of chemotherapy against prostate cancer. FIG. 1A shows the effect of chemotherapy on the growth of androgen-independent PC3-M PCa cells in vitro. Data are mean ± SEM (N=3 sets) of cell viability in a 3-day proliferation assay in the presence of 10 μM KBU2046 or vehicle (control) treated with docetaxel or cabazitaxel. FIG. 1B shows the effect of chemotherapy on the growth of androgen-independent PCa cells in vivo. Mice were implanted with PC3-M cells subcutaneously and tumors were detected (250 mm 3 ), then treated with oral 80 mg KBU2046 / kg (46) or vehicle (CO) daily and intraperitoneal docetaxel (doc) at 0 or 20 mg / kg weekly starting on day 1, and tumor size was measured. Data are mean ± SEM, N = 20 mice per cohort.

[0084] Combining anti-motility and cytotoxic therapy improved efficacy Uncontrolled cell proliferation and uncontrolled cell motility constitute two fundamental processes of cancer. Therapeutic strategies in which both of these processes are targeted are based on rationality. We investigated whether the combination of docetaxel and KBU2046 provides improved anti-metastatic effects while achieving sustained inhibition of tumor growth kinetics of docetaxel compared to either agent alone. The PC3-M orthotopic mouse model allows quantification of primary tumor growth, assessment of distant metastasis to the lung, and has been used previously to measure the effect of treatment on metastasis and genetic changes (Xu et al., Nature Comminications 9, 2454 (2018); Pavese et al., PLoS One 9, e102289 (2014); and Lakshman et al., Clin Exp Metastasis 28, 39-53 (2011)).

[0085] To evaluate the efficacy of a spectrum of docetaxel concentrations, mice were orthotopic implanted with PC3-M-luc cells and treated with 0, 10, or 20 mg / kg docetaxel, and tumor growth was monitored weekly with IVIS imaging. Docetaxel significantly inhibited growth in a dose-dependent manner compared to controls. Mice bearing orthotopic implants of PC3-M-luc cells were subsequently treated with docetaxel, KBU2046, docetaxel+KBU2046, or vehicle as indicated, and the effects on primary tumor growth and lung metastasis were quantified (Figures 2A, 2B, and 2C). Previous studies have shown that as few as 10 human cells can be detected per mouse organ, i.e., kidney, using human Alu element-specific qPCR (Funakoshi et al., Sci Rep 7, 13202 (2017)). We show that there is a linear relationship between the number of PC3-M-luc cells present in mouse lungs and qPCR-based detection of human Alu elements (Pearson, R 2=0.96). Using human Alu element-specific qPCR to quantify PC3-M-luc metastases to the lung, we showed that docetaxel, KBU2046, and docetaxel+KBU2046 reduced metastases by 23% (P<0.05), 20% (P<0.05), and 48% (P<0.001), respectively, compared to control mice. Notably, the combination of these two specifically acting agents achieved a significantly greater reduction in metastases than that observed with either docetaxel (P<0.05) or KBU2046 (P<0.01) alone (Figure 2A). Based on the effect of each treatment alone, the calculated combined effect of docetaxel+KBU2046 was a 42% reduction, with an experimental / calculated ratio of 1.1, i.e., additive to slightly synergistic.

[0086] Assessment of prostate tumor growth by weekly IVIS imaging showed exponential growth kinetics in the docetaxel-naive cohort and slower growth in the docetaxel-treated cohort (Figure 2B). Measurement of actual tumor weight showed that docetaxel and docetaxel+KBU2046 reduced tumor weight by 49% (Fisher's exact test, P<0.05) and 37% (Fisher's exact test, P<0.05), respectively, compared to controls (Figure 2C). KBU2046 as a sole agent had no significant effect and did not significantly impair the efficacy of docetaxel. These findings indicate that combining antimotility and cytotoxic therapies allows dual targeting of cell proliferation and motility, resulting in sustained control of primary tumor growth and robust control of metastatic disease progression.

[0087] Figures 2A-2C show that KBU2046 + docetaxel has a high anti-metastatic effect. Cohorts of N=10 mice bearing orthotopic implants of PC3-M-luc cells were treated with 150 mg / kg KUB2046 (46) daily, intraperitoneal docetaxel 7.5 mg / kg weekly (doc), the combination (doc+46), or oral and intraperitoneal vehicle (CO). KBU2046 treatment began 3 days prior to implantation and docetaxel 1 week later. (A) Lung metastases measured by qPCR against human Alu sequences, expressed as percent control. (B) IVIS imaging of tumors weekly. (C) Tumor weights. * indicates P<0.05 for differences between cohorts indicated by bars. All data are mean ± SEM.

[0088] KBU2046 does not interfere with hormone therapy Hormonal therapy is the mainstay of treatment for metastatic PCa (NCC Network, Prostate Cancer. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Version 4, (2018)). It is therefore important that other therapies do not interfere with it. Initial hormonal treatment involves reduction of testicular androgen production and is called androgen deprivation therapy (ADT). Disease progression is inevitable, termed castration-resistant PCa, and is commonly treated with androgen receptor (AR) antagonists such as enzalutamide (Beer et al., N Engl J Med 371, 424-433 (2014)). To evaluate the effect of KBU2046 on this paradigm, investigations were initiated in androgen-sensitive LNCaP and VCaP human PCa cells that have been extensively characterized for their response to hormone-targeted therapy (Furr et al., Eur Urol 29 Suppl 2, 83-95 (1996) 23; and Tran et al., Science 324, 787-790 (2009)).

[0089] Cells were cultured in androgen-depleted medium and, after 72 h of washing, treated with the synthetic androgens R1881, KBU2046, enzalutamide, and / or vehicle (control), and the resulting effects on the induction of the androgen-responsive gene, prostate-specific antigen (PSA), were measured (Figure 3A). In both cell lines, R1881 significantly increased and enzalutamide decreased PSA expression. In LNCaP cells, KBU2046 had no significant effect. In VCaP cells, KBU2046 significantly decreased PSA expression in R1881-treated cells by 36% (P<0.05). It also significantly increased expression in control cells (P<0.05), although the absolute baseline levels were very low and the relative increase was only 14%. When assessing the effect on cell proliferation, enzalutamide significantly inhibited it compared to control in both LNCaP and VCaP cells (P<0.05), whereas addition of KBU2046 to control or enzalutamide-treated cells had no significant effect (Figures 3C and 3D).

[0090] Several considerations warranted a deeper examination of the effects of KBU2046 on AR function. In VCaP cells, KBU2046 significantly inhibited R1881-mediated PSA gene expression, consistent with inhibition of AR function. In addition, AR is a client protein of HSP90, AR function requires HSP90-mediated chaperone action, and direct inhibitors of HSP90 reduce client protein expression and inhibit AR function (Fang et al., J Biol Chem 271, 28697-28702 (1996); Saporita et al., Prostate 67, 509-520 (2007); and Ni et al., Mol Cell Biol 30, 1243-1253 (2010)). KBU2046 is not a direct inhibitor of HSP90 and therefore does not globally reduce client protein expression. It is a selective HSP90 activity modulator (SHAM) that selectively regulates client proteins that control cell motility. However, the effect of KBU2046 on AR expression and function has never been tested. After ligand binding, functional AR translocates from the cytoplasm to the nucleus, and therefore functional AR can be measured by nuclear localization. By treating LNCaP and VCaP cells with R1881, KBU2046, and / or enzalutamide and measuring AR expression in the resulting nuclear and cytoplasmic cell fractions by Western blot, we showed that KBU2046 does not affect AR expression or nuclear localization in response to R1881, nor its inhibition by enzalutamide (Figure 3E).

[0091] To complete our evaluation of the androgen-related paradigm, we evaluated the effect of KBU2046 on resistance to hormone therapy. Growth of LNCaP / AR-luc cells in castrated mice is routinely used as a model to mimic the transition to a castration-resistant phenotype (Tran et al., Science 324, 787-790 (2009)). In this model, tumor growth is detectable approximately 8 weeks after implantation, and growth kinetics are typically monitored up to 16 weeks after implantation. Here, we investigated the effect of two treatment approaches, starting treatment early at 4 weeks or late at 12 weeks, performed in mice that received subcutaneous or orthotopic implants, respectively (Figures 3F and 3G). KBU2046 did not alter tumor growth in either model. Taken together, these findings support the idea that KBU2046 does not affect androgen signaling or inhibit the efficacy of ADT or AR antagonist therapy.

[0092] Figures 3A-3E help show that KBU2046 does not affect androgen signaling or therapeutic targeting. LNCaP or VCaP cells cultured in hormone-free medium were treated with R1881 (R), KBU2046 (46), and / or enzalutamide (E). (A and B) Effects on AR-responsive gene expression. PSA expression was measured by qRT / PCR, normalized to GAPDH, and expressed as percent of control cells (N=3 pairs); * indicates P<0.05 for bar-separated groups. (C and D) Effects on cell proliferation. A 3-day proliferation assay was performed. Data are number of viable cells, expressed as % of control (N=3 pairs); * indicates P<0.05 compared to control. (E) Effects on AR activation. Western blots of nuclear (N) and cytoplasmic (C) preparations of cell extracts were probed for AR, laminin B1, or α-tubulin. (F and G) Effect on tumor growth following androgen deprivation therapy. Castrated mice were implanted with LNCaP / AR-luc cells subcutaneously (N=6 mice / cohort) or orthotopically (N=15 mice / cohort implanted; N=5 developed tumors), treatment with KBU2046 was initiated as above, and weekly IVIS imaging was performed. Data are tumor size expressed as luminescence intensity. All data are presented as mean ± SEM.

[0093] Combining treatments directed at the bone microenvironment with antimotility therapy improved efficacy Metastasis to bone occurs in more than 90% of people with metastatic PCa (Polascik, Ther Clin Risk Manag 4, 261-268 (2008)). After reaching bone, PCa cells alter the environment in a way that favors their proliferation participating in what is known as the "vicious cycle" (Guise, J Musculoskelet Neuronal Interact 2, 570-572 (2002); Guise et al., Endocr Rev 19, 18-54 (1998); and C. Logothetis et al., Cancer Metastasis Rev 37, 189-196 (2018)). Specifically, PCa cells secrete interleukin (IL)-11 and parathyroid hormone-related protein (PTHrP), which stimulate osteoblasts to secrete receptor activator of nuclear factor kappa-B ligand (RANKL), which induces osteoclasts to degrade bone, and bone matrix degradation releases insulin growth factor (IGF) and transforming growth factor (TGF) β, which act to sustain PCa cell proliferation. We previously demonstrated that KBU2046 inhibits activation of Raf1, specifically its Ser- 338 We showed that by inhibiting phosphorylation of activation motifs, it inhibits cell motility (Xu et al., Nature Communication 9, 2454 (2018)). The central function of Raf1 is related to controlling the reorganization of the actin cytoskeleton, which in turn affects how cells interact with and migrate through the microenvironment (Yotova et al., J Cell Mol Med 16, 2127-2139 (2012)). Cytoskeletal reorganization is required for osteoclasts to continuously migrate through and attach to the bone matrix, by positioning their actin cytoskeleton to form low-pH resorbable cavities where the degradation of bone mineral occurs (Florencio-Silva et al., Biomed Res Int 2015, 421746 (2015)). These facts led us to the idea that osteoclast-mediated bone degradation depends on cytoskeletal reorganization and therefore can be inhibited by KBU2046.

[0094] To explore this paradigm, we first assessed whether KBU2046 inhibits Raf1 phosphorylation in osteoclasts. Following treatment with RANKL, RAW 267.4 cells differentiate into mature osteoclasts. After maturation, osteoclasts become multinucleated, express tartrate-resistant acid phosphatase (TRAP), and have the ability to form actin rings that create occluded cavities in which bone is degraded. KBU2046 inhibits phosphorylation of the activation motif of Raf1 in RANKL-treated RAW267.4 osteoclasts, and does so in a time-dependent manner (Figure 4A). In osteoclasts, Raf1 is known to phosphorylate MEK1 / 2, which in turn phosphorylates ERK1 / 2 (Bradley et al., J Cell Biochem 104, 1439-1451 (2008)), the latter acting to stimulate osteoclast differentiation and bone resorption activity (He et al., PLos One 6, e24780 (2011); and Nakamura et al., J Bone Miner Res 18, 1198-1205 (2003)). The functional relevance of KBU2046-mediated inhibition of Raf1 phosphorylation is demonstrated by showing inhibition of MEK1 / 2 and ERK1 / 2 phosphorylation. In Figure 4A, osteoclasts were not cultured on bone mineral, indicating that KBU2046-mediated inhibition of Raf1 phosphorylation is independent of the presence of bone mineral.

[0095] Treatment of RAW 267.4 cells with KBU2046 induces several structural and functional changes. It reduces RANKL-mediated multinucleated cell formation, and those that do form have fewer nuclei (40x panel, see Figure 4B). The characteristic prominent actin ring (white arrow) surrounding the resorbing cavity (yellow arrow) is readily seen in control cells treated with RANKL ligand, but is lacking in KBU2046-treated cells (100x, panel). Consistent with inhibiting RAW 267.4 cell maturation, TRAP staining is reduced in KBU2046-treated cells (Figure 4C). In the absence of RANKL, undifferentiated RAW 267.4 cells display a phenotype characterized by isolated cells with long, thin cytoplasmic extensions (Figure 4B). However, treatment with KBU2046 significantly reduces cell elongation, indicating that its ability to induce effects on RAW 267.4 cells is independent of RANKL.

[0096] Figures 4A-4D depict KBU2046 inhibition of osteoclast function. Figure 4A compares the effect on Raf1 activation. RAW 267.4 cells were treated with RANKL for 4 days and 10 μM KBU2046 for the indicated time periods and Western blotted for the indicated proteins. (Figure 4B) Effect on cell morphology. RAW 267.4 cells were treated or not with RANKL and KBU2046 for 4 days and stained for actin (green) and DAPI (blue); white arrows indicate actin rings and yellow arrows indicate resorption cavities. Representative immunofluorescence images are shown. (Figure 4C) Tartrate-resistant acid phosphatase (TRAP) expression. Cells were treated as in B and stained for TRAP (indicated by the presence of red) and representative images from light microscopy are shown. (Figure 4D) Bone degradation. RAW 267.4 cells were seeded in osteoassay plates and treated with RANKL for 6 days and KBU2046 (or vehicle control) as indicated, and bone surface area was quantified. Data are the mean ± SEM (N=3 pairs) of bone surface area expressed as a percentage of control cells; * indicates P<0.05 compared to control.

[0097] To determine whether KBU2046 inhibits osteoclast-mediated bone degradation, we used Corning Osteo Assay Surface Plates coated with calcium phosphate bone salt. This platform supports osteoclast proliferation, allows quantitative measurement of bone degradation, and has previously been used to measure bisphosphonate-mediated inhibition of bone degradation (40). RAW 267.4 cells were grown on Osteo Assay Plates in the presence of RANKL and treated with 1 μmol / L, 10 μmol / L KBU2046, or vehicle (control), and the effect on bone destruction was quantified after 6 days. In this manner, we demonstrated that the presence of 10 μmol / L KBU2046 significantly increased bone surface area by 28% ± 1.4% (mean ± SEM) compared to controls (P < 0.05; Figure 4D). To further substantiate the role of Raf1, we showed that Raf1-specific inhibitors, ZM336372, GW5074, and NVP-BHG712, each inhibited osteoclast-mediated bone degradation, as well as RANKL-induced osteoclast maturation. The latter was demonstrated by reduced TRAP staining, reduced multinucleated cell formation, and inhibition of resorption cavity formation. Taken together, these findings indicate that bone matrix degradation may be inhibited through an innovative strategy targeting Raf1.

[0098] We next explored the targeting of bone microenvironment coupled with the targeting of cell motility. Bisphosphonates modify bone microenvironment by chemically binding to calcium phosphate bone mineral, then are taken up by osteoclasts after they digest the bound mineral, and once inside the cell, inhibit osteoclast-mediated bone degradation (Polascik et al., Ther Clin Risk Manag 4, 261-268 (2008)). Zoledronic acid (ZA), a bisphosphonate, is used clinically to reduce fractures in men with metastatic PCa (Saad et al., J Natl Cancer Inst 94, 1458-1468 (2002); Saad et al., J Natl Cancer Inst 96, 879-882 ​​(2004); and James et al., JAMA Oncol 2, 493-499 (2016)). ZA is administered to humans by injection, after which it binds to bone mineral while the remainder is rapidly cleared from the body, and those bound to bone mineral cause inhibition of osteoclast activity and, therefore, reduction of fractures in men with metastatic PCa (N. Pharmaceuticals, Zoledronic Acid package insert FDA approved drug package insert, (2007)). Therefore, we focused our investigation on ZA. Furthermore, we mimicked the pharmacological situation in humans by pretreating osteoassay plates with ZA and washing off unbound ZA before adding RAW 267.4 cells, then treated or not with KBU2046 and measured the effect on bone degradation. ZA was evaluated at 2, 10, and 20 μM, showing a concentration-dependent increase in bone surface area, all of which were significant (P<0.05) compared to the control (Figures 5A and 5B). KBU2046 also significantly increased bone surface area (P<0.05) as well.Importantly, the combination of ZA+KBU2046 resulted in significantly improved efficacy compared with either agent alone (P<0.05), with similar results observed at all ZA concentrations tested, with the combination of 20 μM ZA+KBU2046 increasing bone surface area to 193±1.6% (mean±SEM) of controls. In combination with KBU2046, increasing from 10 to 20 μM ZA did not result in any further improvement in efficacy, consistent with a plateau effect.

[0099] Figures 5A-5H depict the improved efficacy seen with KBU2046 in combination with ZA. In Figure 5A, bone degradation was tested using an osteoassay performed similarly to Figure 4D. Cells were treated with KBU2046 (46), 2, 10, or 20 μM ZA, the indicated combinations, or vehicle (control; CO). Data are means ± SEM (N=3 sets); * indicates P<0.05 compared to control or between conditions indicated by bars. Figure 5B provides representative photomicrographs of osteoassay wells. Black indicates the presence of bone matrix; RL: RANKL. (C-E) Cell viability, cytotoxicity, and apoptosis. RAW 267.4 cells were treated with RANKL and then treated on day 4 with KBU2046, 1 or 10 μM ZA (ZA1 or ZA10), the indicated combinations, or vehicle (CO), and the Triplex assay was performed 24, 48, or 72 hours later per method. Data are mean ± SEM (N=4 pairs); \, #, * indicate P<0.05 compared to control. (F-H) In vivo efficacy. Four cohorts of mice, N=15 / cohort, were given intracardiac injections of PC3-luc cells, resulting in the following successful injections: N=12 control, N=12 ZA, N=13 KBU2046, and N=13 ZA+KBU2046. Treatments were given orally 150 mg KBU2046 / kg daily (starting 3 days before IC injection), intraperitoneally ZA 100 μg / kg weekly (starting 1 week before IC injection), and orally and intraperitoneally vehicle control as indicated. (F) Representative computed tomography images. Image types are lateral and coronal sections of the skull, and transverse and sagittal sections of the femur. Red arrows indicate areas of bone destruction. (C and D) Quantification of bone damage. Gross damage in the jaws was quantified by direct measurement from CT images and expressed as mean ± SEM percent of control. Bone mineral density in the femur was measured from CT images and expressed as mean ± SEM percent of control. * indicates P<0.05 compared to control and for the group indicated by the bar.

[0100] We next investigated the effects of ZA and KBU2046 on osteoclast viability, apoptosis, and cytotoxicity. RAW 267.4 cells were treated with RANKL for the duration of the experiment, then treated with ZA, KBU2046, combination, or vehicle (control) on day 4, and the effects on cell viability, apoptosis, and cytotoxicity were measured by the Triplex assay at 24, 48, and 72 hours after treatment (Figures 5C-E). In Figures 5C, 5D, and 5E, respectively, the order of the bars in each of the three clusters of bars is, from left to right, control, KBU2046, ZA1, ZA10, ZA1+46, and ZA10+46.

[0101] It is important to consider that over the 7 day course of this experiment, RAW 267.4 cells are maturing into well-differentiated osteoclasts under the influence of RANKL. Findings in control cells are consistent with this process, showing a continuous decline in cell viability, accompanied by increased cell cytotoxicity and apoptosis over time. Findings with KBU2046 are consistent with a slowing of the maturation process, including a significant increase in cell viability and decreased cytotoxicity at 72 hours compared to controls (P<0.05). There was a marked and significant increase in apoptosis at 72 hours in KBU2046-treated cells compared to controls (P<0.05), perhaps mirroring previous findings by others linking inhibition of Raf1 signaling to increased apoptotic activity (Alejandro, JD Johnson, Inhibition of Raf-1 alters multiple downstream pathwaysto induce pancreatic veta-cell apoptosis, J Biol Chem 283, 2407-2417 (2008)). The combination of ZA+KBU2046 significantly increased apoptosis and cytotoxicity at 48 hours, but had little or no effect on viability at 72 hours.

[0102] To evaluate the efficacy of this strategy in vivo, we used a human PC3-luc PCa intracardiac injection model (Xu et al., Nature Communications 9, 2454 (2018); and Chu et al., Mol Cancer Res 6, 1259-1267 (2008)). In this model, mice develop extensive metastases to bone, with metastases to the mandible being the most prominent and symptomatic. Mice were treated with either ZA, KBU2046, ZA+KBU2046, or vehicle (control), and CT images of bone were obtained at the end of the experiment, and metastases to the mandible and femur were quantified (Figure 5F-H). Weekly IVIS imaging showed progressive tumor growth in the mandible, femur, and whole body over the 5 weeks of the experiment, across all cohorts of mice. Representative CT images showed typical large destructive damage to the mandible (Figure 5F), and these were quantified by direct measurement (Figure 5G). Metastases to the femur were small and resulted in a generalized loss of bone, with representative images showing reduced density of cancellous bone and thinning of cortical bone (Figure 5F), quantified by density measurements (Figure 5H). In the mandible, KBU2046 inhibited bone destruction by 33 ± 4.2% compared to controls (P < 0.05). ZA also reduced bone destruction, but this effect was not significant. Importantly, ZA did not impair the efficacy of KBU2046. In the femur, bone density was significantly improved by 56 ± 2.7% and 22 ± 2.4% compared to controls with ZA or KBU2046 treatment, respectively (P < 0.05). Furthermore, the combination of ZA + KBU2046 produced a significant (P < 0.05) additive effect compared to either treatment alone, significantly increasing density by 76 ± 3.7% compared to the control-treated cohort. Taken together, these findings indicate that the combined targeting of cell motility and the bone microenvironment has an additive therapeutic effect, and this effect is observed in both in vitro and in vivo model systems.

[0103] Increased cell motility and consequent metastasis are the processes that cause death in the majority of solid tumor cancers (Minn, J. Massague, in CANCER: Principals and Practice of Oncology, VT DeVita, TS Lawrence, SA Rosenberg, Eds. (Lippincott William & Wilkins, New York, 2008), pp. 135-146; and Seyfried et al., CritRev Oncog 18, 43-73 (2013)). Considering all forms of cancer, metastasis is responsible for more than 90% of mortality, and for PCa it is essentially 100% (Norgaard et al., J Urol 184, 162-167 (2010); and Pound et al., JAMA 281, 1591-1597 (1999)). Despite the overwhelming importance of this process, the therapeutic options available to patients remain limited. With KBU2046 and its ability to function as a placebo, selectively inhibiting Raf1 activation, precise targeting of cell motility and consequently metastasis is now possible.

[0104] Increased cancer cell motility is a hallmark of cancer that represents a clinically relevant driver of morbidity and mortality, and constitutes an important therapeutic target that we can now access. Therefore, we believe that it is primarily important to consider it as one of several abnormal functions. In this regard, two criteria become clear. First, this new found therapeutic potential cannot mitigate the effectiveness of established clinical agents. Second, it highlights the strategic importance of treating cancer in a manner that targets multiple functions. Such an approach constitutes a multifunctional therapy. This concept should not be confused with multimodality therapy. The latter uses multiple therapies against different targets and / or pathways, but they are related to a set of similar cellular functions, such as proliferation and / or viability.

[0105] We show here for the first time that anti-motility therapy can be performed in a non-interfering manner in combination with multiple classes of agents previously established to have efficacy in humans. Specifically, we show sustained efficacy in conjunction with several specifically acting cytotoxic chemotherapeutic agents, and also in cell culture-based studies and in two mouse models, one with subcutaneous implantation and one with orthotopic implantation. In the case of ZA, an agent that modifies the bone microenvironment, we also show that anti-motility therapy does not interfere with its activity. In fact, we show that it enhances it. Again, the experiments were extended to cell culture-based and human mouse xenograft-based models of bone destruction. In addition, we showed that anti-motility therapy does not interfere with therapies that target the androgen axis. Also, the studies included cell culture models, as well as subcutaneous and orthotopic mouse models. The studies were performed in the context of androgen deprivation therapy and androgen receptor antagonist therapy, and examined the effects on cell and tumor growth, androgen-responsive gene activation, and androgen receptor activation. Finally, it is important to consider that the combined findings help provide further evidence of the selective nature of KBU2046 and its mechanism of action.

[0106] After showing that antimotility therapy does not inhibit the efficacy of established therapies, we continued to demonstrate its applicability in the context of multimodality therapy. Specifically, we revealed that the combination of docetaxel + KBU2046 not only retains the growth inhibitory effects of docetaxel, but also exhibits additive effects in inhibiting metastasis. Docetaxel is a cytotoxic chemotherapeutic agent that inhibits cell proliferation and induces cell death through apoptosis, and is widely used across several cancer types, including PCa. Such a strategic approach may be applicable to existing clinical scenarios using docetaxel to treat PCa, especially in the setting of newly diagnosed metastases, where the combination would help reduce the formation of secondary metastases. Due to the lack of a robust preclinical systemic model of metastasis formation as a function of targeting the androgen axis, we did not show that KBU2046 provides an additive anti-metastatic effect when combined with hormonal therapy. However, existing data supports that using KBU2046 together with hormonal therapy further reduces the occurrence of metastases. Specifically, we have previously shown that KBU2046 inhibits the motility of androgen-responsive PCa cell lines (Xu et al., supra). Moreover, KBU2046 did not interfere with enzalutamide-mediated AR antagonist therapy, a widely used agent shown to reduce metastasis in humans (Hussain et al., N Engl J Med 378, 2465-2474 (2018)).

[0107] The inventors have also shown that anti-motility therapy can enhance the efficacy of agents that target the bone microenvironment, such as ZA. Osteoclasts modify the bone microenvironment in a manner that requires them to reorganize their actin cytoskeleton to form chambers that interact with bone mineral surfaces and function to degrade bone. Given that Raf1 is a known regulator of this process (Bradley et al., J Cell Biochem 104, 1439-1451 (2008); He et al., PLoS One 6, e24780 (2011); and Nakamura et al., J Bone Miner Res 18, 1198-1205 (2003)), the inventors hypothesized that KBU2046-mediated inhibition of Raf1 phosphorylation on its activation motif would inhibit actin reorganization and, consequently, bone degradation. We have shown that this is the case and have assessed bone degradation in vitro and in animal models of bone destruction. These findings represent a new mechanism and a new strategic approach for inhibiting osteoclast function and bone destruction and have broad implications.

[0108] Considering that KBU2046 affects osteoclast motility and ZA affects the bone microenvironment, i.e., separate but complementary mechanisms, we hypothesized that together they would have at least an additive therapeutic effect in inhibiting osteoclast-mediated bone destruction. Our findings directly supported this notion. Specifically, our in vitro model system of osteoclast-mediated bone destruction showed additive effects of the two agents across the range of ZA concentrations. The combinatorial targeting approach was also supported by our findings using a mouse model of human PCa-mediated bone destruction, where two forms of additive efficacy were observed. In the femur, the combination of both agents showed additional efficacy when compared to either agent alone. Other forms of additive efficacy are related to the anatomical site. In the mandible, KBU2046 showed high efficacy, whereas ZA showed none. In the femur, ZA was highly effective, and KBU2046 was also effective, but much less than ZA. In future studies, it will be important to understand the mechanistic underpinnings for these anatomical differences in agent efficacy. It is interesting to note that ZA use has been associated with osteonecrosis of the jaw in humans. Although the biology of this is not well understood and there are currently no preclinical models, the current findings support the speculation that ZA fails to provide protection in the jaw. Given that coadministration of KBU2046 and ZA resulted in additive efficacy even at low ZA concentrations and that each agent elicits site-specific effects, a multimodal therapy approach may provide a platform for scaling down ZA dosage, minimizing the potential for osteonecrosis while maintaining protection against bone-related events.

[0109] In summary, we have shown that a novel antimotility agent, bisphosphonate-conjugated KBU2046, can be effectively combined with other targeted functional therapies for cancer. Such an approach addresses the paradigm of cancer arising from a relatively broad set of dysfunctions that together constitute the hallmarks of cancer. Such an approach is associated with improved efficacy across several clinically relevant models, together supporting the translation of this approach to human studies.

[0110] Materials and Methods Experimental design These studies were designed to determine whether treating cancer with targeted multifunctional therapies including inhibition of cell motility would provide additional therapeutic benefits compared to existing therapies targeting different cancer-related functions. Cell motility was inhibited with KBU2046, cell proliferation and viability were inhibited with several types of cytotoxic chemotherapeutic agents including docetaxel, taxol, vinblastine, and adriamycin, or hormonal therapy including androgen deprivation or enzalutamide, and the bone microenvironment was modified with zoledronic acid. The effect of the combination of KBU2046 and individual chemotherapeutic agents on cell proliferation was measured by cell proliferation inhibition assays performed on a panel of human cancer cell lines. The effect on tumor growth was measured in athymic mice implanted subcutaneously with PC3-M cells. The effect on tumor growth and metastasis formation was assessed in athymic mice implanted orthotopically with PC3-M cells by measuring primary tumor growth and quantifying lung metastases. The effect of hormone therapy on cell proliferation and PSA expression was measured in in vitro grown LNCaP or VCaP cells, and the effect on tumor growth was evaluated in subcutaneously or orthotopically implanted LNCaP-AR cells. The ability of KBU2046 and ZA to inhibit bone destruction was measured by adding osteoclasts to osteoassay plates and measuring pit formation. Inhibition of bone destruction in athymic mice was measured by CT scanning of animals after intracardiac injection of PC3 cells. Inhibition of Raf1 phosphorylation in osteoclasts was measured by Western blot, and changes in actin cytoskeleton and cell morphology were measured by immunofluorescence microscopy.

[0111] Cell culture and reagents The following prostate cancer cell lines, PC-3, LNCaP, and VCaP, were obtained from the American Type Culture Collection; the constitutively active luciferase-expressing PC3-Luc cell line was obtained from PerkinElmer; the origin of the properties of PC3-M cells was previously described by the inventors (Liu et al., Prostate cancer and prostatic diseases 4, 81-91 (2001)). The constitutively active luciferase-expressing PC3-M-Luc cell line was established by transducing the parental cell line with pGL4.50 luciferase reporter (Promega) and culturing under hygromycin selection for stable integration. The LNCaP / AR-luc cell line was kindly provided by Charles Sawyers (Tran et al., Science 324, 787-790 (2009)).

[0112] The osteoclast precursor mouse macrophage cell line Raw 264.7 was obtained from the American Type Culture Collection. All cells were cultured as previously described (Xu et al., supra; Liu et al., Prostate cancer and prostatic diseases 4, 81-91 (2001); and Korenchuk et al., In Vivo 15, 163-168 (2001)) and maintained in a humidified atmosphere of 5% carbon dioxide at 37°C with medium changes every two weeks. All cell lines were derived from stored stock cells, replenished on a standardized cycle, and routinely monitored for mycoplasma (PlasmoTest™, InvivoGen, San Diego, CA). Cells were obtained from the creators of the lines, grown under quarantine conditions, grown and stored as primary stocks, and confirmed by not using them until they met the following criteria: mycoplasma negative; passed morphological examination; growth characteristics; hormone responsiveness or lack thereof. KBU2046 was synthesized as previously described. (5) Enzalutamide (#S1250), docetaxel (#S1148), and zoledronic acid (#S1314) were purchased from Selleckchem and reconstituted according to the manufacturer's recommendations.

[0113] Cytotoxicity and colony formation assays To evaluate the impact of cytotoxic chemotherapy on PCa growth kinetics, 7-day soft agar colony formation assays and 3-day MTT cell proliferation inhibition assays were performed as described by the inventors (Pavese et al., Cancer Lett 352, 179-186 (2014)). MTT assays were repeated in N=3 sets and soft agar assays in N=2 sets and are presented as the average number of colonies expressed as a percentage of untreated controls.

[0114] Reverse transcription and quantitative PCR analysis RNA was isolated and qRT / PCR was performed as previously described by the inventors (Ding et al., J Biomol Tech 18, 321-330 (2007)). The data obtained were analyzed using the following primer / probe sets (ABI) PSA (Hs02576345_m1) and GAPDH (Hs99999905_m1) for 2 -ΔΔCt Assays were performed in triplicate and repeated.

[0115] Enzalutamide-mediated cell proliferation inhibition assay Enzalutamide-mediated growth inhibition was assessed using a sulforhodamine B (SRB) growth inhibition assay and was performed as previously described ( 49 ). Briefly, LNCaP and VCaP cells were preincubated in charcoal-stripped medium for 48 h and then seeded into 96-well plates (1.9 × 10 4 Cells were stimulated with 1.0 nM R1881 and treated with 10 μM KBU2046 or vehicle control in the presence or absence of 10 μM enzalutamide for 72 hours, followed by SRB assay. Assays were repeated in sets of N=6.

[0116] Subcellular localization of androgen receptor LNCaP and VCaP cells were precultured in charcoal-stripped medium containing 10 μM KBU2046 or vehicle control for 72 hours. Cells were then stimulated with 1.0 nM R1881 and treated with 10 μM KBU2046 or vehicle control in the presence or absence of 10 μM enzalutamide for 24 hours. Nuclear and cytosolic subcellular fractions of cells were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Waltham, MA) according to the manufacturer's instructions. Protein was quantified by BCA assay (Thermo-Fisher Scientific, Waltham, MA) according to the manufacturer's instructions.

[0117] Western blot analysis Western blots were performed as we previously described (Xu et al., Nature Communications 9, 2454 (2018); and Pavese et al., Cancer Lett 352, 179-186 (2014)). All western blots were repeated at least once. Androgen receptor (#5153S), lamin B1 (#13435), and α-tubulin (#3873) phosphor-c-RAF (ser338) (#9427), c-Raf (#53745), MEK 1 / 2 (#12671), phosphor-MEK 1 / 2 (#9121), ERK 1 / 2 (#4695), phosphor-ERK 1 / 2 (#4370), GAPDH (#2118), anti-mouse IgG-HRP-conjugated secondary (#7076), and anti-rabbit IgG-HRP-conjugated secondary (#7074) antibodies were purchased from Cell Signaling Technology. Pierce ECL Western blotting substrate (#32106) and SuperSignal West Femto maximum sensitivity substrate (#34096) were purchased from Thermo Scientific. All primary antibodies were used at a dilution of 1:1000 and the corresponding secondary antibodies at a dilution of 1:5000.

[0118] Immunofluorescence Dylight™ Phalloidin (#12935) was purchased from Cell Signaling Technology.

[0119] Raw 264.7 cells were grown on Chamber Slides (Nunc Lab-Tek) in Differentiation Medium (below) with or without KBU2046 for 4 days. Chambers were washed once with PBS, followed by fixation in chilled 10% neutral buffered formalin (VWR) for 10 minutes and post-fixation washes with PBS. Permeabilization was achieved by incubation in 0.1% Triton X-100 (Sigma) for 5 minutes and then blocked overnight at 4°C with 10% goat serum (Thermo Fisher Scientific), 0.3 mol / L glycine (Sigma), and 1% BSA (Sigma) with 0.1% PBS-Tween 20. Cells were then incubated for 2 hours at 4°C in Dylight Phalloidin (Cell Signaling technology, #12935) antibody diluted 1:40 in 1x PBS with 1% BSA and 0.3% Triton X-100. Slides were then washed once with PBS and coverslips were mounted with ProLong Gold Antifade Reagent containing DAPI (Cell Signaling Technology, #8961) and imaged under a confocal microscope (Nikon / Yokogawa Spinning Disc).

[0120] Pit formation assay Low passage number (p≦4) RAW 264.7 cells were suspended in osteoclast differentiation medium (MEM-α (Gibco) containing 10% FBS, 1% antibiotic-antimycotic, and 50 ng / ml RANKL (EMD Millipore, R0525-10UG)) and seeded at 5000 cells / well in 96-well osteosurface assay plates (Corning). In ZA-treated wells, bone surfaces were precoated with 0, 2, 10, or 20 μM solutions of ZA suspended in sterile water for 30 min at 25°C, followed by gentle washing in PBS to remove free compound before seeding the cells. Cells were treated with 10 μM KBU2046 or control for 7 days, with medium and treatments refreshed on day 4. On day 7, wells were washed with PBS and cellular material removed by incubation in 10% bleach solution for 5 min, followed by extensive washing with ultrapure water. To visualize the resulting osteoclast-mediated pit formation, intact mineralized matrices were stained using a modified von Kossa staining protocol according to the manufacturer's recommendations (Corning). The resulting stained surfaces were imaged and subsequently evaluated using the "BoneJ" plugin in ImageJ. All treatments were performed in triplicate.

[0121] ApoTox-Glo assay ApoTox-Glo triplex assay (Promega) was performed to assess cell viability, cytotoxicity, and caspase-3 / 7 activation according to the manufacturer's instructions. Briefly, 5000 Raw 264.7 cells were seeded into 96-well assay plates and cultured in osteoclast differentiation medium for 4 days, at which point the medium was supplemented with or without KBU2046, and cellular responses were measured after 24, 48, and 72 hours according to the manufacturer's protocol.

[0122] Animal models of systemic effects and metastasis All animal studies adhering to the NIH Guide for the Care and Use of Laboratory Animals were handled under an institutional IACUC approved protocol by Oregon Health and Sciences University, in accordance with all federal, state, and local ethical regulations, and their design and conduct followed approved guidelines (Hollingshead, Antitumor efficacy testing in rodents. J Natl Cancer Inst 100, 1500-1510 (2008)). Animals were housed in a barrier (for immunocompromised mice) facility, with a 12-h light / dark cycle, and provided soy-free food and water ad libitum. Sample size determination for animal studies: Sample size was determined using a sample size estimation formula with a power setting of 80% on average, two-sided α = 0.05, and a prespecified effect size of 30% difference.

[0123] PCa subcutaneous implantation: 2.5 × 10 PC3-M cells in sterile PBS 5 The cells were implanted into the right flank of 6-7 week old male athymic nude mice (Charles River) and tumor growth measurements were performed twice weekly as previously described by the inventors (Gordon et al., Chemotherapy-induced monoamine oxidase expression in prostate carcinoma function as a cytoprotective resistance enzyme and associates with clinical outcomes, PLoS One 9, e104271 (2014)). Treatment with oral gavage 5 days per week with 80 mg / kg KBU2046 or vehicle control (sesame oil) and weekly intraperitoneal injections (IP) of docetaxel (0, 20 mg / kg) resulted in tumors growing to approximately 200-300 mm 18 days after implantation. 3 The study was started when the enrollment criteria of 1000-2000 mice were met. Experimental groups were randomly assigned to cages prior to the start of the study. Tumor measurements were performed in a blinded fashion and tumor burden was calculated using the formula: length × (width) 2 Calculations were performed using ×0.5.

[0124] PCa orthotopic transplant: PC3M-luc 2.5×10 5 Orthotopic implantation into 7-8 week old male athymic mice was performed as we previously described (5, 52 Xu et al., supra; and Pavese et al., J Vis Exp, e50873 (2013)). Daily oral gavage treatment with 150 mg / kg KBU2046 was started 3 days before implantation and continued for the duration of the study. Weekly IP injections with 7.5 mg / kg docetaxel or control were administered starting 1 week after implantation. Primary tumor growth was monitored by weekly IVIS imaging. Upon study completion, primary tumors were excised and weighed, and lungs were excised and snap frozen. To determine the human PCa metastatic burden in each mouse lung, snap frozen tissue was ground, DNA was isolated, and Alu-sequencing was performed as previously described (19). The following custom TaqMan primer-probe set was used: forward primer (101 F) TIFF2025028945000063.tif3128, Reverse primer (206 R) TIFF2025028945000064.tif3128, and hydrolysis probe (144RH) TIFF2025028945000065.tif3128. The 144RH probe was labeled with a 5' fluorescent reporter (6-FAM) and a 3' fluorescent quencher (Black Hole Quencher). The relative content of human DNA was determined by comparing T law, C T (Sample)-C T (negative control). All samples were run and repeated in triplicate.

[0125] Castration-resistant PCa subcutaneous implantation: Male athymic mice aged 6-7 weeks underwent transscrotal bilateral orchiectomy with a single incision in the midline of the scrotum under general anesthesia and were then allowed to recover for 10 days. After the recovery period, mice were implanted with 2.0 × 10 LNCaP / AR-luc as previously described by the inventors. 6Subcutaneous implantation was performed in the right flank of each mouse (Gordon et al., PLoS One 9, e104271 (2014)). Treatment with 80mg / kg KBU2046 or control was administered by oral gavage 5 times per week starting 4 weeks after implantation, prior to the emergence of a castration-resistant phenotype, and continued for 12 weeks. Experimental groups were randomized prior to the initiation of treatment. Tumor measurements were performed twice weekly, and androgen receptor activity was monitored biweekly by IVIS imaging.

[0126] Castration-resistant PCa orthotopic transplantation: Athymic mice were castrated as described above, and after a 2-week recovery period, LNCaP / AR-luc 5.0 × 10 5 Orthotopic implantation was performed as we previously described (Xu et al., supra; and Pavese et al., supra). Biweekly IVIS imaging was performed to assess androgen receptor activity, and treatment with 80 mg / kg KBU2046 or control by oral gavage 5 times per week was initiated 12 weeks after implantation and continued for 4 weeks as the castration-resistant phenotype began to emerge. Experimental groups were randomly assigned to cages before the start of the study.

[0127] PCa intracardiac (IC) injection: PC3-luc cells 4.0 × 10 5Ultrasound-guided IC injections into the left ventricle of 7-8 week-old athymic mice were performed as we previously described (Xu et al., supra). Weekly IP injections of 100 μg / kg ZA or vehicle control (PBS) were started 7 days before IC injection and continued for the duration of the study, while daily oral gavage treatments with 150 mg / kg KBU2046 or control were started 3 days before IC injection. All treatments were randomly assigned to cages before the start of the study. IVIS imaging was performed 30 min after IC injection to confirm systemic distribution of the cells and was performed weekly starting 7 days after injection and continued for 4 weeks. Animals were excluded from analysis if IVIS imaging 30 min after injection showed focal signals only in the thorax, indicating injection failure, with cells not distributed in the circulation. Computed tomography (CT) radiology imaging of the entire cohort was performed with an Inveon X-ray μCT scanner (Inveon, Siemens) at the completion of the study. The obtained images were analyzed in a blinded fashion using Inveon Research Workplace 4.2 visualization / analysis software and ImageJ. Specifically, to evaluate metastasis-associated bone destruction, the images were exported from the Inveon workplace in Digital Imaging and Communications in Medicine (DICOM) format for analysis with ImageJ. Each imported image was adjusted to a common brightness / contrast threshold, and multiplanar reconstructions (MPRs) of the regions of interest (mandible and femur) were performed using the "Stack -> Crop (3D)" and "volume viewer" plug-ins. Using the MPR of the bilateral femurs, a blinded operator obtained step-section serial images of the cancellous bone at both the proximal and distal ends of each femur. Subsequently, a fixed region of interest (ROI) was specified for each step-section image using the "ROI manager" plug-in, and bone mineral density was obtained from its measurements. Bone destruction of the mandible was evaluated in a blinded fashion using the MPR of the mandible. Here, bone loss in the periodontal cavity was calculated by measuring the distance between the molar root margin and the wall of the periodontal cavity in both coronal and transverse planes.

[0128] statistical analysis For all experiments, unless otherwise stated, comparisons between two groups were assessed with a two-tailed Student's t-test of Fisher's exact test, using a significance threshold of P≦0.05. Some experiments used Fisher's exact test, as indicated, using a significance threshold of P≦0.05. In such cases, the mean value of the control group was used as the threshold for assigning the categorical nature of individual results within the different treatment groups (i.e., > / = vs. <mean value).

[0129] 6A-6L: Experimental compounds inhibit osteoclast-mediated bone degradation.

[0130] RAW 267.4 cells were seeded onto osteoassay plates in differentiation medium containing RANKL and the indicated concentrations of experimental compounds (or vehicle control) on day 0, and medium was supplemented with RANKL and treatments on assay day 3. To visualize the resulting osteoclast-mediated pit formation, wells were washed on day 6 to remove cellular material, and intact mineralized matrix was stained with the von Kossa staining protocol and images were acquired. Bone surface area was quantified and data are the mean ± SEM (N=2 pairs) of remaining bone surface area expressed as a percentage relative to control cells; * indicates P<0.05 compared to control. Note: Black color indicates intact bone matrix.

[0131] Figure 7: Experimental compounds inhibit PCa cell motility.

[0132] PC3M cells were seeded with the indicated concentrations of Compound E on day 0 and grown to confluence (48 h), at which point a scratch was made, wells were washed with PBS, media was replenished with treatments, and images were taken at 0 and 20 h after scratching. Data are N=24. * indicates P<0.05 as determined by t-test.

[0133] Figures 8A and 8B: Experimental compounds inhibit the movement of human prostate cancer cells.

[0134] PC3 cells were treated with 10 micromolar compound E (n1=1) for a total of 72 hours, and then placed in the upper wells of uncoated (migration) or collagen I coated (invasion) Boyden chambers for the final 36 hours, and the number of cells in the lower chamber was counted. Data are the mean + / - SEM, N=4 sets, and expressed as % control cells.

[0135] Figure 9: Experimental compounds do not inhibit human prostate cancer cell viability.

[0136] Equal numbers of PC3 cells were seeded under conditions that allowed exponential cell growth, and one day later, treated with 10 micromolar concentrations of compound E (n1=1) for a total of 72 hours, and cell viability was measured using the ApoTox-Glo™ Triplex assay (Promega). Data are the mean + / - SEM, N=4 sets, and expressed as relative fluorescence units.

[0137] FIG. 10: Experimental compounds do not inhibit osteoclast viability.

[0138] Equal numbers of RAW 267.4 cells were seeded under conditions that allow exponential cell growth, treated one day later with RANKL (to stimulate differentiation into mature osteoclasts), and treated with 10 micromolar concentrations of Compound E (n1=1) for a total of 72 hours, and cell viability was measured using the ApoTox-Glo™ Triplex assay (Promega). Data are the mean + / - SEM, N=4 sets, and expressed as relative fluorescence units.

[0139] Figures 11A and 11B: Compound E (n1=1) inhibits bone destruction.

[0140] Mice were started on 0 (vehicle only), 10, 100, or 1000 μg / kg of Compound E (n1 = 1) by intraperitoneal injection weekly, starting 1 week prior to intracardiac (IC) injection of PC3-luc cells (PC3 cells expressing luciferase). IVIS imaging was performed weekly and at the end of the experiment, computed tomography (CT) scans were performed to quantify the degree of jaw bone destruction. A total of N = 10 control mice (administered vehicle only) were injected and for each treatment cohort, N = 10 mice were injected. * indicates P ≤ 0.05 by two-sided Student's t-test for comparison between cohorts indicated by bars.

[0141] Figure 12: Compound E (n1 = 1) extends survival.

[0142] Mice were from Figure 5 and were started on 0 (vehicle only), 10, 100, or 1000 μg / kg of Compound E (n1 = 1) by intraperitoneal injection weekly, starting 1 week prior to intracardiac (IC) injection of PC3-luc cells. Data represent survival of N = 10 mice per cohort and are expressed as a percentage relative to mice at the start of the experiment (i.e., the day of IC injection).

[0143] Figures 13 - 18: KBU2046 does not inhibit the efficacy of chemotherapy.

[0144] For PC3-M PCa and MDA-MB-231 triple-negative breast cancer cells, data are mean ± SD of colony numbers in a 7-day colony formation assay (N = 2 sets). All data are expressed as a percentage relative to untreated control cells. Cells were treated with 10 μM KBU2046 or vehicle (CO) and with the indicated concentrations and types of chemotherapy.

[0145] definition The compounds of the present disclosure may have asymmetric centers and can be produced as racemic mixtures or as individual enantiomers. Individual enantiomers may be obtained by asymmetric synthesis or by resolving racemic or non-racemic mixtures of intermediates at some suitable stage of the synthesis. Individual enantiomers may also be obtained by resolving compounds by conventional means, such as crystallization in the presence of a resolving agent or chromatography using, for example, a chiral high pressure liquid chromatography (HPLC) column. Individual enantiomers and racemic and non-racemic mixtures of enantiomers are within the scope of the present disclosure, and all are intended to be included in the structures shown herein unless otherwise specified.

[0146] The term "comprising" as used in the specification and claims can include "consisting of" and "consisting essentially of" embodiments. As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that require the presence of the specified components / steps and permit the presence of other components / steps. However, such descriptions should also be construed as describing the composition or process as "consisting of" and "consisting essentially of" the recited components / steps, which permits the presence of only the specified components / steps plus any impurities that may result therefrom, and excludes other components / steps.

[0147] The term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount sufficient to effect treatment, as defined below, when administered to a subject (e.g., a mammal such as a human) in need of such treatment. The therapeutically or pharmaceutical effective amount varies depending on the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, and the like, and can be readily determined by one of ordinary skill in the art. For example, a "therapeutically effective amount" or "pharmaceutical effective amount" of a compound of formula I or a pharmaceutical acceptable salt or cocrystal thereof is an amount sufficient to modulate physiological expression or activity, thereby treating a subject (e.g., a human) suffering from an indication, or to improve or alleviate existing symptoms of the indication. For example, a therapeutically or pharmaceutical effective amount may be an amount sufficient to reduce symptoms of a disease or condition that responds to inhibition of activity. In some embodiments, the pharmaceutical effective dose per administration may be from about 1 mg to about 200 mg. In other embodiments, the pharmaceutical effective dose may be from about 5 mg to about 150 mg per dose. In other embodiments, the pharma- ceutically effective dose may be from about 5 mg to about 100 mg per dose, hi further embodiments, the pharma- ceutically effective dose may be from about 1 mg to about 50 mg per dose.

[0148] For the treatment of osteoporosis, a medical professional may provide a dosage on a weekly treatment regimen or the dosage may be divided into equal daily doses. For the treatment of Paget's disease of bone, single or divided doses may be administered every 1, 2, 3, 4, 5, or 6 months.

[0149] "Treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: (i) inhibiting a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition and / or attenuating the severity of a disease or condition); (ii) slowing or halting the onset of one or more clinical symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, preventing or delaying the worsening or progression of a disease or condition, and / or preventing or delaying the spread (e.g., metastasis) of a disease or condition); and / or (iii) relieving the disease, i.e., causing regression of clinical symptoms (e.g., improving a disease state, providing partial or complete remission of a disease or condition, enhancing the effect of another drug therapy, slowing the progression of a disease, improving quality of life, and / or prolonging survival).

[0150] The term "inhibiting" or "inhibition" refers to a decrease, such as a significant decrease, in the baseline activity of a biological activity or process. "Inhibition of a given activity" refers to a reduction in activity as a direct or indirect response to the presence of the compound of formula I or its pharma- ceutically acceptable salt or cocrystal, relative to the activity in the absence of such a compound or its pharma- ceutically acceptable salt or cocrystal. The reduction in activity may result from the direct interaction of the compound with the biological target, or from the interaction of the compound described herein with one or more other factors, which may in turn affect the particular activity. For example, the presence of the compound may reduce the activity by directly binding to the target, by causing (directly or indirectly) another factor that reduces the activity, or by reducing (directly or indirectly) the amount of the factor present in the cell or organism. In some embodiments, the inhibition of the specified activity may be compared in the same subject prior to treatment, or in other controls that do not receive treatment. It is understood that the term "inhibitor" refers to a compound or agent that provides the desired inhibitory activity after administration to a human in need thereof at a pharma- ceutical or therapeutically effective dose.

[0151] "Delaying" the onset of a disease or condition means to postpone, hinder, slow, retard, stabilize, and / or prolong the onset of a disease or condition. This delay can be of various lengths of time, depending on the history of the disease or condition and / or the subject being treated. A method of "delaying" the onset of a disease or condition is one that reduces the probability of the onset of a disease or condition in a given time frame and / or reduces the extent of the disease or condition in a given time frame, compared to the absence of the method. Such comparisons are typically based on clinical trials with a statistically significant number of subjects. The onset of a disease or condition can be detectable using standard methods, such as routine physical examination, mammography, imaging, or biopsy. Onset can also refer to the progression of a disease or condition, including emergence, recurrence, and onset, which may be initially undetectable.

[0152] Numerical values ​​in the specification and claims of this application should be understood to include values ​​that differ from the stated value by less than experimental error using conventional measuring techniques of the type described in this application to determine the same numerical values ​​and values ​​when reduced to a significant fraction of the same number.

[0153] All ranges disclosed herein, including the specification, abstract, and claims, are inclusive of the recited endpoints and are independently combinable (e.g., the range "2 to 10" includes the endpoints 2 and 10, and all intermediate values).

[0154] By "significant" it is meant any detectable change that is statistically significant in a standard parametric test of statistical significance, such as the Student's T-test, where p<0.05.

[0155] "Pharmaceutically acceptable salts" include, for example, salts with inorganic acids and salts with organic acids. Examples of salts include hydrochloride, phosphate, diphosphate, hydrobromide, sulfate, sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate (mesylate), benzenesuflonate (besylate), p-toluenesulfonate (tosylate), 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate (acetate, n is 0 to 4, HOOC--(CH2) n --COOH, etc. In addition, if the compounds described herein are obtained as acid addition salts, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharma- ceutically acceptable addition salt, may be generated by dissolving the free base in a suitable organic solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art will recognize the various synthetic methods that may be used to prepare non-toxic pharma- ceutically acceptable addition salts.

[0156] The term "crystalline form" and related terms herein refer to various crystalline variations of a given substance, including, but not limited to, polymorphs, solvates, hydrates, cocrystals, and other molecular complexes, as well as salts, solvates of salts, hydrates of salts, other molecular complexes of salts, and polymorphs thereof. Crystalline forms of substances can be obtained by several methods known in the art. Such methods include, but are not limited to, melt recrystallization, melt cooling, solvent recrystallization, recrystallization in closed spaces, such as nanopores or capillaries, recrystallization on surfaces or templates, such as polymers, recrystallization in the presence of additives, such as cocrystal pair molecules, desolvation, dehydration, rapid evaporation, rapid cooling, slow cooling, vapor diffusion, sublimation, grinding, and solvent drop grinding.

[0157] The term "co-crystal" or "co-crystal salt" as used herein refers to a crystalline material consisting of two or more unique solids at room temperature, each of which has unique physical properties such as structure, melting point and heat of fusion, hygroscopicity, solubility, and stability. Co-crystals or co-crystal salts can be produced according to co-crystallization methods known per se. The term co-crystal or co-crystal salt also refers to a multi-component system in which there is a host API (active pharmaceutical ingredient) molecule, such as a compound of formula I, and a guest (or co-former) molecule. In certain embodiments, the pharma-ceutically acceptable co-crystal of the compound of formula I or the compound of formula II with the co-former molecule is a crystalline form selected from malonic acid co-crystal, succinic acid co-crystal, decanoic acid co-crystal, salicylic acid co-crystal, vanillic acid co-crystal, maltol co-crystal, or glycolic acid co-crystal. Co-crystals may have improved properties compared to the parent form (i.e., free molecule, zwitterion, etc.) or salt of the parent compound. Improved properties can include increased solubility, increased dissolution, increased bioavailability, increased dose response, reduced hygroscopicity, crystalline forms of normally amorphous compounds, crystalline forms of poorly salted or unsalted compounds, reduced morphological diversity, more desirable morphology, and the like.

[0158] The term "subject" and / or "patient" refers to an animal, such as a mammal, that has been or will be the target of treatment, observation, or experiment. The methods described herein can be useful in both human therapy and veterinary applications. In some embodiments, the subject is a mammal; in some embodiments, the subject is a human; and in some embodiments, the subject is selected from cats and dogs. "Subject in need thereof" or "human in need thereof" refers to a subject, such as a human, that may have or is suspected to have a disease or condition that would benefit from a particular treatment; for example, treatment with a compound of formula I or a pharma- ceutically acceptable salt or cocrystal thereof as described herein. This includes subjects that may be determined to be at risk of or suspected of such a disease or condition, such that treatment would prevent the onset of the disease or condition.

[0159] As used herein, "pharmaceutically acceptable excipient" refers to any and all pharmaceutically acceptable vehicles, including but not limited to carriers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption retarding agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. As long as any conventional media or agent is not incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Auxiliary active ingredients can also be incorporated into the composition.

[0160] The term "carrier" refers to an excipient or vehicle with which a compound is administered, including, but not limited to, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, etc. Carriers are generally described herein and also in "Remington's Pharmaceutical Sciences" by E.W. Martin. Examples of carriers include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethylcellulose, sodium carboxymethylcellulose, crospovidone, glyceryl isostearate, glyceryl monostearate, hydroxyethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxyoctacosanyl hydroxystearate, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 188, poloxamer 237, poloxamer 407, povidone, silicon dioxide, colloidal silicon dioxide, silicone, silicone adhesive 4102, and silicone emulsion. However, it should be understood that the carrier selected for the pharmaceutical composition, and the amount of such carrier in the composition, may vary depending on the method of formulation (e.g., dry granulation formulation, solid dispersion formulation).

[0161] The term "diluent" generally refers to a substance that is used to dilute the compound of interest before delivery.Diluent also serves to stabilize the compound.Examples of diluent can include starch, sugar, disaccharide, sucrose, lactose, polysaccharide, cellulose, cellulose ether, hydroxypropyl cellulose, sugar alcohol, xylitol, sorbitol, maltitol, microcrystalline cellulose, calcium or sodium carbonate, lactose, lactose monohydrate, dicalcium phosphate, cellulose, compressible sugar, calcium hydrogen phosphate anhydrous, mannitol, microcrystalline cellulose, and tricalcium phosphate.

[0162] The term "disintegrant" generally refers to a substance that, when added to a solid formulation, promotes its disintegration or disintegration after administration, allowing the release of active ingredients as efficiently as possible to allow its rapid dissolution. Examples of disintegrants can include corn starch, sodium starch glycolate, croscarmellose sodium, crospovidone, microcrystalline cellulose, modified corn starch, sodium carboxymethyl starch, povidone, pregelatinized starch, and alginic acid.

[0163] The term "precipitation inhibitor" generally refers to a substance that prevents or inhibits the precipitation of an active agent from a supersaturated solution. One example of a precipitation inhibitor includes hydroxypropyl methylcellulose (HPMC).

[0164] The term "surfactant" generally refers to a substance that reduces the surface tension between a liquid and a solid, which may improve the wetting of an active agent or improve the solubility of an active agent. Examples of surfactants include poloxamer and sodium lauryl sulfate.

[0165] The term "lubricant" generally refers to a substance used in tablet and capsule formulations to improve flow during tablet compression and to produce an anti-caking effect. Examples of lubricants can include colloidal silicon dioxide, talc, fumed silica, starch, starch derivatives, and bentonite.

[0166] The term "binder" generally refers to any pharma- ceutically acceptable film that can be used to bind the active and inactive components of the carrier together to maintain cohesive and separate portions. Examples of binders can include hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone, copovidone, and ethyl cellulose.

[0167] The term "lubricant" generally refers to a substance added to a powder blend to prevent the compressed powder mass from sticking to the equipment during the tabletting or encapsulation process. Lubricants can aid in the ejection of the tablet from the die and can improve powder flow. Examples of lubricants include magnesium stearate, stearic acid, silica, fats, calcium stearate, polyethylene glycol, sodium stearyl fumarate, or talc; and lauric acid, oleic acid, and C8 / C 10 Solubilisers such as fatty acids, including fatty acids, may be included.

[0168] Pharmaceutical Compositions and Administration The compound of formula I or its pharmaceutically acceptable salt or cocrystal is usually administered in the form of a pharmaceutical composition. The present disclosure therefore provides pharmaceutical compositions comprising one or more of the described compounds or their pharmaceutically acceptable salts, pharmaceutically acceptable cocrystals, or pharmaceutically acceptable esters as active ingredients, and one or more pharmaceutically acceptable vehicles, such as carriers including excipients, inert solid diluents, and fillers, diluents including sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers, and adjuvants. The pharmaceutical composition may be administered alone or in combination with other therapeutic substances. Such compositions are prepared in a manner well known in the pharmaceutical arts (see, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS Banker & CT Rhodes, Eds.)).

[0169] The pharmaceutical compositions may be administered in single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including, for example, rectal, buccal, intranasal, and transdermal routes, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, as an inhalant, or via an impregnated or coated device such as a stent, or an arterially inserted cylindrical polymer, as described in the patents and patent applications incorporated by reference.

[0170] One mode of administration is parenterally, particularly by injection. Forms in which the compound of formula I or its pharma- ceutically acceptable salt or cocrystal can be incorporated for administration by injection include sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles, as aqueous or oily suspensions, or emulsions. Aqueous solutions in saline may also be used as is customary for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be used. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0171] Sterile injectable solutions are prepared by incorporating the compounds of the present disclosure in the required amount in a suitable solvent with various other ingredients as listed above, as appropriate, followed by sterile filtration. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. In some embodiments, a sterile injectable solution is prepared containing a therapeutically effective amount, for example, 0.1 to 1000 mg of a compound of formula I or a pharma-ceutically acceptable salt or cocrystal thereof, for parenteral administration. In some embodiments, the effective amount will be about 0.1 mg to about 100 mg per dose. In other embodiments, the dosage will be about 5 mg to about 75 mg per dose. However, it will be understood that the amount of compound actually administered will normally be determined by a physician in light of the relevant circumstances, including the condition to be treated, the selected route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

[0172] Oral administration is another route of administration of the compound of formula I or its pharma- ceutically acceptable salt or cocrystal. Administration may be via capsules or enteric coated tablets, etc. In preparing pharmaceutical compositions containing the compound of formula I or its pharma- ceutically acceptable salt or cocrystal, the active ingredient is usually diluted with an excipient and / or enclosed in a carrier, which may be in the form of a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it may be in the form of a solid, semi-solid, or liquid material (as described above), which acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), for example, an ointment containing up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

[0173] Some examples of suitable excipients in oral formulations include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, syrup, and methylcellulose. The formulation may further include: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propyl hydroxybenzoates; sweetening agents; and flavoring agents.

[0174] The pharmaceutical compositions described herein can be formulated to provide rapid, sustained or delayed release of active ingredient after administration to a subject by using procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolution systems, including polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are shown in U.S. Patent Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345. Another formulation for use in the method of the present disclosure uses a transdermal delivery device (patch). Such transdermal patches can be used to provide continuous or discontinuous infusion of a controlled amount of the compound of the present disclosure. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Patent Nos. 5,023,252, 4,992,445, and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0175] In some embodiments, for parenteral administration, each dosage unit contains 0.1 mg to 1 g, 0.1 mg to 700 mg, or 0.1 mg to 100 mg of the compound of formula I or a pharma- ceutically acceptable salt or co-crystal thereof.

[0176] However, for any dosage unit described herein, it will be understood that the amount of compound actually administered will normally be determined by a physician in light of the relevant circumstances, including the condition to be treated, the selected route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

[0177] To prepare solid compositions such as tablets, the primary active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogenous mixture of the compound of Formula I or a pharma- ceutically acceptable salt or co-crystal thereof. When these pre-formulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition such that the composition may be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules.

[0178] The tablets or pills described herein may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action or to protect against the acidic conditions of the stomach.For example, the tablet or pill may comprise an inner dosage form and an outer dosage form, the latter being in the form of an envelope for the former.The two components may be separated by an enteric layer, which serves to resist disintegration in the stomach and allow the inner component to pass intact into the duodenum or to be delayed in release.Various materials may be used for such enteric layers or coatings, including some polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0179] Compositions for inhalation or insufflation may include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions containing the compound of formula I or its pharma- ceutically acceptable salts or cocrystals may include suitable pharma- ceutically acceptable excipients as described above. Preferably, the compositions are administered by oral or nasal respiratory route for local or systemic effect. Compositions, preferably in pharma- ceutically acceptable solvents, may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered from a device that delivers the formulation in an appropriate manner, preferably to the oral or nasal cavity.

[0180] The compounds, pharmaceutical compositions, and methods of treatment herein are in combination with chemotherapy, other bone-directed therapies including bisphosphonates or denosumab, hormone therapy, radiation therapy, surgery, small molecule therapeutics including inhibitors of protein kinases (imatinib, cisplatin, doxorubicin, ifosfamide, methotrexate, vincristine, cyclophosphamide, etoposide, dactinomycin, eribulin mesylate, larotrectinib, pazopanib, trabectedin), interferons, mTOR inhibitors (rapamycin, temsirolimus, everolimus, ridaforolimus), PARP inhibitors (olaparib, rucaparib), , niraparib, talazoparib, veliparib, pamiparib, AZD2281), biologics including antibodies against protein kinases (trastuzumab, denosumab, glenbatumumab, lexatumumab, cixatumumab, ipilimumab, bevacizumab, olaratumab) or immune checkpoint modulators (PD-1 inhibitors such as pembrolizumab, nivolumab, and cemiplimab; PD-L1 inhibitors atezolizumab, avelumab, and durvalumab), and modulators of angiogenesis (PF-4, TSP-1, angiostatin, endostatin).

[0181] For the treatment of bone cancer, including metastatic bone cancer, the compounds herein may be administered in combination treatment with chemotherapeutic agents such as taxol, vinblastine, vincristine, ifosfamide (IFEX®), doxorubicin (ADRIAMYCIN®), etoposide, methotrexate, cisplatin, dactinomycin, denosumab, and cyclophosphamide (CYTOXAN®), or combinations thereof.

[0182] For the treatment of osteoporosis and bone loss, the compounds herein are used in combination with alendronate (FOSOMAX®), risedronate (ACTONEL®), ibandronate (BONIVA®), etidronate (DIDRONEL®), zolendronic acid (zolendronic acid) (RECLAST®, ZOMETA®), denosumab (PROLIA®, XGEVA®), teriparatide (FORTEO®), abaloparatide (TYMLOS®), raloxifine (EVISTA®), teriparatide (FORTEO®), denosumab (PROLIA®), and calcitonin (MIACALCIN®, FORTICAL®), romosozumab, estrogen, conjugated estrogens (PREMARIN®), conjugated estrogens with medroxyprogesterone (PREMPRO®, PREMPHASE®), esterified estrogens (MENEST®), estradiol, etropipate, and calcium and calcium salt supplements.

[0183] Products and Kits Compositions (e.g., including formulations and unit dosage forms) comprising a compound of formula I or a pharma- ceutically acceptable salt or cocrystal thereof can be prepared and placed in a suitable container and labeled for treatment of an indicated condition. Thus, articles of manufacture such as a container comprising a unit dosage form of a compound of formula I or a pharma- ceutically acceptable salt or cocrystal thereof and a label comprising instructions for use of the compound are also provided. In some embodiments, the article of manufacture is a container comprising a unit dosage form of a compound of formula I or a pharma- ceutically acceptable salt or cocrystal thereof and at least one pharma- ceutically acceptable vehicle. The article of manufacture may be a bottle, vial, ampoule, disposable applicator, etc., comprising the pharmaceutical composition provided in this disclosure. The container may be formed from a variety of materials, such as glass or plastic, and in one aspect also comprises a label on or associated with the container indicating instructions for use in the treatment of bone cancer. It should be understood that the active ingredient may be packaged in any material that can improve chemical and physical stability, such as an aluminum foil bag. In some embodiments, the disease or condition indicated on the label can include, for example, treatment of bone cancer that originates from and is native to bone. In other embodiments, the label may be for the treatment of cancer that has metastasized to bone.

[0184] Any of the pharmaceutical compositions provided in this disclosure may be used in the products as if each and every composition were specifically and individually described for use in the products.

[0185] Also provided is a kit comprising a compound of formula I or a pharma- ceutically acceptable salt, a pharma- ceutically acceptable co-crystal, a pharma- ceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer. Also provided is a kit comprising a compound of formula II or a pharma- ceutically acceptable salt, a pharma- ceutically acceptable co-crystal, a pharma- ceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer. In one aspect, the kit comprises a compound of formula I or a pharma- ceutically acceptable salt or co-crystal. In a further aspect, the kit comprises a compound of formula II or a pharma- ceutically acceptable salt or co-crystal. The kit may comprise a label and / or instructions for use of the compound in treating a disease or condition in a subject (e.g., a human) in need thereof. In some embodiments, the disease or condition may be associated with or mediated by a desired activity.

[0186] Sequence information SEQUENCE LISTING <110> Oregon Health & Science University THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS <120> BISPHOSPHONATE-LINKED COMPOUNDS <150> US 62 / 801,858 <151> 2019-02-06 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 1 ggtgaaaccc cgtctctact 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> Synthetic oligonucleotide <400> 2 ggttcaagcg attctcctgc 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> Synthetic oligonucleotide <400> 3 cgcccggcta atttttgtat 20

Claims

1. formula: A compound of the formula: During the ceremony, Z is selected from the group n in each case 1 is an integer selected from the group of 1, 2, 3, 4, 5, and 6; X is (-CH 2 -)n 2 and R 1 is H and C 1 ~C 3 selected from the group of alkyl; R 2 is selected from the group of H and OH; n 2 is an integer selected from the group of 0, 1, 2, and 3; n 3 is an integer selected from the group of 0 and 1; and Y is a bisphosphonate compound; The compound, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

2. A bisphosphonate compound represented by Y, 2. The compound of claim 1, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, selected from the group consisting of:

3. The compound of formula (I) is represented by formula (II): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, n 1 is an integer selected from the group of 1, 2, 3, and 4; and n 3 is an integer selected from the group of 0 and 1, 2. The compound of claim 1.

4. formula:

4. The compound of claim 3, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, having the formula:

5. formula:

4. The compound of claim 3, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, having the formula:

6. formula:

4. The compound of claim 3, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, having the formula:

7. formula:

4. The compound of claim 3, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, having the formula:

8. The compound of formula (I) has the formula (Ib): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, Z is selected from the group n 1 is an integer selected from the group of 1, 2, 3, and 4; X is (-CH 2 -)n 2 and R 1 is H and C 1 ~C 3 selected from the group of alkyl; R 2 is selected from the group of H and OH; n 2 is an integer selected from 0, 1, 2, and 3; n 3 is an integer selected from the group of 0 and 1; and Y is a bisphosphonate compound; 2. The compound of claim 1, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

9. The compound of formula (Ib) may be represented by the formula (Ic): or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, During the ceremony, R 1 is selected from H and methyl; n 1 is an integer selected from the group of 1, 2, and 3; n 3 is an integer selected from the group of 0 and 1; and Z is selected from the group 9. The compound of claim 8, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

10. formula:

10. The compound of claim 9, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, having the formula:

11. formula:

10. The compound of claim 9, or a pharma- ceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, having the formula:

12. 13. A pharmaceutical composition comprising a pharma- ceutical effective amount of the compound of claim 1 or a pharma- ceutical acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof, and a pharma- ceutical acceptable carrier or excipient.

13. 1. A method for treating osteoclast-mediated bone degradation in a subject, comprising: The method comprises administering to a subject in need thereof a pharma- ceutical effective amount of the compound of claim 1 or a pharma- ceutical acceptable salt, co-crystal, polymorph, solvate, hydrate, or enantiomer thereof.

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