RNF4 targeting compounds and uses thereof

Abstract

The present invention provides compounds and pharmaceutical compositions comprising thereof. Further, methods for treating or preventing development of a proliferative disease or disorder in a subject are also provided.

Description

RNF4 TARGETING COMPOUNDS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No.63 / 738,865, titled “RNF4 TARGETING COMPOUNDS AND USES THEREOF”, filed 26 December 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The present invention is in the field conjugates for use in therapy, such as in the treatment of proliferative disease(s) in a subject in need thereof.BACKGROUND OF THE INVENTION

[0003] Despite maj or advances in cancer therapies, the patient’ s response to treatment for aggressive, advanced, or therapy-resistant cancers remains a challenge. Cancer development and progression are intimately linked to increased oncoprotein stabilization. Moreover, the development of resistance to chemotherapy and molecular treatments (e.g. receptor tyrosine kinases inhibitors RTKi, and immune check inhibitors (ICI) in melanoma) is also associated with increased oncoprotein stabilization. These hallmarks are observed in the majority of patients, and overcoming therapy resistance is an unmet need in the clinic. Thus, the development of drugs targeting aggressive and therapy-resistant cancers is required for improving patient outcome and for tailoring precision therapeutics in this large group of cancer patients.

[0004] Recently a novel ubiquitin-dependent pathway that stabilizes and potentiates oncoprotein activity promoting tumorigenesis has been described. The pathway acts upstream and independently of the physiological machinery and degron that mediates the rapid degradation of short-lived oncoproteins. A central enzyme in this pathway is the ubiquitin ligase RNF4 which directly binds, stabilizes, and potentiates multiple oncoproteins. Moreover, in addition to its central role in protein stabilization, RNF4 has multiple roles supporting cancer development. Among these functions are DNA repair, nuclear protein control, enhancement of oncogenic transcription and translation, and tumorigenic impact on the tumor microenvironment including fostering angiogenesis.

[0005] RNF4 belongs to a small group of RING ubiquitin ligases termed SUMO-targeted ubiquitin ligases (STUbL) that ubiquitinate SUMOylated proteins. RNF4 has a tumor suppressive function in the case of acute pre-myelocytic leukemia (APL), in part by theSUMO-dependent ubiquitination and degradation of the leukemia oncoprotein driver PML-RARa.

[0006] RNF4 enhances the tumorigenic properties of cancer cells, is essential for cancer cell survival, and in melanoma confers resistance to molecular therapy in vitro and in vivo. In sarcomas, RNF4 drives the expression of the survival factors; BMP6 and its co-receptor RGMb that are secreted from the tumor cells and act locally. High levels of RNF4 are observed in about -30-40% of colon cancer and melanoma and sarcoma biopsies and are associated with poor prognosis in melanoma, breast cancer, and multiple types of sarcomas. While RNF4 is non-oncogenic on its own, cancer cells need to cope with oncogenic stress, and eliminating RNF4 genetically resulted in the death of aggressive and therapy-resistant cancer cells and tumors. However, RNF4 is less required in non-transformed cells. Thus, RNF4 is potentially an “Achilles’ heel” of multiple tumor entities and therefore an excellent target for precise cancer therapy.

[0007] Accordingly, there is a need for development of efficient RNF4 targeting molecules as a novel strategy for eradicating therapy -resistant cancers.SUMMARY OF THE INVENTION

[0008] In one aspect of the invention, there is provided a compound, a salt, an isomer or a tautomer thereof, wherein the compound is represented by Formula 1 :L represents a linker; Y independently is selected from the group consisting of: NH, S, O and CH; R3 is halo; each R represents H or an optionally substituted alkyl; each n is an integer being independently between 0 and 5; each R1 represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -CONH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -S02R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -C02R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Cl-C6 alkyl), C1-C6 mercaptoalkyl, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, C02H, CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkyl-heteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted Cl -CIO alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; and Ar represents an aryl or a heteroaryl.

[0009] In one embodiment, L comprises [Cl -CIO alkyl-O]m, wherein m is an integer being independently between 1 and 20.

[0010] In one embodiment, A represents phenyl.[Oil] In one embodiment, L is or comprises T-A-T-A-T, wherein each A is independently selected from [Cl -CIO alkyl-O]m, alkyl and heteroalkyl; and T is absent or is independently from -O-, -S-, -NR’-, -C(=O)-, -C(=NR’)-, -C(=S)-, -CONR’-, -C(NR’)NR’-, -C(NR’)O-, -C(NR’)S-, -S-S-, -S-C(=O), -CNNR’-, -CSNR’-, -NR’C(=O)O-, -NR’C(=S)O-, -NR’C(=S)NR’-, -SO2-, -SO-, -OC(=O)-, -OC(=O)O-, -OC(=S)O-, and -OC(=S)NR’-; and wherein at least one A is [C1-C10 alkyl-O]m.

[0012] In one embodiment, the compound is represented by Formula 2 :wherein each k is independently between 0 and 20.

[0013] In one embodiment, the compound is represented by Formula 3 :is -CONR’-; wherein each m and k independently between 1 and 10.

[0014] In one embodiment, Y is O and each R is a C1-C5 alkyl.

[0015] In one embodiment, each of said n independently represents an integer in a range from 1 to 5.

[0016] In one embodiment, the compound is represented by Formula 4:

[0017] In one embodiment, m is between 2 and 5 and k is between 1 and 10.

[0018] In another aspect, there is provided a pharmaceutical composition comprising the compound of the invention, and a pharmaceutically acceptable carrier.

[0019] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the compound.

[0020] In one embodiment, the pharmaceutical composition is for use in reducing Ring Finger Protein 4 (RNF4) abundance, activity, or both and / or (ii) inducing ferroptosis within a cell of a subject, wherein the cell is characterized by abnormal proliferation.

[0021] In one embodiment, reducing is by inducing proteasomal degradation of RNF4.

[0022] In one embodiment, the pharmaceutical composition is for use in the treatment of a proliferative disease in a subject.

[0023] In one embodiment, the proliferative disease is selected from cancer proliferative disease is selected from cancer, pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS) .

[0024] In one embodiment, the cancer is selected from the group consisting of: carcinoma, sarcoma, leukemia, melanoma, tyrosine kinase receptor inhibitors (RTKi) resistantmelanoma, immune-check points inhibitors (ICI) resistant-melanoma, aggressive osteosarcoma and soft-tissue sarcoma.

[0025] In another aspect, there is provided a method for inducing ferroptosis within a cell of a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound represented by Formula 5:L represents a linker; Y independently is selected from the group consisting of: NH, S, O and CH; R3 is halo; each n is an integer being independently between 0 and 5; each R1 represents one or more substituents, each independently comprising alkyl, -NO2, -CN, - OR’, -OH, -CONH2, HC0NH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, - CONH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl),hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Cl-C6 alkyl), C1-C6 mercaptoalkyl, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, CO2H, CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkylheteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted Cl -CIO alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; and Ar represents an aryl or a heteroaryl.

[0026] In one embodiment, the cell is characterized by abnormal proliferation.

[0027] In one embodiment, the cell is selected from a cancer cell and a synovial cell characterized by CSF1 overexpression.

[0028] In one embodiment, the cancer cell comprises any one of carcinoma cell, leukemia cell and melanoma cell.

[0029] In one embodiment, the method is for treating a proliferative disease in said subject.

[0030] In one embodiment, the proliferative disease is selected from cancer, pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS).

[0031] In some embodiments, the cancer is selected from the group consisting of: melanoma, squamous cell carcinoma breast cancer, colorectal cancer, osteosarcoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer and hematological cancers.

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0033] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since variouschanges and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0034] Figures 1A-1T: R4VP degraders force the proteasomal degradation of RNF4, its stabilized proteins and reduce cancer cells survival. (A) Cancer-promoting activities of RNF4. (B) Schematic diagram of RNF4 structure animo acids residues Cys51 and 91 that are required for R4VPs binding are indicated as red triangles. (C) Chemical structure of R4VP. (D) Western-blot analysis of endogenous RNF4 protein level in extracts derived from that B16F10 mouse melanoma cells . Where indicated VHL-only, RV4P alone or together with proteasome inhibitor 80mM MG132 was added, Tubulin serves as a loading control. (E) R4VP treatment results in a dose-dependent reduction in RNF4 levels in B16F10 mouse melanoma cells. (F) Structure of R4VPL3-1. (G-J) Western-blot analysis of endogenous RNF4, p-c-Myc, and p-b-catenin proteins levels in extracts derived from RTKi resistant human melanoma A375R cells treated with VHL-only or R4VPL3-1 at the indicated concentrations (G, H), and time (I, J). Tubulin serves as a loading control, and in all experiments n=3 and ****= p<0.0001, ***=p<0.001, **=p<0.01. (K-M) Proliferation (K) and sphere formation (SFA; L, M) of SSC1 human squamous skin carcinoma cells is attenuated upon treatment with R4VPL3-1 at the indicated doses, but not upon treatment with VHL-only compound. (N-O) SFA of HaCat, a non-tumorigenic skin keratinocyte cell line is only minimally inhibited by R4VPL3L-1. In all experiments, N=3 and ****= p<0.0001, ***= p<0.001, **=p<0.01. ns= non-significance. (P) Endogenous RNF4 protein levels in HaCat and SSC1 cells, Tubulin serves as a loading control. Western-blot analysis of endogenous VHL protein levels in extracts derived from A375R PLX4032-resistant human melanoma cells treated with VHL-r or R4VPL3-1 at the indicated concentrations (Q, R), and time (S, T). In all experiments Tubulin serves as a loading control and ****= p<0.0001, ***= p<0.001, **=p<0.01. ns= non-significance. RNF4 (n=5), p-c-Myc (n=4), p-b-catenin (n=3). Statistical analysis: (H) 2-way Anova Dunnett’s multiple comparisons test. (J, Q, R, T) 1-way Anova Dunnetf s multiple comparisons test. n=3 ****=p<0.0001; ***=p<0.001; *=p<0.1.

[0035] Figures 2A-2I: R4VPL3-1 induces RTKi-resistant melanoma cells death (independent of RNF4). (A-C) Proliferation (A) and SFA (B, C) of human PLX4032-resistant 375R cells, is attenuated upon treatment with R4VPL3-1 at the indicated doses, but not upon treatment with VHL-only compound, and (C) is a representative experiment.(D-G) Cell death using FACS analysis using propidium iodide and the cell death marker Annexin V-FITC. Treatment of A375R cells with R4VPL3-1, but not control results in rapid cell death. (D, E) Time-course of cell death upon exposure to R4VP3-L1. (F, G) Dosedependent four hours treatment with R4VP3-Llof A375R cells induces cell death, (D, F) are representative experiments. (H) In vitro binding of the indicated GST-RNF4 proteins to biotinylated CCW16 (original R4B), or biotinylated-R4VPL3-l where biotin replaces the VHL recruiting moiety. Wild-type GST-RNF4 or the indicated GST-RNF4 mutants were incubated with biotin or biotinylated R4VPL3-1. Covalent RNF4-R4VPL3-1 conjugates were detected by Streptavidin immunoblotting. (I) Expression of increasing amounts of HA-RNF4DM(RNF4C51A,C91A) mutant does not cancel R4VPL3-1 -induced cell death.

[0036] Figures 3A-3L: R4VPL3-1 induces ferroptosis of A375R RTKi resistant melanoma cells. (3A-3C) Cytoscape and KEGG analysis of RNA-seq results identifying statistically significant upregulated and repressed pathways, as well as cellular process effected by two hours treatment lOmM of R4VPL3-1 compared to VHL-r treated A375R cells. In (3C) red circle marks ferroptosis. (D) 5mM R4VPL3-1 induces lipid peroxidation that is inhibited by lOmM a Ferrostatin-1 (Ferr-1), a specific ferroptosis inhibitor, as evident by a-4HNE immune-staining. (E, F) Bodipy™-Cll sensor was used to visualize lipid peroxidation induced by R4VP3L-1 that is inhibited by Ferr-1. (E) are representative confocal images, the Hoechst stain is blue, and the scale bar is 10pm. Quantification of shifts in fluorescence measurements are shown in (F). Cumene hydroperoxide is a lipid peroxidation compound (positive control); SD are shown of three repeats and ****= p<0.0001. (G-H) FACS analysis of R4VPL3-1 induced A375R cell death. lOpM Ferr-1, or lOpM DFX, but not DMSO, 20pM Z-VAD-FMK or lOOpM Chloroquine (Cq), inhibited R4VPL3-1 induced cell death. (G, I) are representative experiments, and quantifications are shown in J, I. Statistical analysis: n=3 and ****= p<0.0001, ***= p<0.001, **=p<0.0, ns= no-significance. One-way Anova Dunnett’s multiple comparisons tests (n=2). (K-L) R4VP3L-1 does not induce cleavage of PARP. Western blot analysis of protein extracts derived from A374R cells treated with either Sterosproine (STS, positive control) or R4VPL3-1 and cleaved-PARP an indicator of apoptotic cell death was determined using PARP and cleaved-PARP antibodies. (K) is a representative experiment, and quantification is shown on (L).

[0037] Figures 4A-4J: Structural determinants of R4VPL3-1 and selectivity involved in its differential anti-cancer activity. (4 A) Schematic diagram of R4VPL3-1 -relatedcompounds; R4B, RNF4 binding moiety. (4B-4E) Testing the activity of R4VPL3-1 -related compounds using SFA toward non-tumorigenic HaCaT cells (4B, 4C) and MEFs (4D, 4E). (4B) and (4D), are representative experiments. (4F-4I) SFA; Biotin-R4VPL3-1 (R4VPL3-1 where the VHL-r moiety was replaced with biotin), has no anti-cancer activity towards A375R (4F, 4G) or HaCat (4H, 41). (4J) Live Cell proliferation assays using a-LamA / C of the indicated transformed epithelial lung cells (see methods). R4VPL3-1 has selective antiproliferative activity against Beas2B lung cells transformed with either BRAFV600C or EGFRL858R activating mutations but not PI3KH1047R mutation. Statistical analysis: Iway Anova Dunnett’s multiple comparisons test (4C, n=3; 4E, n=3; 4G, n=2; 41, n=2); Significance: ****= p<0.0001, ***=p<0.001, **=p<0.0, ns= no-significance.

[0038] Figures 5A-5K: R4VPL3-1 is active towards sarcoma cells, patients-derived primary tumor cells (A-C) R4VL3-1 inhibits cell proliferation (A) and SFA (B, C) of 134B, a highly metastatic human sarcoma cell line. (D-I) R4VL3-lbut not the VHL moiety inhibits cell proliferation of patients-derived primary tumor cells in a dose dependent manner. (I) Table summarizing IC50 of R4VPL3-1 in different patients-derived cells. Patients #1-4 are of sarcoma cells and patient #5 is a lung adenocarcinoma tumor. In all experiments n=3 and ****= p<0.0001, ***= p<0.001, **=p<0.01. ns= non=significance.(J, K) R4VPL3-1 inhibits SFA of undifferentiated polymorphic sarcoma (UPS) (J), and lung adenocarcinoma tumor cells (K).

[0039] Figures 6A-6D: R4VPL3-1 inhibit sarcoma development in-vivo. (A-C) ADME and PK studies for R4VPL3-1 and R4VPL3-3 performed by WuXi® (D) IVIS imaging of our femur bone tumor xenograft model using human RFP-labelled 143B cells. Mice 1 and 2 were untreated and monitored for 1 and 2 weeks, respectively, thereafter; Mice 3-5 were injected s.c. after lOd with either DMSO or 50 mg / Kg R4VPL3-1 and monitored for 3 weeks thereafter.

[0040] Figures 7A-7K: R4VPL3-1 induces death of human skin and melanoma cancer cells but has no effect on MEFs and non-oncogenic cells but is potent against cancer cells. (7 A, 7B) Sphere formation (SFA) of HaCat, a non-tumorigenic skin keratinocyte cell line upon treatment with either VHL or R4VP3-L1 in HaCat (A, B). (7C) Endogenous RNF4 and VHL, p-c-Myc and p— catenin protein levels in the above cells. (7D, 7E) SFA of mouse embryonic fibroblasts (MEFs) in both cells SFA is only minimally inhibited by R4VPL3-1. (7F-7K) Proliferation (7F, 71) and SFA (7G, 7H, 7J, 7K) of SSC1 human squamous skin carcinoma cells (7F-7H) or human PLX4032-resistant 375R humanmelanoma cells (71- 7K) is attenuated upon treatment with R4VPL3-1 at the indicated doses but not upon treatment with DMSO or VHL-r compound.

[0041] Figures 8A-8I: The anti-ferroptotic selanoproteins GPX4 is targeted by R4VP3L-1. (8A) Western blot analysis of protein extracts derived from A374R cells treated with R4VPL3-1 for the indicated times and antibodies. Note the increased protein level and shift of GPX4 upon R4VPL3-1 treatment. (8B-8D) Treatment of A375R cells with R4VP3-L1, but not inactive R4VP3-L1 results in a slower migrating form of GPX4 in A375R (8B), and HaCat cells (8C), and is not prevented by Ferr-1 (8D). (8E) Endogenous GPX4 is bound by R4VP3L-1 but not by the inactive compound in an immune-precipitation of GPX4 in A375R cells. (8F-8I) FACS analysis using PI and annexin 5, RSL-3, an inhibitor of GPX4 and selanoproteins, induces cell death of A375R that is inhibited by Ferr-1 (8F), but does not induce cell death of HaCat cells (8G). Representative experiments are shown in 8F, 8G and (8H, 81) quantification of three biological repeats are shown in 8H, 81, respectively.

[0042] Figures 9A-9E: R4VP force the degradation of RNF4 and reduces survival of cancer cells. (A, B) R4VP but not VHL-only compound, inhibits the proliferation (A) and SFA (B) of A375R melanoma cells. (C) Inactive R4VP lacking a critical Cl atom:has no impact on A375R cells proliferation. (D) R4VP treatment, but not VHL-only inhibit proliferation of human skin cancer cells SSC1.(E) R4VP inhibits SFA of SSC1 cells but had no impact on non-tumorigenic human HeCat cell line (not shown). In all experiments n=3 and ***= p<0.001 **=p<0.01,*=p<0.1 and cell proliferation was measured indirectly by ATP -Lite assay.

[0043] Figure 10: Improvement of RNF4 binding moiety: Chemical structures of Ll-L14 RNF4 binders.

[0044] Figures 11A-11G: Screening of the compounds of the invention for an enhanced RNF4 binding moiety (R4B). We generated fifteen R4B-related compounds and measured their ability to directly bind bacterially purified RNF4 in vitro using a Rhodamine-Iodoacetamide (R-I) labeling and displacement assay enabling quantitativevisualization of RNF4. Using this assay, we determined that the binding of the original R4B molecule (CCW16, LI) to RNF4 resulted in reduced signal in a dose-dependent manner, with a 50% binding inhibition concentration of ~27mM. Using this assay, we compared the binding of LI to newly developed LI -related molecules. Of the compound tested we identify L3 as an RNF4 binder with improved binding affinity with 50% R-I inhibition at 3mM. (11 A) Schematic diagram of a direct Rodamine-Iodacetmide dye displacement assay by the RNF4 binding moiety. (11B) Upper panel: concentration dependent dye displacement by the R4VP-RNF4 binding moiety (R4B), n=3, ***= p<0.001. Lower panel: Representative experiment. (11C-11F) Screening of Rl-displacement of potential RNF4 binders at lOmM (C, D) and 50mM (E, F). (11G) Upper panel: concentration dependent dye displacement by L3, an improved RNF4 binding moiety n=3, ****= p<0.0001). Lower panel is a representative experiment.

[0045] Figures 12A-12BChemical structure of R4VP compounds. (A) R4VPL3 (b) R4VPL3-2.

[0046] Figures 13A-13G: Anti-cancer activities of R4VPL3 and R4VPL3-L2 Based on L3, we synthesized R4VPL3, a VHL-dependent PROTAC using L3 as the RNF4 binder that was more potent than R4VP and retained its selective activity against cancer cells but had no impact on proliferation or SFA of MEFs. (A-D) Dose-dependent effect of R4VPL3 on viability and SFA of A375R melanoma (A, B) and SCC1 (C, D) cells. Lower panels in A and C are representative experiments. Cell proliferation measured indirectly by MTT (B, D). (E, F) R4VPL3 had minimal or no impact on SFA of non-tumorigenic cells; (E) Mouse embryonic fibroblasts (MEFs) and HaCat (F). (G) R4VPL2 had no impact on SFA of human melanoma A375R cells. In all experiments n=3 and ***=p<0.001, **=p<0.01.DETAILED DESCRIPTION OF THE INVENTION

[0047] According to some embodiments, the present invention provides a compound, a salt, an isomer or a tautomer thereof, wherein the compound is represented by any of Formulae shown hereinbelow.

[0048] The present invention is directed to pharmaceutical compositions comprising the compounds. In some embodiments, the compound is targeting or having a binding affinity to Ring Finger Protein 4 (RNF4), or to RNF4 and VHL.

[0049] In some embodiments, the compounds of the invention reduce RNF4 (cellular) abundance, RNF4 activity, or both. In some embodiments, the compounds of the inventioninduce proteasomal degradation of RNF4. In some embodiments, the reduction of RNF4 cellular abundance is by inducing proteasomal degradation of RNF4. In some embodiments, the compounds of the invention reduce the levels of the oncoproteins that are stabilized by RNF4.

[0050] In some embodiments, the compounds and compositions of the present invention are used in methods for treating or preventing a RNF4 related disease or disorder. In some embodiments, the RNF4 related disease / disorder is a proliferative disease.

[0051] In some embodiments, the compounds and compositions of the present invention are used in methods for preventing cell proliferation of over-proliferating cells. In some embodiments, the compounds and compositions of the present invention are used for reducing the level of one or more oncogenes (e.g. c-Myc, B-catenin, c-Jun, and others) within the cell. In some embodiments, the compounds and compositions of the present invention are used in methods for treating or preventing a proliferative disease in a subject.

[0052] In some embodiments, the proliferative disease is selected from cancer, pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS).

[0053] In some embodiments, the cancer comprises any of sarcoma, carcinoma (e.g. colon, breast etc.), leukemia, melanoma, or any combination thereof.

[0054] In some embodiments, the melanoma comprises tyrosine kinase receptor inhibitors (RTKi) resistant-melanoma, immune-check points inhibitors (ICI) resistant-melanoma or melanoma resistant to other therapies.

[0055] In some embodiments, the sarcoma comprises aggressive osteosarcoma and / or soft-tissue sarcoma.

[0056] In some embodiments, the compounds and compositions of the present invention are used for inducing ferroptosis within a cell.

[0057] In some embodiments, the compounds of the present invention are proteolysis targeting chimera (PROTAC) conjugates.

[0058] As used herein, the term “proteolysis targeting chimera (PROTAC)” refers to a heterobifunctional small molecule composed of two active domains associated by a linker capable of removing specific unwanted proteins. PROTACs consist of two covalently linked protein-binding molecules: one capable of engaging an E3 ubiquitin ligase, and another that binds to a target protein meant for degradation.

[0059] The term “linker” as used herein, refers to a chemical moiety utilized to attach one part of a compound of interest to another compound of interest. In some embodiments, the linker of the invention is an ethylene glycol-based linker.

[0060] As used herein, the term “small molecule” refers inter alia to a non-peptidic, non-oligomeric organic compound. Small molecules can be either synthesized in the laboratory or found in nature. In some embodiments, a small molecule is characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 2000 g / mol, preferably less than 1500 g / mol. In some embodiments, small molecules according to the present invention are synthetic small molecules.

[0061] In some embodiments, the compounds of the present invention bind to an E3-ubiquitin ligase. In some embodiments, the E3 -ubiquitin ligase is Von-Hipple Landau (VHL) ubiquitin ligase. In some embodiments, the compounds of the invention reduce VHL (cellular) abundance, VHL activity, or both. In some embodiments, the compounds of the invention induce proteasomal degradation of VHL. In some embodiments, the reduction of VHL cellular abundance is by inducing proteasomal degradation of VHL.Compounds

[0062] According to an aspect of embodiments of the invention there is provided a compound, a salt, an isomer or a tautomer thereof, wherein the compound is represented by Formula 1:L represents a linker;Y independently is selected from the group consisting of: NH, S, O and CH;R3 is halo;each R represents H or an optionally substituted alkyl;each n is an integer being independently between 0 and 5;each R1 represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -CONH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -C0NH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Cl-C6 alkyl), C1-C6 mercaptoalkyl, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkyl-heteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted Cl-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; and Ar represents an aryl or a heteroaryl.

[0063] In some embodiments, the compound is represented by Formula 4:wherein m is between 2 and 5 and k is between 1 and 10.

[0064] In some embodiments, the compound is represented by Formula 4a:between 2 and 4; and k is between 2 and 6 or is between 3 and 5. In some embodiments, the compound is represented by Formula 4a, wherein m is 2 and k is 4 (also termed herein as R4VPL3-1). In some embodiments, the compound is represented by Formula 4a, wherein m is 4 and k is 4 (also termed herein as R4VPL3-3).

[0065] According to another aspect of embodiments of the invention there is provided a method for inducing ferroptosis within a cell, comprising contacting the cell with a compound represented by Formula 5:L represents a linker;Y independently is selected from the group consisting of: NH, S, O and CH;R3is halo;each n is an integer being independently between 0 and 5;each R1represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -CONH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -C0NH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Ci-C6alkyl), Ci-C6mercaptoalkyl, -CONH(CI-C6alkyl), -CON(CI-C6alkyl)2, -CO2H, -CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkyl-heteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; andAr represents an aryl or a heteroaryl.

[0066] In some embodiments, the cell is a cell of a subject. In some embodiments, the method is for treatment of a proliferative disease in the subject. In some embodiments, the method for treatment of the proliferative disease comprises administering to the subject a therapeutically effective amount of a compound represented by Formula 5 or a therapeutically effective amount of a pharmaceutical composition comprising the compound of Formula 5.

[0067] In some embodiments, the compound is represented by Formula 5, wherein R1represents one or more substituents, each independently comprising any one of: OR’, -OH, amino, imino (NRR, R is as disclosed herein) and -SR’.

[0068] In some embodiments, the term “cyclyl” comprises an aryl, a polycyclyl, a heteroaryl, a cycloalkyl, or heterocyclyl or any combinations thereof.

[0069] In some embodiments, the term “polycyclic ring” or “polycyclyl” encompasses a plurality (e.g. 2, 3, 4, 5 or 6) of fused or adjacent rings (e.g. biaryl or bicyclohexyl), wherein each ring is independently selected from aryl, heteroaryl, an optionally unsaturated cycloalkyl, an optionally unsaturated heterocyclyl, or any combination thereof. In some embodiments, the term “polycyclyl” encompasses a polycyclic aromatic ring, a polycyclic aliphatic ring, or a mixed polycyclic ring.

[0070] In some embodiments, the term “mixed polycyclic ring” refers to any plurality of rings covalently bound to each other (e.g. fused rings, dicylyls, spirocyclic rings etc.)comprising at least one aromatic ring (aryl, or heteroaryl) and at least one aliphatic or nonaromatic ring (optionally a heterocyclyl and / or unsaturated cyclyl).

[0071] In some embodiments, the term “cyclyl” comprises C3-C10 cyclyl. In some embodiments, the term “C3-C10 cyclyl” and the term “(C3-C10) ring” are used herein interchangeably and are referred to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or CIO aliphatic ring, aromatic ring, or a ring comprising an unsaturated bond.

[0072] As used herein the term “substituted” encompasses substitution by one or more R, wherein R comprises any one of -NO2, -CN, -OH, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)R’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, carbonyl, C1-C10 haloalkyl, optionally substituted C1-C10 alkyl, -NH2, -NH(Ci-Cio alkyl), -N(Ci-Cio alkyl)2, C1-C10 haloalkoxy, hydroxy(Ci-Cio alkyl), hydroxy(Ci-Cio alkoxy), alkoxy(Ci-Cio alkyl), alkoxy(Ci-Cio alkoxy), amino(Ci-Cio alkyl), -CONH(Ci-Cio alkyl), -CON(Ci-Cio alkyl)2, -CO2H, -CO2R’, -OCOR’, -C(=O)R’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, a heteroatom, cycloalkyl, heterocyclyl, aryl, heteroaryl, (C1-C10 alkyl)alkyl-cycloalkyl, (C1-C10 alkyl)alkyl-aryl, (Ci-C10 alkyl)alkyl-heteroaryl, or any combination thereof, and wherein each of cycloalkyl, heterocyclyl, aryl, heteroaryl is substituted or non- substituted, including any combination thereof.

[0073] In some embodiments, the compound of the invention comprises any one of the compounds disclosed herein, including any enantiomers thereof. In some embodiments, the compound of the invention comprises a mixture of enantiomers (e.g. a racemic mixture).

[0074] The compounds described hereinabove may be applied or otherwise utilized either as is, or as an acceptable salt, enantiomer, diastereomer, solvate, or hydrate.

[0075] Non limiting examples of salts include but are not limited to: cations derived from alkali or alkaline earth metals (e.g. sodium, potassium, magnesium), cations derived from ammonia and amines (e.g. ammonium, diethylammonium, ethanolammonium, isopropylammonium) and trimethyl sulfonium salts.

[0076] In some embodiments, the compounds described herein are chiral compounds (i.e. possess an asymmetric carbon atom). In some embodiments, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. In some embodiments, a chiral compound described herein is in form of a racemic mixture. In some embodiments, a chiral compound is in form of a single enantiomer, with an asymmetric carbon atom having the R configuration. In some embodiments, a chiralcompound is in form of a single enantiomer, with an asymmetric carbon atom having the S configuration as described hereinabove.

[0077] In some embodiments, a chiral compound is in form of a single enantiomer with enantiomeric purity of more than 70%. In some embodiments, a chiral compound is in form of a single enantiomer with enantiomeric purity of more than 80%. In some embodiments, a chiral compound is in form of a single enantiomer with enantiomeric purity of more than 90%. In some embodiments, a chiral compound is in form of a single enantiomer with enantiomeric purity of more than 95%.

[0078] In some embodiments, the compound of the invention comprising an unsaturated bond is in a form of a trans-, or cz -isomer. In some embodiments, the composition of the invention comprises a mixture of cis- and / ra / z.s-i somers, as described hereinabove.

[0079] In some embodiments, the compounds described herein can exist in unsolvated form as well as in solvated form, including hydrated form. In general, the solvated form is equivalent to the unsolvated form and is encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0080] The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the conjugate described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid and the like.

[0081] The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water.

[0082] In some embodiments, a compound as described hereinabove reduces proliferation and / or viability of cells with abnormal proliferation (i.e. over-proliferating cells). In some embodiments, the cells with abnormal proliferation are characterized by overexpression of RNF4, as compared to the same cell types of a healthy subject. In some embodiments, the cells with abnormal proliferation are characterized by overexpression of CSF1, as compared to the same cell types of a healthy subject. In some embodiments, the cells with abnormal proliferation are characterized by overexpression of GPX4, as compared to the same cell types of a healthy subject. In some embodiments, the cells with abnormal proliferation are characterized by overexpression of RNF4, as compared to the same cell types of a healthy subject. In some embodiments, the cells with abnormal proliferation are characterized by overexpression of RNF4 and GPX4, as compared to the same cell types of a healthy subject.

[0083] In some embodiments, the compound described hereinabove reduces proliferation and / or induces cell death (e.g. by ferroptosis) of cells with abnormal proliferation at a concentration ranging from 0.01 pM to 2 pM, 0.05 pM to 2 pM, 0.1 pM to 2 pM, 0.01 pM to 3 pM, 0.05 pM to 3 pM, 0.1 pM to 3 pM, including any range therebetween. Each possibility represents a separate embodiment of the invention.

[0084] In some embodiments, the compound described hereinabove has a ferroptosis-related IC50 of cells with abnormal proliferation ranging from 0.01 pM to 2 pM, 0.05 pM to 2 pM, 0.1 pM to 2 pM, 0.01 pM to 3 pM, 0.05 pM to 3 pM, 0.1 pM to 3 pM, including any range therebetween.

[0085] In some embodiments, the compound described hereinabove selectively reduces proliferation and / or induces cell death (e.g. by ferroptosis) of cells with abnormal proliferation at a concentration below 3 pM, such as 0.01 pM to 3 pM, 0.05 pM to 3 pM, 0.1 pM to 3 pM, including any range therebetween. As used herein the terms “selectively” and “selectivity” in relationship to selective induction of cell death are used herein interchangeably and refer to the capability of the compounds to induce cell death solely in abnormally proliferating cells, whereas the viability of normal cells (i.e. devoid of overproliferation) is not reduced by more than 10%, relative to untreated normal cells. The cell viability is determined in-vitro in a cell culture after incubation with the compound at the above-disclosed concentration below 3 pM (e.g. for a time period between land 4h, see Cell Viability Assay and additional methods disclosed herein) and wherein the concentration refers to a concentration in the cell culture medium.

[0086] In some embodiments, a compound as described hereinabove induces proteasomal degradation ofRNF4, VHL or both. In some embodiments, a compound as described herein above reduces GPX4 activity. In some embodiments, a compound as described hereinabove is configured for covalent binding to GPX4. In some embodiments, a compound as described herein above induces proteasomal degradation of RNF4 and / or reduces GPX4 activity, and / or induces lipid peroxidation within a cell. In some embodiments, the cell is a cancer cell, a cancerous cell, a malignant cell, or any combination thereof. In some embodiments, the cell is a cell of a subject.Pharmaceutical Compositions comprising the disclosed compounds

[0087] According to an aspect of embodiments of the invention there is provided a pharmaceutical composition comprising one or more compounds as described herein and a pharmaceutically acceptable carrier.

[0088] According to an aspect of embodiments of the invention there is provided a pharmaceutical composition comprising therapeutically effective amount of one or more compounds as described herein.

[0089] According to another aspect, the invention provides a pharmaceutical composition comprising as an active ingredient, a therapeutically effective amount of a compound the present invention, and a pharmaceutically acceptable carrier and / or diluent.

[0090] In some embodiments, the pharmaceutical composition is for use in the reduction of Ring Finger Protein 4 (RNF4) abundance, RNF4 activity, or both. In some embodiments, reduction of RNF4 abundance is by inducing proteasomal degradation of RNF4. In some embodiments, the pharmaceutical composition is for use in any one of: (i) reduction of RNF4 abundance, activity, or both; (ii) reduction of GPX4 activity; and (iii) induction ferroptosis within a cell, including any combination thereof. In some embodiments, the cell is an isolated cell or a cell of a subject. In some embodiments, the cell characterized by abnormal proliferation.

[0091] In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of a disorder associated with cancer or any proliferative disease within a subject. In some embodiments, the pharmaceutical composition is for use in inducing ferroptosis within a cell characterized by abnormal proliferation.

[0092] In some embodiments, the cancer is selected from the group consisting of: melanoma, squamous cell carcinoma breast cancer, colorectal cancer, osteosarcoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer and hematological cancers. In some embodiments, the cancer is selected from the group consisting of: carcinoma, sarcoma, leukemia, melanoma, tyrosine kinase receptor inhibitors (RTKi) resistantmelanoma, immune-check points inhibitors (ICI) resistant-melanoma, aggressive osteosarcoma and soft-tissue sarcoma.

[0093] In some embodiments, the proliferative disease is selected from cancer, pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS) .

[0094] The compounds described hereinabove may be administered or otherwise utilized either as is, or as a pharmaceutically acceptable salt, an enantiomer, a tautomer, a diastereomer, a protonated or non-protonated form, a solvate, a hydrate, or a prodrug thereof.

[0095] The phrase "pharmaceutically acceptable salt" refers to a charged species of the parent compound and its counter ion, which is typically used to modify the solubilitycharacteristics of the parent compound and / or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0096] The phrase "pharmaceutically acceptable salts" is meant to encompass salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.

[0097] Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compound as described herein to be converted into either base or acid addition salts.

[0098] In some embodiments, the neutral forms of the compounds described herein are regenerated by contacting the salt with a base or acid and isolating the parent compounds in a conventional manner. The parent form of the compounds differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0099] The term "prodrug" refers to an agent, which is converted into the active compound (the active parent drug) in vivo. Prodrugs are typically useful for facilitating the administration of the parent drug. The prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve a sustained release of the active compound in vivo.

[0100] In some embodiments, the compounds described herein possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.

[0101] As used herein and in the art, the term "enantiomer" describes a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion / reflection (mirror image) of each other. Enantiomers are said to have “handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems.

[0102] In some embodiments, the compounds described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0103] The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the conjugate described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid and the like.

[0104] The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water.

[0105] In some embodiments, the "pharmaceutical composition" refers to a preparation of one or more of the compounds described herein (as active ingredient), or physiologically acceptable salts or prodrugs thereof, with other chemical components including, but not limited to, physiologically suitable carriers, excipients, lubricants, buffering agents, antibacterial agents, bulking agents (e.g., mannitol), antioxidants (e.g., ascorbic acid or sodium bisulfite), anti-inflammatory agents, anti-viral agents, chemotherapeutic agents, anti-histamines and other.

[0106] In some embodiments, the purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject. The term "active ingredient" refers to a compound, which is accountable for a biological effect.

[0107] The terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which may be interchangeably used, refer to a carrier or a diluent that does notcause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0108] Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a drug. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0109] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0110] In some embodiments, pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The dosage, as described and specified herein, may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.l).

[0111] In some embodiments, the pharmaceutical composition may be formulated for administration in either one or more of routes depending on whether local or systemic treatment or administration is of choice, and on the area to be treated. As further described herein throughout, administration may be done orally, dentally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (including ophtalmically, vaginally, rectally, intranasally).

[0112] Formulations for topical and / or dental administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0113] Compositions for oral administration may include powders or granules, suspensions, dental compositions, or solutions in water or non-aqueous media, sachets, pills, caplets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.

[0114] Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents and other suitable additives. Slow release compositions are envisaged for treatment.

[0115] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0116] The pharmaceutical composition may further comprise additional pharmaceutically active or inactive agents such as, but not limited to, an antibacterial agent, an antioxidant, a buffering agent, a bulking agent, a surfactant, an anti-inflammatory agent, an anti-viral agent, a chemotherapeutic agent and anti-histamine.

[0117] Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

[0118] It will be recognized that these embodiments are susceptible to various modifications and alternative forms well known to those of skill in the art.

[0119] As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human and / or for a proliferating cell as described herein. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In some embodiments, pharmaceutically acceptable carrier is non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the“Inactive Ingredient Guide, “ U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents that may be useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety.

[0120] According to an embodiment of the invention, pharmaceutical compositions contain 0.1% - 95% of the compound of the present invention. According to another embodiment of the invention, pharmaceutical compositions contain 1-70% of the compound. According to another embodiment of the invention, the pharmaceutical composition or formulation to be administered may contain a quantity of the compound, according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.

[0121] According to one embodiment, the pharmaceutical compositions of the present invention are administered in the form of a pharmaceutical composition comprising at least one of the active components of this invention (a compound as described hereinabove) together with a pharmaceutically acceptable carrier or diluent. In another embodiment, the compositions of this invention can be administered either individually or together in any conventional sub-retinal or transdermal dosage form.

[0122] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0123] The pharmaceutical compositions also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[0124] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.

[0125] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.

[0126] As used herein, the term "therapeutically active molecule" or "therapeutic agent" means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. This term includes pharmaceuticals, e.g., small molecules, treatments, remedies, biologies, devices, and diagnostics, including preparations useful in clinical screening, prevention, prophylaxis, healing, imaging, therapy, surgery, monitoring, and the like. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a bioactive effect, for example.

[0127] The term “therapeutically effective amount” refers to the concentration of the compound(s), or their combination, normalized to body weight, that is effective to treat a disease or disorder in a mammal. The term “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the bioactive agent required.

[0128] In some embodiments, a pharmaceutical composition of the invention comprises pharmaceutically active agents. In some embodiments, pharmaceutically active agents are added prior to transplantation. Pharmaceutically active agents include but are not limited to any of the specific examples disclosed herein. Those of ordinary skill in the art will recognize also numerous other compounds that fall within this category and are useful according to the invention.Methods of use

[0129] According to some aspects, there is provided a method for treating, ameliorating, reducing and / or preventing a RNF4 related disorder in a subject. According to some aspects, there is provided a method for treating, ameliorating, reducing and / or preventing of a proliferative disease in a subject.

[0130] According to some aspects, there is provided a method for any one of: (i) reduction ofRNF4 abundance, activity, or both; (i) reduction of RNF4 abundance, activity, or both; (ii) reduction of GPX4 activity; and (iii) induction ferroptosis within a cell, including any combination thereof. In some embodiments, the cell is an isolated cell or a cell of a subject. In some embodiments, the cell characterized by abnormal proliferation. In some embodiments, the method is an in-vivo method. In some embodiments, the method is an in-vitro method.

[0131] According to some aspects, there is provided a method for treating, ameliorating, reducing and / or preventing a condition associated with increased cell proliferation in a subject in need thereof, the method comprising the step of: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the compound of the invention, thereby treating, ameliorating, reducing and / or preventing the condition associated with increased cell proliferation in the subject in need thereof. In some embodiments, the condition associated with increased cell proliferation in a subject in need thereof is a GPX4-related disease.

[0132] According to some aspects, there is provided a method for treating, ameliorating, reducing and / or preventing a GPX4-related disease in a subject, the method comprising the step of: administering to the subject the compound of the invention or a pharmaceutical composition comprising same.

[0133] In some embodiments, the method disclosed hereinabove further comprises a preliminary step performed before the administering step, the preliminary step comprises selecting a subject. In some embodiments, the preliminary step comprises selecting a subject having increased GPX4 abundance, activity, or both. In some embodiments, the preliminary step comprises selecting a subject having increased RNF4 abundance, activity, or both. In some embodiments, the preliminary step comprises selecting a subject having increased RNF4 and GPX4 abundance, activity, or both.

[0134] In some embodiments, the selecting comprises selecting a subject suffering from a GPX4 related disease. In some embodiments, the selecting comprises selecting a subject suffering from a RNF4 related disease. In some embodiments, the selecting comprises selecting a subject suffering from cancer. In some embodiments, the selecting comprises selecting a subject suffering from (i) GPX4 expressing cancer; (ii) RNF4 expressing cancer; (iii) RNF4 and GPX4 expressing cancer or any combination of (i) - (iii). In some embodiments, expressing is overexpressing. In some embodiments, the selecting comprises selecting a subject confirmed to suffer from a cancer comprising GPX4 and / or RNF4expression. In some embodiments, the selecting comprises selecting a subject confirmed to suffer from a cancer comprising GPX4 and / or RNF4 expressing cancer.

[0135] RNF4 expression can be determined by any method known in the art, for example using an anti-RNF4 antibody, as disclosed in US 11,407827.

[0136] In some embodiments, the method comprises receiving a biological sample from the subject. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample comprises cancer cells. In some embodiments, the biological sample comprises cells expressing GPX4 and / or RNF4. In some embodiments, the biological sample comprises cells expressing GPX4 and RNF4. In some embodiments, the method comprises measuring expression of GPX4 and / or RNF4 in the sample. In some embodiments, method comprises selecting a subject with a sample that comprises cancer cells that express GPX4 and / or RNF4. In some embodiments, method comprises selecting a subject with a sample that comprises cancer cells that express GPX4, RNF4 and VHL. In some embodiments, express GPX4 and / or RNF4 is express GPX4 and / or RNF4 above a predetermined threshold. In some embodiments, overexpression is expressing GPX4 above a predetermined threshold. In some embodiments, overexpression is expressing RNF4 above a predetermined threshold. In some embodiments, the threshold is GPX4 expression in control cells. In some embodiments, the threshold is RNF4 expression in control cells. In some embodiments, control cells are healthy cells. In some embodiments, control cells are non-cancerous cells. In some embodiments, control cells are non-cancerous cells from the same tissue or cell type as the cancerous cells. In some embodiments, the confirming comprises receiving the sample and confirming in the sample. In some embodiments, the method comprises receiving a report containing the subject’s cancer’s RNF4 and / or GPX4 expression and selecting a subject with a cancer comprising RNF4 and / or GPX4 expression. In some embodiments, confirming comprises receiving the report.

[0137] In some embodiments, there is provided a method for treating cancer or premalignancy condition in a subject in need thereof, the method comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of a compound of the invention and pharmaceutical acceptable carrier, thereby treating, ameliorating, reducing and / or preventing cancer, a proliferative disease selected from pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS), or pre-malignancy condition in a subject in need thereof. In some embodiments, the subject is further treated with an additional anticancer therapy such as chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery.

[0138] In one embodiment, the compound is the compound of Formula 5. In one embodiment, the compound is the compound of Formula 1 or any of the Formulae disclosed herein. In one embodiment, the compound of the present invention is provided to the subject per se. In one embodiment, one or more of the compound of the present invention are provided to the subject per se. In one embodiment, the compound of the present invention is provided to the subject as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. In one embodiment, one or more of the compound of the present invention are provided to the subject as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

[0139] In some embodiments, the disease associated with increased cell proliferation is cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, squamous cell carcinoma breast cancer, colorectal cancer, osteosarcoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer and hematological cancers.

[0140] As used herein "cancer" or "pre-malignancy" are diseases associated with cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma and germ cells tumors. In one embodiment, carcinoma refers to tumors derived from epithelial cells including but not limited to breast cancer, prostate cancer, lung cancer, pancreas cancer, and colon cancer. In one embodiment, sarcoma refers of tumors derived from mesenchymal cells including but not limited to sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas. In one embodiment, lymphoma refers to tumors derived from hematopoietic cells that leave the bone marrow and tend to mature in the lymph nodes including but not limited to Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma and immunoproliferative diseases. In one embodiment, leukemia refers to tumors derived from hematopoietic cells that leave the bone marrow and tend to mature in the blood including but not limited to acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia and adult T-cell leukemia. In one embodiment, blastoma refers to tumors derived from immature precursor cells or embryonic tissue including but not limited to hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma and glioblastoma-multiforme. In one embodiment, germ cell tumors refer to tumors derived from germ cells including but not limited to germinomatous or seminomatous germ cell tumors (GGCT, SGCT) andnongerminomatous or nonseminomatous germ cell tumors (NGGCT, NSGCT). In one embodiment, germinomatous or seminomatous tumors include but not limited to germinoma, dysgerminoma and seminoma. In one embodiment, non-germinomatous or non-seminomatous tumors refers to pure and mixed germ cells tumors including but not limited to embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, tearoom, polyembryoma, gonadoblastoma and teratocarcinoma.

[0141] In some embodiments, the present invention is directed to methods of prevention or treatment of a neural cancer disease. Non-limiting types of neural cancer include acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma, medulloblastoma, meningioma, metastatic brain tumor, primary brain lymphoma, spinal cord tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor, Schwannoma, juvenile pilocytic astrocytoma, pineal tumor and rhabdoid tumor. In one embodiment, astrocytoma refers to tumor derived from astrocytes including but not limited to grade I - pilocytic astrocytoma, grade II - low-grade astrocytoma, grade III - anaplastic astrocytoma and grade IV - glioblastoma. In one embodiment, other types of glioma include but not limited to brain stem glioma, ependymoma, mixed glioma, optic nerve glioma and subependymoma.

[0142] In some embodiments, As used herein, "cancer or pre-malignant cell proliferation" is a molecular process which requires the involvement of the RING finger protein 4 (RNF4). In some embodiments, cancer cell transition from benign to malignant is RNF4-dependent. In another embodiment, RNF4 (and further optionally GPX4) is an indicator of malignancy of tissues selected from, but not limited to, breast epithelium, skin cells, colorectal tissue, bone, and muscle tissue. In another embodiment, RNF4 is a biomarker correlating with poor patient prognosis.

[0143] In some embodiments, as known to one skilled in the art, malignancy-associated RNF4 is detected by an assay, including immune-assays, western-blot, immune-histochemistry, mRNA level, and the like.

[0144] The term "subject" as used herein refers to an animal, more particularly to nonhuman mammals and human organism. Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses. Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, and pig. In one embodiment, the subject is a human. Human subjects may also include fetuses. In one embodiment, a subject in need thereof isa subject afflicted with and / or at risk of being afflicted with a condition associated with increased cell proliferation.

[0145] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[0146] As used herein, the term “prevention” of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the presently described subject matter, the term "prevention" relates to a process of prophylaxis in which a subject is exposed to the presently described peptides prior to the induction or onset of the disease / disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease / disorder to be prevented. The term "suppression" is used to describe a condition wherein the disease / disorder process has already begun but obvious symptoms of the condition have yet to be realized. Thus, the cells of an individual may have the disease / disorder, but no outside signs of the disease / disorder have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.

[0147] As used herein, the term "condition" includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.Definitions

[0148] As used herein, the term "alkyl" describes an aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 21 to 100 carbon atoms, and more preferably 21-50 carbon atoms. Whenever a numerical range; e.g., “21-100”, is stated herein, it implies that the group, in this case the alkyl group, may contain 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, etc., up to and including 100 carbonatoms. In the context of the present invention, a "long alkyl" is an alkyl having at least 20 carbon atoms in its main chain (the longest path of continuous covalently attached atoms). A short alkyl therefore has 20 or less main-chain carbons. The alkyl can be substituted or unsubstituted, as defined herein.

[0149] The term "alkyl", as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl.

[0150] The term "alkenyl" describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[0151] The term "alkynyl", as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[0152] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring ( / .< ., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted, as indicated herein.

[0153] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted, as indicated herein.

[0154] The term "alkoxy" describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.

[0155] The term "aryloxy" describes an -O-aryl, as defined herein.

[0156] Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule. Additional substituents are also contemplated.

[0157] The term "halide", "halogen" or “halo” describes fluorine, chlorine, bromine or iodine.

[0158] The term “haloalkyl” describes an alkyl group as defined herein, further substituted by one or more halide(s).

[0159] The term “haloalkoxy” describes an alkoxy group as defined herein, further substituted by one or more halide(s).

[0160] The term “hydroxyl” or "hydroxy" describes a -OH group.

[0161] The term "thiohydroxy" or “thiol” describes a -SH group.

[0162] The term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group, as defined herein.

[0163] The term "thioaryloxy" describes both an -S-aryl and a -S-heteroaryl group, as defined herein.

[0164] The term “amine” describes a -NR’R’ ’ group, with R’ and R’ ’ as described herein.

[0165] The term "heteroaryl" describes a monocyclic or fused ring ( / .<?., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.

[0166] The term "heteroalicyclic" or "heterocyclyl" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.

[0167] The term "carboxy" or "carboxylate" describes a -C(=O)-OR' group, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon) as defined herein.

[0168] The term “carbonyl” describes a -C(=O)-R' group, where R' is as defined hereinabove.

[0169] The above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).

[0170] The term “thiocarbonyl” describes a -C(=S)-R' group, where R' is as defined hereinabove.

[0171] A "thiocarboxy" group describes a -C(=S)-OR' group, where R' is as defined herein.

[0172] A "sulfinyl" group describes an -S(=O)-R' group, where R' is as defined herein.

[0173] A "sulfonyl" or “sulfonate” group describes an -S(=O)2-R' group, where Rx is as defined herein.

[0174] A "carbamyl" or “carbamate” group describes an -OC(=O)-NR'R" group, where R' is as defined herein and R" is as defined for R'.

[0175] A "nitro" group refers to a -NO2 group.

[0176] A "cyano" or "nitrile" group refers to a -C=N group.

[0177] As used herein, the term “azide” refers to a -N3 group.

[0178] The term “sulfonamide” refers to a -S(=O)2-NR'R" group, with R' and R" as defined herein.

[0179] The term “phosphonyl” or “phosphonate” describes an -O-P(=O)(OR')2 group, with R' as defined hereinabove.

[0180] The term “phosphinyl” describes a -PR'R" group, with R' and R" as defined hereinabove.

[0181] The term “alkaryl” describes an alkyl, as defined herein, which substituted by an aryl, as described herein. An exemplary alkaryl is benzyl.

[0182] The term "heteroaryl" describes a monocyclic or fused ring ( / .<?., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.

[0183] As used herein, the terms "halo" and "halide", which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine or iodine, also referred to herein as fluoride, chloride, bromide and iodide.

[0184] The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide(s).General

[0185] As used herein the term “about” refers to ± 10 %.

[0186] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0187] The term “consisting of means “including and limited to”.

[0188] The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additionalingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0189] The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0190] The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

[0191] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0192] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0193] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0194] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0195] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical oraesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0196] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0197] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0198] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.Materials and MethodsSynthesis

[0199] The compounds of the invention were synthesized as described below.

[0200] Materials were purchased from commercial vendors and used without further purification. All reactions were generally carried out under inert atmosphere unless otherwise noted. TLC was performed on Merck Kieselgel 60 F254 plates, and spots were visualized under UV light. Products were purified by column chromatography on silica gel (100-200 mesh, Merck).TH and13C NMR spectra were recorded on Briiker ADVANCE 400 (400 MHz and 100 MHz), instrument using deuterated solvents as detailed and at ambient probe temperature (300 K). Chemical shifts are reported in parts per million (ppm) and are referred to the residual solvent peak. The following notations are used: singlet (s); doublet (d); triplet (t); quartet (q); multiplet (m); broad (br). Coupling constants are quoted in Hertz and are denoted as J.

[0201] Mass spectra were recorded on a Micromass® Q-Tof (ESI) spectrometer.

[0202] HPLC was performed on a Thermo instrument (Dionex Ultimate 3000) using analytical Thermo Scientific (Hypersil Gold, Cl 8, 3 pm, 4.6 * 150 mm) columns at flow rate of 1.2 ml / min. Preparative HPLC was performed on a Thermo Scientific instrument ultimate 3000 using Waters XSelect C18 (10pm, 19 x 250 mm) and semi preparative HPLCwas performed on a Thermo Scientific instrument (Spectra System SCM1000) using XBridge BEH300 C4 (10 pm, 150 x 10 mm) and Jupiter C4 (10 pm, 300 A, 250 x 10 mm) column, at flow rate of 15 and 4 mL / min S2 respectively. Buffer A: 0.1% TFA in water; buffer B: 0.1% TFA in acetonitrile. (Method: 0-60% B in 33 min).>

[0203] General procedure 1 for the synthesis of Amine 1-14: 4-(4-methoxyphenoxy) aniline (500 mg, 2.32 mmol) was dissolved in anhydrous DCM (10 mL) in a round bottom flask, aldehyde (2.32 mmol) was added and the solution was stirred at room temperature for 2h under nitrogen. Sodium triacetoxyborohydride (737.6 mg, 3.48 mmol) was added and the reaction mixture was stirred overnight. After completion, the reaction mixture was extracted with ethyl acetate. The organic layers were washed with brine and dried over anhydrous Na2SO4. After evaporation the crude residue was purified on a silica gel column (10-40% EtOAc / hexanes).

[0204] General procedure 2 for the synthesis of Ligand 1-14: To a solution of corresponding amine (0.5 mmol) in dry DCM (10 mL), 2-chloroacetyl chloride (67 mg, 0.6 mmol) and triethylamine (84 pL, 0.6 mmol) were added and the reaction mixture was stirred overnight at room temperature. After completion, the crude residue was purified by silica gel column (10-50% EtOAc / hexanes).

[0205] N-benzyl-4-(4-methoxyphenoxy)aniline (Amine 1): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500 mg, 2.32 mmol) and benzaldehyde(246.2 mg, 2.32 mmol) and reduction withsodium triacetoxyborohydride afforded Amine 1 (425 mg, 60%) NMR (400 MHz, CDCh): 87.38-7.37(m, 2H), 7.35- 7.33(m, 2H), m, 2H), 6.86(s, 1H), 6.84- 6.83(m, 2H), 6.81(s, 1H), 6.62- 6.60(m, , 3H).13C NMR (100 MHz, CDCh): 8 154.9, 152.3, 149.3, 144.3, .1, 118.9, 114.6, 113.8, 55.7, 49.0.loro-N-(4-(4-methoxyphenoxy)phenyl)acetamide (Ligand 1):Following the GP-2, the reaction of N-benzyl-4-(4- methoxyphenoxy)aniline (Amine 1) (152.7 mg, 0.5 mmol) with2-chloroacetyl chloride (67 mg, 0.6 mmol) afforded Ligand 1 (162 mg, 85%) as a colourless liquid. 'H NMR (400 MHz, CDCh): 87.31-7.30(m, 3H), 7.26-7.23(m, 2H), 7.03-7.01(m, 2H), 6.96-6.93(m, 4H), 6.90- 6.88(m, 2H), 4.90(s, 2H),3.91(s, 2H), 3.84(s, 3H).13C NMR (100 MHz, CDCh): 8 166.4, 158.9, 156.5, 148.8, 136.6, 134.7, 129.5, 129.0, 128.5, 127.7, 121.5, 117.8, 115.1, 55.6, 53.8, 42.2.

[0207] N-(2,4-dimethoxybenzyl)-4-(4-methoxyphenoxy)aniline (Amine 2): Followingafforded Amine 2 (600 mg, 71%) as a white solid. 'H NMR (400 MHz, CDCh): 6 7.21-7.19(m, 1H), 6.91- 6.89(m, 2H), 6.86(s, 1H), 6.84(s, 2H), 6.81(s, 1H), 6.63(s, 1H), 6.61(s, 1H), 6.49- 6.48(m, 1H), 6.46- 6.43(m, 1H). 4.22(s, 2H), 3.84(s, 3H), 3.80(s, 3H), 3.78(s, 3H).13C NMR (100 MHz, CDCh): 6 160.3, 158.5, 154.9, 152.4, 149.1, 144.6, 129.4, 120.1, 118.9, 114.6, 114.2, 103.9, 98.6, 55.7, 55.4, 55.4, 43.9.

[0208] 2-chloro-N-(2,4-dimethoxybenzyl)-N-(4-(4-methoxyphenoxy)phenyl)g, . g g, 82%) as a brown liquid. 'H NMR (400 MHz, CDCh): 67.13(d, 1H, J= 8.3), 6.93(s, 1H), 6.91(s, 2H), 6.88(s,lH), 6.86(s, 1H), 6.84(s, 1H), 6.80(s, 1H), 6.78(s, 1H), 6.36(dd, 1H, J = 8.3, 2.3), 6.29(d, 1H, J= 2.3), 4.82(s, 2H), 3.83(s, 2H), 3.74(s, 3H), 3.71(s, 3H), 3.52(s, 3H).13C NMR (100 MHz, CDCh): 6 166.3, 160.5, 158.6, 158.5, 156.4, 149.1, 134.9, 131.5, 129.6, 121.2, 117.6, 115.0, 104.2, 98.2, 55.6, 55.3, 55.1, 47.7, 42.2.

[0209] 4-(((4-(4-methoxyphenoxy)phenyl)amino)methyl)-N,N-dimethylaniline (Amine 3): Following the GP-1, the reaction of 4-(4- methoxyphenoxy)aniline (500 mg, 2.32 mmol) and 4- (Dimethylamino)benzaldehyde (346.1 mg, 2.32 mmol) andreduction with sodium triacetoxyborohydride afforded Amine 3 (566 mg, 70%) as a white solid. 'H NMR (400 MHz, CDCh): 6 7.24(s, 1H), 6.91(s, 1H), 6.89- 6.88(s, 1H), 6.86(s, 1H), 6.84- 6.81(m, 3H), 6.74(s, 1H), 6.72(s, 2H), 6.62- 6.60(m, 2H), 4.17(s, 2H), 3.77(s, 3H), 2.94(s, 6H).13C NMR (100 MHz, CDCh): 8 154.9, 152.4, 149.0, 144.6, 128.8, 120.2, 118.9, 114.6, 113.8, 112.9, 112.8, 55.7, 48.6, 40.8.

[0210] 2-chloro-N-(4-(dimethylamino)benzyl)-N-(4-(4-methoxyphenoxy)phenyl)acetamide (Ligand 3): Following the GP-2, the reaction of 4 (((4-(4-methoxyphenoxy)phenyl)amino)methyl)-N,N-dimethyl aniline (Amine 3) (174 mg, 0.5 mmol) with 2-chloroacetylchloride (67 mg, 0.6 mmol) afforded Ligand 3 (170 mg, 80%) as a brown solid.XH NMR (400 MHz, CDCh): 67.06(s, 1H), 7.03(s,lH), 6.99(s, 1H), 6.97(s, 1H), 6.90(s, 2H), 6.88(s, 2H), 6.85(s, 1H), 6.83(s, 1H), 6.62(s, 1H), 6.60(s, 1H). 4.75(s, 2H), 3.84(s, 2H), 3.79(s, 3H), 2.90(s, 6H).13C NMR (100 MHz, CDCh): 6 166.2, 158.8, 156.5, 150.0, 149.0, 134.8, 130.2, 129.7, 124.5, 121.5, 117.8, 115.0, 112.5, 55.7, 53.3, 42.3, 40.6.

[0211] N,N-diethyl-4-(((4-(4-methoxyphenoxy)phenyl)amino)methyl)aniline (Amine 4): Following the GP-1, the reaction of 4-(4- methoxyphenoxy)aniline (500 mg, 2.32 mmol) and 4- (Diethylamino)benzaldehyde (411.2 mg, 2.32 mmol) and reductionwith sodium triacetoxyborohydride afforded Amine 4 (570 mg, 65%) as a brown solid. 'H NMR (400 MHz, CDCh): 87.23- 7.21(m, 2H), 6.91- 6.89(m, 2H), 6.87- 6.85(m, 2H), 6.83-6.80(m, 2H), 6.68- 6.65(m, 2H), 6.63- 6.61(m, 2H), 4.15(s, 2H), 3.78(s, 3H), 3.36- 3.32(m, 4H), 1.18- 1.14(m, 6H).13C NMR (100 MHz, CDCh): 6 154.9, 152.4, 149.0, 147.2, 144.7, 129.1, 128.0, 120.2, 118.8, 114.6, 113.7, 111.9, 55.7, 48.6, 44.4, 12.6.

[0212] 2-chloro-N-(4-(diethylamino)benzyl)-N-(4-(4-methoxyphenoxy)phenyl)acetamide (Ligand 4): Following the GP-2, the reaction of N,N- diethyl-4-(((4-(4-methoxyphenoxy)phenyl)amino)methyl)aniline (Amine 4) (188.3 mg, 0.5 mmol) with 2-chloroacetyl chloride (67mg, 0.6 mmol) afforded Ligand 4 (193 mg, 75%) as a white solid. 'H NMR (400 MHz, CDCh): 8 7.03(s, 1H), 7.00(s, 2H), 6.98(s, 1H), 6.92-6.89(m, 4H), 6.87-6.85(m, 2H), 6.56-6.54(m, 2H), 4.73(s, 2H), 3.83(s, 2H), 3.81(s, 3H), 3.34-3.29(m, 4H), 1.13(t, 6H, J = 6.8).13C NMR (100 MHz, CDCh): 8 166.0, 158.7, 156.5, 149.0, 147.3, 135.0, 130.4, 129.7, 123.1, 121.4, 117.8, 115.0, 111.6, 55.7, 53.4, 44.3, 42.3, 12.5.

[0213] 4-(4-methoxyphenoxy)-N-(4-methylbenzyl)aniline (Amine 5): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500 mg, 2.32 mmol) and 4-Methylbenzaldehyde (278.8 mg, 2.32mmol) and reduction with sodium triacetoxyborohydride afforded Amine 5 (600 mg, 81%) as a white solid. 'H NMR (400 MHz, CDCh): 8 7.28-7.26(m, 2H), 7.17- 7.15(m, 2H), 6.91- 6.88(m, 2H), 6.86- 6.81(m, 4H), 6.61- 6.59(m, 2H),4.26(s, 2H), 3.78(s, 3H), 2.35(s, 3H).13C NMR (100 MHz, CDCh): 6 154.9, 152.3, 149.2, 144.4, 136.9, 136.3, 129.3, 127.6, 120.1, 118.9, 114.7, 113.8, 55.7, 48.7, 21.1.

[0214] 2-chloro-N-(4-(4-methoxyphenoxy)phenyl)-N-(4-methylbenzyl)acetamide (Ligand 5): Following the GP-2, the reaction of 4-(4- methoxyphenoxy)-N-(4-methylbenzyl)aniline (Amine 5) (159.7mg, 0.5 mmol) with 2-chloroacetyl chloride (67 mg, 0.6 mmol) afforded Ligand 5 (164 mg, 83%) as a colourless liquid. 'H NMR (400 MHz, CDCh): 6 6.97- 6.96(m, 4H), 6.88(s, 1H), 6.86(s, 1H), 6.81(s, 1H), 6.79-6.78(m, 2H), 6.77(s, 1H), 6.75(s, 1H), 6.73(s, 1H), 4.81(s, 1H), 3.86(s, 2H), 3.79(s, 3H), 2.30(s, 3H).13C NMR (100 MHz, CDCh): 6 166.3, 158.9, 156.5, 148.9, 137.3, 134.7, 133.7, 129.6, 129.2, 129.0, 121.5, 117.8, 115.1, 55.7, 53.5, 42.2, 21.2.

[0215] N-(4-(tert-butyl)benzyl)-4-(4-methoxyphenoxy) aniline (Amine 6): Following the GP-1, the reaction of 4-(4- methoxyphenoxy)aniline (500 mg, 2.32 mmol) and 4-tert-Butylbenzaldehyde (376.4 mg, 2.32 mmol) and reduction with sodium triacetoxyborohydride afforded Amine 6 (750 mg, 89%) as a white solid. 'H NMR (400 MHz, CDCh): 87.39- 7.37(m, 2H), 7.33- 7.30(m, 2H), 6.91- 6.89(m, 2H), 6.87(s, 1H), 6.85- 6.84(m, 2H), 6.81(s, 1H), 6.63- 6.60(m, 2H), 4.26(s, 2H), 3.78(s, 3H), 1.33(s, 9H).13C NMR (100 MHz, CDCh): 6 154.9, 152.3, 150.3, 149.2, 144.4, 136.3, 127.4, 125.6, 120.1, 118.9, 114.6, 113.8, 55.7, 48.6, 34.5, 31.4.

[0216] N-(4-(tert-butyl)benzyl)-2-chloro-N-(4-(4-methoxyphenoxy)phenyl)acetamide (Ligand 6): Following the GP-2, the reaction of N-(4- (tert-butyl)benzyl)-4-(4-methoxyphenoxy)aniline (Amine 6) (180.6 mg, 0.5 mmol) with 2-chloroacetyl chloride (67 mg, 0.6mmol) afforded Ligand 6 (171 mg, 78%) as a white solid. 'H NMR (400 MHz, CDCh): 67.30(s, 1H), 7.28(s, 1H), 7.15(s, 1H), 7.12(s, 1H), 7.01(s, 1H), 6.98(s, 1H), 6.95(s, 1H), 6.93(s, 1H), 6.92(s, 1H), 6.89(s, 1H), 6.87(s, 1H), 6.85(s, 1H), 4.82(s, 2H), 3.86(s, 2H), 3.81(s, 3H), 1.29(s, 9H).13C NMR (100 MHz, CDCh): 6 166.4, 158.9, 156.5, 150.6, 149.0, 135.0, 133.5, 129.6, 128.7, 125.4, 121.5, 117.8, 115.0, 55.7, 53.6, 42.1, 34.5, 31.3.

[0217] 4-(4-methoxyphenoxy)-N-(4-(piperidin-l-yl)benzyl)aniline (Amine 7):Following the GP-1, the reaction of 4-(4- methoxyphenoxy)aniline (500 mg, 2.32 mmol) and 4-(l- Piperidinyl)benzaldehyde (439 mg, 2.32 mmol) and reductionwith sodium triacetoxyborohydride afforded Amine 7 (681 mg, 76%) as a white solid.XH NMR (400 MHz, CDCh): 67.3 l(s, 2H), 6.98(s, 4H), 6.91(s, 4H), 6.68(s, 2H), 4.25(s, 2H), 3.84(s, 3H), 3.22(s, 4H), 1.78(s, 4H), 1.65(s, 2H).13C NMR (100 MHz, CDCh): 6 154.9, 152.4, 151.6, 149.1, 144.6, 129.7, 128.6, 120.2, 118.9, 116.6, 114.7, 113.8, 55.7, 50.8, 48.5, 25.9, 24.4.

[0218] 2-chloro-N-(4-(4-methoxyphenoxy)phenyl)-N-(4-(piperidin-l-yl)benzyl)7.04(s, 1H), 6.99(s, 1H), 6.97(s, 1H), 6.90(s, 1H), 6.89-6.86(m, 2H), 6.84 (s, 2H), 6.83- 6.82(m, 2H), 6.79(s, 1H), 4.75(s, 2H), 3.83(s, 2H), 3.79(s, 3H), 3.1 l(t, 4H, J= 5.3), 1.69-1.65(m, 4H), 1.56-1.55(m, 2H).13C NMR (100 MHz, CDCh): 6 166.18, 158.8, 156.5, 151.6, 148.9, 134.8, 130.0, 129.7, 127.0, 121.4, 117.8, 116.1, 115.0, 55.7, 53.3, 50.4, 42.3, 25.8, 24.3.

[0219] 4-(4-methoxyphenoxy)-N-(oxazol-4-ylmethyl)aniline (Amine 8): Following2.32 mmol) and reduction with sodium triacetoxyborohydride afforded Amine 8 (516 mg, 75%) as a brown solid.XHNMR (400 MHz, CDCh): 87.87(s, 1H), 7.58(s, 1H), 6.90- 6.81(m, 6H), 6.64- 6.61(m, 2H), 4.26(s, 2H), 3.77(s, 3H).13C NMR (100 MHz, CDCh): 6 155.1, 152.1, 151.3, 149.8, 143.6, 138.4, 135.5, 119.9, 119.1, 114.7, 114.3, 55.7, 40.9.

[0220] 2-chloro-N-(4-(4-methoxyphenoxy)phenyl)-N-(oxazol-4-ylmethyl)acetamide (Ligand 8): Following the GP-2, the reaction of 4-(4- methoxyphenoxy)-N-(oxazol-4-ylmethyl)aniline (Amine8) (148.2 mg, 0.5 mmol) with 2-chloroacetyl chloride (67 mg, 0.6 mmol) afforded Ligand 8 (140 mg, 75%) as a white solid. 'H NMR (400 MHz, CDCh): 6 7.78(s, 1H), 7.63(s, 1H), 7.11-7.09(m, 2H), 6.97-6.95(m, 2H), 6.88-6.87(m, 4H), 4.72(s,2H), 3.83(s, 2H), 3.76(s, 3H).13C NMR (100 MHz, CDCh): 6 166.4, 159.0, 156.5, 150.9, 148.8, 137.6, 135.7, 135.0, 129.4, 121.5, 117.9, 115.1, 55.7, 45.7, 42.0.

[0221] 4-(4-methoxyphenoxy)-N-(thiazol-2-ylmethyl)aniline (Amine 9): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500 mg, 2.32 mmol) and 5-Thiazolecarboxaldehyde (265.5 mg,2.32 mmol) and reduction with sodium triacetoxyborohydride afforded Amine 9 (550 mg, 76%) as a white solid. 'H NMR (400 MHz, CDCh): 6 8.73(s, 1H), 7.81(s, 1H), 6.91- 6.82(m, 6H), 6.64- 6.62(m, 2H), 4.54(s, 2H), 3.78(s, 3H).13C NMR (100 MHz, CDCh): 6 155.1, 152.9, 151.9, 150.3, 143.0, 141.1, 120.0, 119.2, 114.7, 114.5, 55.7, 41.5.

[0222] 2-chloro-N-(4-(4-methoxyphenoxy)phenyl)-N-(thiazol-2-ylmethyl)acetamide (Ligand 9): Following the GP-2, the reaction of 4-(4- methoxyphenoxy)-N-(thiazol-2-ylmethyl)aniline (Amine9) (156.2 mg, 0.5 mmol) with 2-chloroacetyl chloride (67 mg, 0.6 mmol) afforded Ligand 9 (181 mg, 93%) as a white solid.XH NMR (400 MHz, CDCh): 8 8.71(s, 1H), 7.56(s, 1H), 6.97-6.94 (m, 2H), 6.91(s, 1H), 6.88-6.86(m, 4H), 4.97(s, 2H), 3.81(s, 2H), 3.76(s, 1H).13C NMR (100 MHz, CDCh): 8 166.4, 159.4, 156.6, 154.5, 148.7, 143.4, 133.8, 132.9, 129.4, 121.6, 118.0, 115.1, 55.7, 45.8, 41.8.

[0223] N-(cyclopentylmethyl)-4-(4-methoxyphenoxy)aniline (Amine 10): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500 mg, 2.32 mmol) and cyclopentanecarboxaldehyde (227.7 mg, 2.32mmol) and reduction with sodium triacetoxyborohydride afforded Amine 10 (550 mg, 80%) as a white solid. 'H NMR (400 MHz, CDCh): 66.90- 6.81(m, 6H), 6.59- 6.57(m,2H), 3.78(s, 3H), 3.01- 2.99(m, 2H), 2.17- 2.13(m, 1H), 1.84-1.82(m, 2H), 1.65- 1.57(m, 4H), 1.29- 1.24(m, 2H).13C NMR (100 MHz, CDCh): 8 154.9, 152.5, 148.9, 144.9, 120.2, 118.8, 114.6, 113.6, 55.7, 50.2, 39.5, 30.7, 25.3.

[0224] 2-chloro-N-(cyclopentylmethyl)-N-(4-(4-methoxyphenoxy)phenyl)acetamide. , . y , 0.6 mmol) afforded Ligand 10 (181 mg, 97%) as a colourless liquid.XH NMR (400 MHz, CDCh): 8 7.12(s, 1H), 7.09(s, 1H), 7.00(s, 1H), 6.98(s, 1H), 6.93(s, 1H), 6.91(s, 1H), 6.90(s, 1H), 6.88(s, 1H), 3.80(s, 2H), 3.78(s, 3H), 3.64(d, 2H, J = 7.7), 2.05-1.97(m, 1H), 1.62-1.60(m, 4H), 1.48-1.45(m, 2H), 1.26-1.20(m, 2H).13C NMR (100 MHz, CDCh): 8166.4, 158.8, 156.5, 148.9, 135.0, 129.3, 121.5, 117.9, 115.0, 55.6, 54.3, 42.2, 37.8, 30.2, 25.2.

[0225] N-(cyclopropylmethyl)-4-(4-methoxyphenoxy)aniline(Amine 11): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500mg, 2.32 mmol) and cyclopropanecarboxaldehyde (162.6Hmg, 2.32 mmol) and reduction with sodium triacetoxyborohydride afforded Amine 11 (400 mg, 64%) as a white solid. 'H NMR (400 MHz, CDCh): 86.91(s, 1H), 6.88(s, 1H), 6.87(s, 1H), 6.85- 6.83(m, 2H), 6.81(s, 1H), 6.60- 6.57(m, 2H), 3.78(s, 3H), 2.93(d, 2H, J= 6.9), 1.14- 1.06(m, 1H), 0.56- 0.54(m, 2H), 0.25- 0.23(m, 2H).13C NMR (100 MHz, CDCh): 6 154.9, 152.4, 149.0, 144.7, 120.2, 118.8, 114.6, 113.8, 55.7, 49.8, 10.9, 3.5.

[0226] 2-chloro-N-(cyclopropylmethyl)-N-(4-(4-methoxyphenoxy)phenyl)acetamide (Ligand 11): Following the GP-2, the reaction of Amine11 (134.7 mg, 0.5 mmol) with 2-chloroacetyl chloride (67 mg, o -cl0.6 mmol) afforded Ligand 11 (116 mg, 67%) as a brown liquid.3H NMR (400 MHz, CDCh): 6 7.15(s, 1H), 7.12(s, 1H), 6.98(s, 1H), 6.96(s, 1H), 6.92(s, 1H), 6.90(s, 1H), 6.88(s, 1H), 6.86(s, 1H), 3.80(s, 2H), 3.76(s, 3H), 3.52-3.50(m, 2H), 0.93-0.89(m, 1H), 0.40-0.39(m, 2H), 0.11-0.10(m, 2H).13C NMR (100 MHz, CDCh): 8 166.0, 158.8, 156.5, 148.9, 135.1, 129.7, 121.4, 117.9, 115.0, 55.6, 54.3, 42.2, 9.6, 3.7.

[0227] 4-(4-methoxyphenoxy)-N-neopentylaniline (Amine 12): Following the GP-1,mmol) afforded Ligand 12 (172 mg, 95%) as a colourless liquid.XH NMR (400 MHz, CDCh): 8 7.17(s, 1H), 7.15(s, 1H), 6.99(s, 1H), 6.97(s, 1H), 6.91(s, 1H), 6.89(s, 2H), 6.87(s, 1H), 3.85(s, 2H), 3.78(s, 1H), 3.62(s, 1H), 0.85(s, 9H).13C NMR (100 MHz,CDCh): 6 167.1, 158.3, 156.4, 149.0, 137.2, 129.1, 121.3, 117.8, 115.0, 61.2, 55.6, 42.3, 34.0, 28.4.

[0229] N-isobutyl-4-(4-methoxyphenoxy)aniline (Amine 13): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500 mg, 2.32 mmol)and isobutyraldehyde (167.3 mg, 2.32 mmol) and reduction with sodium triacetoxyborohydride afforded Amine 13 (450 mg, 76%) as a white solid. 'H NMR (400 MHz, CDCh): 66.91- 6.88(m, 3H), 6.86(s, 1H), 6.85- 6.81(m, 3H), 6.58- 6.56(m, 2H), 3.78(s, 3H), 2.91(d, 2H, J= 6.7), 1.92- 1.85(m, 1H), 1.00- 0.98(m, 6H).13C NMR (100 MHz, CDCh): 6 154.9, 152.5, 148.8, 144.8, 120.2, 118.8, 114.6, 113.6, 55.7, 52.5, 28.1, 20.5.

[0230] 2-chloro-N-isobutyl-N-(4-(4-methoxyphenoxy)phenyl)acetamide (Ligand 13):Following the GP-2, the reaction of N-isobutyl-4-(4- methoxyphenoxy)aniline (Amine 13) (128.6 mg, 0.5 mmol)with 2-chloroacetyl chloride (67 mg, 0.6 mmol) afforded Ligand 13 (137 mg, 79%) as a colourless liquid.XH NMR (400 MHz, CDCh): 6 7.1 l(s, 1H), 7.09(s, 1H), 6.99(s, 1H), 6.96(s, 1H), 6.92(s, 1H), 6.90(s, 1H), 6.89(s, 1H), 6.88(s, 1H), 3.80(s, 2H), 3.77(s, 3H), 3.52(s, 1H), 3.50(s, 1H), 1.78-1.71(m, 1H), 0.88-0.86(m, 6H).13C NMR (100 MHz, CDCh): 6 166.7, 158.8, 156.5, 148.9, 135.2, 129.2, 121.5, 117.9, 115.0, 56.9, 55.6, 42.1, 26.6, 19.9.

[0231] 4-(4-methoxyphenoxy)-N-propylaniline (Amine 14): Following the GP-1, the reaction of 4-(4-methoxyphenoxy)aniline (500 mg, 2.32 mmol) and propionaldehyde (134.7 mg, 2.32 mmol)and reduction with sodium triacetoxyborohydride afforded Amine 14 (430 mg, 72%) as a white solid. 'H NMR (400 MHz, CDCh): 66.91-6.88(m, 2H), 6.87(s, 1H), 6.85- 6.81(m, 3H), 6.59- 6.57(m, 2H), 3.78(s, 3H), 3.06(t, 2H, J = 7), 1.67- 1.61(m, 2H), 1.00(t, 2H, J= 7.4).13C NMR (100 MHz, CDCh): 6 154.9, 152.4, 148.9, 144.7, 120.2, 118.8, 114.6, 113.7, 55.7, 46.5, 22.8, 11.7.

[0232] 2-chloro-N-(4-(4-methoxyphenoxy)phenyl)-N-propylacetamide (Ligand 14):chloroacetyl chloride (67 mg, 0.6 mmol) afforded Ligand 14 (160 mg, 96%) as a brown liquid.rH NMR (400 MHz, CDCh): 6 7.08(s, 1H), 7.06(s, 1H), 6.96(s, 1H), 6.94(s, 1H), 6.91(s, 1H), 6.88(s, 1H), 6.86(s, 1H), 6.84(s, 1H), 3.77(s, 2H), 3.76(s, 3H), 3.58(t, 2H, J= 6.0), 1.51-1.45(m, 2H), 0.83(t, 3H, J= 7.3).13C NMR(100 MHz, CDCh): 6 166.2, 158.8, 156.5, 148.9, 135.0, 129.3, 121.4, 117.9, 115.0, 55.6, 51.6, 42.0, 20.6, 11.1.

[0233] Synthesis of N-benzyl-N-(4-(4-methoxyphenoxy)phenyl)acetamide (inactive Ligand):

[0234] To a solution of N-benzyl-4-(4-methoxyphenoxy)aniline (amine 1)(152.7 mg, 0.5 mmol) in dry DCM (10 mL) acetyl chloride (39 mg, 0.6 mmol) and triethylamine (84 pL, 0.6 mmol) were added and the reaction mixture was stirred overnight at room temperature. After completion, the crude residue was purified by silica gel column (1 :4 EtOAc / hexanes) to afford inactive Ligand (149 mg, 86%) as a colourless oil^HNMR (400 MHz, CDCh): 8 7.26-7.21(m, 5H), 6.99-6.97(m, 2H), 6.90-6.85(m, 6H), 4.85(s, 2H), 3.80(s, 3H), 1.90(s, 3H). °CNMR(100MHz, CDCh): 6170.6, 158.1, 156.3, 149.2, 137.5, 137.0, 129.4, 128.8, 128.3, 127.3, 121.2, 117.7, 115.0, 55.6, 52.8, 22.7.

[0235] Synthesis of 4-(((R)-l-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5 yl) benzyl) carbamoyl) pyrrolidin-l-yl)-3,3-dimethyl-l-oxobutan-2-yl)amino)-4-oxobutanoic acid (VHL acid):

[0236] a solution of VHL ligand hydrochloride (250 mg, 0.53mmol) in DMF(5 mL), HATU (244 mg, 0.64 mmol) and 4-(tert-butoxy)-4-oxobutanoic acid(112 mg, 0.64mmol) were added and stirred for 5 min at room temperature. To this, DIEA (277pL, 1.6 mmol) was added and continued the reaction for 2 h. After 2 h, the reaction mixture was diluted with ethyl acetate and ice-cold water. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. To the solution of crude product in DCM(5mL), TFA(5mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Finally, the dried crude product was kept under high vacuum for overnight. The crude product was further purified using preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system. The pure product VHL acid (127mg,45%) was obtained as a white solid after the lyophilization. 'H NMR (400 MHz, D6-DMSO): 39.06 (s, 1H), 8.64 (t, 1H, J= 6.0), 8.00 (d, 1H, J= 9.2), 7.49-7.42 (m, 4H), 4.59 (d, 1H, J= 9.2), 4.52-4.46 (m, 2H), 4.40 (broad, 1H), 4.29-4.24 (m, 1H), 3.74-3.66 (m, 2H), 2.56-2.55 (m, 4H), 2.46-2.38 (m, 3H), 2.15-2.06(m, 1H), 1.98-1.92(m, 1H), 0.98 (s, 9H).13C NMR (400 MHz, D6-DMSO): 3 174.3, 172.4, 171.3, 170.0, 159.0, 158.6, 152.0, 147.9, 140.0, 129.1, 127.9, 117.1, 114.2, 69.3, 59.1, 56.9, 42.1, 38.3, 35.8, 30.2, 29.7, 26.8, 16.3. HRMS (m / z): [M+H]+calcd. for C26H35N4O6S, 531.2277; found 531.2277.

[0237] Synthesis of Nl-(4-(4-(4-(N-benzyl-2-chloroacetamido)phenoxy)phenoxy) butyl) -N4-((R)-l-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl) pyrrolidin-l-yl)-3,3-dimethyl-l-oxobutan-2-yl)succinimide (R4VP):

[0238] To a solution of CCW 24(120.9 mg, 0.227 mmol) in DCM(5mL), TFA (5 mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF (5 mL), VHL acid (100 mg, 0.227 mmol), HATU (95 mg, 0.250 mmol) and DIEA (160 pL, 0.908 mmol) were added and continued the reaction for 2h at room temperature and was monitored by TLC. The reaction mixture was diluted with cold water and ethyl acetate. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system with a reverse phase C18 column to afford the R4VP (78.1 mg, 36%) as white solid. 'H NMR (400 MHz, CDCh): 39.00 (s, 1H), 7.39 (t, 1H, J = 6.0 ), 7.29 (q, 4H, J = 13.2), 7.20-7.18 (m, 4H), 7.13-7.10 (m, 2H), 6.90-6.88 (m, 2H), 6.84-6.76 (m, 6H), 6.24 (t, 1H, J= 4.8), 4.92 (s, 1H), 4.78 (s, 2H), 4.62 (t, 1H, J= 8.0), 4.48-4.44 (m, 2H), 4.39-4.37 (m, 1H), 4.30-4.25 (m, 1H), 3.98 (d, 1H, J= 11.6), 3.87 (t, 2H, J= 6.0), 3.80 (s, 2H), 3.57-3.53 (m, 1H), 3.22-3.19 (m, 2H), 2.47-2.30 (m, 8H), 2.14-2.08 (m, 1H), 1.73-1.69 (m, 2H), 1.62-1.58 (m, 2H), 0.89 (s, 9H).13C NMR (100 MHz, CDCh): 3172.3, 171.7, 170.8, 170.2, 170.1, 165.7, 159.4, 159.0, 157.9, 154.7, 151.1, 148.0, 144.6, 138.1, 135.4, 133.6, 132.5, 128.5, 128.4, 128.0, 128.0, 127.5, 127.2, 126.7, 120.5,116.8, 114.6, 69.2, 66.8, 57.8, 57.4, 55.8, 52.9, 42.1, 40.9, 38.5, 35.4, 33.9, 30.2, 30.1, 28.7, 25.5, 25.3, 25.1, 13.5. HRMS (m / z): calcd. for CsiHeoCINeOsS, 951.3882; found 951.3876.

[0239] Preparation of tert-butyl (4-(4-(4-(N-benzylacetamido)phenoxy) phenoxy)butyl)carbamate (1): To a solution of CCW 22 (200mg, 0.43 mmol) in DCM(5mL), acetyl chloride (40.7 mg, 0.52mmol) and triethylamine (Sigma, mmol) were added and the reaction mixture was stirred for overnight. Upon reaction completion the crude was purified by silica gel chromatography (30% EtOAc / hexanes) to yield 180 mg (82.5 %). 'H NMR (400 MHz, CDCh): 6 7.23- 7.16(m, 5H), 6.96-6.92(m, 2H), 6.87-6.80(m, 6H), 4.82(s, 4H), 4.51(s, 2H), 3.92(t, 2H, J= 6.8), 3.17- 3.16(m, 2H), 1.78(t, 2H, J = 6.8), 1.68-1.62(m, 2H), 1.41(s, 9H).13C NMR (100 MHz, CDCh): 6 170.5, 158.1, 157.7, 156.0, 155.6, 154.3, 150.1, 149.0, 129.4, 129.3, 128.7, 128.3, 127.3, 121.2, 117.9, 117.6, 115.7, 115.5, 73.4, 67.8, 52.7, 40.2, 28.4, 26.5, 22.6. HRMS (m / z): [M+H]+calcd. for C3OH37N205, 505.2702; found: 505.2670.

[0240] Preparation of Nl-(4-(4-(4-(N-benzylacetamido)phenoxy)phenoxy)butyl)-N4-((R)-l-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl) pyrrolidin-l-yl)-3,3-dimethyl-l-oxobutan-2-yl)succinimide (inactive PROTAC):

[0241] To the solution of tert-butyl (4-(4-(4-(N-benzylacetamido)phenoxy)phenoxy)butyl)carbamate (l)(100 mg, 0.2 mmol) in DCM(5mL), TFA(5mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF(5mL), VHL-acid (106 mg, 0.2 mol), HATU (91 mg, 0.24 mmol) and DIPEA (108pl, 0.6 mmol) were added and continued the reaction for 2h at room temperature and was monitored by TLC. The reaction mixture was diluted withcold water and ethyl acetate. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system with a reverse phase C18 column to afford the inactive PROTAC (92 mg, 50%) as white solid.XH NMR (400 MHz, CDCh): 69.03(s, 1H), 7.31- 7.26(m, 4H), 7.18(sbr, 4H), 7.11- 7.09(m, 2H), 6.89- 6.87(2H), 6.79- 6.74(m, 5H), 6.20(s, 1H), 4.77(s, 3H), 4.53- 4.44(m, 3H), 4.38- 4.36(m, 1H), 4.29- 4.24(m, 2H), 4.00- 3.97(m, 1H), 3.87- 3.84(m, 2H), 3.56- 3.54(m, 1H), 3.21- 3.20(m, 2H), 2.47(s, 6H), 2.41- 2.3 l(m, 2H), 1.84(s, 3H), 1.72- 1.68(m, 2H), 1.63- 1.58(m, 2H), 0.88(s, 9H).13C NMR (400 MHz, CDCh): 6 173.4, 172.8, 172.0, 171.8, 171.2, 158.4, 155.6, 152.2, 149.2, 139.2, 136.8, 136.3, 129.4, 129.2, 128.9, 128.5, 128.3, 127.6, 121.4, 117.8, 115.6, 70.2, 67.9, 58.8, 58.4, 56.8, 53.3, 43.2, 39.6, 36.4, 34.9, 31.2, 31.1, 26.6, 26.3, 26.1, 22.3, 14.3. HRMS (m / z): [M+H]+calcd. for CsiHeiNeOsS, 917.4272; found: 917.4265.

[0242] Preparation of L3 PROTAC:Preparation of 4-(4-((4-(dimethylamino)benzyl)amino)phenoxy)phenol (3):

[0243] To a slution of 4-(4-aminophenoxy) phenol (302 mg, 1.5 mmol) in 5 mL DCM, 4 (dimethylamino)benzaldehyde (224 mg, 1.5 mmol) was added and the solution was stirred at room temperature for 2h under nitrogen. Sodium triacetoxyborohydride (477 mg, 2.25 mmol) was added and the reaction mixture was stirred overnight. After completion, the reaction mixture was extracted with ethyl acetate. The organic layers were washed with brine and dried over anhydrous Na2SO4. After evaporation the crude residue was purified on a silica gel column (40% EtOAc / hexanes) and compound 3(421 mg, 84%) was obtained as yellow liquid. (1H NMR (400 MHz, CDCh): 8 7.30- 7.28(m, 2H), 6.91- 6.85(m, 4H), 6.79- 6.76(m, 4H), 6.67- 6.65(m, 2H), 4.22(s, 2H), 2.99(s, 6H).13C NMR (100 MHz, CDCh): 6152.3, 150.9, 150.1, 149.2, 144.5, 129.4, 128.8, 127.1, 120.2, 119.0, 116.6, 116.1, 114.0, 113.0, 48.7, 40.8.Preparation of tert-butyl (4-(4-(4-((4 (dimethylamino)benzyl)amino)phenoxy)phenoxy)butyl) carbamate (4):

[0244] To a solution of 3(679 mg, 2 mmol) in 10 mL Acetone tert-butyl (4-bromobutyl) carbamate(756 mg, 3 mmol), potassium carbonate(622 mg, 4.5 mmol) were added and the solution was put in reflux for 72h. After that the mixture was concentrated and purified by a silica gel column (30% EtOAc / hexanes) and compound 4(435 mg, 43%) was obtained as yellow liquid. 'H NMR (400 MHz, CDCh): 8 7.23(s, 1H), 7.1 l(d, 1H, J =8.6), 6.89-6.88(m, 1H), 6.87-6.86(m, 2H), 6.84-6.83(m, 1H), 6.82-6.80(m, 2H), 6.79-6.78(m, 1H), 6.74-6.69(m, 2H), 6.62-6.60(m, 1H), 4.48(s, 2H), 3.94-3.91(m, 2H), 3.18(t, 2H, J= 6.3), 2.94-2.92(m, 6H), 1.81-1.76(m, 2H), 1.67-1.63(m, 2H), 1.44(s, 9H).13C NMR (100 MHz, CDCh): 6156.0, 154.2, 152.4, 150.0, 149.7, 149.0, 144.6, 128.7, 127.8, 120.2, 118.8, 115.3, 113.7, 112.8, 79.1, 68.0, 53.9, 53.4, 48.5, 40.7, 28.4, 26.6. HRMS (m / z): [M+H] + calcd. for C30H40N3O4, 506.3019; found: 506.3007.

[0245] Preparation of tert-butyl (4-(4-(4-(2-chloro-N-(4- (dimethylamino)benzyl)acetamido) phenoxy)phenoxy)butyl) carbamate (5):

[0246] To a solution of 4 (101 mg, 0.2 mmol), chloro acetyl chloride (27 mg, 0.24 mmol), Triethyl amine (24 mg, 0.24 mmol) were added and the reaction mixture was stirred for overnight. Upon reaction completion the crude was purified by silica gel chromatography (2% MeOH / DCM) to give compound 5(99 mg, 85%) as yellow liquid.1H NMR (400 MHz, CDCh): 67.06-7.03(m,2H), 6.98-6.96(m, 2H), 6.89-6.83(m, 6H), 6.62-6.60(m, 2H), 4.75(s, 2H), 3.98(t, 2H, J=6.1), 2.92(s, 6H), 1.84-1.80(m, 2H), 1.71-1.65(m, 2H), 1.44(s, 9H).13C NMR (100 MHz, CDCh): 6 166.1, 156.0, 155.8, 150.0, 148.9, 134.8, 130.2, 129.7, 124.3, 121.4, 117.7, 115.6, 112.3, 79.2, 67.9, 53.3, 42.2, 40.5, 40.2, 28.4, 26.8, 26.6. HRMS (m / z):[M+H] + calcd. for C32H41CIN3O5, 582.2735; found: 582.2744.Preparation of N l-(4-(4-(4-(2-chloro-N-(4-(dimethylamino)benzyl)acetamido) phenoxy)phenoxy) butyl)-N4-((R)-l-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-l-yl)-3,3-dimethyl-l-oxobutan-2-yl)succinamide (L3 PROTAC):

[0247] To the solution of 5 (35 mg, 0.06 mmol) in 2mL DCM, TFA(2mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF(5mL), VHL-acid (37 mg, 0.07 mmol), HATU (27 mg, 0.07 mmol) and DIPEA (39 mg, 0.3 mmol) were added and continued the reaction for 2h at room temperature and wasmonitored by TLC. The reaction mixture was diluted with cold water and ethyl acetate. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system with a reverse phase Cl 8 column to afford L3 PROTAC (27 mg, 45%) as a white solid.XH NMR (400 MHz, CDCh): 8 9.02(s, 1H), 7.43(s, 1H), 7.41(s, 1H), 7.37-7.34(m, 6H), 7.07-7.05(m, 1H), 6.98-6.96(m, 2H), 6.93-6.91(m, 2H), 6.89-6.86(m, 2H), 6.85(s, 1H), 6.21(s, 1H), 4.85(s, 2H), 4.72-4.68(m, 1H), 4.60-4.5 l(m, 2H), 4.46-4.43(m, 1H), 4.37-4.32(m, 2H), 3.95(t. 2H, J= 6), 3.86(s, 2H), 3.62-3.58(m, 1H), 3.31-3.26(m, 2H), 3.15(s, 6H), 2.54-2.52(m, 4H), 2.48-2.42(m,2H), 2.19-2.14(m, 1H), 1.80-1.75(m, 2H), 1.71-1.64(m, 2H), 0.95(s, 9H). °CNMR(100MHz, CDCh): 6 172.5, 171.8, 171.0, 166.8, 161.3, 159.2, 155.8, 151.6, 148.8, 146.4, 143.7, 138.9, 134.4, 133.0, 130.9, 129.5, 129.3, 128.3, 121.6, 119.6, 118.0, 115.7, 70.2, 67.9, 58.6, 58.2, 56.7, 53.3, 45.5, 43.2, 41.8, 39.5, 34.9, 31.3, 31.2, 26.4, 14.9. HRMS (m / z): [M+H] + calcd. for C53H65CIN7O8S, 994.4304; found: 994.4299.Preparation of (2R,4R)-l-((R)-2-(tert-butyl)-18-(4-(4-(2-chloro-N-(4 (dimethylamino)benzyl) acetamido) phenoxy)phenoxy)-4,13-dioxo-7,10-dioxa-3,14-diazaoctadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PEG L3 PROTAC):

[0248] To the solution of 5 (35 mg, 0.06 mmol) in 2mL DCM, TFA(2mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF(5mL), (S, R, S)-AHPC-PEG2-acid (43 mg, 0.07 mmol), HATU (27 mg, 0.07 mmol) and DIPEA (39 mg, 0.3 mmol) were added and continued the reaction for 2h at room temperature and was monitored by TLC. The reaction mixture was diluted with cold water and ethyl acetate. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system with a reverse phase C18 column to afford PEG L3 PROTAC(39 mg, 60%) as a white solid. 'H NMR (400 MHz, CDCh): 6 9.29(s, 1H), 8.21(sbr, 3H), 7.53- 7.5 l(m, 2H), 7.46- 7.43(m, 5H), 7.40- 7.38(m, 2H), 7.02- 6.98(m, 3H), 6.96(s, 1H), 6.93- 6.90(m, 3H), 4.90(s, 2H), 4.71- 4.65(m, 2H), 4.61- 4.59(m, 2H), 4.41- 4.36(m, 1H), 4.16- 4.13(m, 1H), 3.97(t, 2H, J= 6), 3.91(s, 2H), 3.76- 3.73(m, 4H), 3.65- 3.63(m, 4H), 3.33- 3.32(m, 2H), 3.23(s, 6H), 2.60(s, 4H), 2.57- 2.52(m, 4H), 2.37- 2.28(m, 1H), 1.83-1.78(m, 2H), 1.73- 1.68(m, 2H), 1.03(s, 9H).13C NMR (100 MHz, CDCh): 6 173.4, 171.6, 167.2, 160.7, 160.3, 159.3, 155.8, 152.7, 148.7, 144.3, 142.4, 139.7, 138.8, 134.4, 134.2, 131.1, 129.3, 129.2, 128.3, 121.6, 120.5, 118.0, 116.8, 115.7, 114.0, 70.2, 70.0, 67.8, 67.1, 66.8, 59.3, 58.2, 53.3, 46.4, 43.1, 41.7, 39.5, 36.9, 36.2, 35.9, 35.2, 26.5, 26.4, 26.3, 25.9, 13.9. HRMS (m / z): [M+H] + calcd. for C57H73CIN7O10S, 1082.4823; found: 1082.4863.Preparation of Nl-(4-(4-(4-(2-chloro-N-(4-(dimethylamino) benzyl) acetamido) phenoxy) phenoxy) butyl)-N16-( (R)-l-( (2R, 4R)-4-hydroxy-2-( (4- (4-methylthiazol-5-yl) benzyl) carbamoyl) pyrrolidin-l-yl) -3, 3-dimethyl-l-oxobutan-2-yl) -4, 7, 10, 13 tetraoxahexadecanediamide (PEG L4 PROTAC):

[0249] To the solution of 5 (35 mg, 0.06 mmol) in 2mL DCM, TFA(2mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with DCM and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF(5mL), (S, R, S)-AHPC-PEG 4-acid (49 mg, 0.07 mmol), HATU (27 mg, 0.07 mmol) and DIPEA (39 mg, 0.3 mmol) were added and continued the reaction for 2h at room temperature and was monitored by TLC. The reaction mixture was diluted with cold water and ethyl acetate. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system with a reverse phase C18 column to afford PEG L4 PROTAC (35 mg, 50%) as a white solid. 'H NMR (400 MHz, CDCh): 89.05(s, 1H), 7.68- 7.61(m, 2H), 7.59- 7.57(m, 1H), 7.52- 7.47(m, 3H), 7.38- 7.36(m, 6H), 7.19- 7.14(1H), 6.99- 6.87(m, 5H), 4.85(s, 2H), 4.70- 4.59(m, 1H), 4.57- 4.51(m, 3H), 3.94(t, 2H, J = 6), 3.86(s, 2H), 3.73- 3.70(m, 4H),3.66- 3.61(m, 13H), 3.32- 3.27(m, 2H), 3.16(s, 6H), 2.55- 2.52(m, 7H), 2.43- 2.36(m, 1H), 2.24- 2.22(m, 1H), 1.79(t, 2H, J = 6), 1.68(t, 2H, J= 6), 0.96- 0.93(m, 9H). °CNMR(100 MHz, CDCh): 6 172.9, 172.7, 171.6, 171.2, 166.9, 160.8, 160.4, 159.3, 155.9, 152.0, 148.6, 143.1, 139.2, 137.8, 134.3, 132.6, 132.2, 132.1, 131.0, 129.4, 129.2, 128.8, 128.7, 128.3, 121.6, 120.1, 118.0, 115.7, 70.1, 70.0, 69.9, 67.8, 67.2, 66.9, 58.9, 58.0, 57.0, 53.3, 45.9, 43.1, 41.8, 39.3, 36.4, 36.3, 36.1, 35.1, 26.5, 26.4, 26.0, 14.6. HRMS (m / z): [M+H] + calcd. for C61H81CIN7O12S, 1170.5352; found: 1170.5333.Preparation of tert-butyl 4-(4-((4-(4-(4-(2-chloro-N-(4 (dimethylamino)benzyl)acetamido) phenoxy)phenoxy) butyl)amino)-4-oxobutanoyl)piperazine-l-carboxylate (7):

[0250] To the solution of 5 (292mg, 0.5 mmol) in 5mL DCM, TFA(5mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF(5mL), compound 6 (172 mg, 0.6 mmol), HATU (228 mg, 0.6 mmol) and DIPEA (261 pL, 1.5 mmol) were added and continued the reaction for 2h at room temperature and was monitored by TLC. The reaction mixture was diluted with cold water and DCM. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by silica gel chromatography (5% MeOH / DCM) to give compound 7 (281 mg, 75%) as a white solid. 'H NMR (400 MHz, CDCh): 8 7.47- 7.45(m, 2H), 7.40-7.38(m, 2H), 6.99- 6.97(m, 2H), 6.94- 6.92(m, 2H), 6.89- 6.88(m, 4H), 4.86(s, 2H), 3.95(t, 2H, J= 6), 3.87(s, 2H), 3.58(t, 2H, J= 6), 3.47(s, 4H), 3.40(t, 2H, J= 6), 3.33- 3.3 l(m, 2H), 3.18(s, 6H), 2.73- 2.70(m, 2H), 2.59- 2.56(m, 2H), 1.82- 1.79(m, 2H), 1.72- 1.68(m,2H), 1.46(s, 9H), 1.36- 1.34(m, 1H), 1.23- 1.21(m, 1H).13C NMR (100 MHz, CDCh): 6 173.5, 170.9, 167.0, 160.7, 160.4, 159.4, 156.0, 154.6, 148.6, 142.8, 138.4, 134.3, 131.0, 129.2, 121.6, 120.4, 118.0, 115.7, 80.7, 67.8, 53.3, 46.2, 45.3, 41.8, 41.7, 39.5, 31.2, 28.8, 28.3, 26.5, 26.0, 20.5. HRMS (m / z): [M+H] + calcd. for C40H53CIN5O7, 750.3634; found: 750.3632.

[0251] Preparation of (2R,4R)-l-((R)-2-(4-(4-(4-((4-(4-(4-(2-chloro-N-(4-(dimethylamino)benzyl) acetamido)phenoxy)phenoxy)butyl)amino)-4-oxobutanoyl)piperazin-l-yl)-4-oxobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (piperazine L3 PROTAC):

[0252] To the solution of 7 7(39 mg, 0.05 mmol) in 2mL DCM, TFA(2mL) was added dropwise and stirred the reaction for 1 h at room temperature. After 1 h, the reaction mixture was distilled under reduced pressure. The crude was diluted with dichloromethane and distilled under reduced pressure (3 times). Then to the solution of this crude product in DMF(5mL), VHL-acid (32 mg, 0.06 mmol), HATU (23 mg, 0.06 mmol) and DIPEA (26pL, 0.15 mmol) were added and continued the reaction for 2h at room temperature and was monitored by TLC. The reaction mixture was diluted with cold water and ethyl acetate. The organic layer was separated, dried over Na2SO4 and distilled under reduced pressure. Then crude was purified by preparative HPLC with a gradient of 0-60% ACN / H2O in 30 min system with a reverse phase C18 column to afford piperazine L3 PROTAC (29 mg, 50%) as a white solid. 'H NMR (400 MHz, CDCh): 8 8.87(s, 1H), 7.52- 7.40(m, 1H), 7.28-7.26(m, 6H), 7.18(s, 2H), 6.91- 6.89(m, 2H), 6.86- 6.83(m, 2H), 6.81- 6.78(m, 4H), 6.21-6.15(m, 1H), 4.76(s, 2H), 4.46- 4.41 (m, 2H), 4.31- 4.25(m, 1H), 3.95- 3.93(m, 1H), 3.86(t, 2H, J = 6), 3.78(s, 2H), 3.54- 3.51(m, 2H), 3.47- 3.39(m, 4H), 3.21(t, 2H, J = 6), 3.05(s, 6H), 2.71- 2.59(m, 2H), 2.55- 2.45(m, 7H), 2.33- 2.27(m, 1H), 2.10(s, 1H), 1.72- 1.70(m, 2H), 1.62- 1.60(m, 2H), 0.88(s, 9H).13C NMR (100 MHz, CDCh): 6 172.8, 172.7, 171.7, 171.2, 171.0, 170.9, 170.8, 166.8, 161.2, 160.8, 159.2, 155.9, 151.6, 148.7, 143.8, 139.0, 136.5, 134.4, 130.9, 129.4, 129.3, 128.2, 121.6, 119.4, 117.9, 117.2, 115.7, 114.3, 70.1, 67.8, 58.8, 58.0, 56.9, 53.3, 45.4, 44.9, 43.1, 41.9, 41.6, 39.3, 36.6, 35.1, 31.1, 30.0, 26.4, 14.9. HRMS (m / z): [M+H] + calcd. for C61H77CIN9O10S, 1162.5203; found: 1162.5209.MaterialsAntibodies:

[0253] aRNF4 antibody (1:200, sc-517643) and mouse a-tubulin (1:2000, SC-5286) were obtained from Santa Cruz Biotechnology. a-HA antibody was a kind gift from Ami Aronheim. p-c-Myc (1:500, #94015), p-P-catenin (1:300, #9564) and Ferroptosis Antibody Sampler Kit (#29650) were obtained from Cell Signaling Technology.Cultured cell line transfections and infections:

[0254] A375R, HaCat, SCC1, HEK293, MEF and 143B were maintained in DMEM with 100 U / ml penicillin, 0.1 mg / ml streptomycin, Glutamine 4mM, and 10% FBS. A375R was cultured in DMEM with 10% FCS, penicillin / streptomycin, and 2 pM PLX4032. Cells were transfected using CalFectin transfection reagent (Sinagen Laboratories) according to manufacturer instructions. BRAF inhibitor PLX4032 was purchased from Selleckchem.Plasmids:

[0255] pcDNA3 HA-hRNF4 were generated by Thomas et al. 2016. pcDNA3 RNF4C51Aand pcDNA3 RNF4C91A: mutants were generated by quickchange sitedirected mutagenesis (SDM), using the RNF4C51A site directed mutagenesis (SDM) primers (#1,2) and RNF4C91A SDM primers (#3,4). Point mutations were performed on RNF4 where cystein at 51 and 91 were replaced by alanin to generate RNF4C51A, RNF4C91Aand RNF4C51A-C91A.Primers: Primers used in this study are listed below (Table 1)

[0256] Table 1 : Primers used in this study>>>>MethodsCell Viability Assay (ATP -Lite assay)

[0257] Cell viability was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega). In brief, cells were seeded in triplicates for each time-point, in white 96-well plates with transparent bottom and cultured for the indicated time. 30 microlitersCellTiter-Glo® solution were added to each well and viability was quantified by monitoring luminescenceintensity using Plate-Reader.Sphere formation assay

[0258] A375R, SCC1, MEF, 143B and HacaT cells (lOOOcells / well) were seeded in 6-well plates in 2ml DMEM medium, maintained at 37°C in humidified incubator. After 8 days, cells were washed with PBSX1 genetly once and fixed for 1 hour with 5% formaldehyde, washed thrice with PBSxl, stained with 0.05% crystal violet for 20 min, photographed and counted.Cell lysates preparation (RIPA lysis buffer)

[0259] Cells were washed with ice-cold PBSxl and harvested in RIPA buffer supplemented with protease and phosphatase inhibitors. Samples followed by 1 pulse sonication of 37% in a microtip for 10 seconds and then centrifuge for 20 minutes at 13,000rpm. Protein concentrations were determined by Bradford Reagent. Proteins were resolved over SDS-PAGE, identified using indicated antibodies, and visualized using chemiluminescence (ECL, Image-Quant LAS4000). Quantification was performed using ImageQuant software, and fold changes in protein levels were calculated relative to either tubulin.SDS-PAGE and western blot analysis

[0260] Protein samples were boiled with SDS-PAGE sample buffer at 95°C for 5 minutes and then resolved on a 10% or 12.5% SDS-polyacrylamide gel with Tri s-Gly cine- SDS buffer and transferred to nitrocellulose membrane after electrophoretic separation. Membranes were blocked for 1 hour at room temperature and incubated overnight at 4°C with the designated primary antibody. Next, membranes were subjected to HRP-conjugated secondary antibodies at 1:10,000 for 1 hour, washed with TBSTxl, and imaged by Enhanced Chemiluminescence (ECL, Image-Quant LAS4000, GE Healthcare).Site-Directed Mutagenesis (SDM)

[0261] Sdm was performed using QuikChange II Site Directed Mutagenesis Kit. Mutagenic primers were designed according to the desired mutation required and ordered. The SDM reaction mix was prepared with 5pl of 10X reaction buffer, 50ng of plasmid DNA, 1.25pl of forward primer, 1.25pl of reverse primer, Ipl of dNTP mix, Ipl of Pfu Ultra HF DNA polymerase (2.5 U / pl), to a total volume of 50pl (ddH2O). PCR conditions used were 95°C x 30 sec, and 18 cycles of 95°C x 30 sec, 55°Cx1 min and 68°Cx1 min / kb. The amplified reaction was cooled and Ipl of Dpn I restriction enzyme (lOU / pl) was added to digest parental DNA. Dpn treated DNA was incubated at 37°C for Ihr,transformed to electro-competent e.coli and transformation plates were incubated at 37°C overnight. Later, DNA was extracted from each colony using Plasmid Mini Prep Kit and sequenced to identify the mutated clones.RNA-seq and data analyses

[0262] A set of three conditions using A375R cells; The aim was to investigate DEGs between A375R WT cells and cells treated with 10pm for 2h with R4VPL3-1 (full PROTAC) or with VHL only(partial molecule). Each treatment group contains 3 replicates. Each group were subjected to RNA extraction according to the MACHEREY-NAGEL NucleoSpin RNA, Mini kit for RNA purification protocol. The quality of the RNA was evaluated using the TapeStation 4200 (Agilent) with the RNA kit (cat no. 5067- 5576). The RINe values of all samples were in the range of 9.3-10, indicating high quality. Libraries were constructed simultaneously using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, cat no. E7760), according to the manufacturer protocol. 800ng total RNA was used as starting material. mRNA pull-down was performed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, cat no. E7490). RNA-seq library QC was performed by measuring library concentration using Qubit (Invitrogen) with the dsDNA HS Assay Kit (Invitrogen, cat no. Q32854) and size determination using the TapeStation 4200 with the High Sensitivity D1000 kit (cat no. 5067-5584). All libraries were then mixed into a single tube with equal molarity. The RNA-seq data was generated on Illumina NextSeq2000, using P2 100 cycles (Readl-100; Indexl-8; Index2-8) (Illumina, cat no. 20046811). Single reads (100 bps) were aligned to the Homo sapiens (GRCh38.109) reference genome using STAR (V2.5.3a) with mismatch ratio allowed < 0.2, the minimum and maximum intron sizes were set to 20 and 1,000,000, respectively. The number of reads per gene was counted using HTSeq-count (v2.0.2) with ‘reverse’ mode. Normalization and differential expression analyses were conducted using DESeq2 R package (vl.36.0). The similarity between samples was evaluated within DESeq2 package using a euclidean distance matrix (shown in a heatmap with a clustering dendogram) and a principal component analysis (PC A). The latter is drawn from the most variable genes (50, 500, 1000, 2000, 5000, 10000 genes). The threshold for significantly differentially expressed genes is determined by two factors: FDR adjusted p-value < 0.05 (for mouse B16 experiment a p-value < 0.01 threshold was specifically chosen since the DE analysis yielded over 4000 differential genes) and the ‘base-mean independent filtering’ threshold, which is calculated by the DESeq2 algorithm for each comparison. FDR was calculated using the default approach of DESeq2, Benjamini -Hochberg (BH). Since in both experiments, no majorbatch effect was indicated, a simple single-factor model was applied and lists of DEGs were created for all the possible contrasts.Brief summary of the results

[0263] We developed advanced Protein targeting chimeras-like compounds (PROTACs-like) as a ferroptosis-inducing compounds that lead to the elimination of RNF4 a ubiquitin ligase and its stabilized oncoproteins termed R4VPs termed R4VPL3-1 and R4VPL3-3 and their potential derivatives, (Fig. 1C, and IF).

[0264] The compounds of the invention (also used herein as “R4VPs”) are double-headed molecules (conjugates) that on one hand bind to the protein of interest (POI) and on the other hand bind to an E3-ligase recruiter, that are connected via a short linker. This proximity results in the ubiquitination and subsequent proteasomal degradation of the POI. In this case our R4VPs, bind the VHL E3 ubiquitin ligase on one hand and RNF4 on the other hand. RNF4 is the key enzyme in an oncoprotein stabilization pathway and is also pivotal for DNA repair making an important target in cancer (Figs. 1 A, B). R4VPs induce the proteasomal degradation of RNF4 and reduces the levels of the oncoproteins that RNF4 stabilizes (Fig. 1).

[0265] Biologically, R4VPs compounds have activity toward a wide range of cancer cells. R4VPs induces rapid lipid-peroxidation and subsequent ferroptosis of multiple cancer cells but are harmless to non-tumorigenic and primary cells like MEFs (Fig. 1, Fig. 11, Fig. 2, Fig. 3). Ferroptosis is a distinct form of cell death that involves an increase in free cellular iron, and oxidative stress culminating to lipide peroxidation of the plasma membrane, swelling, rupture of the membranes and cell death. Ferroptosis can be inhibited by Ferrostatin 1 (Ferr-1).

[0266] Surprisingly, R4VPs-induced ferroptosis is likely independent of RNF4, but is inhibited by Ferr-1 (Fig. 3). Remarkably, R4VPs preferentially targets tumor-driving mutations, particularly those in the EGFR pathway, while not affecting PI3K-transformed cells (Fig. 4). We characterized the activity and selectivity of R4VPs in-depth including, their mode of action and impact on gene expression on human cancer cells (Fig.3 A-C, Fig 4).

[0267] A key feature of R4VPs is their activity toward hard to treat cancer and specifically sarcomas. R4VPs effectively induce cell death in human therapy -resistant melanoma and sarcomas including primary patient-derived sarcoma tumor cells (Figs. 3 and 5). Initial EDMA and PK studies suggest that R4VPs are stable in the plasma of CD1 mice with half-life greater than 8 hours, and are degraded by liver microsomes. Moreover, in a pilot sarcoma xenograft experiment treatment of R4VPs of human sarcoma tumors cells injected to the femur of SCID mice results in significant inhibition of tumor development (Figure 6).

[0268] Thus, R4VPs can be used as: 1. ferroptosis selective inducers in experimental systems in cellulo and in vivo. 2. therapeutic compound by local administration to affected joints exhibiting PVNS, and 3. therapeutic compound in multiple carcinoma entities (colon breast and others), Leukemias, melanomas including Receptor tyrosine kinase inhibitors resistant melanomas, aggressive osteosarcomas and soft-tissue sarcomas and other hard-to-treat cancers.EXAMPLE 1R4VP promotes the proteasomal degradation of RNF4:

[0269] RNF4 is highly expressed in multiple types of cancer cells and therefore was selected as a potential E3 recruiter. RNF4 binding moiety was discovered via a direct binding screen. As RNF4 is critical for cancer cell survival as a starting point, we tested whether eliminating RNF4 is a strategy for cancer cell eradication. As a starting point, we linked the CCW16 molecule (termed here R4B) to the established VHL moiety that recruits the VHL ligase using a short linker, creating a novel PROTAC that we termed R4VP (Fig.lC).

[0270] We evaluated the steady-state protein levels of endogenous RNF4 in murine B16F10 melanoma cells treated with R4VP. Treatment with R4VP (3h) but not VHL moiety alone resulted in a dose-dependent reduction in endogenous RNF4 level, that was inhibited in the presence of the proteasome inhibitor MG132 (Figure ID, IE).Anti-cancer activities of R4VP

[0271] We tested for anti-cancer activities of R4VP towards multiple cancer and non-tumorigenic cells; R4VP inhibited the proliferation and sphere formation (SFA) of SCC1 human squamous skin cancer cells and PLX4032, (a Vemurafenib® analog)-resistant melanoma cells (A375R). An inactive R4VP lacking the critical chloro atom that mediates the covalent reaction of R4VP to RNF4 showed no anti -proliferative activity. Notably, R4VP did not affect the non-tumorigenic human keratinocytes HeCat cell line (Figs. 11C- 111).Generation of advanced R4VP PROTACs

[0272] To improve the activity of R4VP we performed a focused in vitro screen of fifteen derivatives of CCW16 we (termed L1 / R4B). As detailed under Figs. 10, and 11 we identified L3 as an improved binder of RNF4. Since the structure of the linker region of PROTACs is critical for its function, we modified the original R4VP linker region generating different R4VPs [R4VPL3-1, R4VPL3-2, and R4VPL3-3 (Figure IF, Fig. 12). While R4VPL3-2 was inactive, R4VPL3-1 and R4VPL3-3 were highly active with potent anti-cancer activities but did not affect non-tumorigenic cells and MEFs. (Fig. 13).Induced protein degradation and anti-cancer activity of R4VPL3-1

[0273] Focusing on R4VPL3-1 we tested the impact of R4VPL3-1 on the protein level of endogenous RNF4 and its stabilized oncoproteins p-c-Myc and p-b-catenin. We observed a dose and time-dependent decrease in the levels of RNF4 and its stabilized proteins, where 5mM of R4VPL3-1 were sufficient for a 90% reduction in the level of RNF4, p-c-Myc, and p-b-catenin (Figure 1F-J). Next, we tested for R4VPL3-1 activity SCC1, a human squamous carcinoma ski cell line. R4VP3-L1 inhibited proliferation and SFA of these cells' with IC50 of ~0.3 mM (Fig. 1K-M). In contrast, in these concentrations, R4VPL3-1 showed only minimal effect on non-tumorigenic HaCat cells a human skin keratinocyte cell line.R4VPs are dual PROTCAS that in addition to degradation of RNF4 also induce the degradation of VHL an anti-ferroptotic E3 ubiquitin ligase:

[0274] R4VPs connect between two ubiquitin ligases, RNF4 and VHL. Therefore, we also tested whether R4VPs may co-force the degradation of VHL. Indeed, we found that R4VPL3-1 induces dose- and time-dependent reduction in the abundance of VHL protein (Fig.l Q-1T). Thus, this PROTAC eliminates both RNF4 canceling oncoprotein addiction and VHL that is a potent anti ferroptotic E3 ligase impinging on two major cancer cells vulnerabilities.R4VPL3-L1 induced rapid cell death of RTKi-resistant melanoma cells.

[0275] R4VPL3-1 also inhibited the proliferation and SFA of aggressive human A375R melanoma cells (Figure 2A-C). To understand the inhibitory effects of R4VPL3-1 on cell viability and SFA of A375R cells we performed FACS analysis using Annexin and Propidium Iodide (PI) staining. R4VPL3-L1 induced rapid cell death of A375R cells resulting in >50% cell death in a time and dose-dependent manner (Fig.2D-2G) .

[0276] We first mapped the binding site(s) of R4VPL3-1 to RNF4 using a biotin-tagged R4VPL3-1 where the VHL recruiting moiety was replaced with biotin (bio-R4VPL3-l . We identified that bio-R4VP3-l binds to Cys 51 and 91 within RNF4 using MS analysis (Fig.3H, indicated in Fig. IB). We generated RNF4 double mutants GST-RNF4C51S, C92S, as well as single mutants GST-RNF4C51S and GST-RNF4C51S. While single mutated had reduced binding to R4VPL3-1, the double mutant GST-RNF4C51S, C92S did not bind to the bio-R4VPL3-l in a “pull-down” binding assay in vitro (Fig. 2H) consistent with the MS analysis. Moreover, mutating other Cys residues within the RING domain of RNF4 did not abolish its interaction with bio-R4VPL3-l (Fig. 2H).

[0277] Next, we tested whether overexpression of RNF4 double mutant HA-RNF4C51 A, C92A termed RNF4DM protects A375R cells from R4VPL3-1 -induced cell death. We found that increasing doses of RNF4DM did not prevent R4VPL3-1 -induced cell death (Fig. 21). Therefore, we concluded that while R4VPL3-1 induces proteasomal degradation of RNF4 and reduces the levels of its stabilized proteins, the cell death induced by R4VPL3-1 treatment is not solely attributed to RNF4 proteasomal degradation.R4VPs are highly selective and do not impact non-tumorigenic cells:

[0278] We found that R4VPL3-1 had no activity towards non-tumorigenic HaCat keratinocyte cell line, or primary mouse embryonic fibroblasts (MEFs, Fig. 7A-7E) We observed that in contrast to A375R melanoma cells in HaCat cells the levels of RNF4, VHL, and RNF4 stabilized proteins p-c-Myc and p-b-catenin, were not reduced upon treatment with R4VPL3L-1 (Fig. 7C). Importantly R4VPL3-1 has significant anti-cancer activity towards human squamous cancer cells (SCC1), inhibiting proliferation and SFA of SCC1 cells with an IC50 of ~0.3 uM (Figs. 7F-7H).EXAMPLE 2R4VP3-1 induces ferroptosis.

[0279] To identify the cellular pathways involved in R4VPL3-1 -induced cell death and anti-cancer activities we performed RNA-seq comparing A375R cells treated for two hours by VHL-recruiter moiety or R4VPL3-1 (Fig. 3A-C). We identified 316 differentially regulated genes (DEGs) specific to R4VPL3-1 treatment. Of these genes, 191 showed enhanced expression and 125 exhibited reduced expression. KEGG analysis identified ferroptosis and necroptosis as prominent pathways transcriptionally regulated by R4VPL3-1. Ferroptosis is a form of regulated cell death characterized by lipid peroxidation and iron accumulation.

[0280] To validate that the cause of cell death induced by R4VPL3-1 is indeed due to ferroptosis we treated cells with either Z-VAD-FMK, a pan-caspase inhibitor, Chloroquine, a lysosomal inhibitor, and Ferrostatin-1 (Ferr-1), a specific ferroptosis inhibitor along withtreatment with R4VPL3-1. While Z-vad and Chloroquine did not inhibit R4VPL3-1-induced ferroptosis, Ferr-1 completely prevented R4VPL3-1 -induced cell death (Fig. 3D).

[0281] Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation and increase in free cellular iron. To determine whether R4VPL3-1 induces lipid peroxidation, a hallmark of ferroptosis, we treated A375R cells with 3mM R4VPL3-1, or 3mM R4VPL3-1 along with the ferroptosis inhibitor Ferrostatin-1 (lOmM Ferr-1). We used established markers for lipid peroxidation; A major product that is formed by lipid peroxidation is 4-Hydroxynonenal (4HNE), and we used a-4HNE antibody to monitor lipid-peroxidation. We identified that R4VPL3-1 lipid peroxidation as evident by positive a-4HNE immuno-staining, that was inhibited by pre-treatment with lOmM Ferr-1 (Fig. 3D). Our results, collectively, suggest that R4VP3L-1 induces ferroptotic cell death.

[0282] Cell death analysis using FACS further corroborated that R4VPs induced Ferroptosis cell death that is inhibited by the iron chelator DFX and Ferr-1 but not inhibitors of other cell death. Neither control (DMSO), Necrostatin, Z-VAD, or chloroquine failed to inhibit R4VPL3-1 -induced cell death. However, Ferr-1, or the iron chelator deferasirox (DFX) completely prevented R4VPL3-1 -induced cell death (Fig. 3G-3J). Along this line we observed that while exposure of the cells to 2mM sterosproine (STS), induced the cleavage of PARP that is a hallmark of apoptosis, R4VPL3-1 did not cause cleavage of PARP (Fig. 3K, 3L). We concluded that R4VPL3-1 induces ferroptotic cell death and likely no other cell death pathways.R4VPs induces lipid peroxidation:

[0283] In addition to our previous report, we find that lipid peroxidation induced by R4VPs is prevented by pre-treatment of cells with the anti -ferroptotic inhibitor Ferr-1: Moreover, we visualized live lipid peroxidation using the BODIPY-581 / 591-C11™ sensor to visualize lipid peroxidation. Upon lipid peroxidation, the Cl 1 -sensor shifts from nonoxidized state (red) to oxidized (green). As shown in Figure 3E, in control (DMSO) treated cells, the sensor is in its non-oxidized state, and the cells are mostly red, exhibiting only minimal oxidized signal (green). Upon exposure to lOOuM Cumene hydroperoxide, a known lipid-oxidation agent, the sensor molecules shift to green. Likewise, treatment with 3uM R4VPL3-1 resulted in a similar and significant spectral shift of the sensor molecules that was not observed by pre-treatment with lOuM Ferr-1 (Fig. 4E and quantified in 4F).Molecular determinant for R4VPL3-1 selectivity.

[0284] We were intrigued by the differential effects of R4VP and R4VPL3-1 toward killing cancer cells, but not affecting non-tumorigenic HaCat cells or primary MEFs. We,therefore, tested for inhibition of SFA upon treatment with the different R4VPL3-1 components, as well R4VPL3-1 where we replaced the VHL recruiter moiety with Biotin (Fig. 4A). RNF4 binding moiety, R4B inhibited SFA of both non-tumorigenic HaCat and primary MEFs as well as cancer cells. In contrast, R4VPL3-1 had potent activity towards cancer cells but had no activity against non-tumorigenic cells (Fig.4B-E). Moreover, replacing the VHL recruiting moiety with biotin resulted in an inactive compound that had no activity against cancer cells and non-tumorigenic cells (Fig. 4F-4H).

[0285] R4VPL3-1 had selective and potent activity toward cancer cells but had no activity against non-tumorigenic cells at concentration from 0.1 mM to 2mM (Fig. 8B-8E). Moreover, Biotin-R4VPL3-1 was an inactive compound with no activity against both cancer cells and non-tumorigenic cells (Fig. 4F-4I), suggesting that the VHL moiety of R4VPL3-ls critical for R4VPs anti -cancer activity.

[0286] Interestingly, we found that R4VPL3-1 has differential activity toward cancer cells depending on the oncogenic driving mutation used to induce cell transformation. We transformed Beas2B lung epithelial cells by lentiviral infection with activated oncogenes: BRAFV600A, EGFRL858R,or PI3KH1017R. We observed that R4B inhibited cell proliferation of all three kinds of tumor cells. However, the full PROTAC R4VPL3-1 inhibited the proliferation of BRAFV600A, EGFRL858R, transformed lung cancer cells, but did not inhibit PI3KH1017Rtransformed cells (Fig. 5J). Taken together this set of experiment suggests that the activity of R4VPL3 towards aggressive cancer cells requires the VHL recruiting moiety and is cancer-pathway specific.R4VPL3-1 activity in sarcomas

[0287] One unmet clinical need is the treatment for sarcomas. We observed that RNF4 is essential for sarcoma tumorigenicity, and high levels of RNF4 are observed in multiple sarcoma entities, and is associated with poor sarcoma patient survival. While other proteins may mediate R4VPs action we decided to test the impact of R4VPs on the metastatic human sarcoma cell line 134B. R4VP treatment resulted in the inhibition of proliferation and SFA of these cells (Figs. 5A-5C). Furthermore, R4VPL3-1 was potent towards primary patient-derived sarcoma and lung cells isolated during onco-surgical resections, inhibiting proliferation and sphere formation in culture with IC50 ~0.5mM (Fig. 5D-5K). Thus, R4V0L3-1 is highly active against sarcoma cells including diverse primary patients-derived tumor cells.

[0288] To this end, we developed ferroptosis-inducing PROTACs that eliminate multiple cancer cells including RTKi-resistant human melanoma cells, primary patient-derivedsarcoma, and lung cancer cells, depending on the tumor driving mutation. In contrast, non-tumorigenic cell lines and primary MEFs are not affected by R4VPs. R4VP treatment induces the proteasomal degradation of RNF4 and its stabilized p-oncoproteins. It also induces RNF4-independent ferroptosis, a cell death that was inhibited by co-treatment by the ferroptosis inhibitor Ferrostatin-1.

[0289] In our study, we used CCW16 (R4B) as a basis for a focused screen and discovered an RNF4 binder with higher affinity to RNF4. We also observed that the small changes within the linker region of R4VPs have significant effects on R4VPs activity.

[0290] Our MS analysis of the full R4VPL3-1 PROTAC identified that R4VPL3-1 binds to Cys 51 and 91 outside the RING domain of RNF4, an observation that was followed by molecular verification in a binding assay. Moreover, the VHL recruiting moiety of R4VPL3-1 was found to be essential for all R4VPL3-1 anti-cancer activities. When the VHL recruiting moiety was substituted with biotin, or the well-established recruiter moiety that binds to the E3 Crbn, these compounds were inactive. While RV4Ps efficiently induce the proteasomal degradation of RNF4, this case is not trivial, as in this case R4VPs bring into proximity two E3 ubiquitin ligases, and the outcome is empiric, with VHL E3 dominant in this case, resulting in RNF4 degradation.

[0291] All together, our results observations suggest that in R4VPL3-1 sensitive cancer cells ferroptosis inhibitors (not exclusively RNF4) are targeted by R4VPs to induce degradation thereof. R4VPs have activity against specific cancer cells and did not affect non-transformed cells. This differential activity may be dependent on the addiction of specific cancer cells to RNF4, or dependent on the presence of potential other ferroptosis inhibitors, or cell-type sensitivity or resistance to ferroptosis.

[0292] Sarcoma of different types including bone and soft tissue sarcomas present a clinical challenge. Albeit extensive efforts, and since the introduction of Cys-platinum in the early 1980s, no major improvement in patients survival was observed. Thus, our observation that R4VPL3-1 is effective in inhibiting proliferation and SFA formation of multiple sarcoma entities including patient-induced sarcomas is highly surprising.

[0293] Further, our observation that R4VP is a potent ferroptosis inducing PROTAC strongly suggests that compounds of the invention can be utilized for elimination of sarcomas and other aggressive cancers.Identification of GPX4 a major anti-ferroptotic and selanoproteins as a protein target bound by R4VPs.

[0294] We identified that selanoprotein and in particular GPX4 are targets of R4VPL3-1. They are modified and likely inactivated but not degraded by R4VPs. R4VPL3-1 covalently binds to GPX4 and induces a molecular shift in SDS-PAGE that is known to inactive these enzymes. Selanoproteins contain a Cys residue that is critical for and is likely blocked by R4VPs that targets Cys residues canceling its activity. The protein levels of (GPX4) in cells treated with R4VPL3-1 increased and migrated slower in the SDS-PAGE gel. Treatment with inactive R4VPL3-1 lacking the Chloro-atom that was biologically inactive and did not of GPX4, and neither in a molecular shift. (Figs. 8B-8D). Indeed, in a binding assay R4VPL3-1, but not the inactive mutant R4VPL3-1 chloro did bind to GPX4 (Fig. 8E). Moreover, the ferroptotic cell death induced by R4VPL3-1 was highly similar to the one induced by RSL3, a well-known ferroptosis inducer. Both compounds induced ferroptotic cell death that is inhibited by Ferr-1, and both R4VPL3-1, RSL3 did not induce cell death of HaCaT cells (Figs. 8F, 8G).

[0295] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0296] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

CLAIMS1. A compound, a salt, an isomer or a tautomer thereof, wherein said compound is represented by Formula 1 :L represents a linker;Y independently is selected from the group consisting of: NH, S, O and CH;R3is halo;each R represents H or an optionally substituted alkyl;each n is an integer being independently between 0 and 5;each R1represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -C0NH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -C0NH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Ci-C6alkyl), Ci-C6mercaptoalkyl, -CONH(CI-C6alkyl), -CON(CI-C6alkyl)2, -CO2H, -CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkyl-heteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; andAr represents an aryl or a heteroaryl.

2. The compound of claim 1, wherein L comprises [Cl -CIO alkyl-O]m, wherein m is an integer being independently between 1 and 20.

3. The compound of claim 1 or 2, wherein said A represents phenyl and wherein L comprises ethylene glycol oligomer.

4. The compound of any one of claims 1 to 3, wherein L is or comprises T-A-T-A-T, each A is independently selected from [Cl -CIO alkyl-O]m, alkyl and heteroalkyl; and T is absent or is independently from -O-, -S-, -NR’-, -C(=O)-, -C(=NR’)-, -C(=S)-, -CONR’-, -C(NR’)NR’-, -C(NR’)O-, -C(NR’)S-, -S-S-, -S-C(=O), -CNNR’-, -CSNR’-, -NR’C(=O)O-, -NR’C(=S)O-, -NR’C(=S)NR’-, -SO2-, -SO-, -OC(=O)-, -OC(=O)O-, -OC(=S)O-, and -OC(=S)NR’-; and wherein at least one A is [C1-C10 alkyl-O]m.

5. The compound of claim 4, wherein said compound is represented by Formula 2:wherein each k is independently between 0 and 20.

6. The compound of any one of claims 1 to 5, wherein said compound is represented by Formula 3:is -CONR’-; wherein each m and k independently between 1 and 10.

7. The compound of any one of claims 1 to 6, wherein Y is O.

8. The compound of any one of claims 1 to 7, wherein each R is a C1-C5 alkyl.

9. The compound of any one of claims 1 to 8, wherein each of said n independently represents an integer in a range from 1 to 5.

10. The compound of any one of claims 1 to 9, wherein said compound is represented by Formula 4:

11. The compound of claim 10, wherein m is between 2 and 5 and k is between 1 and 10.

12. A pharmaceutical composition comprising the compound of any one of claims 1 to 11, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, comprising a therapeutically effective amount of the compound.

14. The pharmaceutical composition of claim 12 or 13, for use in any one of: (i) reducing Ring Finger Protein 4 (RNF4) abundance, activity, or both; (ii) reducing Glutathione peroxidase 4 (GPX4) activity and (iii) inducing ferroptosis within a cell, wherein the cell is characterized by abnormal proliferation.

15. The pharmaceutical composition for use of claim 14, wherein said (i)comprises inducing proteasomal degradation of RNF4.

16. The pharmaceutical composition of claim 12 or 13, for use in the treatment of a cell proliferation disease in a subject.

17. The pharmaceutical composition for use of claim 16, wherein said cell proliferation disease is selected from cancer, pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS).

18. The pharmaceutical composition for use of claim 17, wherein said cancer is selected from the group consisting of: carcinoma, sarcoma, leukemia, melanoma, tyrosine kinase receptor inhibitors (RTKi) resistant-melanoma, immune-check points inhibitors (ICI) resistant-melanoma, aggressive osteosarcoma and soft-tissue sarcoma.

19. A method for treating Glutathione peroxidase 4 (GPX4)-related disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound represented by Formula 5:L represents a linker;Y independently is selected from the group consisting of: NH, S, O and CH;R3is halo;each n is an integer being independently between 0 and 5;each R1represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -C0NH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -C0NH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Ci-C6alkyl), Ci-C6mercaptoalkyl, -CONH(CI-C6alkyl), -CON(CI-C6alkyl)2, -CO2H, -CO2R, - OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkyl-heteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; andAr represents an aryl or a heteroaryl.

20. The method of claim 19, wherein the Glutathione peroxidase 4 (GPX4) -related disease is cancer.

21. A method for treating cancer, comprises selecting a subject having increased Glutathione peroxidase 4 (GPX4) abundance, activity, or both and administering to said subject a compound represented by Formula 5:L represents a linker;Y independently is selected from the group consisting of: NH, S, O and CH;R3 is halo;each n is an integer being independently between 0 and 5;each R1 represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -CONH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate,phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -C0NH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -C02R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Cl-C6 alkyl), C1-C6 mercaptoalkyl, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, C02H, CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkylheteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted Cl -CIO alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; and Ar represents an aryl or a heteroaryl.

22. A method for inducing ferroptosis within a cell of a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound represented by Formula 5:L represents a linker;Y independently is selected from the group consisting of: NH, S, O and CH;R3is halo;each n is an integer being independently between 0 and 5;each R1represents one or more substituents, each independently comprising alkyl, -NO2, -CN, -OR’, -OH, -C0NH2, HCONH-, oxo, carbonyl, amino, imino, thioxo, phosphate, phosphonate, phosphine, phosphite, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -C0NH-NH2, -NHCOR’, -NHCSR’, -NHCNR’, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’R’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -COR’, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, amino(Ci-C6alkyl), Ci-C6mercaptoalkyl, -CONH(CI-C6alkyl), -CON(CI-C6alkyl)2, -CO2H, -CO2R, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, alkyl-aryl, alkyl-heteroaryl or a combination thereof; and wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; andAr represents an aryl or a heteroaryl.

23. The method of claim 22, wherein said cell is characterized by abnormal proliferation.

24. The method of claim 22 or 23, wherein said cell is selected from a cancer cell and a synovial cell characterized by CSF1 overexpression.

25. The method of claim 24, wherein said cancer cell comprises any one of carcinoma cell, leukemia cell and melanoma cell.

26. The method of any one of claims 18-19 and 22 to 25, wherein the method is for treating a proliferative disease in said subject.

27. The method of claim 26, wherein said proliferative disease is selected from cancer, pigmented villonodular synovitis (PVNS) and pigmented villonodular tumor of the tendon sheath (PVNTS).

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